Biological Ice Nucleation and Its Applications [1st ed.] 9780890541722

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D epartm ent o f A gricultural C hem istry, U niversity o f T okyo, B unkyoku, T okyo 113, Japan

S o ic h i A r a i,

N . A s h w o r t h , D epartm ent o f H orticulture, P urdue U niversity, W est L a ­ fayette, Indiana 47907, U .S.A .

E dw ard

D ep artm en t o f H orticulture, O regon State U niversity, C orvallis, O regon 97331, U .S.A .

M ic h a e l J . B u r k e ,

D ep artm en t o f H orticulture, O regon State U niversity, C orvallis, O regon 97331, U .S.A .

T. H . H . C hen,

J o n P . C o sta n z o ,

D ep artm en t o f Z oology, M iam i U niversity, O xford, O hio 4 5 056,

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G regory M . F ah y,

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R a y F a ll,

A. F a l l o n , A m ersham International, W hite L ion R oad, A m ersham , B ucks, HP7 9L L , U.K .

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R obert

L.

G reen,

Idetek, Inc., 1245 R eam w ood A ve., Sunnyvale, C alifornia

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U S D A A gricultural R esearch S ervice and D epartm ent o f P lant Pathology, U niversity o f W isconsin, M adison, W isconsin 53706, U.S.A.

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W illia m T . T u c k e r ,

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K in g

P r e fa c e

T he phenom enon o f ice nucleation has both intrigued and dem anded the atten ­ tion o f biologists from a w ide range o f disciplines. P lant physiologists and crop scientists found it relev an t because o f the agricultural im portance o f frost-sensitivity. W hen ice nucleators in this context w ere found to be a sm all group o f epiphytic bacteria— the m ost po ten t o f heterogeneous ice-nucleating agents— they excited the interest o f m icrobiologists and biochem ists. A pplications suggested them selves, ranging from snow m aking, to food technology, to the use o f ina genes as reporters o f transcription and transduction. M oreover, bacterial epiphytes w ere discovered to be an im portant source o f ice nuclei in the atm osphere, and thus o f interest to m e­ teorologists. Ice nucleation also governs the w inter survival o f som e ectotherm ic anim als. M any freeze-intolerant insects avoid lethal freezing by depressing the tem perature at w hich internal ice nucleation can occur; conversely, som e freeze-tolerant form s synthesize ice-nucleating pro tein s to ensure that freezing happens at high subzero tem peratures. Sim ilarly, the tem perature o f nucleation is a critical factor in the cryopreservation o f cells and tissues. A lthough the p h enom enon o f heterogeneous ice nucleation had long been re c ­ ognized, it w as not know n until the 1970s that the m ost active ice nuclei in nature w ere o f biological origin. T his discovery led to a series o f conferences on this sub­ je c t (1982, San F rancisco, C alifornia, organized by L. M . K ozloff, S. E. L indow , and R. C. Schnell; 1984, Flagstaff, A rizona, by G. C aple and G. Layton; 1987, N ew port, O regon, by M . J. B urke and S. E. L indow ; 1989, Saskatoon, C anada, by L. V. G usta; 1991, M ad iso n , W isconsin by S. S. H irano and C. D. U pper; 1993, L aram ie, W yom ing, by G . V ali). T he idea and im petus for this book originated, at least in part, from discussions at these conferences. T he scientific literature related to ice nucleation in biological system s is un­ usually scattered ow ing to the diversity o f disciplines w ith interest in this subject. R eports have com e from the fields o f m eteorology, b acteriology, plant physiology, crop science, physiology o f cold to lerance in ectotherm ic anim als (particularly in ­ sects), and the application o f ice nucleation to m edicine, cryobiology, food science, and snow m aking. T he p u rp o se o f this book is to integrate for the first tim e in fo r­ m ation from each o f these areas to serve not only as a reference for researchers in v

the field b u t also one that will appeal to those with a m ore peripheral interest in the topic. W e hope th at this book w ill aid in the synthesis o f principles o f ice nucleation spanning a range from theoretical to applied aspects in bacterial, plant, and anim al system s. In addition, students new to this subject m ay use it as an accessib le starting point. S ince each o f these disciplines has developed its own, largely in d ependent, body o f literature, com plete w ith different sets o f term s for phenom ena that are com m on to all, we have included a glossary as an aid to interpretating the literature. D uring the p rep aratio n o f this book, we soon recognized, how ever, that it w as not possible to reconcile all points o f view w ith com m on definitions. W e thank our colleagues for their contributions. C hristina V ertucci m ade valu­ able suggestions for im proving the glossary. W e especially appreciate the efforts o f our editor, Joyce L oper, for her thorough review and cogent suggestions for im ­ proving this book. W e also gratefully acknow ledge C rop D evelopm ent C entre, D N A P lan t T echnology C orp., G en en co r International, Inc., N ational Institutes o f H ealth, N ational S cience F oundation, and the U nited States D epartm ent o f A g ricu l­ ture for th e ir support o f research in our laboratories.

Richard E. Lee, Jr., Gareth J. Warren, and Lawrence V. Gusta July, 1994

C o n te n ts

CHAPTER 1

1

P r in c i p le s o f I c e N u c l é a t io n

G a b o r Vali CHAPTER 2

29

T h e D is c o v e r y o f B a c t e r ia l I c e N u c l é a t i o n a n d I t s R o le in t h e I n j u r y o f P l a n t s b y F r o s t

C hristen D. U p p e r and G ab o r Vali CHAPTER 3

41

E c o l o g y o f I c e N u c l e a t i o n - A c t i v e B a c t e r ia

S u san S. H ira n o an d C h risten D. U p p e r CHAPTER 4

63

B io c h e m i s t r y o f B a c t e r i a l I c e N u c l e i

R ay F all and P aul K. W o lb e r CHAPTER 5

85

I d e n t i f ic a t i o n a n d A n a l y s i s o f

ina

G e n e s a n d P r o te in s

G areth J. W arren CHAPTER 6

101 M o l e c u l a r M o d e l i n g o f t h e T h r e e - D i m e n s i o n a l S t r u c t u r e o f B a c te r ia l I n a P r o te in s

A n d rey V. K a jav a CHAPTER 7

1 1 5 F r e e z in g T o l e r a n c e in P la n t s : A n O v e r v i e w

T. H . H. C h en , M . J. B u rk e, and L. V. G u sta vii

CHAPTER 8

137

I c e N u c le a tio n A c t iv it y A s s o c ia t e d w it h P la n ts a n d F u n g i

E d w a r d N . A s h w o rth a n d T h o m a s L . K ie ft CHAPTER 9

163

D e e p S u p e r c o o lin g in W o o d y P la n ts a n d th e R o le o f C e ll W a ll S tr u c tu r e

M ic h a e l W is n ie w s k i CHAPTER 10

183

D e e p S u p e r c o o lin g in B u d s o f W o o d y P la n ts

H . A . Q uam m e CHAPTER 11

201

T h e R o le s o f I c e N u c l e a t o r s in C o ld T o le r a n t I n v e r t e b r a t e s

J o h n G . D u m a n , T . M a r k O ls e n , K i n g L u n Y e u n g , a n d F re d Je rv a CHAPTER 12

221

S u p e r c o o lin g a n d I c e N u c le a t io n in V e r t e b r a t e E c t o t h e r m s

J o n P . C o s ta n z o a n d R i c h a r d E . L e e , J r . CHAPTER 13

239

C o n t r o l o f E p ip h y t ic I c e N u c le a t io n - A c t iv e B a c t e r ia fo r M a n a g e m e n t o f P la n t F r o st In ju r y

S te v e n E . L in d o w CHAPTER 14

257

B io lo g ic a l C o n tr o l o f I n s e c t P e sts U s in g I c e - N u c le a t in g M ic r o o r g a n is m s

R i c h a r d E . L e e , J r ., M a r c i a R . L e e , a n d J a n e t M . S tro n g -G u n d e rs o n CHAPTER 15

271

I c e N u c le a tio n G e n e s a s R e p o r te r s

N i c k o l a s J. P a n o p o u l o s CHAPTER 16

283

T r a n s d u c t io n o f

ina

G e n e s fo r B a c t e r ia l I d e n t if ic a t io n

P . K . W o lb e r , R . L . G r e e n , W . T . T u c k e r , N . M . W a t a n a b e , C . A . V a n c e , R . A . F a llo n , C . L in d h a rd t, a n d A . J. S m ith

viii

CHAPTER 17

299

A p p l i c a t i o n s o f B a c t e r i a l I c e N u c l e a t i o n A c t iv it y in F o o d P r o c e s s in g

M ic h ik o W a ta n a b e a n d S o ic h i A ra i CHAPTER 18

315

T h e R o le o f N u c le a t io n in C r y o p r e s e r v a t io n

G re g o ry M . F a h y CHAPTER 19

337

A p p lic a t io n s o f B io lo g ic a l I c e N u c le a t o r s in S p r a y -I c e T e c h n o lo g y

R i c h a r d J. L a D u c a , A . F r a n k l i n R i c e , a n d P a t r i c k J. W a r d 351

G lo ss a r y

363

In d ex

B io lo g ica l Ice N u cléa tio n a n d Its A p p lication s

CH APTER

1

P r in c ip le s o f I c e N u c lé a t io n Gabor Vali

H o m o g e n e o u s a n d H e te r o g e n e o u s I c e N u c le a tio n T h e rm o d y n a m ic e q u ilib riu m

b e t w e e n d i f f e r e n t p h a s e s o f w a t e r e x i s t s a lo n g

w e l l - k n o w n p r e s s u r e - t e m p e r a t u r e lin e s . C h a n g e s o f p h a s e ( liq u id

v a p o r, v ap o r

s o lid , a n d liq u id s o l i d ) ta k e p l a c e a t c o n d i t i o n s s lig h tly , o r s i g n i f i c a n t l y , a w a y f r o m t h e s e lin e s . T h e d e v i a t i o n s a r e u s u a l l y m i n o r f o r c h a n g e s f r o m t h e m o r e o r d e r e d to t h e le s s o r d e r e d s ta te s ( l i q u i d —> v a p o r , s o lid —» liq u id , a n d s o lid —» v a p o r ) b u t c a n b e v e r y l a r g e f o r th e r e v e r s e p r o c e s s e s , e v e n th o u g h th e s e c h a n g e s a r e to w a r d s t a t e s o f l o w e r f r e e e n e r g y . P h a s e c h a n g e s to w a r d l o w e r e n e r g y s ta te s o c c u r v i a m e t a s t a b l e s t a t e s ; t h e m o s t s i g n i f i c a n t o n e s , a n d t h e o n ly o n e s a d d r e s s e d in th is d i s c u s s i o n , a r e s u p e r s a t u r a t e d v a p o r a n d s u p e r c o o l e d liq u id w a te r . In g e n e r a l , m e t a s t a b l e s t a t e s a r e r e a c h e d f r o m t h e l i q u i d e i t h e r b y l o w e r i n g t e m ­ p e r a t u r e s o r b y c h a n g e s in p r e s s u r e . H o w e v e r , th e m e l t i n g p o i n t o f ic e v a r ie s so little w it h p r e s s u r e ( f o r t h e r a n g e o f p r e s s u r e s e n c o u n t e r e d a t th e s u r f a c e o f th e e a r th o r in t h e a t m o s p h e r e ) t h a t th e m e t a s t a b l e li q u i d s ta te is r e a c h e d , in p r a c tic a l te r m s , o n l y b y c o o l i n g . H e n c e i t is a v e r y g o o d a p p r o x i m a t i o n to r e f e r to w a t e r a t te m p e ra tu re s b e lo w 0 ° C a s s u p e rc o o le d . W a te r v a p o r b e c o m e s s u p e rs a tu ra te d w h e n c o o le d o r r a i s e d in p r e s s u r e p a s t t h e e q u i l i b r i u m li n e , w ith th e s a m e t e r m u s e d to d e s c r ib e t h i s s t a t e w h e t h e r t h e s ta b le p h a s e is t h e li q u i d ( a b o v e 0 ° C ) o r t h e s o lid ( b e lo w 0 ° C ) . F i g u r e 1 d e p i c t s t h e s e p h a s e c h a n g e s a n d t h e n a m e s m o s t c o m m o n l y u s e d to d e s c r i b e th e m . T h e p r o c e s s o f c o n v e r s i o n f r o m a m e t a s t a b l e s ta te to th e s ta b le p h a s e is in itia te d by

nucleation,

th e f i r s t a p p e a r a n c e o f a v e r y s m a ll v o l u m e o f th e n e w p h a s e . F o l ­

lo w in g th a t, t h e p h a s e c h a n g e is c o m p l e t e d b y t h e g r o w t h o f t h e s ta b le p h a s e u n til e i t h e r a ll o f t h e m a s s is c o n v e r t e d o r t h e t e m p e r a t u r e s t a b iliz e s a t th e p h a s e e q u i ­ l ib r iu m p o i n t . T h e g r o w t h p r o c e s s is c o n t r o l l e d b y th e r a t e o f r e m o v a l o f t h e l a te n t h e a t b e i n g r e l e a s e d . T h e n u c l e a t i o n s t e p is t h e t o p i c o f t h is c h a p te r . T h e k e y t o e n v i s a g i n g h o w n u c l e a t i o n t a k e s p l a c e a t th e m o l e c u l a r le v e l is th e f a c t th a t e v e n in d is p e r s e d p h a s e s t h e r e a r e s m a ll, t r a n s i e n t a g g r e g a te s o f t h e c o n ­ d e n s e d p h a s e . F o r e x a m p l e , in a v a p o r , s m a ll c l u s t e r s o f m o l e c u l e s e x is t in a d d itio n to th e s i n g l e m o l e c u l e s m a k i n g u p m o s t o f t h e g a s e o u s s ta te . T h e n u m b e r d i s t r i b u ­ tio n o f t h e a g g r e g a t e s o f d i f f e r e n t s iz e s is d e f i n e d a s a f u n c t i o n o f th e f r e e e n e r g y

1

2

Vali

o f th e c l u s t e r s b y a B o l t z m a n n t y p e d is tr ib u tio n . A s a f i r s t a p p r o x i m a t i o n , th e c lu s te r s c a n b e c o n s i d e r e d a s m i n u t e e n t i t i e s o f li q u id . S i m i l a r l y , in a li q u i d th e r e a r e c l u s t e r s o f m o l e c u l e s in a s o l i d l i k e c o n f ig u r a tio n . S in c e th e f r e e e n e r g y o f a c l u s t e r is h i g h e r th a n t h e s u m o f t h e e n e r g i e s o f th e m o l e c u l e s m a k i n g u p t h e c l u s ­ te r, th e r e is a n a t u r a l t e n d e n c y f o r t h e m to d i s i n t e g r a t e , le a d i n g to v e r y s h o r t lif e ­ tim e s . T h e e n e r g e t i c s o f c l u s t e r f o r m a t i o n c h a n g e s f o r v a p o r o r liq u id in a m e t a s t a b l e s ta te . T h e b u l k f r e e e n e r g y p e r m o l e c u l e is th e n l o w e r f o r m o l e c u l e s i n s i d e th e c l u s t e r th a n o u t s i d e it. T h e l o w e r b u l k e n e r g y is o p p o s e d b y th e e n e r g y in v o l v e d in c r e a t i n g t h e i n t e r f a c e b e t w e e n t h e c l u s t e r a n d th e d i s p e r s e d p h a s e , b u t t h e b a l a n c e c h a n g e s in f a v o r o f t h e b u l k e n e r g y a s t h e e m b r y o b e c o m e s la r g e r . B e y o n d a c e r ­ ta in s iz e , f u r t h e r i n c r e a s e s a c t u a l l y le a d to l o w e r in g to ta l f r e e e n e r g i e s , s o th a t g r o w th b e c o m e s m o r e lik e ly th a n d i m i n u t i o n . B e c a u s e o f t h is p o s s i b i l i t y , c l u s t e r s in a m e t a s t a b l e p h a s e a r e c a l l e d lik e ly t h a n d e c a y is c a l l e d th e

nucleus f o r nucleation.

embryos. T h e s iz e a t w h ic h critical size ; th e e m b r y o a t

g ro w th b e c o m e s m o re th a t p o in t b e c o m e s a

t h e f u r t h e r g r o w t h o f t h e c o n d e n s e d p h a s e ; a n d t h e e v e n t is c a lle d

LU

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O UJ C />

cc UJ CL

cn

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F ig u r e 1 . P h a s e tr a n s itio n s o f w a te r . A b o x o r c ir c le o n th e a rro w in d ic a te s th a t th e tr a n s itio n p r o c e e d s

v ia n u c le a tio n f ro m th e m e ta s ta b le f o rm o f th e p a re n t p h a se .

Principles of Ice Nucleation

3

T h e c r i t i c a l q u e s t i o n r e g a r d i n g n u c l e a t i o n , th e n , is W h a t c o n t r o l s th e g r o w t h o f e m b r y o s in t h e m e t a s t a b l e p h a s e ? T w o c a s e s m u s t b e d i s t i n g u i s h e d . T h e f i r s t is th e s im p le c a s e o f h a v i n g o n l y m o l e c u l e s o f t h e s u b s t a n c e ( w a te r ) to c o n s id e r . In th is c a s e , th e f o r m a t i o n a n d g r o w t h o f e m b r y o s is c h a r a c t e r i z e d b y a n i n c r e a s e in th e ir a v e r a g e s iz e a s c o n d i t i o n s m o v e f u r t h e r f r o m th e p h a s e e q u i l i b r i u m c u r v e a n d b y a d d i t i o n a l r a n d o m f l u c t u a t i o n s in e m b r y o s iz e d u e to t h e r a p id a t t a c h m e n t a n d d e ­ ta c h m e n t o f m o l e c u l e s a s s o c i a t e d w ith th e r m a l m o t i o n . F o r m a t i o n o f a c r i t i c a l s iz e e m b r y o in t h is c a s e is c a l l e d

homogeneous nucleation.

T h e o t h e r s itu a tio n to c o n ­

s id e r is t h a t in w h ic h e m b r y o s f o r m o n t h e s u r f a c e o f s o m e f o r e ig n m a te r ia l w it h w h ic h t h e m e t a s t a b l e s u b s t a n c e ( s u p e r c o o l e d w a t e r o r s u p e r s a t u r a t e d v a p o r ) is in c o n ta c t. A t t a c h m e n t o f a n e m b r y o to a f o r e i g n s u r f a c e , a

substrate ,

c a n i n c r e a s e its

s ta b ility b y i n c r e a s i n g t h e v o l u m e - t o - s u r f a c e r a t i o a n d b y r e p l a c i n g p a r t o f th e e m b r y o - p a r e n t p h a s e i n t e r f a c e w ith a n e m b r y o - s u b s t r a t e i n te r f a c e . T h is is t h e c a s e of

heterogeneous nucleation.

T h e r o l e o f th e s u b s t r a t e is t o m a k e t h e f o r m a t i o n o f a

c r itic a l e m b r y o p o s s i b l e a t a s m a l l e r s u p e r c o o l i n g , o r s m a l l e r s u p e r s a t u r a t i o n , th a n th a t n e e d e d f o r h o m o g e n e o u s n u c le a tio n . In b o t h t h e h o m o g e n e o u s a n d th e h e t e r o g e n e o u s c a s e , th e g r o w th o f ic e e m ­ b r y o s is g o v e r n e d b y t h e d y n a m i c s o f a d d i t i o n s o f w a t e r m o l e c u l e s to t h e e m b r y o . T h e p r o c e s s is n o t m o n o t o n i c , b u t o n e o f f lu c t u a t i o n s : m o l e c u l e s a tta c h a n d d e ta c h fro m th e e m b ry o a t ro u g h ly e q u a l a n d v e ry ra p id ra te s . M o s t m o le c u le s g e t a d d e d o r r e m o v e d s in g ly , a l t h o u g h s o m e g r o u p s o f f e w m o l e c u l e s m i g h t a tta c h o r d e ta c h a s a u n it. T h i s r a n d o m p r o c e s s g iv e s a f i n i t e p r o b a b i l i t y f o r l a r g e f l u c t u a t i o n s f r o m th e a v e r a g e s iz e to o c c u r a n d f o r th e c r it i c a l s iz e f o r n u c le a t i o n to b e r e a c h e d a n d e x c e e d e d , e v e n t h o u g h a d e c r e a s e in s i z e is m o r e li k e l y , o n t h e a v e r a g e , th a n a n in c r e a s e . O n c e t h e c r i t i c a l s iz e h a s b e e n r e a c h e d , t h e e n e r g y d e c r e a s e a s s o c ia te d w it h a n i n c r e a s i n g v o l u m e m a k e s t h e p r o b a b i l i t y o f g r o w t h m o r e lik e ly b u t s till n o t a b s o lu te ly c e r ta in . T h e q u a n t i t a t i v e d e s c r i p t i o n o f th e n u c le a t i o n p r o c e s s o u tlin e d a b o v e c o n s i s t s o f tw o p a r ts : c a l c u l a t i o n o f th e f r e e e n e r g y c h a n g e a s s o c i a t e d w ith th e f o r m a t i o n o f a n e m b r y o , a n d th e c a l c u l a t i o n o f t h e r a te o f c r itic a l e m b r y o f o r m a t i o n ( th e p r o b a b i l i t y o f n u c l e a t i o n ) . T h e f r e e e n e r g y o f a n e m b r y o h a s a n e g a t i v e t e r m p r o p o r t i o n a l to th e v o l u m e o f t h e e m b r y o a n d a p o s i t i v e te r m p r o p o r t i o n a l to th e s u r f a c e a r e a o f th e e m b r y o . A d i f f e r e n c e in d e p e n d e n c e o f th e s e t w o t e r m s o n t h e n u m b e r o f m o l e ­ c u le s in t h e e m b r y o ( to t h e th ir d a n d s e c o n d p o w e r s , r e s p e c t i v e l y , f o r h o m o g e ­ n e o u s n u c l e a t i o n ) l e a d s to a m a x i m u m

in th e f r e e e n e r g y

v s. s iz e c u r v e — th e

e n e r g y b a r r i e r to n u c l e a t i o n — a t th e c r itic a l siz e . F o r h e t e r o g e n e o u s n u c l e a t i o n th e f o r m u l a t i o n o f th e e n e r g y t e r m is m o r e c o m p le x . W i t h t h e e n e r g y v s . e m b r y o s i z e f u n c t i o n e s t a b l i s h e d , th e

nucleation rate

( th e

n u m b e r o f e m b r y o s p e r u n i t v o l u m e r e a c h i n g c r i t i c a l s iz e in a u n it o f tim e ) is c a l c u l a t e d a s t h e p r o d u c t o f a p r e e x p o n e n t i a l f a c t o r t h a t d e p e n d s o n th e k i n e t i c s o f m o l e c u l a r a t t a c h m e n t s t o t h e e m b r y o , a n d a f a c t o r t h a t c o n t a i n s th e e n e r g y o f a c r itic a l e m b r y o in t h e e x p o n e n t . In s c h e m a t i c f o r m , AE = v o lu m e e n e rg y + s u rfa c e e n e rg y

(1 )

and n u c l e a t i o n r a te ,

J-

k i n e t i c f a c t o r ■ e _A£*/tT

w ith A E* d e n o t i n g t h e m a x i m u m o f e q u a t i o n 1,

k

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th e B o ltz m a n n c o n s ta n t, an d

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te m p e r a t u r e in d e g r e e s K e lv in . E x p l i c i t f o r m s o f t h e s e e q u a t i o n s c a n b e f o u n d in

4

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m a n y t e x t s ( e .g ., A b r a h a m , 1 9 7 4 ; F le t c h e r , 1 9 6 2 , 1 9 7 0 ; H o b b s , 1 9 7 4 ; P r u p p a c h e r a n d K le tt, 1 9 7 8 ) u s i n g d i f f e r e n t a s s u m p t i o n s a n d r e f i n e m e n t s . I n g e n e r a l , o n e c a n r e f e r to t h is t h e o r e t i c a l f o r m u l a t i o n a s th e G i b b s - V o l m e r th e o r y o r , in a g e n e r ic s e n s e , a s t h e t h e r m o d y n a m i c / k i n e t i c a p p r o a c h . T h e m a in id e a s o f t h e th e o r y w e r e d e v e l o p e d a r o u n d t h e tu r n o f t h e c e n t u r y . I t p r o v i d e s a u s e f u l f r a m e w o r k f o r th e e x a m i n a t i o n o f n u c l e a t i o n p h e n o m e n a , th o u g h in th e c a s e o f h e t e r o g e n e o u s n u ­ c le a tio n s o m a n y v a r i a b l e s m u s t b e i n t r o d u c e d th a t r e s u l t s o f g e n e r a l v a l i d i t y a r e r a r e ly o b t a i n e d . F u n d a m e n t a l l i m i t a t i o n s o f th e t h e r m o d y n a m i c / k i n e t i c th e o r y a r is e f r o m h a v i n g to p r e s c r i b e s p e c i f i c e m b r y o g e o m e t r i e s ( s p h e r e s f o r t h e h o m o g e n e o u s c a s e , s p h e r i c a l c a p s o r d is k s f o r t h e h e t e r o g e n e o u s c a s e ) a n d f r o m t h e u s e o f v o l ­ u m e a n d s u r f a c e e n e r g y v a lu e s , w h i c h a r e , s tr ic tly s p e a k in g , v a l i d o n l y f o r t h e b u lk p h a s e s . O t h e r a p p r o a c h e s to t h e t h e o r y o f n u c le a tio n a r e b e i n g d e v e l o p e d , p r i n c i ­ p a lly b y e x t e n d i n g m o l e c u l a r i n t e r a c t i o n m o d e ls to m u l t i m o l e c u l a r c l u s t e r s , b u t th e a p p l i c a t i o n o f s u c h m o d e ls to w a t e r a n d ic e is s till v e r y lim ite d .

E m p ir ic a l R e s u lts o n H o m o g e n e o u s Ic e N u c le a tio n T h e s u p e r s a t u r a t i o n r e q u i r e d f o r c o n d e n s a t i o n v i a h o m o g e n e o u s n u c l e a t i o n is a r o u n d 4 5 0 % ( i.e ., a s a t u r a t i o n r a t i o 4 .5 tim e s h i g h e r th a n th e e q u i l i b r i u m v a l u e a t th e s a m e t e m p e r a t u r e ) . H o m o g e n e o u s f r e e z in g n u c le a tio n t a k e s p l a c e a t a p p r o x i ­ m a te ly - 4 0 ° C . H o m o g e n e o u s d e p o s i t i o n ( f r o m v a p o r , a t t e m p e r a t u r e s c o l d e r th a n 0 ° C ) , in f a c t , t a k e s p l a c e w ith t h e s e q u e n c e o f h o m o g e n e o u s c o n d e n s a t i o n f o l l o w e d by

hom ogeneous

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n u c le a tio n .

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f r e e z in g n u c l e a t i o n is o f p r a c t i c a l i m p o r ta n c e , s in c e th e - 4 0 ° C

hom ogeneous te m p e ra tu re re ­

q u ir e d is c o m m o n l y f o u n d a t t h e e a r t h s u r f a c e a n d in th e a t m o s p h e r e a n d c a n b e r e a d ily p r o d u c e d in th e l a b o r a to r y . T h e r e q u i r e m e n t s q u o t e d a b o v e f o r h o m o g e n e o u s ic e n u c l e a t i o n n e e d to b e q u a l i f i e d in tw o w a y s . F ir s t, t h e i r m e a n i n g m u s t b e m a d e m o r e p r e c i s e b y s p e c i f y ­ in g th e m in t e r m s o f a n u c l e a t i o n r a t e . S e c o n d , d i s a g r e e m e n t s a m o n g e x p e r i m e n t a l r e s u lts a n d u n c e r t a i n t i e s in t h e o r e t i c a l v a lu e s n e e d to b e c o n s i d e r e d . N u c l e a t i o n r a t e c a n b e v i e w e d e i t h e r a s th e n u m b e r o f c r i t i c a l e m b r y o s t h a t f o r m in a v o l u m e o v e r a g i v e n le n g th o f t i m e o r a s th e p r o b a b i l i t y p e r u n i t t i m e a n d u n it v o l u m e t h a t a c r i t i c a l e m b r y o w ill f o r m . T h e la tte r v ie w is m o r e u s e f u l , s in c e th e f o r m a t i o n o f o n e e m b r y o is u s u a l l y s u f f i c i e n t f o r p h a s e t r a n s f o r m a t i o n to p r o c e e d in th e e n t i r e v o l u m e . I n itia l ic e g r o w t h in s u p e r c o o le d w a t e r is s o r a p i d th a t th e f o r m a t i o n o f c r i t i c a l e m b r y o s b e y o n d th e f ir s t is p r a c t i c a l l y e x c l u d e d , e x c e p t f o r s a m p le s c o o l e d e x t r e m e l y f a s t. T h u s , th e p r o b a b i l i t y t h a t a v o l u m e c o o le d w a t e r w ill f r e e z e in ti m e A i is a t te m p e ra tu re

T.

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T h e c o n s e n s u s o f e m p ir ic a l r e s u lts is c h a r a c t e r i z e d ( G o t z e t a l.,

1991 , 1 4 2 ) b y th e e q u a tio n

J(T) w h e re th e

J

= 6 .8 x 10 -50 e -3 9 r

is in u n i t s o f c u b i c m e t e r s p e r s e c o n d a n d

e q u a t i o n d e m o n s t r a t e s , th e r a te o f in c r e a s e o f

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in d e g r e e s C e l s i u s . A s th e

w ith d e c r e a s i n g t e m p e r a t u r e is v e r y

r a p id , in c r e a s i n g a t a f a c t o r o f n e a r l y 5 0 f o r e a c h 1 ° C l o w e r i n g o f te m p e r a t u r e . T h is is w h y a s i n g l e t e m p e r a t u r e , —4 0 ° C , is q u i t e u s e f u l f o r d e f i n i n g t h e p o i n t o f h o m o g e n e o u s ic e n u c l e a t i o n , a n d w h y th e r e w ill b e o n ly s m a ll, t h o u g h q u a n t i f i ­ a b le , c h a n g e s in t h a t t e m p e r a t u r e d u e to v a r ia tio n s in s a m p l e v o l u m e o r in t h e r a te

Principles of Ice Nucleation

threshold temperature

o f c o o lin g . T h e w h ic h

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1 c m -3 s_1 -

5

is c u s t o m a r i l y d e f i n e d a s th e t e m p e r a t u r e a t

1 0 6 m ~ 3 s -1 .

E x p e r i m e n t a l t e c h n i q u e s u s e d f o r t h e s tu d y o f h o m o g e n e o u s ic e n u c le a t i o n w ill b e d i s c u s s e d l a t e r in t h i s c h a p t e r . A t t h i s p o in t, o n l y t h e c a v e a t w ill b e m a d e th a t th e t h r e s h o l d t e m p e r a t u r e o f h o m o g e n e o u s n u c l e a t i o n is k n o w n w ith in , p e r h a p s , ± 3 ° C . T h e u n c e r t a i n t y a r i s e s f r o m d i f f i c u l t i e s in r e l a t i n g o b s e r v a t i o n s to a n u c l e a ­ tio n r a te , f r o m c o r r e c t i o n s f o r t h e v o l u m e d i s t r i b u t i o n s o f e m u l s i o n s , f r o m c o o l i n g r a te e f f e c t s , a n d f r o m a v a r i e t y o f a r t i f a c t s . I f h o m o g e n e o u s f r e e z i n g is t o b e c o n s i d e r e d in a p r a c t i c a l s itu a tio n , a p p l i c a t i o n o f e q u a tio n 3 , a n d th e d e fin itio n o f

J

p r e c e d i n g i t in t h e te x t, o f f e r a b a s i s f o r p r e ­

d i c t i n g t h e r a t e o f n u c l e a t i o n f o r a g i v e n te m p e r a t u r e . S tr ic tly s p e a k in g , t h a t t e m ­ p e ra tu re c a n n o t b e r e a c h e d in s ta n ta n e o u s ly , a n d th a t m a k e s th e a p p lic a tio n o f e q u a t i o n 3 m o r e d i f f i c u l t . H o w e v e r , t h e s tr o n g v a r i a t i o n o f

J

w ith

T

m a k e s re fin e ­

m e n ts a i m e d a t a l l o w i n g f o r th e t r a n s i e n t s le s s i m p o r t a n t . A s a s im p le r r e s u lt, it is u s u a lly s u f f i c i e n t to v i e w t h e t e m p e r a t u r e o f - 4 0 ° C a s t h e l i m i t f o r th e s u p e r c o o l ­ in g o f p u r e w a te r . I t is i n s t r u c t i v e to c o n s i d e r th e p h y s i c a l s c a l e o n w h ic h t h e n u c le a t i o n e v e n t ta k e s p la c e (i.e ., th e c r itic a l e m b r y o siz e ). F o r h o m o g e n e o u s f r e e z in g a t - 4 0 ° C , - 2 0 ° C , a n d —5 ° C , t h e c a l c u l a t e d v a l u e s o f t h e c r it i c a l r a d i u s a r e 0 .8 , 1 .8 , a n d 7 .0 n m , r e s p e c t i v e l y , w ith r o u g h l y 7 0 , 6 5 0 , a n d 4 5 , 0 0 0 m o l e c u l e s o f w a t e r in t h e e m b r y o . T lje l a r g e n u m b e r o f m o l e c u l e s in t h e c r it i c a l e m b r y o a t w a r m e r t e m p e r a t u r e s m a k e s it e v id e n t w h y th e p ro b a b ility o f h o m o g e n e o u s n u c le a tio n b e c o m e s n e g lig i­ b ly s m a ll a t j u s t a f e w d e g r e e s a b o v e - 4 0 ° C . A s a lr e a d y m e n t i o n e d , h o m o g e n e o u s ic e n u c l e a t i o n is c o n d i t i o n e d o n h a v in g n o f o r e i g n m a t e r i a l in t h e s a m p l e o f li q u id o r v a p o r — o r , to r e la x th a t c o n d i t i o n a little , n o n e t h a t c o u l d i n f l u e n c e th e f o r m a t i o n o f ic e e m b r y o s . T h a t is a s tr i n g e n t r e ­ q u i r e m e n t , o n e t h a t c a n h a r d l y e v e r b e s a tis f ie d w i t h o u t q u e s t i o n . It is m o s t li k e ly to b e s a ti s f i e d f o r s m a l l v o l u m e s o f w a t e r in s o m e i n e r t g a s o r liq u id . C l o u d d r o p ­ le ts in t h e u p p e r t r o p o s p h e r e a n d c a r e f u l l y p r e p a r e d la b o r a t o r y s a m p le s m a y c o m e c lo s e . I n b i o l o g i c a l s y s t e m s , t h e i n t e r n a l w a t e r o r f l u i d v o l u m e s a r e in e v ita b ly in c o n t a c t w i t h o t h e r s u r f a c e s , s o t h a t n u c l e a t i o n is j u s t i f i a b l y a s s u m e d t o b e h e t ­ ero g e n e o u s.

M o d e s o f H e te r o g e n e o u s Ic e N u c le a tio n W h e n ic e f o r m s a t t e m p e r a t u r e s a b o v e - 4 0 ° C , o r a t s u p e r s a t u r a t i o n s le s s th a n 4 5 0 % , it is d u e to t h e p r e s e n c e o f s o m e m a t e r i a l o t h e r th a n w a t e r ( i.e ., b y h e t e r o g e ­ n e o u s n u c l e a t i o n ) . F o r e i g n b o d i e s s e r v e a s s ite s o n w h i c h ic e e m b r y o s g r o w m o r e r e a d i l y t h a n p u r e l y b y t h e r a n d o m a g g r e g a t i o n o f w a t e r m o l e c u l e s w ith o n e a n ­ o th e r . F o r e m b r y o f o r m a t i o n o n a f o r e i g n s u r f a c e , t h e t e m p e r a t u r e o r s u p e r s a t u r a ­ t io n

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o f w a te r

m o l e c u l e s w i t h t h e f o r e i g n s u r f a c e . T h e l o c a t i o n a t w h i c h a n ic e e m b r y o f o r m s o n a s u r f a c e , w i t h t h e p o t e n t i a l f o r t h a t e m b r y o t o g r o w to c r it i c a l s iz e , is c a lle d a

nucleating site.

S i m i l a r l y t o t h e h o m o g e n e o u s c a s e , h e t e r o g e n e o u s n u c le a t i o n is

g o v e r n e d b y tw o m a j o r f a c t o r s : th e f r e e e n e r g y c h a n g e i n v o lv e d in f o r m i n g th e e m b r y o a n d t h e d y n a m i c s o f f l u c t u a t i n g e m b r y o g r o w t h . I m p o r t a n t l y , in h e t e r o g e ­ n e o u s n u c l e a t i o n , t h e c o n f i g u r a t i o n a n d e n e r g y o f in t e r a c t i o n a t th e n u c le a t i n g s ite b e c o m e t h e d o m i n a t i n g in f l u e n c e s . A s i m p l e d e s c r i p t i o n o f h e t e r o g e n e o u s n u c l e a t i o n is t h a t t h e e m b r y o f o r m s a

s p h e r ic a l c a p o n a f l a t a n d u n i f o r m s u r f a c e , m u c h lik e a d r o p o f w a t e r w o u ld s it o n a h y d r o p h o b i c s u r f a c e . W ith t h a t m o d e l , th e e n e r g y o f in te r a c t i o n b e t w e e n th e e m ­ b r y o a n d t h e s u r f a c e c a n b e c h a r a c t e r i z e d b y th e c o n t a c t a n g l e . N o t e t h a t th is m o d e l d o e s n o t i n v o l v e s p e c i f i c s ite s . T h e s p h e r i c a l c a p m o d e l is r e a d i l y f i t t e d to e q u a t i o n 1: th e v o l u m e a n d v o l u m e e n e r g y o f t h e e m b r y o a r e a d ju s te d f o r t h e n e w g e o m e t r y , a n d th e s u r f a c e e n e r g y t e r m is d i v i d e d in to tw o p a r ts , n a m e l y , e m b r y o - t o - m o t h e r p h a s e a n d e m b r y o - t o - f o r e i g n s u r f a c e , w ith th e s iz e o f e a c h s u r f a c e c a l c u l a t e d f r o m th e g e o m e t r y o f t h e s p h e r i c a l c a p . T h e r e s u l t is p l e a s i n g l y s im p le : t h e c r it i c a l f r e e e n e r g y c h a n g e ( t h e m a x i m u m in t h e e n e r g y v s . s iz e c u r v e ) is a f r a c t i o n o f t h a t r e ­ q u ir e d f o r h o m o g e n e o u s n u c l e a t i o n , a n d th e f r a c tio n is a u n i q u e f u n c t i o n o f th e c o n t a c t a n g l e . T h e s m a l l e r th e c o n t a c t a n g le , th e lo w e r t h e e n e r g y b a r r i e r t o n u ­ c le a tio n . T h e h o m o g e n e o u s c a s e a p p e a r s a s a l im it f o r a c o n t a c t a n g l e o f 1 8 0 ° . T h e im p a c t o f a s m a l l c o n t a c t a n g le c a n b e i llu s tr a te d b y c o m p a r i n g t h e c r i t i c a l e m b r y o siz e o f 6 0 0 m o le c u le s (a s s u m in g a 3 0 ° c o n ta c t a n g le ) re q u ir e d f o r n u c le a tio n a t - 5 ° C w it h th e p r e v i o u s l y c i t e d f i g u r e o f 4 5 , 0 0 0 m o le c u le s r e q u i r e d f o r h o m o g e n e o u s f r e e z in g . M o r e c o m p l e x f o r m s o f th e t h e o r y a s s ig n d i f f e r e n t i n t e r a c t i o n e n e r g i e s to th e n u c le a t i n g s i t e t h a n to t h e r e s t o f t h e s u r f a c e , a llo w th e s u r f a c e to b e c u r v e d , u s e d i f f e r e n t a s s u m p t i o n s f o r th e e m b r y o

s h a p e , a n d in c l u d e s u r f a c e i r r e g u l a r i t i e s

( s te p s , d i s l o c a t i o n s , c a v i t i e s , e t c .) a s p o s s i b l e n u c le a tio n s ite s . A s m e n t i o n e d b e ­ f o r e , s u c h e l a b o r a t i o n s , w h i l e c o n c e p t u a l l y a p p e a lin g , u s u a lly i n v o l v e s o m a n y d e ­ g r e e s o f f r e e d o m in t h e a s s u m p t i o n s t h a t n o g e n e r a l i z a t i o n s a r e r e a c h e d . H e n c e th e r e m a i n d e r o f t h i s d i s c u s s i o n f o c u s e s o n e m p ir ic a l r e s u lts . A l t h o u g h f r e e z i n g ( t h e l i q u i d - t o - s o l i d tr a n s itio n ) is t h e o n l y h o m o g e n e o u s ic e n u c le a t i o n p r o c e s s o f p r a c t i c a l i n t e r e s t in t h e b i o s p h e r e a n d in t h e a tm o s p h e r e , a v a r ie ty o f h e t e r o g e n e o u s p r o c e s s e s m a y b e c o n s id e r e d . B o t h t h e l i q u i d - s o l i d a n d v a p o r - s o l i d t r a n s i t i o n s a r e o f p r a c t i c a l in te r e s t, a n d o b s e r v a t i o n s h a v e s h o w n t h a t f r e e z in g n u c l e a t i o n d e p e n d s o n h o w t h e s u p e r c o o le d liq u id a n d t h e n u c l e a t i n g p a r ­ tic le c o m e t o g e t h e r . H e n c e th e p a t h w a y s , o r s id e r e d .

D e fin itio n s

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h a v e to b e c o n ­ a

“ N u c le a tio n

T e r m i n o l o g y ” ( s e e J. A e r o s o l S c i. 1 6 [ 1 9 8 5 ] :5 7 5 - 5 7 6 ; G o t z e t a l., 1 9 9 1 , 2 6 5 - 2 6 7 ) f ir s t a d o p t e d b y th e a t m o s p h e r i c s c i e n c e c o m m u n ity b u t c e r t a i n l y n o t r e s t r i c t e d to a tm o s p h e r i c p r o c e s s e s . ( T w o w o r d c h a n g e s , in p a r e n th e s e s , h a v e b e e n m a d e s o t h a t th e d e f i n i t i o n s m a y a p p l y b e y o n d t h e a t m o s p h e r i c s itu a tio n in w h i c h th e n u c l e i a r e a e r o s o l p a r t i c l e s .) 1.

Deposition nucleation — T h e

f o r m a t i o n o f ic e in a ( s u p e r s a t u r a t e d ) v a p o r e n v i ­

ro n m e n t 2.

Freezing nucleation — T h e

f o r m a t i o n o f ic e in a ( s u p e r c o o l e d ) li q u i d e n v i r o n ­

m ent 2 .1

Condensation freezing — T h e

s e q u e n c e o f e v e n ts w h e r e b y a c o n d e n s a t i o n

n u c l e u s i n i t i a t e s f r e e z i n g o f th e c o n d e n s a t e 2 .2

Contact freezing — N u c l e a t i o n

o f a s u p e r c o o le d d r o p l e t s u b s e q u e n t to a

n u c l e a t i n g p a r t i c l e c o m i n g in t o c o n t a c t w ith it. 2 .3

Immersion freezing — N u c l e a t i o n

o f s u p e r c o o le d w a t e r b y a n u c l e u s s u s ­

p e n d e d in t h e b o d y o f w a t e r . W h e n t h e m o d e o f n u c l e a t i o n is k n o w n , it is a d v a n t a g e o u s t o r e f e r to t h a t s p e ­ c if ic m o d e b y e m p l o y i n g t h e c o r r e s p o n d i n g te r m . I f t h e m o d e is u n k n o w n , o r a c o l l e c t i v e d e s c r i p t i o n is d e s i r e d , o n e c a n r e f e r to “ ic e n u c l e i ,” o r “ ic e n u c l e a t i o n . ”

Principles of Ice Nucleation

7

T h e m a in c h a r a c t e r i s t i c o f d e p o s i t i o n n u c le a t i o n is t h a t th e n u m b e r o f n u c le a t i n g s ite s ( p e r u n i t s u r f a c e a r e a o f th e m a t e r i a l , o r p e r a e r o s o l p a r t i c l e ) in c r e a s e s r a p id ly w ith s u p e r s a t u r a t i o n . T h e r e l e v a n t p a r a m e t e r is s u p e r s a t u r a t i o n o v e r ic e . T e m p e r a ­ t u r e p e r se d o e s n o t h a v e a n y i n f l u e n c e , e x c e p t t h r o u g h th e d e p e n d e n c e o f th e s a t u ­ r a tio n v a p o r p r e s s u r e o f ic e . S o m e a c t i v i t y is p r e s e n t e v e n b e l o w w a te r s a tu r a tio n , a n d th e r e is n o d i s c o n t i n u o u s in c r e a s e in t h e n u m b e r o f n u c le i a t w a te r s a tu r a tio n . T h e d e p e n d e n c e o n s u p e r s a t u r a t i o n o v e r ic e is v e r y s tr o n g : a p o w e r la w r e l a t i o n ­ s h ip b e t w e e n n u m b e r a n d s u p e r s a t u r a t i o n h a s e x p o n e n t s r a n g i n g f r o m 4 to 1 2. T h e s e r e l a t i o n s h i p s a r e v a l i d b e l o w a t e m p e r a t u r e t h a t a p p e a r s to b e c h a r a c t e r i s t i c f o r e a c h s u b s t a n c e . A b o v e th a t t e m p e r a t u r e , ic e n u c l e a t i o n r e q u i r e s th a t th e v a p o r p r e s s u r e e x c e e d s a t u r a t i o n w ith r e s p e c t to w a te r , s u g g e s t i n g t h a t th e m o d e o f n u ­ c le a t i o n e v e n f r o m t h e v a p o r e n v i r o n m e n t is f r e e z i n g , w ith a t r a n s i e n t c o n d e n s a t i o n o f w a te r . T h i s l i m i t i n g t e m p e r a t u r e w a s d e t e r m i n e d f o r C d l 2 (—2 2 ° C ) b y B r y a n t e t a l. ( 1 9 5 9 ) , f o r le u c i n e ( - 2 0 ° C ) b y M a y b a n k a n d B a r t h a k u r ( 1 9 6 6 ) , a n d f o r s i l v e r io d id e ( - 8 ° C ) , k a o l i n i t e ( - 1 3 ° C ) , a n d o t h e r s u b s t a n c e s b y S c h a lle r a n d F u k u t a (1 9 7 9 ). I m m e r s i o n f r e e z i n g is c o m m o n l y o b s e r v e d v e r y n e a r 0 ° C f o r la r g e v o l u m e s o f w a t e r in c o m m o n c o n t a i n e r s o r in p u d d l e s , l a k e s , r i v e r s , e tc . S m a ll v o lu m e s o f w a ­ te r u n d e r l a b o r a t o r y c o n d i t i o n s c a n b e o b s e r v e d to f r e e z e a s a r e s u l t o f n u c le i s u s ­ p e n d e d in t h e w a t e r a t t e m p e r a t u r e s r a n g i n g f r o m —1 ° C to n e a r - 3 0 ° C , d e p e n d i n g o n t h e ty p e a n d q u a n t i t y o f s u s p e n d e d m a tte r . C o n d e n s a t i o n f r e e z in g a n d c o n t a c t f r e e z i n g h a v e b e e n f o u n d t o b e a s o r m o r e e f f e c t i v e th a n i m m e r s i o n f r e e z i n g f o r s o m e n u c l e a t i n g m a t e r i a l s in a e r o s o l f o r m , b u t t h e s e e x p e r i m e n t s h a v e b e e n r e ­ s tr ic te d b y e x p e r i m e n t a l d i f f i c u l t i e s to t e m p e r a t u r e s a t le s s t h a n —1 0 ° C . I t is n o t k n o w n h o w a c t i v i t i e s o f m a t e r i a l s d i f f e r w ith r e s p e c t to th e th e th r e e f r e e z in g m o d e s a t s m a l l e r s u p e r c o o l i n g s . T h i s is a n

i m p o r t a n t p o i n t , s in c e d i f f e r e n c e s

a m o n g t h e f r e e z i n g m o d e s c a n y ie l d u s e f u l i n f o r m a t i o n o n th e f a c to r s t h a t g o v e r n th e f o r m a t i o n o f ic e e m b r y o s o n t h e p a r t i c u l a r s u r f a c e s . P o s s i b l e e x p l a n a t i o n s f o r e n h a n c e d a c t i v i t y in t h e c o n t a c t m o d e a r e t h a t n u c l e a t i n g s ite s m a y p a r t i a l l y d i s ­ s o lv e w h e n i m m e r s e d in w a te r , t h a t e m b r y o s in e q u i l i b r i u m w ith th e v a p o r e n v i ­ ro n m e n t

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c h a r a c t e r i s t i c s o f t h e s u r f a c e l a y e r o f w a t e r e n h a n c e e m b r y o f o r m a tio n . I n f o r m a ­ ti o n a b o u t t h e c o n d e n s a t i o n f r e e z i n g s e q u e n c e is s o s c a n t th a t t h e r e is little b a s is f o r th e o r i z i n g ; it is w o r t h m e n t i o n i n g , t h o u g h , t h a t p e r h a p s s o m e c o n n e c t i o n e x is ts b e tw e e n c o n d e n s a t i o n f r e e z i n g a n d e v a p o r a t i o n f r e e z i n g , th e l a tte r h a v in g b e e n o b ­ s e r v e d w h e n d r o p l e t s a r e in t h e i r f in a l s ta g e s o f e v a p o r a t i o n . I n b i o l o g i c a l s y s t e m s , i m m e r s i o n f r e e z i n g is th e m o s t lik e ly p a th o f ic e i n i t i a ­ tio n . S o f a r , t h e d i s t i n c t i o n a m o n g t h e f r e e z i n g m o d e s h a s b e e n s h o w n to b e i m p o r ­ ta n t o n ly in l a b o r a t o r y te s t s m i m i c k i n g a t m o s p h e r i c c o n d i t i o n s ( i.e ., w ith a e r o s o l p a r t i c l e s a n d d r o p l e t s o f w a t e r ) . I t is c o n c e i v a b l e t h a t c o n t a c t f r e e z i n g a ls o a r is e s in b i o l o g i c a l s y s t e m s — f o r e x a m p l e , b y th e g r o w t h o f a w a t e r v o lu m e u n t i l it j u s t c o m e s i n to c o n t a c t w i t h t h e n u c l e a t i n g s ite , o r b y a c t u a l m o v e m e n t o f e i t h e r t h e w a t e r v o l u m e o r t h e n u c l e a t i n g s u r f a c e . C o n d e n s a t i o n f r e e z i n g c o u ld b e ta k in g p l a c e j u s t a s in t h e a tm o s p h e r e . I t is a l s o p o s s i b l e t h a t th e s e t o f d e f i n i t i o n s g iv e n a b o v e w ill h a v e to b e b r o a d e n e d t o i n c l u d e s p e c i f i c c i r c u m s t a n c e s a p p l i c a b l e to b i o l o g i c a l s y s te m s . I n v ie w o f th e k n o w n r o l e o f im m e r s i o n f r e e z i n g in b i o l o g i c a l s y s te m s , a tte n tio n in s u b s e q u e n t s e c t i o n s o f th i s o v e r v i e w w ill f o c u s o n t h a t m e c h a n is m .

8

Vali H is t o r ic a l S u r v e y o f F r e e z in g N u c le a t io n E x p e r im e n t s T h e s u p e r c o o l i n g o f w a t e r w a s o b s e r v e d in 1 7 21 b y F a h r e n h e i t ( to a l o w e s t

t e m p e r a t u r e o f —9 ° C ) in a s e a le d g l a s s v e s s e l — a n e x p e r i m e n t f r e q u e n t l y r e p e a t e d a f t e r w a r d s , w i t h t h e u s u a l r e s u l t t h a t t h e w a te r f r o z e a t t h e i n s t a n t t h e v e s s e l w a s o p e n e d o r m o v e d . T h e s e o b s e r v a t i o n s l in k e d f r e e z in g to m e c h a n i c a l d i s t u r b a n c e s a n d s h o w e d t h a t t h e c r y s t a l l i z a t i o n r a t e o f w a te r , o n c e s ta r te d , is s o r a p i d t h a t it is im p o s s i b l e to c o u n t t h e c e n t e r s o f c r y s t a l l i z a t i o n in th e s a m e w a y a s in e x p e r i m e n t s w it h s u p e r s a t u r a t e d s o l u t i o n s . T h e s e w e r e th e b e s t k n o w n o b s e r v a t i o n s r e l a t i n g to th e f r e e z i n g o f w a t e r f o r o v e r 2 0 0 y e a r s ; h o w e v e r , a s A l t b e r g ( 1 9 3 8 ) r e v i e w s , e x ­ p e r i m e n t s a s e a r l y a s 1 7 8 8 a l r e a d y g a v e i n d ic a tio n s th a t t h e d e g r e e o f s u p e r c o o l i n g a c h ie v e d f o r w a t e r is li m i t e d b y i m p u r i t i e s s u s p e n d e d in it. T h a t f a c t b e c a m e m o r e f ir m ly e s t a b l i s h e d w i t h o t h e r s u b s t a n c e s t h r o u g h th e n i n e t e e n t h c e n t u r y , u n til e x ­ p e r i m e n t e r s f i n a l l y r e t u r n e d to w a t e r in th e 1 9 3 0 s a n d 1 9 4 0 s . R e s e a r c h w ith w a t e r d u r i n g t h a t p e r i o d w a s p a r t o f a b r o a d in c r e a s e in p h a s e c h a n g e e x p e r i m e n t s a n d o f s i g n i f i c a n t d e v e l o p m e n t s in n u c l e a t i o n th e o r y . M o d e r n n u c l e a t i o n e x p e r i m e n t s w i t h w a t e r h a v e tw o t r a d i t i o n a l r o o t s . E x p e r i ­ m e n ts w ith b u l k s u p e r c o o l e d w a t e r f o l l o w e d w o r k o n t h e s o l i d i f i c a t i o n o f s u p e r ­ s a tu r a te d s o l u t i o n s a n d s u p e r c o o l e d liq u id s , a s m e n tio n e d a b o v e . S a m p l e s w e r e h e ld in t e s t t u b e s o r s i m i l a r v e s s e l s . S o li d i f i c a t i o n w a s o b s e r v e d a s a f u n c t i o n o f th e c o m p o s i t i o n o f t h e s a m p le , th e ti m e o r th e t e m p e r a tu r e a t w h i c h s o l i d i f i c a t i o n t o o k p la c e , t h e e f f e c t s o f s h a k i n g , th e le n g t h o f s to r a g e o f th e s a m p l e , e tc . A n o t h e r lin e o f i n v e s t i g a t i o n h a s its o r i g i n s in t h e u s e o f th e c lo u d c h a m b e r i n v e n t e d b y C . T . R . W ils o n a r o u n d 1 8 9 5 f o r m a k i n g t h e t r a c k s o f e le m e n ta r y p a r t i c l e s v is ib le . T h i s a p ­ p a r a tu s le d to t h e s e r ie s o f “ c l o u d c h a m b e r e x p e r i m e n t s ,” w h i c h h a v e th e e s s e n t i a l f e a tu r e o f tin y d r o p l e t s o f w a t e r s u s p e n d e d in a ir d u r in g t h e o b s e r v a t i o n s . C l o u d c h a m b e r e x p e r i m e n t s a r e o f p r i m a r y r e le v a n c e to a t m o s p h e r i c p r o b l e m s , a n d th e y m a y b e u s e f u l in t h e t e s t i n g o f b i o lo g ic a l ic e n u c le i i f t h e n u c l e i a r e d i s ­ p e r s e d a l o n g w i t h t h e w a t e r t o f o r m t h e c lo u d . T h is is n o t v e r y d e s i r a b l e in g e n e r a l , a n d th e r e s u l t s o f th e e x p e r i m e n t s c a n b e d i f f i c u l t to in te r p r e t. I t w i l l s u f f i c e h e r e to m e n t i o n t h a t t h e p r i n c i p a l a d v a n t a g e o f t h e c l o u d c h a m b e r t e c h n i q u e is t h a t t h e i n ­ d i v i d u a l d r o p l e t s a r e o n ly a f e w m i c r o m e t e r s in d ia m e te r , a n d h e n c e t h e p r o b a b i l i t y f o r th e d r o p l e t s t o c o n t a i n f o r e i g n m a t t e r ( p o te n tia l n u c le i) is q u i t e s m a ll. A s a r e ­ s u lt, h o m o g e n e o u s n u c l e a t i o n c a n b e in v e s t i g a t e d b y th is a p p r o a c h , a s s e e n b o t h in t h e e a r ly e x p e r i m e n t s ( C w i l o n g , 1 9 4 7 ; S c h a e f e r , 1 9 4 8 ; F o u r n i e r d ’A l b e , 1 9 4 9 ; M o s s o p , 1 9 5 5 ) a n d r e c e n t l y ( H a g e n e t a l., 1 9 8 1 ). T h e lo w t e m p e r a t u r e s n e e d e d f o r th e s e e x p e r i m e n t s w e r e r e a c h e d b y r a p i d e x p a n s i o n o f h u m i d i f i e d a ir ; t h e d e g r e e o f e x p a n s io n d e te rm in e s th e lo w e s t te m p e ra tu re re a c h e d . A c o n c o m ita n t d is a d v a n ta g e o f th e r a p i d e x p a n s i o n is t h a t th e s a m p l e r e m a in s a t th e l o w e s t t e m p e r a t u r e o n ly f o r a b r i e f i n t e r v a l o f tim e . T h e d e r i v a t i o n o f a n u c le a tio n r a te f r o m t h e s e e x p e r i m e n t s t h e r e f o r e d e p e n d s o n t h e a p p l i c a t i o n o f s o m e th e o r e tic a l m o d e l to t h e d a ta . G r e a t e r c o n t r o l o v e r t h e v a r i a b l e s c a n b e a c h ie v e d in e x p e r i m e n t s w i t h v o l u m e s o f w a t e r s u p p o r t e d in v e s s e l s , s u s p e n d e d in liq u id s , s u p p o r t e d o n s u r f a c e s , e t c ., a t th e c o s t o f in c re a s in g th e p o s s ib ility o f in te rfe re n c e b y th e s u p p o rtin g s u rfa c e s . T h e r e is a n i n t e r m e d i a t e a p p r o a c h , t h a t o f le ttin g d r o p s f a ll i n to c o l d a i r ( e .g ., K u h n s a n d M a s o n , 1 9 6 8 ) . O v e r a l l , s o l i d a n d liq u id s u p p o r t s h a v e b e e n m o s t w id e ly e m p l o y e d . D e v e l o p m e n t o f th is a p p r o a c h to o k o f f w ith t h e w o r k o f M e y e r a n d P fa ff (1 9 3 5 ) a n d o f T a m m a n n a n d B u c h n e r (1 9 3 5 ) a n d g o t a m a jo r im p u ls e fro m th e w o r k o f D o r s e y ( 1 9 3 8 , 1 9 4 8 ) . D o r s e y w e n t c o n s i d e r a b l y b e y o n d h i s p r e d e c e s ­

Principles of Ice Nucléation

9

s o r s in t h e v a r i e t y a n d n u m b e r o f w a t e r s a m p l e s te s t e d , a ll in s e a le d g la s s “ b u l b s .” D o r s e y ’s r e s u l t s c l e a r l y c o n f i r m e d t h a t t h e f r e e z i n g ( n u c l e a t i o n ) te m p e r a t u r e d e ­ p e n d s o n t h e s o u r c e o f t h e w a t e r ( d i s t i l l e d , c o n d u c t i v i t y , W a s h i n g t o n C ity , a q u a r ­ iu m , s tr e a m , o r s n o w

and

ic e ) ; t h a t m e c h a n i c a l d is t u r b a n c e s r a r e ly

r e s u l t in

f r e e z i n g ; a n d t h a t r e p e a t e d te s ts w ith a s a m p l e r e s u l t in f r e q u e n t r e p e t i t i o n s o f th e p r e v i o u s l y o b s e r v e d f r e e z i n g t e m p e r a t u r e s , a l t h o u g h n o t w i t h o u t la r g e u n p r e d i c t ­ a b le c h a n g e s . W h e n S c h a e f e r ( 1 9 4 6 ) a n d V o n n e g u t ( 1 9 4 7 ) r e p o r t e d t h a t ic e c r y s t a l s c a n b e p r o d u c e d in a la b o r a t o r y c l o u d a n d t h a t s i l v e r i o d i d e ( A g l ) p a r t i c l e s a d d e d to th e c l o u d c a u s e ic e to f o r m a b u n d a n t l y , r e s e a r c h o n th e n u c l e a t i o n o f ic e a c c e l e r a t e d e n o r m o u s l y . B y th e e n d o f t h e 1 9 5 0 s , e s s e n t i a l l y a ll e x p e r i m e n t a l m e t h o d s n o w k n o w n h a d b e e n i n t r o d u c e d , a n d t h e o r e t i c a l d e v e l o p m e n t s f r o m p h y s ic a l c h e m i s t r y a n d m e t a l l u r g y h a d b e e n r a p i d l y a p p l i e d to ic e . M a n y o f th e p r a c t i c a l a n d t h e o r e t i ­ c a l p r o b l e m s t h a t h a d n o t b e e n s o l v e d b y th e n r e m a i n u n s o l v e d . O n e o f t h e f a c t o r s g u i d i n g th e d e v e l o p m e n t o f n e w e x p e r i m e n t a l m e t h o d s w a s th e r e a l i z a t i o n t h a t th e f r e e z i n g o f a n y o n e s a m p l e r e v e a l s in f o r m a t i o n o n ly a b o u t th e m o s t a c t i v e n u c l e u s in it, th e o n e p r o d u c i n g n u c l e a t i o n a t th e w a r m e s t te m p e r a ­ tu r e . T o g a in a m o r e c o m p l e t e i d e a o f t h e n u c le i c o n t a i n e d in a s a m p le , a n d to im ­ p r o v e th e s ta ti s t i c a l v a l i d i t y o f a n y r e s u l t , it w a s n e c e s s a r y to d e a l w ith la r g e n u m b e r s o f s u b u n i t s o f a n y s a m p le . T h i s f a c t f o r c e d e x p e r i m e n t e r s to g o b e y o n d te s t tu b e s , s e a l e d g la s s tu b e s , e tc . R a u ( 1 9 4 4 ) a n d D o r s c h a n d H a c k e r ( 1 9 5 0 ) p r o ­ d u c e d e x t e n s i v e , i f s o m e w h a t c o n f u s i n g , d a t a s e ts w ith d r o p s p la c e d o n m e ta l p la te s th a t w e r e c o o l e d a t r a t e s o f th e o r d e r o f o n e d e g r e e p e r m in u te . B ig g ( 1 9 5 3 ) a n d L a n g h a m a n d M a s o n ( 1 9 5 8 ) p r o t e c t e d t h e i r s a m p l e d r o p s f r o m p o s s ib le c o n ­ ta m i n a t i o n a n d f r o m t h e i n f l u e n c e o f a m e ta l s u p p o r t b y s u s p e n d i n g t h e m a t th e i n te r f a c e o f t w o i m m i s c i b l e liq u id s . M u c h a t t e n t i o n w a s p a id in t h e s e e x p e r i m e n t s to th e e f f e c t o f d r o p v o l u m e o n th e t e m p e r a t u r e o f n u c l e a t i o n a n d to th e s ta tis tic a l d i s t r i b u t i o n o f f r e e z i n g te m p e r a t u r e s . A ls o , o f c o u r s e , e x p e r i m e n t s w e r e c a r r i e d o u t w ith p o t e n t i a l n u c l e a t i n g s u b s t a n c e s ; w i t h s i l v e r i o d i d e a b o v e a ll, b u t w ith m a n y n a tu r a l l y o c c u r r i n g a n d s y n t h e t i c s u b s t a n c e s a s w e ll. I n t e r p r e t a t i o n s o f t h e r e s u lts in t e r m s o f t h e t h e r m o d y n a m i c / k i n e t i c th e o r y a p p l i e d to ic e n u c le a tio n ( F le tc h e r , 1 9 5 8 ) m e t w i t h s o m e s u c c e s s b u t u s u a l l y r e m a i n e d s h o r t o f g e n e r a l v a lid ity . A n o th e r d ire c tio n o f n u c le a tio n s tu d ie s fo c u s e d o n h o w fo re ig n s u rfa c e s in d u c e ic e n u c l e a t i o n . I n s t e a d o f d e a l i n g w ith p a r t i c l e s s u s p e n d e d in w a te r , a d i r e c t e x a m i ­ n a tio n o f th e s u r f a c e s w a s d e s i r e d . T h i s w a s a c h i e v e d b y p l a c i n g c a r e f u lly p r e p a r e d c r y s ta l s u r f a c e s o n a m i c r o s c o p e c o ld s ta g e a n d e n c l o s i n g th e s a m p le in a s m a ll c h a m b e r in w h i c h h u m i d i t y c o u l d b e c o n t r o l l e d . S u c h s t u d i e s o n w e ll- c h a r a c t e r i z e d su rfa c e s

(M o n tm o ry ,

1956;

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a l.,

1959;

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M aso n ,

1963,

C a s la v s k y a n d V e d a m , 1 9 7 1 ) le d to th e r e c o g n i t i o n t h a t s u r f a c e f e a tu r e s s u c h a s s te p s a n d s c r e w d i s l o c a t i o n s a r e f a v o r e d s ite s f o r n u c l e a t i o n . T h e s e f i n d in g s le d to m u c h s p e c u l a t i o n a b o u t t h e m i c r o s c o p i c f a c to r s i n f l u e n c i n g ic e n u c le a tio n , b u t th e r e l e v a n c e o f t h e s e s t u d i e s is lim ite d b y th e f a c t t h a t n u c le a t i o n ta k e s p l a c e o n a s c a le m u c h s m a l l e r th a n t h e s u r f a c e f e a t u r e s t h a t c a n b e i d e n t i f i e d b y d i r e c t e x a m i ­ n a tio n . T h e d e v e l o p m e n t s d e s c r i b e d a b o v e w e r e m o t i v a t e d b y q u e s t i o n s r e l a t e d to th e a t m o s p h e r e : th e i n i t i a t i o n o f ic e in c l o u d s , th e f o r m a t i o n o f p r e c i p i t a t i o n , a n d a r t i ­ f ic ia l c l o u d s e e d i n g . T h e o v e r l a p o f in t e r e s t s w i t h t h e b i o l o g i c a l s c ie n c e s b e c a m e e s t a b l i s h e d — p e r h a p s n o t u n i q u e l y , b u t i m p o r t a n t l y — th r o u g h th e w o r k o f S a lt ( 1 9 5 8 , 1 9 6 6 , a n d o t h e r s ) , w h o r e a l i z e d t h e a p p l i c a b i l i t y o f n e w f i n d in g s o n h e te r o -

10

Vali

g e n e o u s ic e n u c l e a t i o n to th e w i n t e r s u r v i v a l o f in s e c ts . T h e e x p e r i m e n t s o f S a lt e s t a b l i s h e d t h a t t h e f u n d a m e n t a l a s p e c t s o f ic e n u c le a tio n c l e a r l y m a n i f e s t t h e m ­ s e lv e s in b i o l o g i c a l s y s t e m s a s w e ll, a lt h o u g h a n u m b e r o f a d d i t i o n a l c o m p l i c a t i n g f a c to r s a l s o h a v e to b e d e a l t w ith . N o n e t h e l e s s , it is n o w c l e a r f r o m c u m u l a t i v e e v i d e n c e t h a t t h e p r i n c i p l e s d i s c u s s e d in th is c h a p t e r h a v e g e n e r a l a p p l i c a b i l i t y to a tm o s p h e ric , b io lo g ic a l, o r o th e r s y s te m s o f c o m p a ra b le te m p e ra tu re a n d p re s s u re re g im e s .1

P r a c tic a l M e th o d s o f M e a s u r e m e n t o f I m m e r s io n F r e e z in g N u c le i M a n y v e rs io n s o f th e s o -c a lle d d ro p -fre e z in g a s s a y 2 h a v e b e e n d e v e lo p e d . T h e e s s e n c e o f t h e s e te s t s is t h a t th e n u c l e a t i n g m a te r ia l is d is p e r s e d in w a t e r ; t h e w a t e r s a m p le is d i v i d e d in t o m a n y s u b u n i t s ( d r o p s ) ; th e s e t o f d r o p s is c o o l e d ; a n d t h e i r f r e e z in g is o b s e r v e d . T h e b a s ic r e a s o n f o r te s tin g m a n y s u b u n i t s o f a s a m p l e h a s b e e n m e n t i o n e d b e f o r e . T h e t e s ts p r o v i d e i n f o r m a tio n a b o u t t h e a b u n d a n c e s o f n u ­ c le a tin g s ite s w i t h d i f f e r e n t c h a r a c t e r i s t i c t e m p e r a t u r e s ( a p r e c i s e d e f i n i t i o n o f th e te r m w ill f o l l o w ) . T h e s e te s ts , in th e m s e l v e s , d o n o t r e v e a l a n y o t h e r p r o p e r t y o f th e n u c l e a t i n g m a t e r i a l . T h e s y s t e m s to b e e x a m i n e d h e r e h a v e c e r ta in b a s ic c h a r a c t e r i s t i c s , a n d t h e i r u s e im p lie s c e r t a i n a s s u m p t i o n s . P e r h a p s o f b r o a d e s t s ig n i f i c a n c e is t h e a s s u m p t i o n th a t th e te s t s y s t e m s i n v o l v e e s s e n t i a l l y “ b u l k ” w a te r , w it h w id e ly d i s p e r s e d n u c l e a t i n g m a te r ia l. T h e p r o p e r t i e s o f th e w a t e r a r e n o t a lte r e d b y a p r e v a l e n c e o f in t e r f a c i a l f o r c e s , a s w o u l d b e th e c a s e , f o r e x a m p l e , i f o n e d e a l t w ith a d s o r b e d w a t e r la y e r s . T h is r e q u i r e m e n t d o e s n o t e x c l u d e t h e p o s s ib ility o f d e l i b e r a t e l y a d d i n g d i s s o l v e d m a te r ia ls , n o r d o e s it s i g n i f y t h a t t h e e m b r y o is a s s u m e d to h a v e t h e p r o p e r t i e s o f b u lk w a t e r ( t h e c o n t r a r y is , in f a c t, t h e lik e ly c a s e ) . T h e n u c l e a t i n g m a t e r i a l h a s to b e d is p e r s e d a m o n g t h e d r o p s . G i v e n t h e s m a ll s iz e s o f n u c l e a t i n g s it e s (< 1 n m ) , p ie c e s o f t h e n u c l e a t i n g m a t e r i a l m u c h la r g e r th a n th e n u c l e a t i n g s it e s c a n b e s u s ­ p e n d e d in w a t e r d r o p s o f te n s o f m i c r o n s to m illim e te r s in s i z e w i t h o u t v io l a t i n g th e a s s u m p t i o n o f b u l k w a t e r p r o p e r t i e s . C le a r ly , th e p o s s i b i l i t y f o r c o o p e r a t i v e a c tio n a m o n g p i e c e s o f t h e n u c l e a t i n g s u b s t a n c e b e c o m e s li m i t e d ; th e r e a r e s o m e h in ts , b u t n o c l e a r e v i d e n c e , t h a t c o o p e r a t i v e a c tio n o f s o m e k in d m a y b e im p o r t a n t w it h s o m e s a m p l e s . In o r d e r to a l l o w q u a n t i t a t i v e in t e r p r e t a t i o n o f t h e r e s u l t s , th e d is p e r s i o n o f t h e n u c le i s h o u l d b e m a c r o s c o p i c a l l y u n i f o r m ( i.e ., e a c h d r o p s h o u ld c o n ta in t h e s a m e a m o u n t o f n u c l e a t i n g m a te r ia l) . A s w a s s h o w n in a n u m b e r o f te s ts w ith d i f f e r e n t m a t e r i a l s , th e d e g r e e o f d is p e r s io n d o e s n o t i n f l u e n c e t h e r e s u l t s d e r iv e d f r o m t h e te s t s ( V a li, 1 9 7 1 ; R o g e r s e t a l., 1 9 8 7 ; D u b r o v s k y a n d S k o lo u d , 1 9 8 9 ; s e e a l s o c o m m e n t s b e l o w o n t h e v o l u m e e f f e c t) . F r o m a p r a c t i c a l p o i n t o f v ie w , t h e g e n e r a l c o n c e r n w ith t h e d r o p - f r e e z i n g a s s a y s is t h a t t h e i m m e d i a t e e n v i ­ r o n m e n t o f t h e d r o p s n o t i n f l u e n c e t h e r e s u l t s ( i.e ., th a t th e s u p p o r t i n g o r c a r r y i n g

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t h e a t m o s p h e r i c s c i e n c e s is d e r i v e d

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th e th e o rie s o f

B e rg e r o n ( 1 9 3 5 ) a n d F in d e is e n ( 1 9 3 8 ) c o n n e c tin g p r e c ip ita tio n to ic e f o r m a tio n in c lo u d s . W o r ld W a r II a n d t h e a t t e n d a n t i n c r e a s e i n a v i a t i o n , i n c l u d i n g f l i g h t s i n i n c l e m e n t w e a t h e r , b r o u g h t t h e s e q u e s t i o n s t o g r e a t e r i m p o r t a n c e a n d a l s o b r o u g h t t o l i g h t p r o b l e m s r e l a t e d t o t h e i c i n g o f a i r c r a f t in s u p e r c o o le d c lo u d s . 2T h e te r m

drop-freezing assay

w ill b e e m p lo y e d h e r e , e v e n th o u g h s a m p le s m a y , in f a c t, b e h e ld in te s t

tu b e s o r o t h e r c o n t a i n e r s o r m a y b e o t h e r e n t i t i e s . T h e e s s e n t i a l f e a t u r e — t h a t t h e r e b e m a n y i d e n t i c a l u n i t s o f t h e s a m p l e — is w e l l c o n v e y e d b y t h e r e f e r e n c e to d r o p s .

11

Principles of Ice Nucléation

m e d iu m b e i n e r t, n e i t h e r i n c r e a s i n g n o r d e c r e a s i n g th e in h e r e n t a c tiv ity o f th e n u ­ c le i s u s p e n d e d in th e s a m p l e ) . B e y o n d t h is r e q u i r e m e n t , t h e s y s te m m u s t b e s ta b le o v e r th e p e r i o d o f te s t i n g ( t e n s o f m i n u t e s to h o u r s ) . A n o t h e r g e n e r a l q u e s t i o n is th e r o l e o f tim e v e r s u s t e m p e r a t u r e in le a d i n g to a n u c le a t i o n e v e n t . A s d i s c u s s e d in th e f i r s t s e c t i o n o f t h i s c h a p t e r , a n u c le a t i n g site c a n b e c h a r a c t e r i z e d in t e r m s o f its r a t e o f n u c l e a t i o n a s a f u n c t i o n o f te m p e r a t u r e . S in c e t h is d e f i n i t i o n is f o r a r a te , tim e is a n i n t r i n s i c v a r i a b l e in th e p r o c e s s . H o w ­ e v e r , it h a s b e e n s h o w n t h a t n u c l e a t i n g s ite s c a n b e d e f i n e d b y a

temperature,

characteristic

th e t e m p e r a t u r e a t w h i c h t h e n u c l e a t i o n r a te h a s s o m e f ix e d v a l u e 3.

T h is is s i m i l a r to th e d e f i n i t i o n o f t h r e s h o l d t e m p e r a t u r e f o r h o m o g e n e o u s n u c l e a ­ tio n . T h e in c r e a s e o f t h e n u c l e a t i o n r a t e is s o r a p id w ith d e c r e a s i n g te m p e r a t u r e s t h a t th e c h a r a c t e r i s t i c t e m p e r a t u r e is a r e a s o n a b l y g o o d p r e d i c t o r o f th e a c tu a l t e m ­ p e r a t u r e o f n u c l e a t i o n f o r w id e v a r i a t i o n s in t i m e - t e m p e r a t u r e h is to r y . T h u s , o b ­ serv ed

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c h a r a c t e r i s t i c t e m p e r a t u r e s f o r e x p e r i m e n t s p e r f o r m e d w ith c o n t i n u o u s c o o l i n g a t s o m e m o d e r a t e r a te . A b e t t e r a p p r o x i m a t i o n c a n b e a c h i e v e d b y r e p l a c i n g th e o b ­ s e rv e d fre e z in g te m p e ra tu re ,

T,

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is th e a b s o l u t e v a l u e o f t h e r a t e o f c o o l i n g in d e g r e e s C e l s i u s p e r m in u te . F o r tim e t e m p e r a t u r e h i s t o r i e s m o r e c o m p l e x th a n a l i n e a r o n e , t h e i n t e r p r e ta tio n o f t h e r e ­ s u lts is le s s s t r a i g h t f o r w a r d . T h e b a s i s f o r t h e c o o l i n g - r a t e c o r r e c t i o n a n d th e g e n e r a l is s u e o f tim e d e p e n d e n c e a r e t r e a t e d in m o r e d e t a i l b y V a li (1 9 9 4 ) . D r o p - f r e e z i n g a s s a y s c a n b e c a r r i e d o u t in a n u m b e r o f d i f f e r e n t f o r m s . T h e r e a r e d i f f e r e n c e s in t h e v o l u m e s o f th e s a m p l e s u s e d , in t h e w a y th e d r o p s a r e s u p ­ p o r t e d o r s u s p e n d e d , in t h e w a y c o o l i n g is e f f e c t e d , a n d in th e w a y f r e e z in g is d e ­ te c te d . P e r h a p s t h e s i m p l e s t m e t h o d is to p l a c e d r o p s o f , a p p r o x i m a t e l y , m il l i m e t e r s iz e ( te n s o f m i c r o l i t e r s in v o l u m e ) o n s o lid s u r f a c e s , t h e t e m p e r a t u r e o f w h i c h c a n b e v a r ie d in a c o n t r o l l e d w a y . A l t e r n a t i v e l y , t h e w a t e r m a y b e c o n t a i n e d in s m a ll c e n t r i f u g e tu b e s o r l a r g e r te s t tu b e s . T h e l a t t e r is e s p e c i a l l y u s e f u l i f la r g e p ie c e s o f m a te r ia l, s u c h a s w h o l e le a v e s , a r e to b e te s te d . C o o l i n g o f th e tu b e s is a c c o m ­ p lis h e d b y i m m e r s i n g t h e m in a t e m p e r a t u r e - c o n t r o l l e d b a th . A t th e o t h e r e n d o f th e s c a le , t h e w a t e r s a m p l e m a y b e d is p e r s e d a s a n e m u l s i o n , y i e l d i n g th e a d v a n ­ ta g e o f a c o m p a c t s a m p l e a n d a r e d u c e d d a n g e r o f i n t e r f e r e n c e f r o m th e s u p p o r t i n g s u rfa c e o r c o n ta in e r. D e t e c t i o n o f f r e e z i n g c a n b e b a s e d o n th e c h a n g e in o p a c ity , r e le a s e o f l a te n t h e a t, v o l u m e e x p a n s i o n , o r t h e c h a n g e in e le c t r i c c o n d u c t i v i t y th a t a c c o m p a n i e s th e p h a s e c h a n g e . T h e s i g n a l s d e r i v e d f r o m a n y o f t h e s e p h y s i c a l c h a n g e s a r e r o u g h ly p r o p o r t i o n a l to t h e a m o u n t o f ic e f o r m i n g d u r i n g t h e b r i e f in itia l p e r io d w h e n th e l a t e n t h e a t r e l e a s e is m u c h m o r e r a p id t h a n th e r a te o f t r a n s f e r o f h e a t f r o m th e d r o p to its s u r r o u n d i n g s . A f t e r t h a t in itia l f r e e z i n g , th e r a t e o f s o l i d i f i c a t i o n is g o v e r n e d b y th e r a te o f h e a t r e m o v a l f r o m th e d r o p . S i n c e t h e i n itia l ic e f r a c tio n is p r o p o r ­ t io n a l to t h e t e m p e r a t u r e ( in d e g r e e s C e l s i u s ) , d e t e c t i o n o f f r e e z in g a t s m a ll s u p e r ­ c o o l i n g s is m o r e d i f f i c u l t th a n d e t e c t i o n a t l a r g e s u p e r c o o l i n g s . A f e w e x a m p l e s o f s p e c i f i c e x p e r i m e n t a l a r r a n g e m e n t s w ill s e r v e to illu s tr a te h o w d r o p - f r e e z i n g a s s a y s a r e d o n e in p r a c tic e .

F o r h e te r o g e n e o u s n u c le a tio n , th e te r m

onset tem perature

is u s e d t o d e s c r i b e t h e h i g h e s t t e m p e r a t u r e

a t w h ic h s o m e m a te ria l e x h ib its a p p re c ia b le n u c le a tin g a c tiv ity . T h e te rm r i g o r o u s d e f i n i t i o n . In c o n t r a s t ,

characteristic temperature

h a s n o t b e e n g iv e n a

r e l a t e s to a s p e c i f i c n u c l e a t i n g s ite . A g i v e n

s u b s ta n c e m a y h a v e a la r g e r a n g e o f d if f e r e n t s ite s , w ith c o r r e s p o n d in g c h a r a c te r is tic te m p e r a tu re s .

12

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D r o p s o n P la te s

R a u ( 1 9 4 4 ) , B r e w e r a n d P a l m e r ( 1 9 5 1 ) , a n d V a li a n d S t a n s b u r y ( 1 9 6 6 ) d e s c r i b e s o m e e x a m p l e s o f th e a p p l i c a t i o n o f t h i s m e th o d . In c o m p a r i s o n w i t h e a r l i e r m e t h ­ o d s o f e n c l o s i n g t h e s a m p l e s in g la s s v e s s e ls , tu b e s , o r c a p i l l a r i e s , p l a c i n g d r o p s o n a p l a t e is s i m p l e r . T h e p l a t e c a n b e c o o l e d in a n y n u m b e r o f w a y s , t h e d r o p s a r e r e a d ily o b s e r v e d , a n d m a n y e x p e r i m e n t s c a n b e c a r r i e d o u t in a r e l a t i v e l y s h o r t tim e d u e to t h e e a s e o f p r e p a r a t i o n . T h e s u p p o r t i n g p la te , o f c o u r s e , h a s to b e in e r t ( n o t c a u s i n g n u c l e a t i o n ) ; t h is a p p e a r s to b e p o s s ib le , to t e m p e r a t u r e s o f a b o u t - 2 0 o r - 2 5 ° C , b y c a re fu l p o lis h in g a n d c le a n in g o f c h ro m e o r g o ld s u rfa c e s , o r by c o a tin g o f t h e s u r f a c e s w i t h th in la y e r s o f o ils o r v a r n is h e s . T h e d r o p s c a n b e p r o ­ d u c e d b y c o n d e n s a t i o n f r o m a h u m i d a i r s tr e a m o r b y s o m e m e c h a n i c a l d is p e n s e r . T h e l a t t e r is p r e f e r a b l e f o r c o n t r o l l i n g th e v o lu m e s o f t h e d r o p s a n d t h e i r s p a c in g . V is u a l d e t e c t i o n o f f r e e z i n g is t h e s im p l e s t , b u t is d i f f i c u l t a t t e m p e r a t u r e s c l o s e to 0 °C . F ig u re 2 s h o w s a s e t o f d ro p s o n a p la te a t a p o in t w h e n s o m e h a v e fro z e n an d so m e a re

s till s u p e r c o o l e d .

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m e th o d a n d t h e d a t a p r o c e s s i n g — v i d e o i m a g e in te r f a c e d to a c o m p u t e r is th e m o s t p r a c t i c a l a t th is t i m e — i t is p o s s i b l e to te s t s e v e r a l s a m p l e s a t t h e s a m e tim e , f o r c o m p a r i s o n o r c o n t r o l . I f f r e e z i n g e v e n t s a r e r e c o r d e d w ith r e f e r e n c e to th e l o c a ­ tio n o f e a c h d r o p , t h e d a t a c a n b e s e p a r a t e l y a n a ly z e d f o r e a c h s a m p l e . I n a d d i t i o n to e n s u r i n g t h a t t h e s u p p o r t i n g s u r f a c e d o e s n o t c a u s e n u c l e a t i o n a t t e m p e r a t u r e s c o m p a r a b l e to t h a t o f t h e n u c le i c o n t a i n e d in th e s a m p l e s , c a r e is re -

F i g u r e 2 . A s e t o f d r o p s , s o m e still s u p e r c o o le d , o th e r s ( w h ite ) fro z e n . A ll d r o p s a r e f ro m

s a m p l e . D r o p s a r e 0 . 1 0 |il i n v o l u m e .

th e s a m e

Principles of Ice Nucleation

13

q u i r e d to e n s u r e t h a t ic e d o e s n o t s p r e a d f r o m o n e f r o z e n d r o p to a n o th e r . T h e d a n ­ ger

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d i s a d v a n t a g e s o f th e m e t h o d . O t h e r r i s k s , t h o u g h m i n o r , a r e e v a p o r a t i o n o f th e d r o p s d u r i n g t h e te s ts , a i r b o r n e p a r t i c l e s f a l l i n g o n t o t h e d r o p s , a n d c o o l i n g r a t e s so f a s t t h a t s i g n i f i c a n t t e m p e r a t u r e g r a d i e n t s a r is e w ith in t h e d r o p s . T h e u s e o f a s o lid p l a t e a s th e s u p p o r t i n g s u r f a c e is a c o m m o n a r r a n g e m e n t. A p la te o f a b o u t 5 c m s q u a r e c a n h o ld u p to 1 0 0 d r o p s w i t h o u t a p p a r e n t d a n g e r o f in t e r f e r e n c e . T h e d r o p s a r e e i t h e r p l a c e d o n th e p o l i s h e d m e ta l s u r f a c e , o r o n a th in p l a s t i c f ilm o r a l u m i n u m f o i l s tr e t c h e d o v e r th e p la te . I n a ll c a s e s , a h y d r o p h o b i c c o a t i n g is n e e d e d to e n s u r e t h a t th e d r o p s d o n o t s p r e a d a n d to m i n i m i z e t h e p o s ­ s ib ility o f t h e s o lid s u p p o r t i n g s u r f a c e n u c l e a t i n g t h e d r o p . T h e f ilm c a n b e r e ­ p la c e d

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t h e r m o e l e c t r i c c o o l e r s , o r c o n d u c t i o n to a h e a t s in k ( d r y ic e o r liq u id n itr o g e n ) . T h e t e m p e r a t u r e o f th e p l a t e is ta k e n a s th e d r o p te m p e r a t u r e . F o r d r o p s n o g r e a t e r th a n a f e w m i l l i m e t e r s , th e t e m p e r a t u r e g r a d i e n t s in t h e d r o p s a r e o n ly a f e w h u n d r e d t h s o f a d e g r e e , a l t h o u g h a th in l a y e r a t t h e d r o p s u r f a c e m a y h a v e a g r e a te r te m p e r a ­ tu r e d i f f e r e n c e . A v e r s io n o f th e d r o p - f r e e z i n g a s s a y , r e q u i r i n g lit t l e s p e c i a l i z e d e q u i p m e n t , w a s d e s c rib e d b y L in d o w (1 9 8 2 ). D ro p s a re p la c e d o n a s h e e t o f p a ra ffin -c o a te d a lu m i­ n u m f o il w h o s e e d g e s a r e b e n t u p to f o r m a tr a y . T h e t r a y is th e n p la c e d o n a n o p e n c o ld b a th . T h i s m e t h o d is l i m i t e d to a s in g l e t e m p e r a t u r e o r to a s e t o f f i x e d t e m ­ p e r a tu r e s . A p a r t i c u l a r l y e l e g a n t v e r s i o n o f t h e d r o p - f r e e z i n g a s s a y is th a t o f P a r o d y M o r r e a l e e t a l. ( 1 9 8 6 ) . D r o p s a r e p l a c e d o n a t h e r m o p i l e , a n d th e w h o le a s s e m b ly is c o o l e d b y liq u id n i t r o g e n v i a a l o n g m e ta l c o l u m n . F r e e z i n g o f e a c h d r o p is a c ­ c o m p a n i e d b y a v o l t a g e p u l s e f r o m th e t h e r m o p i l e . T h i s s y s t e m h a s g r e a t s e n s i t i v ­ ity , s o th a t t h e f r e e z i n g o f d r o p s a s s m a ll a s 1 |i l c a n b e d e te c te d . A lim ita tio n o f th e m e t h o d is t h a t it c a n b e u s e d o n ly w ith d r o p s o f a s i n g l e s a m p le a n d o f t h e s a m e v o l u m e in a n y g iv e n r u n . T h e d a t a p r o d u c e d c o n t a i n s t h e tim e a n d t e m p e r a t u r e a t w h ic h t h e d r o p s f r o z e , b u t th e ti m e a t w h i c h e a c h p a r t i c u l a r d r o p f r o z e c a n n o t b e r e c o v e r e d f r o m th e d a ta . T est T u b es

A c o m b i n a t i o n o f t h e e a r l y e x p e r i m e n t s w ith s e a l e d g la s s v e s s e ls a n d o f th e d r o p s - o n - a - p l a t e t e c h n i q u e is th e p l a c e m e n t o f t h e s a m p l e s in te s t tu b e s t h a t a r e c o o l e d in a l i q u i d b a th . T h i s m e t h o d o f f e r s th e a d v a n t a g e o f th e m u ltip le s u b u n its o f t h e s a m p l e s , a s w ith t h e d r o p s , a n d a t t h e s a m e ti m e p r o v i d e s g r e a te r i s o l a t i o n o f th e in d i v i d u a l u n its . H i r a n o a n d U p p e r ( 1 9 8 6 ) d e s c r i b e t h e u s e o f t e s t tu b e s to h o ld s m a ll l e a v e s , b e a n p o d s , c e r e a l h e a d s , e tc ., i m m e r s e d in 0 .0 1 M b u f f e r . T h e tu b e s w e r e te s te d a t a s e r i e s o f t e m p e r a t u r e s , w ith u n f r o z e n s a m p l e s t r a n s f e r r e d to b a th s a t s u c c e s s i v e l y l o w e r t e m p e r a t u r e s . M a k i n o ( 1 9 8 2 ) s e a le d s a m p le s in to 1 0 -(il m i ­ c r o p i p e t t e s , s u b m e r g e d t h e tu b e s in a c o o l i n g b a th , a n d d e t e c t e d th e f r e e z in g e v e n ts b y t h e c h a n g e in v o l u m e w i t h i n th e t u b e s a s th e t e m p e r a t u r e o f th e b a th w a s g r a d u a lly l o w e r e d . A n a p p a r a t u s u s i n g s a m p l e tu b e s is s h o w n in F i g u r e 3 . T h e s a m p le c o n t a i n e r s a r e m i c r o c e n t r i f u g e t u b e s . W i t h i n t h e h o l d e r p la te , e a c h tu b e h a s a t h e r m o c o u p l e to u c h i n g its s id e ; t h e s e s e n s e th e l a t e n t h e a t o f f u s i o n a n d p r o v i d e s ig n a ls f o r r e ­ c o r d i n g t h e f r e e z i n g e v e n t s . T h e a r r a y o f tu b e s is im m e r s e d in a b a th , th e t e m p e r a ­ tu r e o f w h i c h is c o n t r o l l e d b y t h e p r o g r a m m e d m i x i n g o f tw o s tr e a m s o f liq u id s ,

14

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o n e c o o l e d to a b o u t - 3 5 ° C

a n d t h e o t h e r k e p t a t a b o u t + 5 ° C . I n g e n e r a l , th is

m e t h o d o f f e r s f l e x i b i l i t y f o r t e s t i n g d i f f e r e n t k in d s o f s a m p l e s a n d p r o v i d e s a s i m ­ p le a n d s o u n d p r o c e d u r e , b u t t h e c o s t o f th e e q u i p m e n t is a d r a w b a c k . E m u ls io n s

D i s p e r s i n g t h e w a t e r in th e f o r m o f a n e m u l s i o n f u lf ills , in a v e r y e f f e c t i v e w a y , th e p r i n c i p a l r e q u i r e m e n t s t a t e d e a r l i e r th a t t h e s a m p le b e s u b d i v i d e d in m a n y s m a ll u n its . E s p e c i a l l y f o r h o m o g e n e o u s n u c le a t i o n te s ts , o r f o r o t h e r s i n v o l v i n g t e m p e r a t u r e s o f - 2 5 ° C o r lo w e r , t h e s m a ll d r o p l e t s iz e s o f e m u l s i o n s is a m a j o r a d ­ v a n ta g e . C r i t i c a l f a c t o r s in t h e m e t h o d a r e t h e c h o i c e o f th e c a r r i e r f l u i d a n d th e m e t h o d o f p r e p a r a t i o n . S i l i c o n e o ils , o t h e r o ils , a n d h e p ta n e h a v e b e e n u s e d a s c a r ­ r i e r f l u i d s , u s u a l l y w ith a s m a ll a m o u n t o f a d d e d d is p e r s i n g a g e n t ( s o r b i t a n t r i ­ s te a r a te ) . T h e e m u l s i o n is p r e p a r e d w ith s tr o n g m e c h a n i c a l a g i t a t i o n . T h e r e s u l t i n g d r o p l e t s a r e f r o m a f e w |xm t o a b o u t 1 0 p.m in d ia m e te r . C l e a r l y , o n l y s m a l l p a r t i ­ c le s o f a n u c l e a t i n g m a t e r i a l c a n b e c o n t a i n e d in th e d r o p s , a l t h o u g h R a s m u s s e n e t a l. ( 1 9 7 5 ) , F r a n k s e t a l. ( 1 9 8 3 ) , a n d C la u s s e e t a l. ( 1 9 9 1 ) s u c c e e d e d in d i s p e r s i n g c e ll s u s p e n s i o n s . A w e l l - p r e p a r e d e m u l s i o n c a n r e m a in s ta b le f o r m a n y h o u r s . T h e s m a ll v o l u m e s o f t h e e m u l s i o n s a l l o w c o o l i n g in a n y n u m b e r o f w a y s . D e t e c t i o n o f f r e e z in g c a n a l s o t a k e d i f f e r e n t f o r m s . W o o d a n d W a lto n ( 1 9 7 0 ) p l a c e d th e e m u l ­ s io n in a s m a ll c a v i t y in a m e ta l p l a t e a tta c h e d to a h e a t s in k . F r e e z i n g o f t h e d r o p ­ le ts w a s r e c o r d e d t h r o u g h a m i c r o s c o p e . M o r e r e c e n tly , c a l o r i m e t e r s ( T a b o r e k , 1 9 7 5 ) o r c o m m e r c i a l l y a v a i l a b l e d i f f e r e n t i a l th e r m a l a n a l y z e r s ( e .g ., R a s m u s s e n e t a l., 1 9 7 5 ) a n d d i f f e r e n t i a l s c a n n i n g c a l o r i m e t e r s ( e .g ., M i c h e l m o r e a n d F r a n k s , 1982) h a v e b e e n u sed .

F i g u r e 3 . A n a r r a y o f m i c r o c e n t r i f u g e t u b e s s u p p o r t e d i n p l a t e w i t h e m b e d d e d t h e r m o c o u p l e s n e x t to

e a c h t u b e . T u b e s c o n t a i n 2 5 0 jil o f w a t e r . F o r a t e s t , t h i s a s s e m b l y is i m m e r s e d i n a c o o l i n g l i q u i d .

Principles of Ice Nucléation

15

O th e r F o r m s o f D r o p -F r e e z in g A ssa y

F r o m t h e p o i n t o f v i e w o f b i o l o g i c a l ic e n u c l e a t i o n , i t is o f s p e c ia l i m p o r t a n c e t h a t t h e e s s e n c e o f t h e d r o p - f r e e z i n g a s s a y c a n b e c a p t u r e d w ith th e t e s t i n g o f a l a r g e n u m b e r o f e x t e r n a l l y i d e n t i c a l s p e c i m e n s o f a n y s h a p e o r f o r m . D is k s c u t f r o m l e a v e s , l a r v a e , b u d s , o r o t h e r e n t i t i e s c a n b e t r e a t e d e x a c tly a s “ d r o p s ” in th e a n a l y s e s o f th e d a ta . O f c o u r s e , th e m a n n e r o f c o o l i n g o f th e u n its a n d th e d e t e c t i o n o f f r e e z i n g h a v e to b e m a t c h e d to th e t y p e o f s a m p l e s te s te d . S tr ic tly f r o m t h e p o in t o f v ie w o f t h e m e a s u r e m e n t , n e g l e c t i n g a n y p o s s i b l e p h y s i o l o g i c a l i n f lu e n c e s , th e s a m e u n its c a n b e i m m e r s e d in c l e a n w a t e r d r o p s o r tu b e s f i l l e d w ith w a te r . T h e a d d i t i o n a l w a t e r v o l u m e a c t s a s a s i g n a l a m p l i f i e r , m a k i n g th e d e t e c t i o n o f f r e e z i n g e a s ie r w h ile le a v in g th e in itia tio n o f fre e z in g u n a ffe c te d . E v a lu a tio n o f D r o p -F r e e z in g A s s a y s

In e s s e n c e , th e te c h n iq u e s d e s c rib e d c a n b e v ie w e d a s c o n s is tin g o f v o lu m e

V,

N0 d r o p s o f T as a

a ll d r a w n f r o m t h e s a m e s a m p l e , a n d s u b j e c t e d to te m p e r a t u r e

f u n c tio n o f ti m e

t.

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N(T)

or

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th e n u m b e r o f d r o p s r e ­

m a in in g u n f r o z e n a s a f u n c t i o n o f t e m p e r a t u r e o r o f tim e . T h e r e a r e n u m e r o u s a l t e r n a t i v e m e a s u r e s f o r c h a r a c t e r i z i n g th e r e s u l t s o f a f r e e z in g e x p e r i m e n t . O n e c a n n o te th e r a n g e o f t e m p e r a t u r e s o v e r w h ic h th e n u ­ c le a tio n e v e n t s t o o k p l a c e . O n e c a n o b t a i n th e a v e r a g e o r m o d a l f r e e z in g t e m p e r a ­ tu r e , o r th e t e m p e r a t u r e s a t w h i c h g i v e n p e r c e n t i l e s o f t h e d r o p s w e r e o b s e r v e d to b e f r o z e n . F r e q u e n t l y , a n o n s e t t e m p e r a t u r e ( a t w h i c h f r e e z i n g is f ir s t o b s e r v e d ) is r e p o r t e d . O n e c a n p r e s e n t a h i s t o g r a m o f t h e f r e e z i n g e v e n t s o r p l o t th e f r a c t i o n o f th e s a m p l e r e m a i n i n g u n f r o z e n a s a f u n c t i o n o f t e m p e r a t u r e o r tim e . T e s ts d o n e a t o n e o r a f e w f i x e d t e m p e r a t u r e s y i e l d t h e f r a c t i o n o f d r o p s f r o z e n a t th o s e t e m p e r a ­ tu r e s . E a c h o f th e d e s c rip to rs m e n tio n e d c a n s e rv e s p e c ific p u rp o s e s o f e v a lu a tio n , b u t a ll a r e d e p e n d e n t o n th e d r o p v o l u m e s u s e d a n d o n t h e p r o p o r t i o n s o f n u c le a t i n g m a te r ia l to w a te r . T e s t s w ith d i f f e r e n t v o l u m e s o r d i f f e r e n t d i lu tio n s c a n n o t b e q u a n tita tiv e ly c o m p a re d u s in g th e s e m e a s u re s . W ith th e c a v e a t th a t t i m e - d e p e n d e n t e f f e c t s a r e tr e a t e d a s s e c o n d - o r d e r , a m o r e g e n e r a l i n t e r p r e t a t i o n o f t h e d r o p - f r e e z i n g a s s a y s c a n b e o b t a i n e d b y u s in g th e d i f ­ f e r e n tia l a n d c u m u l a t i v e n u c l e u s c o n c e n t r a t i o n s i n t r o d u c e d b y V a li ( 1 9 7 1 ) . T h e

k(T) d e s c r i b e s th e c o n c e n t r a t i o n o f n u c le i a c tiv e T, a n d th e c u m u l a t i v e s p e c t r u m K(T) d e s c r ib e s a t t e m p e r a t u r e s w a r m e r th a n T:

d if f e r e n t i a l c o n c e n t r a t i o n s p e c t r u m

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th e to ta l n u m b e r o f d ro p s . T h e u n its o f

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la r g e e n o u g h t o a p p r o a c h t h e s e v a l u e s , o r i f t h e to ta l n u m b e r o f d r o p s in th e e x ­ p e r i m e n t is s m a l l , a m o r e a c c u r a t e v a l u e o f

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o n th e v o l u m e o f t h e d r o p s a n d o n t h e n u m b e r o f d r o p s u s e d in t h e e x p e r i m e n t ; f o r

k(T),

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m in i m u m a n d m a x i m u m v a l u e s o b t a i n e d f r o m a n e x p e r i m e n t a r e I [ ^ ( T ) ] min —

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f r o m 0 .3 3 to 1 53 c m " 3.

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th e t e m p e r a t u r e i n t e r v a l s u s e d to g r o u p th e d a ta , a im in g f o r h i g h r e s o l u t i o n in t e m ­ p e r a t u r e c a n l e a d to l a r g e u n c e r t a i n t i e s in th e v a lu e s o f

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F i g u r e 1. P re d ic te d s e c o n d a r y s tru c tu re s o f th e In a W an d In a Z p ro te in s. T h e la y o u t o f th e p re d ic tio n s is iso m e tric w ith th a t o f th e a m in o a c id s e q u e n c e s (se e C h a p te r 5). R e p rin te d w ith p e rm is s io n fro m W a rre n e t al. (1 9 8 6 ).

M o le c u la r M o d e lin g

103

m u st o rd er w a te r m o le c u le s on its su rface in an ice-lik e m an n er. T he arra n g e m en t o f d o n o r-a c c ep to rs o f h y d ro g e n b o n d s in such a p ro tein m ust be co m p le m e n ta ry to the a rra n g e m en t o f d o n o r-a c c e p to rs on ice. H o w ev er, this is n o t sufficien t fo r e f­ fe ctiv e ice n u c leatio n , sin c e th ere ex ist p ro tein stru ctu res (the “ a n tifreezes” ) w ith ice-lik e sites co n sistin g o f th ree to fiv e co m p a c tly a rran g ed d o n o r-accep to rs o f h y ­ d ro g en b o n d s th at d o n o t in itiate b u t ra th e r in h ib it ice fo rm atio n (Y an g e t al., 1988). O th e r p h y sical c o n d itio n s m u st be satisfied fo r a p ro tein m o lecu le to a c t as an ice n u cleato r: the ic e -lik e site o f the m o lecu le m u st be larg er than 100 A (10 n m ), the ra d iu s k n o w n ro u g h ly as th e size o f th e c ritical n u cleu s (F letcher, 1970). A lso, the m o re rig id th e ice -lik e p ro te in stru ctu re, th e m o re effectiv e it is e x p ected to be in n u c le a tin g ice. E v en in th e a b sen ce o f ex p e rim e n ta l d ata on In a p ro tein stru ctu re, th ese tw o co n strain ts (fo llo w in g fro m reg u la rity and fu n ctio n ) sev erely re strict the ra n g e o f p lau sib le m o d e ls and h e n c e fa c ilitate m o le c u la r m o d elin g . M o reo v er, several co m m o n p rin c ip le s o f 3 -D p ro te in stru ctu res also co n stra in the m o d elin g o f p ro tein stru ctu res: 1) th e a b sen ce o f sig n ific a n t co v a le n t an d steric ten sio n s, 2) clo se p a c k ­ ing, and 3) in v o lv e m e n t o f all d o n o r-a c c ep to rs o f h y d ro g e n bon d s in b o n d in g w ith each o th er o r w ith w a te r m o lecu les. T h is so ca lle d “p ro h ib itio n on d e h y d ra tio n ” w as d raw n fro m th e o b se rv a tio n th at th e re are p ra c tic a lly no u n p aired d o n o r-a c c e p ­ tors in th e n o n p o la r in te rio rs o f k n o w n p ro tein stru ctu res. F o r ex am in atio n o f the h y d ro g e n b o n d in g , the fo llo w in g c riterio n c o u ld be used: the A -H - • • B b o n d is fo rm ed w h en th e d ista n c e A B is b e tw een 2.6 an d 3.2 A and the an g le H A B is less th an 30° (V e n k a c h a tala m , 1968).

Secondary Structure Prediction In nearly all p ro tein s, th e local fo ld in g o f th e ch a in lead s to th e fo rm atio n o f a h elices o r P -sh eets, an d th e se assem b le to give th e m o lecu les th eir 3-D stru ctu res. It can be assu m e d th at th e ice n u cleatio n p ro tein is also an ag g reg ate o f seco n d ary stru ctu re ele m e n ts. W h e n th e a lg o rith m o f G a rn ie r e t al. (1 9 7 8 ) to p red ict p ro tein seco n d ary stru c tu re fro m th e a m in o acid seq u en ce w as ap p lied to the ice n u cleatio n p ro tein s (W arren e t al., 1986), th e ir n o n re p e titiv e N - and C -term in al d o m ain s w ere p red icted to c o n sist o f b o th a -h e lic e s and (i-strands ty p ical o f g lo b u lar p ro tein a r­ ra n g e m e n t (F ig . 1). T h e c o rre c t p re d ic tio n o f th e te rtia ry stru ctu re o f these d o m ain s is ra th e r d iffic u lt d u e to th e irre g u la r am in o acid seq u en ces. It w ill be m o re a p p ro ­ p riate to o b tain d ire c t e x p e rim e n ta l ev id e n c e o f th e ir 3-D stru ctu res. In co n trast, the rep etitiv e p o rtio n s w ere p re d ic te d to c o n sist o f a lte rn a tin g p -stran d s and ran d o m co il reg io n s (F ig . 1). O th e r alg o rith m s (C h o u an d F asm an , 1974; P titsy n and F in k elstein , 1983) d isp lay sim ila r results. It sh o u ld be noted, h o w ev er, that the a l­ g o rith m s fo r p re d ic tin g se c o n d a ry stru ctu re w ere w o rk ed o u t w ith g lo b u lar p ro ­ teins; they sh o u ld be a p p lie d w ith cau tio n to large, re p e titiv e am in o acid seq u en ces th at p ro b ab ly a ssu m e a n o n g lo b u la r stru ctu re. R e g u la r p rim ary stru ctu res re q u ire a sp ecial ste re o c h e m ica l a n a ly sis o f th eir p a ck in g in all p o ssib le re g u la r c o n fo rm a ­ tio n s (a -h e lix , P -stru ctu re, an d ch a in s in a p o ly p ro lin e co n fo rm atio n ). S te re o ­ ch em ical an a ly sis has re v e a le d th at the ice n u cleatio n p ro tein s are very u n lik ely to self-a g g re g ate into a c o m p le x co n sistin g o f a -h e lic e s b e cau se o f th e ab sen ce o f the p e rio d icity ( H - X - X - H - X - X - X ) n (w h ere H is a n o n p o la r resid u e and X is any h y ­ d ro p h ilic re sid u e ) w h ich is ty p ical for the a -h e lic a l fib rillar pro tein s (F raser and M acR ae, 1973). It w as also im p o ssib le to u n ite p o ly p e p tid e ch ain s to form a p o ly ­

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f p ro lin e h elix b e c a u se o f th e a b sen ce o f co llag en -lik e p e rio d ic ity ( G ly - X - X ) n in the seq u en ce. T h e [3 co n fo rm a tio n w as fo u n d to be the o n ly re g u la r co n fo rm a tio n o f th e ice n u c le a tio n p ro te in s th at a llo w ed the assem bly o f d iffe re n t re p e titiv e 3-D stru ctu res, sin c e th e P -stran d s can in teract w ith each o th er by in te rp e p tid e h y d ro g en b onds, la rg ely in d e p e n d e n t o f th e am in o acid sequence. T h u s, on th e b a sis o f all th ese p red ictio n s, one can c o n c lu d e th a t th e rep etitiv e p o rtio n o f th e ice n u c le a tio n p ro te in s m o st p robably co n sists o f (3-strands.

Models of the 3-D Structure of Ina Proteins T h ree a tte m p ts h a v e been m a d e to p re d ic t the 3-D stru ctu re o f th e ice n u cleatio n p ro tein s (W arren e t al., 1986; M izu n o , 1989; K ajava and L in d o w , 1993). W a rre n e t al. (1 9 8 6 ) p ro p o se d tw o stru ctu ral m o d els th a t h a v e tria n g u la r and h e x ag o n al sh a p e s (F ig . 2). O n e o f th e m o d els (Fig. 2B ) re p re se n ts an u p -a n d -d o w n (3-sheet c o n sistin g o f 3 x 8 re sid u e p -stran d s. T his P -stru ctu re is m u ch lo n g e r in the h y d ro g en b o n d d ire c tio n than in th e P -stran d one. T he P -sh e e t is fo ld ed in a tria n ­ g u lar p rism . T h e o th e r m o d el (F ig. 2 C ) is form ed by rig h t- an d le ft-h a n d e d h elical fo ld s u n ited by th e a n tip a ra lle l p -stru c tu re in teractio n s into a h e x a g o n a l d o u b le h e­ lix. T h ese a rra n g e m e n ts o f th e p ro tein w ere suggested by th e a u th o rs b e cau se 1) they are c o n siste n t w ith th e re su lts o f seco n d ary structure p re d ic tio n , w h ich su g g est th at m ain ly p -stra n d s ap p e a r in th is p o ly p ep tid e; 2) it w as p o ssib le to in co rp o rate the th ree lev els o f stru c tu ra l rep etitio n into the m odels; and 3) p ro te in fo ld in g o f the m o d els re se m b le s th e sp ace g ro u p P 6 -3 /m m c o f ice Ih (d e fin ed b elo w ).

A

B

C

F i g u r e 2 . S tru c tu ra l m o d e ls s u g g e s te d b y W a rre n e t al. (1 9 8 6 ) in w h ic h th e ic e n u c le a tio n p ro te in d is p la y s a s y m m e try r e la te d to th a t o f ic e (n o t to sc a le ). A , Ice; B , 4 8 - re s id u e u n it o f tr ia n g u la r m o d e l; C , 4 8 - re s id u e u n its o f b o th c h a in s o f a n tip a r a lle l d o u b le -h e lix m o d e l. T h e ir in d iv id u a l stru c tu re s , th e s y m m e trie s o f th e s e s tr u c tu r e s , a n d th e ir e x te n d e d s y m m e trie s are sh o w n at le ft, c e n te r, a n d rig h t. R e p rin te d w ith p e r m is s io n fro m W a rre n e t al. (1 9 8 6 ).

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T h e m o d el, h o w ev er, w as p resen ted only sc h em atically , w ith o u t any d e ta ils at th e m o le c u la r level. T h e c o n stru c tio n o f th eir m o le c u la r stru ctu res rev eals so m e d isc re p a n c ies b e tw een th e m o d e ls an d p rin cip les o f p ro tein stru ctu res. F o r e x a m ­ ple, the p -stru c tu re s o f the m o d e ls are flat, w h ereas P -stru ctu res o f p ro tein s alw ay s fo rm a rig h t-h a n d e d tu rn (C h o th ia , 1973). T h e tria n g u la r and h ex ag o n al p rism s, b o th co n stru c te d from th e tw iste d P -stran d s, m u st be left-h an d ed along the p rism axis, and this m u st d isto rt th e ir sy m m e try relativ e to th a t o f ice. A n o th er d isa d v a n ­ tag e o f these a rra n g e m e n ts is th e lack o f clo se p a c k in g o f the p ro tein stru ctu res in ­ side th e p rism s. T h e re are no su ch “o p e n ” fo rm s am o n g ex p erim en tally d e term in ed p ro tein stru ctu res. T h e first m o d e l d e v e lo p e d a t th e ato m ic level w as su g g ested by M izu n o (1 9 8 9 ) (Fig. 3). T h e m o d el w as d e d u c e d fro m th e assu m p tio n th at the 3-D stru ctu re o f the ice n u cleatio n p ro te in s m u s t b e a h elix , w ith each o c ta p ep tid e rep eat a ssu m in g an id en tical co n fo rm a tio n . (T h e m o d el n eg lects the tw o h ig h e r o rd ers o f p erio d icity [16 and 4 8 re sid u e s] th a t a re fo u n d in th e Ina p ro tein s). T h is assu m p tio n facilitated the g e n e ratio n o f m o d e le d stru c tu re s an d th eir en erg y calcu latio n as w ell as the search for ice -lik e re g io n s on th e p ro tein surface. A d etailed an a ly sis o f ic e -lik e sites req u ires d e te rm in a tio n o f the ice cry stals that sh o u ld be tak en fo r the c o m p a riso n w ith the p ro tein tem p lates. T h e m o st fav o rab le ice cry stals, w ith an o p tim a l te trah ed ral o rien tatio n o f h y d ro g en b o n d s o f w ater m o lecu les, can e x ist in tw o fo rm s: ice Ih (h e x ag o n al) and ice Ic (cubic). Ice Ih is th e m o st c o m m o n stru ctu re. A t th e sam e tim e ice Ic c o u ld act as an in term ed iate in th e n u cleatio n o f ice Ih, b e c a u se 1) ice Ic can be irre v e rsib ly tra n sfo rm ed into ice Ih w h en freezin g o c cu rs n e a r 0 °C (E ise n b e rg and K a u zm an n , 1969); and 2) ice Ih and

F i g u r e 3 . S k e le ta l a x ia l p r o je c tio n o f th e tu rn o f a h e lix s u g g e s te d b y M iz u n o (1 9 8 9 ) fo r th e ice n u c le a tio n p ro te in s . T h e h e lix tw is t a n d h e ig h t p e r o c ta p e p tid e a re 6 0 .6 ° a n d 1.41 Â , re s p e c tiv e ly . T h e in s id e su rfa c e o f th e h e lix c o n s is ts o f n o n p o la r le u c in e s id e c h a in s . C o n to u rs a ro u n d th e a to m s o f th e in s id e su rfa c e d e m o n s tr a te v a n d e r W a a ls v o lu m e s . A w a te r m o le c u le (W ) in th e c e n te r o f th e n o n p o la r c a v ity s h o w s its in a b ility to fo rm all n e c e s s a ry h y d r o g e n b o n d s.

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ice Ic h a v e a s im ila r su rface, an d ice Ic fo rm ed d u ring n u c le a tio n c o u ld p erm it the su b se q u e n t g ro w th o f ice Ih. A n X -ray diffractio n study o f ice stru c tu re s fo rm ed in p ro te in a c e o u s g e ls sh o w ed th at e ith e r h ex ag o n al or cu b ic ice I co u ld b e co m e the p re d o m in a n t p h a se , d e p e n d in g on co o lin g co n d itio n s (D o w ell e t al., 1962). T h e re ­ fore, p la n e s b o th o f ice Ih an d ice Ic, d en sely p opulated by h y d ro g e n -b o n d in g sites, w ere co n sid e re d . A s a re su lt o f M iz u n o ’s c a lcu latio n s, tw o helices fo rm e d by rep etitio n o f id e n ti­ cal o c ta p e p tid e c o n fo rm a tio n s w ere selected. T he helices h av e in tern al an d e x tern al su rfaces a p p ro x im a te d by a h e x a g o n a l shape. O ne o f th e h e lices (F ig. 3) h as an ep itax ial fit to ice Ih , th e o th er, a fit to ice Ic. A lth o u g h , acc o rd in g to M iz u n o (1 9 8 9 ), th e e x te rn a l su rfa c e o f th ese tw o helices hav e ice -lik e sites, th e p ro tein stru ctu res th e m se lv e s are u n u su al (irre g u la r c o n fo rm atio n s o f th e o c ta p e p tid e se g ­ m ents, in te rse g m e n ta l in te ra c tio n s, an d the helical a rc h ite c tu re as a w h o le). A s a rule, a c a lc u la te d m in im u m en e rg y stru ctu re is highly d e p e n d e n t on initial c o n fo r­ m atio n s an d in p u t c o n strain ts. T h e c o n d itio n that the ice p ro te in ch ain form a helix has led to stru c tu re s w ith a h e x a g o n a l shape, sim ilar to th e m o d e ls o f W a rre n e t al. (1 9 8 6 ). T h u s, M iz u n o ’s m o d e ls h a v e a sim ilar d isad v an tag e c o n c e rn in g th e cav ities in sid e th e stru c tu re . A ste re o c h e m ica l an aly sis reveals th e in ab ility o f w ater m o le ­ cules (F ig. 3) in sid e th ese n o n p o la r cav ities to form all th e n e c essary h y d ro g e n bonds; i.e., so m e h y d ro g e n b o n d in g g ro u p s w ould rem ain d e h y d ra te d . T h ese sh o rtc o m in g s o f p re v io u s m o d e ls en co u rag ed K a ja v a an d L in d o w (1 9 9 3 ) to u n d e rta k e a n o th e r atte m p t to p re d ic t the 3-D structure o f ice n u c le a tio n p ro tein s. T h e go al o f th e n ew m o d el w as to d em o n strate an ic e-lik e site c o n sistin g o f Pstran d s— th e th e o re tic a lly p re d ic te d e lem en ts o f an Ina p ro te in ’s se c o n d ary stru c ­ ture. F ig u re 4 sh o w s th a t the a n tip a ra lle l P -strands, o rien ted in a su ch a m a n n e r that they c o n ta c t e a c h o th e r via side ch ain s, have donors and a c c e p to rs in an arran g e-

F i g u r e 4 . S p a c e - f illin g m o d e ls o f tw o a n tip a ra lle l P -s tra n d s h a v in g a n ic e - lik e a rra n g e m e n t o f th e d o n o r -a c c e p to rs o f h y d r o g e n b o n d s (le ft) a n d a fra g m e n t o f ic e Ic (rig h t). T h e (3-strands c o n ta c t e a c h o th e r b y s e rin e a n d th r e o n in e s id e c h a in s . D o n o r-a c c e p to rs o f th e P -s tra n d s a n d o f th e ic e , w h ic h fo rm sim ila r p a tte r n s , a re h a tc h e d . T h e ic e -lik e p a tte rn c a n b e e x te n d e d b y its re p e titio n s a lo n g th e c h a in a n d in th e d ir e c tio n p e r p e n d ic u la r to th e c h a in s .

M o le c u la r M o d e lin g

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m e n t sim ilar to th a t in ice Ic. T o c reate this ice-lik e p attern , the p -stran d s sh o u ld h av e p a rtic u la r sid e c h ain s, n am ely , sid e ch a in s o f serin e and th reo n in e (in som e cases also a sp artic and g lu ta m ic acid s, asp a ra g in e an d g lu tam in e). T he am in o acid se q u en ces o f ice n u c le a tio n p ro te in s h av e a n o m alo u sly larg e co n ten ts o f se rin e and th re o n in e re sid u e s, c o n siste n t w ith the p o ssib ility o f th e ir ad o p tin g the m o d eled stru ctu re. S in g le ch a in s in P -c o n fo rm a tio n s are h ig h ly flex ib le. T o fix th eir co n fo rm atio n , th e (3-strands sh o u ld p a rtic ip a te in th e fo rm atio n o f in te rp ep tid e h y d ro g en b o n d s (fo rm in g a (3-structure). T h e re p e titiv e d o m ain o f an ice n u cleatio n p rotein, h o w ­ ever, has an e x tre m e ly lo w p ro p o rtio n o f h y d ro p h o b ic resid u es (ab o u t 30% ), an d a ste re o c h e m ica l an aly sis in d ic a te s th at a p o ly p e p tid e ch a in w ith such a d istrib u tio n o f re sid u e s c a n n o t be in v o lv e d in a stru ctu re co n sistin g o f e x ten siv e (each m o re th an tw o |3-strands) ^ -la y e rs (F ig. 5, left) b e cau se o f th e d eh y d ra tio n o f h y d ro p h ilic re sid u e s in th e zo n e o f in te rla y e r co n tact. In th is case, a bro ad , rig id ice-lik e te m ­ plate, w h ich a t th e sam e tim e satisfies p rin cip les o f p ro te in stru ctu re m o d elin g , can be co n stru c te d fro m tw o -stra n d e d P -stru ctu res stack ed w ith each o th er (F ig. 5, right). A n o th e r c o n stra in t fo r m o d e lin g w as th at the P -stran d s th at take p art in this a r­ ra n g e m e n t b e lo n g to th e sam e p o ly p e p tid e chain. S in ce a d ja c e n t P -strands alo n g the ch ain are m o st lik ely to in te ra c t w ith each o th er, in ferred fro m the 3-D stru ctu re o f o th e r p ro tein s, th e m o st p ro b a b le tw o -stran d ed p -stru c tu re is a P -hairpin. T h e a rra n g e m en t o f stack ed P -h a irp in s sh o w n in F ig u re 6 w as ch o sen as the m o st p ro b ­ ab le fo r the fo llo w in g reaso n s: 1) T h is fo ld in g pattern c o rre sp o n d s to th e 8-, 16-, an d 4 8 -re sid u e p e rio d icities o f th e p rim ary stru ctu re. 2) C o n se rv a tiv e g ly cin e resid u e s, w h ich are the b e st resid u es fo r b en d in g o f the c h ain , are lo c a te d in e v ery turn o f th e su g g ested stru ctu re. H ig h ly c o n serv ativ e se rin e and th re o n in e re sid u e s o ccu r o nly in th e m id d le o f these P -strands and can effe c tiv ely p a rtic ip a te in th e ice-lik e tem p late as in F ig u re 4. 3) T h e re is n o g ly cin e in th e “ g ly cin e p o sitio n ” in e v ery sixth o ctap ep tid e; th is o c ­ ta p e p tid e th u s d o es n o t p ro d u c e a sh a rp bend.

c

c

D

m

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Q

x

E

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F i g u r e 5 . S c h e m a tic re p re s e n ta tio n o f p o s s ib le (5-layer a rra n g e m e n ts v ie w e d a lo n g th e c h a in s . H a tc h e d re g io n s d e n o te o rd e re d w a te r n e a r ic e -lik e te m p la te . C ro s s e s a n d p o in ts o n th e c h a in s sh o w o p p o s ite [5-strand o r ie n ta tio n s n e e d e d f o r fo rm a tio n o f th e ic e - lik e su rfa c e . B la c k h a lf-c irc le s m e a n sid e c h a in s w h ic h a re d e h y d r a te d b e tw e e n [i-la y e rs. R e p rin te d w ith p e rm is s io n fro m K a ja v a an d L in d o w (1 9 9 3 ).

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4) T h e le n g th s o f th e fJ-strands in th is m odel co in cid e w ith th e a v e rag e len g th s o f (3-strands (six to e ig h t re sid u e s) w hich o ccur in k n o w n p ro tein stru ctu res (S te rn b e rg an d T h o rn to n , 1977). 5 ) A n a ly sis o f th e p rim a ry stru c tu re s rev eals th at the 4 8 -re sid u e u nits h av e four hig h ly c o n se rv a tiv e o c ta p e p tid e s and tw o o ctap ep tid es w ith a lo w co n serv atio n p la c e d a lo n g th e ch ain as sh o w n in F ig u re 6. O ne can e x p e c t th at rep eats w ith the g re a te st fid e lity w ill p a rtic ip a te in the form ation o f th e fu n c tio n a lly im p o r­ tan t ice -lik e su rface. T h e su g g e ste d fo ld in g pattern, u n lik e all o th ers, has only th e h ig h ly c o n se rv e d re sid u e s on th e suggested ice -lik e su rfa c e (Fig. 6). T he o th e r sid e o f th e u n its m ig h t in te ra c t via hy d ro g en and io n ic b o n d s w ith th e p o ­ la r h e a d s o f m e m b ra n e lip id s w h ich , in turn, could in d u ce an d su p p o rt su ch a flat p ro te in stru c tu re (see “In flu e n c e o f th e B acterial M e m b ra n e on P rotein S tru c tu re ” la te r in this ch ap ter). In sp e c tio n o f k n o w n p ro tein stru ctu res reveals th at so m e, fo r ex a m p le , ty p e 1 m o d u le o f fib ro n e c tin (B aro n e t al., 1990) and N -term in al d o m a in o f C D 4 (R y u et

F i g u r e 6 . S c h e m a tic re p re s e n ta tio n o f le v e ls o f o rg a n iz a tio n o f th e In a Z p r o te in in th e K a ja v a a n d L in d o w (1 9 9 3 ) m o d e l. A r e p re s e n ta tiv e 4 8 - re s id u e re p e a tin g u n it (p o s itio n s 4 8 8 - 5 3 5 ) in th e In a Z p ro te in is d e p ic te d a t th e lo w e r left. T h e w id e a rro w s in d ic a te [5-strands w h e r e a s th e th in lin e s c o n n e c tin g th e s e a r ro w s r e p re s e n t h y d r o g e n b o n d s. T h e c a p ita l le tte rs c o r re s p o n d to th e m o st c o n s e rv e d r e s id u e s . O c ta p e p tid e s w ith h ig h c o n s e rv a tio n th ro u g h o u t th e m o le c u le a re d e p ic te d in b la c k . T h e s p a tia l f o ld in g o f a re g io n c o n s is tin g o f tw o o f th e se 4 8 -re s id u e u n its re p e a te d a lo n g th e c h a in is d e p ic te d a t th e lo w e r rig h t. T h re e h ig h ly c o n s e rv a tiv e re g io n s ( b la c k a rro w s ) a re lo c a te d o n th e s a m e s id e o f th e p r e d ic te d s tru c tu re fo r e a c h 4 8 a m in o a c id u n it. A n a p p r o x im a te s c a le d e p ic tio n o f a n a g g r e g a te o f th r e e In a Z p ro te in m o le c u le s v ie w e d fro m a b o v e th e p la n e o f th e m e m b ra n e is d e p ic te d a t th e to p . T w o c o n tig u o u s 4 8 - re s id u e re p e a te d b lo c k s a re in d ic a te d in b la c k . T iltin g o f th e tr a p e z o id a l b lo c k s is n o t s h o w n . R e p rin te d w ith p e rm is s io n fro m K a ja v a a n d L in d o w (1 9 9 3 ).

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al., 1990), h av e stack ed [3-hairpins sim ila r to th o se p ro p o sed above. T h e fo ld in g p a ttern o f a 4 8 -re sid u e u n it has a m irro r to p o lo g y if co m p ared w ith a n e ig h b o rin g u n it alo n g th e ch a in (Fig. 6). M o d e lin g o f th e m o le c u la r stru ctu re rev ealed th at th e c o n fo rm a tio n o f (3-hairpins in a d ja c e n t u nits co u ld be the sam e and sim ilar to th a t in o th e r k n o w n p ro tein s; i.e., th e (3-strands in a slightly rig h th an d ed tw ist an d th eir (3-turns c o n stru cted w ith a La L- o r P o ^ -co n fo rm atio n s

F i g u r e 7 . S p a c e - f illin g m o d e ls o f a 4 8 - a m in o a c id p o rtio n o f th e p ro p o s e d th re e -d im e n s io n a l s tru c tu re o f ic e n u c le a tio n p r o te in s w h ic h h a s a tr a p e z o id a l s tru c tu re ( b o tto m le ft) a n d th e (1 1 0 ) su rfa c e o f ic e Ic ( b o tto m rig h t). H a tc h e d a to m s a n d th o s e d e p ic te d in b la c k in b o th th e In a p ro te in a n d in ic e Ic r e p re s e n t c o r re s p o n d in g d o n o r -a c c e p to rs o f h y d ro g e n b o n d s. T h e z ig z a g g e d lin e s su p e rim p o s e d o n th e p r o te in s tru c tu re ( b o tto m le ft) re p re s e n t c h a in s o f w a te r m o le c u le s in th e la ttic e o f ic e Ic to fa c ilita te v ie w in g . T h e to p o f th e fig u re s h o w s tiltin g o f th e 4 8 -a m in o a c id tr a p e z o id a l b lo c k s o f a n ic e p ro te in u p o n in te r d ig ita tio n o f th e r e c ta n g u la r 4 8 a m in o a c id b lo c k s fro m tw o d iffe re n t ic e p ro te in m o le c u le s w h e n v ie w e d fro m a b o v e th e p la n e o f th e m e m b ra n e . T h e z ig z a g lin e s o n th e p ro te in su rfa c e (to p le ft) sh o w th e o r ie n ta tio n o f th e ic e - lik e p a tte rn . A n e n la rg e d re p re s e n ta tio n o f th e tw o ice c ry sta l p la n e s c o rre s p o n d in g to th e tw o ic e - lik e p r o te in p la n e s c o m p ris e d o f re c ta n g u la r a n d tra p e z o id a l b lo c k s is d e p ic te d in th e c irc le .

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F i g u r e 8 . S te re o v ie w o f ic e c ry s ta l o v e r ic e -lik e s ite o f In a Z p ro te in . T h in lin e s re p re s e n t h y d ro g e n b o n d s o f th e ic e c r y s ta l; th ic k lin e s r e p re s e n t p o ly p e p tid e c h a in s. B la c k d o ts o n th e h y d ro g e n b o n d n e tw o rk re p re s e n t w a te r m o le c u le s . O p e n c irc le s d e n o te p ro te in d o n o r -a c c e p to rs o f h y d ro g e n b o n d s, w h ic h fo rm a p a tte rn s im ila r to th a t o n th e (1 1 0 ) p la n e o f ic e Ic. L e tte rs a n d n u m b e rs d e n o te id e n tity a n d p o s itio n , r e s p e c tiv e ly , o f th e r e s id u e s w ith in In a Z p ro te in . R e p rin te d w ith p e r m is s io n fro m K a ja v a a n d L in d o w (1 9 9 3 ).

(E fim o v , 1986; W ilm o t and T h o rn to n , 1988) ( a , a L, p m ean c o n fo rm a tio n s o f a re sid u e fro m a c o rre sp o n d in g re g io n o f th e R am ach an d ran p lo t). T h e stru ctu res o f the u n its are c lo se ly p a c k e d an d h av e no forb id d en van d e r W a a ls co n ta c ts o r d e ­ h y d rated d o n o r-a c c e p to rs o f h y d ro g e n bonds. B oth ad ja c e n t u n its u se th e N H and C O g ro u p s o f th e b a c k b o n e an d serin e, th reo n in e, and a sp artic acid sid e c h a in s to o rg a n iz e th e ic e -lik e te m p la te (F ig. 7). A lattice-m atch o f th e ice-lik e p ro te in su r­ face w ith th e (1 1 0 ) fa c e o f ice Ic is rep resen ted in F ig u re 8. A g o o d fit o f th e van d er W aals su rfa c e s o f th e p ro tein and the ice crystal sh o u ld ad d itio n a lly d ecrease th e in te rfa c ial e n erg y . T h e d iffe re n c e s o f the p o ly p e p tid e co n fo rm atio n o f the fo ld s from q u a si-m irro r sy m m etry are lo c a liz e d in the cro sso v e rs b etw een one g ro u p o f P -h airp in s an d the next. B e c a u se o f th is d iffe re n c e, ty p e 1 units h av e a re c ta n g u la r sh ap e and ty p e 2 u nits h av e a tra p e z o id a l sh ap e (F igs. 6 and 7). T he stru ctu ral m o d el w ith th e tw o q u a si-m irro r-sy m m e tric a l u n its a lte rn a tin g along the chain lead s to th e e x p e ctatio n o f an a d d itio n a l 9 6 -re sid u e p e rio d icity . H ow ever, an an aly sis o f the p rim ary stru c ­ ture o f ice n u c le a tio n p ro te in s d o e s n o t su p p o rt this c o n se q u e n c e o f th e m o d el. If it is a ssu m e d th a t the N - an d C -term in al do m ain s o f ice n u c le a tio n p ro tein s are g lo b u la r an d th a t th e c e n tral p o rtio n co n sists o f “re c ta n g u la r” an d “tra p e z o id a l” stru ctu res a lte rn a tin g w ith each o th er, the w hole stru ctu re o f th e In aZ p ro tein w o u ld a p p e a r sc h e m a tic a lly as in F ig u re 6. T here is an in te rru p tio n o f th e 4 8 -re si­ du e reg u la rity in th e In a Z p ro te in , w h ere th e sequ en ce co n ta in s 16 ad d itio n a l re si­ dues. In th is ca se , an ice -n u c le atio n b lo ck m ig h t h av e an a d d itio n a l (fo u rth ) Ph airp in (F ig . 6). It is w o rth n o tin g th at In aW p rotein has a sim ila r + 1 6 in te rru p tio n , IceE and In a X p ro te in s have no such in terruption, and the 4 8 -re sid u e reg u la rity o f InaA p ro te in is in te rru p te d th ree tim es. T h e re c ta n g u la r b lo c k s are o rie n te d to o n e side and the tra p e z o id a l b lo ck s to the o th er sid e re la tiv e to th e c e n tral ax is o f th e reg u lar dom ain.

Aggregation of the Ina Proteins Ice n u c le a tio n a c tiv ity in a p o p u la tio n o f bacterial cells o c c u rs at tem p e ra tu res from —12°C to —2 °C , c o rre sp o n d in g to n u clean t m asses th a t ra n g e fro m ab o u t 1 to

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50 ice p ro tein s, re sp e c tiv e ly (G o v in d a ra ja n and L in d o w , 1988b). Ina p ro tein s m u st th e re fo re be a b le to a g g re g a te in an u n lim ited fa sh io n w ith ad d itiv e effects. L ittle is kn o w n a b o u t th e g e n eral sh a p e o f th e ag g reg ate. T h e g en eral sh ap e for th e a g g re ­ g ate w as estim a te d by c a lc u la tio n s o f th e d e p e n d e n c e o f n u cleatio n te m p e ra ­ tu re /m o le c u la r m ass fo r v a rio u s sh ap es o f th e n u c le a to r u sin g the h ete ro g e n e o u s ice n u cleatio n th eo ry (B u rk e an d L in d o w , 1990). T h e b e st fit b etw een the m easu red an d calcu lated d a ta w as o b ta in e d fo r a flat, d isk -lik e n u c le a to r w ith a d iam eter d e ­ p en d in g on the n u m b e r o f m o le c u le s in the a g g reg ate. T h is h y p o th etical n u c le a to r g en e ra te s ice e m b ry o s o n ly o n its u p p e r su rface an d w o u ld ap p ear as a h y d rated p atch o n the m e m b ra n e su rface. P atch es (1 ,0 0 0 -2 ,0 0 0 A in d iam eter) th at m ight re p re se n t n u c le a tio n sites h a v e been o b serv ed on n e g a tiv e ly stained Ice* b a c te ria u sin g an e lectro n m ic ro sc o p e (W elch an d S p eid el, 1989). T h e m e c h a n ism o f th e a d ju stm e n t o f sep arate m o le c u le s into an ag g reg ate w ith a large c o n tin u o u s ice-lik e site is n o t e v id e n t in th e p rism m odels (W arren e t al., 1986; M izu n o , 1989). K a ja v a an d L in d o w (1 9 9 3 ) p ro p o se d th at In a p ro tein s fo rm a flat ag g re g a te on the su rfa c e o f th e m e m b ran e by v irtu e o f in terd ig itatio n o f 48resid u e u nits (F ig. 6). T h e ir m o d elin g has rev ealed th at a re ctan g u lar b lo ck can be

F i g u r e 9 . T w o p r o je c tio n s o f K a ja v a a n d L in d o w ’s s k e le ta l m o d e l o f fra g m e n ts o f fo u r In a Z p ro te in s w h e n v ie w e d fro m a b o v e th e p la n e o f th e m e m b ra n e (to p ) a n d a lo n g th e p la n e o f th e m e m b ra n e (b o tto m ). T h e s tru c tu re , c o n s is tin g o f 14 4 8 -re s id u e u n its , re fle c ts its r e fin e m e n t a fte r e n e rg y m in im iz a tio n . O n e in te r d ig ita te d b a n d (c e n tra l b a n d o n th e u p p e r p ro je c tio n a n d p ro tru d in g fro m th e fla t s tru c tu re o n th e lo w e r p ro je c tio n ) is c o m p o s e d o f tilte d tr a p e z o id a l b lo c k s w h ile o th e r b a n d s are c o m p o s e d o f re c ta n g u la r b lo c k s .

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d o ck ed w ell b e tw e e n o th e r re c ta n g u la r blocks and ca n n o t be c lo se -p a c k ed b etw een trap ezo id al b lo c k s. In te rd ig ita te d rec ta n g u la r blocks in th e zo n e o f in te rm o le c u la r co n tacts h a v e th e sam e a rra n g e m e n t o f the co n serv ativ e serin e, th reo n in e, an d g lu ­ tam in e re sid u e s as th a t w ith in th e 4 8 -resid u e units, in d ic a tin g th a t re c ta n g u la r ice­ lik e te m p la te s c a n m e rg e in to a co n tin u o u s ice-like b and. B e c a u se o f a p re fe re n ce fo r “re c ta n g le -re c ta n g le ” p a c k in g , the p ro tein s in teract in an a n tip a ra lle l o rie n ta ­ tion; th eir sp e c ific sta g g e rin g is d ictated by the d istrib u tio n o f 4 8 - an d 6 4 -resid u e u n its (F ig. 6). M o d e lin g has d e m o n strated that an in te rd ig itated a rra n g e m en t o f trap ezo id al b lo c k s c a n n o t lie in th e sam e p lan e b ecau se o f stru c tu ra l co n strain ts. M o le c u la r m o d e lin g has also re v e a le d th at the space b etw een a d ja c e n t re c ta n g u la r b lo ck s alo n g th e c h a in is in a d e q u a te for insertion o f th e re c ta n g u la r b lo c k from an o th e r m o le c u le w h en b o th re c ta n g u la r and trap ezo id al b lo c k s lie in th e sam e plane. B ro a d e n in g o f th is in te rb lo c k space o ccu rs w h en tra p e z o id u nits are tilted relativ e to th e p la n e o f re c ta n g le b lo ck s (Figs. 7 and 9). K a ja v a an d L in d o w (1 9 9 3 ) th e re fo re p ro p o se d a q u a te rn a ry stru ctu re o f ice p ro te in s w ith a lte rn a tin g flat “re c ta n g u la r” b a n d s an d w av y “trap e z o id a l” bands. T h e p la n e o f the trap ezo id al b locks is tilte d 120° w ith re sp e c t to th e plane o f the re c ta n g u la r b lo c k s (F igs. 7 and 9). T h is a rra n g e m e n t p erm its th e efficien t d o ck in g o f d iffe re n t m o lecu les and m ain tain s an ic e -lik e su rface th a t is co m m o n to one ice cry stal o v e r the w h o le ag ­ g reg ate e v en th o u g h e ach b and has a d iffe re n t o rien tatio n o f ice -lik e te m p lates. T he ag g reg ate w o u ld h av e n u cleatio n sites m ore than 25 0  in len g th and 2 5 n  in w idth (w h e re n is th e n u m b e r o f m o lecu les in the a g g reg ate). T h is is co n siste n t w ith the siz e s o f n u c le a tio n sites as p red icted by F le tc h e r (1 9 7 0 ). T he p ro p o se d q u a te rn a ry stru c tu re w ith an ice-lik e su rfa c e th a t is c o m m o n to o n e ice cry sta l can b e co n stru c te d o n ly from the units co n sistin g o f th ree (i-hairpins b ecau se th e tiltin g o f th e tra p e z o id b lo ck s d ep en d s on th e n u m b e r o f (3-hairpins in th e unit. M o re o v e r, u n its w ith an e v e n n u m b er o f (3-hairpins h a v e su ch an a rra n g e ­ m en t o f in te ru n it c o n n e c tio n s th at b ro ad en in g o f the sp ace b e tw e e n re c ta n g u la r u nits d o es n o t lead to th e tiltin g o f th e trap ezo id al blocks. T h u s, w ith in th e fra m e o f this m o d el, th e th re e fo ld p e rio d ic ity in ice n ucleation p ro tein s is re la te d to th e c o n ­ tinuity o f th e ic e -lik e tem p late an d is unrelated to the space g ro u p s o f ice I as it w as in the p re v io u s m o d e ls (W arren e t al., 1986; M izuno, 1989).

Influence of the Bacterial Membrane on Protein Structure E x p e rim e n ta l d a ta (L in d o w e t al., 1989), and the kn o w n fu n c tio n an d h y d ro p h ilicity o f th e ice n u c le a tio n p ro te in , im p ly th at it is located on th e su rfa c e o f th e b a c ­ terial m e m b ra n e ra th e r th an b u rie d in this m em b ran e. It is n o t c le a r h o w th e flat m e m b ran e su rfa c e m ig h t fa c ilitate fo rm atio n o f the stru ctu re w ith a th ree- o r six ­ fo ld sy m m e try (W a rre n e t al., 1986; M izu n o , 1989). A t th e sam e tim e, ex iste n c e o f the flat q u a te rn a ry stru c tu re o f ice n u cleatio n p ro tein s (K a ja v a an d L in d o w , 1993) can n o t be im a g in e d w ith o u t su p p o rt by the m em brane. T h is stru c tu re su g g ests p o ssib le p a th w a y s fo r fo ld in g o f th e m olecule. Ju d g in g fro m th e ir p rim ary s tru c ­ tures, the a p e rio d ic N - an d C -te rm in a l do m ain s m ig h t self-a sse m b le in so lu tio n into g lo b u les an d th en p a rtic ip a te in a n c h o rin g to m em b ran es an d th e ag g re g a tio n o f In a p ro tein m o le c u le s. T h e re p e titiv e p o rtio n o f the p ro tein , b e c a u se o f a lack o f n o n p o la r re sid u e s, c a n n o t fo rm a h y d ro p h o b ic co re and h en c e p ro b a b ly has no fix ed 3-D s tru c tu re in so lu tio n . A lth o u g h sm all toxin m o le c u le s also c o n sist o f th ree [3-hairpins (e.g ., A lm assy e t al., 1983) and have ra tio s o f h y d ro p h ilic to h y ­

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d ro p h o b ic re sid u es sim ilar to th o se o f th e rep etitiv e d o m a in s o f ice n u cleatio n p ro ­ teins, th ey m a in ta in th e ir n a tiv e stru ctu re in so lu tio n by m ean s o f fo u r to five d isu lfid e b rid g es (w h ich a re a b se n t in ice n u cleatio n p ro tein s). T h e rep etitiv e p art o f ice n u c le a tio n p ro te in s th e re fo re n eed s assistan ce to o b tain a fix ed stru ctu re, and the su rfa c e o f th e b acterial o u te r m e m b ran e m ay p ro v id e such sup p o rt. T h e flat stru ctu re d esc rib e d a b o v e ap p e a rs as a resu lt o f se lf-a sso c ia tio n o f the re p e titiv e p a rt o f In a p ro te in s on th e m e m b ra n e . T h e 4 8 -re sid u e b lo ck n earest the N - o r C term in al d o m ain m ay fo ld first b ecau se o f stab ilizatio n fro m the n eig h b o rin g g lo b ­ ule. T h e a p p e a ra n c e o f th e first b lo c k m ig h t d e te rm in e th e o rd er o f a ltern atio n o f re c ta n g u la r an d tra p e z o id a l b lo c k s alo n g th e m o lecu le. T h e p referred in terd ig itatio n o f re c ta n g u la r u n its su g g ests th at the asso ciatio n o f tw o an tip arallel m o lecu les o f In aZ p ro tein is th e first ste p in ag g re g a te assem b ly . T h is b im o lecu lar in teractio n p ro v id e s a p o ssib le b asis fo r th e a p p ro x im ately se c o n d -o rd e r d ep en d e n c e o f n u ­ c leatio n activ ity o n p ro tein co n c e n tra tio n (S o u th w o rth e t al., 1988; see also C h ap ter 5). In th e final ste p o f se lf-a sse m b ly , in terd ig itatio n o f re c ta n g u la r b lo ck s m ay lead to a tiltin g o f th e trap e z o id a l b lo ck s, th u s p e rm ittin g fu rth e r in terd ig itatio n o f the “tra p e z o id a l” sides. T h u s, due to th e sig n ific a n t c o n stra in ts on m o le c u la r m o d e lin g it w as p o ssib le to su g g e st the d e ta ile d stru c tu ra l m o d els fo r the b a c te ria l ice n u cleatio n p ro tein s. It sh o u ld be m e n tio n e d th at th e re su lts o f d eletio n s, in sertio n s (G reen and W arren , 1985; G reen e t al., 1988) an d sin g le su b stitu tio n s o f a m in o acids (G u rian -S h erm an e t al., 1993) in th e re p e titiv e p a rt o f the p ro tein w e re also tak en into c o n sid eratio n d u rin g the m o d elin g . H o w e v e r p lau sib le a stru ctu ral m o d el m ay be, it needs a d d i­ tio n al e x p e rim e n ta l su p p o rt. T h e g ro w in g b io lo g ical an d c o m m ercial im p o rtan ce o f ice n u cleatio n p ro te in s h o p e fu lly w ill lead to th e a p p e a ra n ce o f new ex p erim en tal e v id e n c e w h ich w ill su p p o rt, refu te, o r d e m an d m o d ific a tio n o f the su g g ested m odels.

Literature Cited A b e , K „ W a ta b e , S ., E m o ri, Y ., W a ta n a b e , Y ., a n d A rai, S . 198 9 . A n ic e n u c le a tio n a c tiv e g e n e o f E rw in ia a nanas: S e q u e n c e s im ila r ity to th o s e o f P se u d o m im a s s p e c ie s a n d re g io n s re q u ire d f o r ice n u c le a tio n a c tiv ity . F E B S L e tt. 2 5 8 : 2 9 7 -3 0 0 . A lm a s s y , R. J., F o n te c illa -C a m p s , C . J., S u d d a th , F . L ., a n d B u g g , C . E. 1983. S tru c tu re o f v a ria n t-3 s c o rp io n n e u r o to x in fro m C e n tru ro id e s sc u lp tu ra ru s E w in g , re fin e d a t 1.8 A re so lu tio n . J. M o l. B io l. 1 7 0 :4 9 7 -5 2 7 . B a ro n , M ., N o rm a n , D ., W illis , A ., a n d C a m p b e ll, I. D. 1990. S tru c tu re o f th e fib ro n e c tin ty p e 1 m o d u le . N a tu re 3 4 5 :6 4 2 -6 4 6 . B u rk e , M . J., a n d L in d o w , S. E . 1 9 9 0 . S u rfa c e p ro p e rtie s a n d s iz e o f th e ice n u c le a tio n s ite in ic e n u ­ c le a tio n a c tiv e b a c te ria : T h e o re tic a l c o n s id e ra tio n s . C r y o b io lo g y 2 7 :8 0 -8 4 . C h o u , P. Y ., a n d F a s m a n , G . D . 19 74 . P re d ic tio n o f p ro te in c o n f o rm a tio n . B io c h e m is try 1 3 :2 2 2 -2 4 5 . C h o th ia , C . 1973. C o n f o r m a tio n o f tw is te d P p le a te d s h e e ts in p ro te in s . J. M o l. B io l. 7 5 :2 9 5 -3 0 2 . D o w e ll, L . G ., M o lin e , S . W ., a n d R in fre t, A. P . 1962. A lo w -te m p e ra tu re X -ra y d iffra c tio n s tu d y o f ic e s tru c tu re s f o rm e d in a q u e o u s g e la tin g e ls . B io c h e m . B io p h y s . A c ta 5 9 :1 5 8 -1 6 7 . E fim o v , A . V . 1 9 8 6 . S ta n d a rd c o n f o rm a tio n s o f a p o ly p e p tid e c h a in in irre g u la r re g io n s o f p ro te in s. M o l. B io l. (U S S R ) 2 0 :2 0 8 -2 1 6 . E is e n b e rg , D ., a n d K a u z m a n n , W . 19 6 9 . T h e S tru c tu re a n d P ro p e rtie s o f W a te r. O x fo rd U n iv e rsity P re ss , N e w Y o rk . F le tc h e r, N . H . 1 9 7 0 . P a g e s 9 7 - 1 0 3 in : T h e C h e m ic a l P h y s ic s o f Ice. C a m b rid g e U n iv e rsity P re ss, C a m b rid g e . F ra s e r R . D ., a n d M a c R a e , T . P. 1 9 7 3 . C o n fo rm a tio n in F ib ro u s P ro te in s a n d R e la te d S y n th e tic P o ly p e p tid e s . A c a d e m ic P re ss , N e w Y o rk . G a m ie r, J., O s g u th o r p e , D . J „ a n d R o b s o n , B . 197 8 . A n a ly s is o f a c c u ra c y a n d im p lic a tio n s o f sim p le m e th o d s fo r p r e d ic tin g s e c o n d a r y s tr u c tu r e o f g lo b u la r p ro te in s . J. M o l. B io l. 1 2 0 :9 7 -1 2 0 .

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G o v in d a ra ja n , A . G ., a n d L in d o w , S. E . 198 8 a. P h o sp h o lip id r e q u ire m e n t f o r e x p r e s s io n o f ic e n u clei in P se u d o n u m a s sy rin g a e . J. B io l. C h e m . 2 6 3 :9 3 3 3 -9 3 3 8 . G o v in d a ra ja n , A . G ., a n d L in d o w , S . E . 19 8 8 b . S iz e o f b a c te ria l ic e - n u c le a tio n site s m e a s u re d in situ b y r a d ia tio n in a c tiv a tio n a n a ly s is . P ro c . N a tl. A c a d . S ci. U S A 8 5 :1 3 3 4 -1 3 3 8 . G re e n , R . L ., C o r o tto , L . V , a n d W a rre n , G . J. 1988. D e le tio n m u ta g e n e s is o f th e ic e n u c le a tio n g e n e fro m P se u d o n u m a s sy rin g a e S 2 0 3 . M o l. G e n . G e n e t. 2 1 5 :1 6 5 -1 7 2 . G re e n , R . L ., a n d W a r r e n , G . J. 198 5 . P h y s ic a l a n d fu n c tio n a l re p e titio n in a b a c te ria l ic e n u c le a tio n g e n e . N a tu r e 3 1 7 :6 4 5 - 6 4 8 . G u ria n -S h e rm a n , D ., P a n o p o u lo s , N . J., a n d L in d o w , S. E . 1993. Is o la tio n a n d c h a r a c te r iz a tio n o f hyd ro x y la m in e -in d u c e d m u ta tio n s in th e E rw in ia h erb ico la ic e n u c le a tio n g e n e th a t s e le c tiv e ly re d u c e w a rm te m p e r a tu re ic e n u c le a tio n a c tiv ity . M o l. M ic ro b io l. 9 :3 8 3 -3 9 1 . K a ja v a , A . V ., a n d L in d o w , S. E . 19 9 3. A m o d e l o f th e th re e -d im e n s io n a l s tr u c tu r e o f ic e n u c le a tio n p ro te in s. J. M o l. B io l. 2 3 2 :7 0 9 -7 1 7 . L in d o w , S . E ., L a h u e , E ., G o v in d a r a ja n , A . G ., P a n o p o u lo s, N . J., a n d G ie s , D . 1 9 8 9 . L o c a liz a tio n o f ic e n u c le a tio n a c tiv ity a n d th e ic e C g e n e p ro d u c t in P seu d o n u m a s sy rin g a e a n d E sc h e ric h ia coli. M o l. P la n t-M ic r o b e In te ra c t. 2 :2 6 2 -2 7 2 . M a k i, L. R ., G a ly a n , E . L ., C h a n g -C h ie n , M ., a n d C a ld w e ll, D . R. 19 7 4 . Ic e n u c le a tio n in d u c e d by P se u d o n u m a s sy rin g a e . A p p l. M ic ro b io l. 2 8 :4 5 6 -4 5 9 . M iz u n o , H. 19 8 9. P re d ic tio n o f th e c o n fo rm a tio n o f ice n u c le a tio n p ro te in b y c o n f o rm a tio n a l e n e rg y c a lc u la tio n . P ro te in s 5 :4 7 -6 5 . P h e lp s, P ., G id d in g s , T . H ., P ro c h o d a , M „ a n d F a ll, R . 1986. R e le a se o f c e ll- fr e e ic e n u c le i b y E rw in ia h e rb ico la . J. B a c te rio l. 1 6 7 :4 9 6 -5 0 2 . P titsy n , O . B ., a n d F in k e ls te in , A . V. 198 3 . T h e o ry o f p ro te in s e c o n d a ry s tr u c tu r e a n d a lg o rith m o f its p re d ic tio n . B io p o ly m e r s 2 2 :1 5 -2 5 . R y u , S .-E ., K w o n g , P. D ., T ru n e h , A ., P o rte r, T . G ., A rth o s, J., R o s e n b e rg , M „ D a i, X ., X u o n g , N .-H ., A x e l, R ., S w e e t, R. W ., a n d H e n d ric k s o n , W . A. 1990. C ry sta l stru c tu re o f a n H IV -b in d in g r e c o m ­ b in a n t f r a g m e n t o f h u m a n C D 4 . N a tu r e 3 4 8 :4 1 9 -4 2 6 . S o u th w o rth , M . W ., W o lb e r , P. K , a n d W a rre n , G . J. 1988. N o n lin e a r r e la tio n s h ip b e tw e e n c o n c e n tr a ­ tio n a n d a c tiv ity o f a b a c te ria l ic e n u c le a tio n p ro te in . J. B io l. C h e m . 2 6 3 :1 5 2 1 1 -1 5 2 1 6 . S te rn b e rg , M . J. E ., a n d T h o rn to n , J. M . 1977. C o n fo rm a tio n o f p r o te in s — A n a ly s is o f [5 p le a te d s h e e ts. J. M o l. B io l. 1 1 0 :2 8 5 -2 9 6 . V e n k a c h a ta la m , C . M . 19 6 8. S te re o c h e m ic a l c rite ria fo r p o ly p e p tid e s a n d p r o te in s . V . C o n f o r m a tio n o f a s y s te m o f 3 lin k e d p e p tid e u n its. B io p o ly m e rs 6 :1 4 2 5 -1 4 3 6 . W a rre n , G ., a n d C o r o tto , L . 1989. T h e c o n s e n s u s s e q u e n c e o f ic e n u c le a tio n p r o te in s fro m E rw in ia h e rb ico la , P se u d o n u m a s flu o r e s c e n s , a n d P seu d o n u m a s sy rin g a e. G e n e 8 5 :2 3 9 -2 4 3 . W a rre n , G ., C o r o tto , L ., a n d W o lb e r, P. 198 6 . C o n s e rv e d re p e a ts in d iv e r g e d ic e n u c le a tio n stru c tu ra l g e n e s fro m tw o sp e c ie s o f P se u d o n u m a s. N u c le ic A c id s R es. 1 4 :8 0 4 7 -8 0 6 0 . W a rre n , G . J ., L in d e m a n n , J., S u slo w , T . V ., a n d G re e n , R. L. 1987. Ice n u c le a tio n d e f ic ie n t b a c te r ia as fro s t p r o te c tio n a g e n ts . P a g e s 2 1 5 -2 2 7 in: A p p lic a tio n s o f B io te c h n o lo g y to A g ric u ltu ra l C h e m is try . H . M . L e B a r o n , R . O . M u m m a , R . C . H o n e y c u tt, a n d J. H. D u e s in g , e d s . A m e r ic a n C h e m ic a l S o c i­ e ty , W a s h in g to n , D C . W e lc h , J. F ., a n d S p e id e l, H . K. 198 9. V is u a liz a tio n o f p o te n tia l b a c te ria l ic e n u c le a tio n site s. C ry o L e tt. 1 0 :3 0 9 -3 1 4 . W ilm o t, C . M „ a n d T h o rn to n , J. M . 19 8 8 . A n a ly s is a n d p re d ic tio n o f th e d if fe r e n t ty p e s o f [3-tum s in p ro te in s. J. M o l. B io l. 2 0 3 :2 2 1 -2 3 2 . W o lb e r, P. K „ D e in in g e r , C . A ., S o u th w o rth , M . W ., V a n d e k e rc k h o v e , J., v a n M o n ta g u , M ., a n d W a r­ re n , G . J. 19 8 6 . I d e n tific a tio n a n d p u r ific a tio n o f a b a c te ria l ic e -n u c le a tio n p ro te in . P ro c . N atl. A c a d . S c i. U S A 8 3 :7 2 5 6 -7 2 6 0 . Y a n g , D. S . C ., S a x , M „ C h a k ra b a r tty , A ., a n d H ew , C . L. 1988. C ry s ta l s tr u c tu r e o f a n a n tifre e z e p o ly p e p tid e a n d its m e c h a n is tic im p lic a tio n s . N a tu re 3 3 3 :2 3 2 -2 3 7 . Z h a o , J., a n d O rs e r, C . S. 199 0 . C o n s e r v e d re p e titio n in th e ic e n u c le a tio n g e n e in a X fro m X a n lh o m o n a s c a m p e s tris p v . tra n slu cen s. M o l. G e n . G e n e t. 2 2 3 :1 6 3 -1 6 6 .

CHAPTER 7

Freezing Tolerance in Plants: An Overview T. H. H. Chen, M. J. Burke, and L. V. Gusta

F o o d and fib er p ro d u c tio n , th e m o st im p o rtan t g lo b al industry, in terco n n ects n a ­ tions th ro u g h trad e, p rim a rily n a tio n s th at h av e a su rp lu s w ith th o se th at hav e the fin an cial ability to p u rch ase. A d v e rse clim atic an d m e teo ro lo g ical co n d itio n s, e.g ., fro st o r d ro u g h t, w h ich c a u se lo sse s in both y ield an d q u ality , can h av e a d ram atic e ffe c t on th e e c o n o m ic s o f a c o u n try . E v en in a larg e co u n try su ch as C an ad a, cro p lo sses d u e to an a b io tic stress h a v e both a reg io n al an d natio n al im pact. F o r e x a m ­ ple, an u n se a so n a l fro st in m id -A u g u st o f 1992 in w e stern C a n a d a resu lted in o v e r a b illio n d o lla r loss in cro p p ro d u c tio n . N o t only y ield b u t also the qu ality o f the cro p w as red u c e d . W h e a t g rain w as d ev a lu e d fro m h ig h -q u ality b read flo u r to feed w heat. T h is n o t o n ly re su lted in a m ajo r loss to th e p ro d u c e r b u t also affected the g rain h a n d lin g se rv ic e s, ra ilro a d s, in tern atio n al trad e, m illers, farm sales, etc. T h e g ro w in g seaso n in th e te m p e ra te zo n e is g en e ra lly d efin ed by tem p eratu re and th e len g th o f th e fro st-fre e p erio d . E v en th o u g h g lo b al w arm in g m ay re su lt in an in c rease in te m p e ra tu re, th e risk o f an u n seaso n al fro st m ay also in crease d u e to e a rlie r seed in g an d e a rlie r e m e rg e n c e. F ru it trees m ay also flo w er e a rlie r an d be injured by frosts. B e cau se fro st p la y s a m a jo r ro le in p la n t d istrib u tio n and cro p y ield and q u ality , the stu d y o f fro st p ro te c tio n an d a v o id an ce is no w rec e iv in g in ten siv e in terest b oth at the in d u strial an d re se a rc h lev els. F ro st in ju ry is p rim arily d u e to the fo rm atio n o f ice in p la n t tissu e s a t su b z e ro tem p eratu res. M e th o d s o f p rev en tin g fro st in ju ry h av e re c e iv e d c o n sid e ra b le a tten tio n sin ce the re c e n t d isco v ery o f b io lo g ical ice n u cleato rs. In th e a b sen ce o f nu cleato rs, w ater w ill n o t c ry stallize w hen co o le d several d eg re e s b e lo w zero. B io lo g ic a l ice nu cleato rs, su ch as b a cteria o r fu n g i, are th o u g h t to be th e m ain e x trin sic ag en ts th a t in itiate fre ezin g at tem p eratu res c lo se to zero. R e c e n t e v id e n c e su g g e sts th a t in trin sic n u cleato rs, w hich also m ay be p re se n t in p lan ts, initiate fre e z in g a t w arm su b zero te m p eratu res. In co n trast, certain flo w e r bu d s, e.g ., th o se o f azalea, an d the w o o d o f the h a rd w o o d s lack b o th intrinsic and ex trin sic n u cleato rs. A z a le a flo w e r bu d s w ill su p e rc o o l fro m - 1 5 to —20°C ; th e xylem ray p a re n c h y m a cells o f A m erican elm w ill su p e rc o o l to - 4 5 ° C in m id w in ter. T his overview and nex t three chapters pro v id e in sig h t into the recent progress m ad e in u n derstanding h o w plants eith er avoid ice form ation o r tolerate its presence. 115

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Plant Cold Hardiness P lan ts in n a tu re face se v e ra l ty p es o f freezing stress in c lu d in g u n se a so n a l frosts (u su ally in th e g ro w in g sea so n ), and lo w -tem p eratu re e x tre m e s (u su ally w h en the p la n t is d o rm a n t an d n o t a ctiv ely g ro w in g ). D ep en d in g on th e m in im u m tem p era­ ture, p la n ts m ay b e p a rtially d am a g e d o r killed, w ith re su lta n t re d u c e d y ield and q u ality o r c o m p le te cro p failu re. D u rin g active g row th in th e sp rin g an d sum m er, certain crops are killed at the m om ent o f ice form ation, e.g., cucum bers ( - 2 to -3 ° C ), w hereas cereal grains can tolerate the presence o f ice to tem peratures as low as -9 °C . Som e w inter crops can acclim ate to autum n tem peratures as low as -3 0 ° C . T h e m ost h ard y p la n ts, su ch as w o o d y p e re n n ia ls, can n o t to lerate - 3 ° C d u rin g a c tiv e grow th. W hen fully acclim ated, how ever, these plants tolerate tem peratures as low as - 1 9 6 ° C (G uy e t al., 1986). In te n d e r p la n ts, fre ezin g in ju ry occurs w hen ice fo rm s in th e tissu e, reg ard less o f the in itia tio n te m p e ra tu re o f freezin g . T he p resen ce o f ice resu lts in e ith e r m e­ ch an ical d a m a g e a n d /o r d e h y d ra tio n injury to the tissue. I f ice d o es n o t form at su b z e ro te m p e ra tu re s , w ater in th e cells is said to be su p e rc o o le d , an d n o ap p aren t in ju ry o cc u rs. T h u s fo r te n d e r p lan ts to su rv iv e freezin g te m p e ra tu re, th e y m ust so m e h o w a v o id o r esc a p e ice fo rm atio n . O n the o th er h a n d , h ard y p lan ts tolerate ice fo rm a tio n in th e ir tissu e if ice is ex clu d ed from the c y to p la sm . T h e ab ility o f h ard y p la n ts to su rv iv e fre e z in g is d e p e n d e n t on m any fa c to rs, in c lu d in g site o f ice n u cleatio n , ic e n u c le a tio n te m p e ra tu re, rate o f co o lin g d u rin g cry sta lliz a tio n , ra te o f ice g ro w th , m in im u m te m p e ra tu re o f ex p o su re, d u ratio n o f e x p o su re to freezing co n d itio n s, etc. (L e v itt, 1980). T h e in h e re n t a b ility o f te m p e ra te plants to acclim ate to c o ld an d th eir ra te o f ac­ c lim atio n are th e tw o m a jo r facto rs th at lim it lo w -te m p e ra tu re su rv iv al. C old a c c lim a tio n is th o u g h t to b e a co m p le x g enetic trait in d u c e d b y lo w tem p eratu re and re su lts in b o th m o rp h o lo g ic a l and m o lecu lar ch an g es. R e c e n t a d v an ces in the field o f p la n t c ry o b io lo g y an d th e id en tificatio n and c h a ra c teriz a tio n o f g e n e s asso ­ ciated w ith c o ld a c c lim a tio n h a v e g reatly en h an ced o u r u n d e rsta n d in g o f p lant fre e z in g re sista n c e .

Freezing Injury in Plants D u rin g th e g ro w in g seaso n , rad iatio n frosts on clear, w in d le ss n ig h ts are the m o st c o m m o n ty p e o f fre e z in g stress. L arg e leaves o rie n te d p a ra lle l to th e sk y ra p ­ idly lo se h e a t to th e o p en sk y th ro u g h b lack b ody rad iatio n an d can co o l to tem ­ p e ra tu re s s u b sta n tia lly b e lo w a m b ie n t (C handler, 1958). A d v e c tiv e co o lin g , w h ereb y le a f an d a ir te m p e ra tu res d ro p at sim ilar rates, is c a u se d by th e in flo w o f co ld air an d c a n a lso o c c u r d u rin g th e g ro w in g season. A s m e n tio n e d earlier, c e r­ tain te n d e r p la n t tissu e s, su ch as in cu cu m b ers and to m ato es, are d am a g e d a t the m o m e n t o f ice fo rm a tio n . T h e re fo re , in o rd er fo r these p la n ts to esc a p e u n se a so n ­ able fro st, ice n u c le a tio n m u st b e a v o id ed by low ering the fre e z in g p o in t o f th e tis­ sue w a te r o r by m a x im iz in g th e ex te n t and d u ratio n o f su p e rc o o lin g . P e re n n ia l p la n ts th a t su rv iv e h a rsh w in ter co n d itio n s d e v e lo p fre e z in g to leran ce in the au tu m n . T h e te m p e ra tu re an d the site o f ice n u c le a tio n h a v e a p ro fo u n d e f­ fect on the u ltim a te level o f fre e z in g tolerance. G en erally , th e m o re a p la n t su p e r­ co o ls b e fo re it fre e z es, th e m o re in ju ry it w ill sustain. S im in o v itc h an d S carth (1 9 3 8 ) sh o w e d th a t th e p ro b a b ility o f lethal in tracellu lar fre e z in g in creases in hardy

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cells a llo w ed to su p e rc o o l su b sta n tia lly b e fo re freezin g . T h ey d em o n strated th at in itiatio n o f ice fo rm a tio n c lo se to 0 °C red u ces th e p ro b a b ility o f injury. O lien (1 9 6 4 ) fu rth er d e m o n stra te d th at su p e rco o lin g p ro m o te s n o n eq u ilib riu m freezin g , w hich is in ju rio u s to th e tissu es. R ap id n o n e q u ilib riu m freezin g th at fo llo w s sig ­ n ific a n t su p e rc o o lin g leads to a larg e G ib b s free e n e rg y fo r ice fo rm atio n , w hich p ro v id e s the e n e rg y fo r d e stru c tiv e w o rk on tissu es a t th e ice-liq u id interface. W in ­ ter w h eat cro w n tissu e n u cleated w ith ice at - 3 ° C is less h ard y than at ju s t b elo w 0 °C (G u sta and F o w le r, 1977). R a ja sh e k a r e t al. (1 9 8 3 ) d e te rm in e d th at leav es o f Solanum acaule n u cleated at - 1 ° C survived a controlled freeze test to —7°C. If the leaves w ere nucleated at - 3 ° C , h o w ev er, th ey w e re k illed a t th a t tem p eratu re. S im ilar fin d in g s h av e also been re ­ p o rted fo r flo w e r b u d s o f P runus spp. (A n d rew s e t al., 1986; G ro ss e t al., 1984). T h ese stu d ies d e m o n stra te d th a t in m o st p lan t sp ecies if fre ezin g in itiates clo se to 0°C , the p la n t is a b le to su rv iv e a c o ld e r te m p eratu re th an if ex ten siv e su p erco o lin g is a llo w ed to occur. O n ce n u c le a tio n o ccu rs in a p lan t, ice q u ick ly m o v es into the ex tra c e llu lar sp ace an d p ro p a g a te s th ro u g h larg e v essels w ith a h ig h w a te r c o n ten t (L evitt, 1980). F ro m th e v essels, ice sp read s th ro u g h o u t th e e x tra c e llu lar sp aces (A sh w o rth , 1990; C h an d ler, 1958; L e v itt, 1980) an d co n tin u es to g ro w un til it reach es parts o f the p la n t co n ta in in g n o w ater o r w a rm e r reg io n s. It m u st be n o ted th at the tem p eratu re o f p la n t p a rts can v ary by 5 °C d u rin g a ra d iativ e fro st. M e asu rem en ts o f rates o f ice g ro w th in w o o d y stem tissu es re v e a le d rates o f ice p ro p a g a tio n as h ig h as 6 0 to 74 cm /m in , i.e., c o m p a ra b le to th e rate o f ice g ro w th in U -tu b e s filled w ith pure w ater (S ak ai an d L a rc h e r, 1987). In field c o n d itio n s, fre e z in g in itiates fro m a few ice n u ­ c leatio n sites o r ice seed in g sites and th en sp read s q u ick ly th ro u g h o u t the p la n t th ro u g h vessels. In h ard y p lan ts, th is is p ro b ab ly an effe c tiv e w ay o f p rev en tin g su p e rc o o lin g and re d u c in g th e risk o f in tracellu lar ice fo rm atio n . In p a rtially fro z e n tissu es, ice c ry stallizatio n o c c u rs e x tracellu larly , and ice g ro w s th ro u g h m o v e m e n t o f w a te r v ap o r, film s, an d c h a n n e ls in and aro u n d cell w alls an d th e e x tra c e llu la r sp aces (O lien, 1967). W h en th e freezin g rate is su ffi­ cien tly slo w to a llo w w ater to d iffu se to ice loci ex tra c e llu larly , th e v o lu m e o f c y ­ to p lasm g ra d u a lly d ec re a ses, c o n c e n tra tin g the cell sap, w h ich in tu rn d ep resses the freezin g p o in t o f th e in tra c e llu la r w ater (B u rk e e t al., 1976). E q u ilib riu m is reach ed w hen th e ch em ical p o te n tia l o f th e cell w ater eq u a ls th e ch em ical p o tential o f th e ex tra c e llu la r ice. T h e ice n u c le a tio n tem p eratu re (su p e rc o o lin g p o in t) o f the in tra ­ cellu la r flu id s also d e c re a ses u p o n cell d e h y d ratio n . A t te m p e ra tu res clo se to 0 °C , cells are in e q u ilib riu m w ith e x tra c e llu lar ice an d are u n lik ely to u n d erg o in tra c e llu la r n u c leatio n . E x tra c e llu la r freezin g re su lts in freeze d e h y d ratio n , cell v o lu m e re d u ctio n , an d c o n c e n tra tio n o f cell so lu tes in c lu d in g salts, w hich, if too e x ten siv e, m ay in ju re the m em b ra n e s. U n d e r rap id fre e z in g co n d itio n s, such as w hen ex te n siv e su p e rc o o lin g p re c e d e s n u cleatio n , w ater d iffu sio n to th e e x tra c e llu ­ lar ice d o es n o t p ro c e e d q u ic k ly e n o u g h to c o n c e n tra te the cell sap. S uch rap id ly co o led cells o ften freeze in tra c e llu la rly d u e to ice se e d in g from ex tra c e llu lar ice o r from in tra c e llu la r ice n u c leatio n . T h e ab ility o f the p ro to p la sm to to lerate th e strain ex erted by e x tra c e llu la r ice fo rm a tio n d eterm in es th e fre e z in g to leran ce o f a plant. F re ezin g to le ra n c e o r to le ra n c e to ex tra c e llu lar fre e z in g is th erefo re a form o f a v o id an ce, i.e., a v o id a n c e o f in tra c e llu la r ice fo rm atio n . L e v itt (1 9 8 0 ) c la ssifie d fre e z in g in ju ry as follow s: 1) p rim ary d ire c t injury d u e to in tra c e llu la r fre e z in g ; 2 ) se c o n d a ry free z e-in d u c e d d eh y d ra tio n injury due to ex-

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I tra c e llu la r fre e z in g , and 3) in ju ry d u e to o th er tertiary fre e z e -in d u c e d stresses. In his early w o rk , L e v itt d e sc rib e d th e p rin cip al m ech an ism o f fro st h ard in ess, i.e., to leran ce o f ex tra c e llu la r fre e z in g an d ex tracellu lar ice an d a v o id a n c e o f in tracellu ­ lar ice (L e v itt an d S carth , 1936). A lth o u g h this w o rk w as d o n e in th e 1930s, it is still re le v a n t to d a y . L e v itt (1 9 8 0 ) d escrib es fo u r “m o m en ts o f fre e z in g in ju ry ” or p o in ts in th e fre e z e-th a w cy c le w h ere p lan t cells are p o ssib ly in ju red : 1) d u rin g ice g ro w th as th e c e lls d e h y d ra te , 2) at the p o in t w here a lo w -te m p e ra tu re lim it has been re a c h e d an d a te m p e ra tu re-d e p e n d e n t structural tra n sitio n has o ccu rred , 3) d u rin g th a w in g as ice m elts an d cells reh y d rate, and 4 ) fo llo w in g th a w in g w hen b io c h e m ic a l d y s fu n c tio n s in d u c e d in the frozen state tak e effect. A lthough e x tra c e llu la r fre e z in g is n e c essary for the survival o f h ard y p lan ts, freeze-in d u ced d eh y d ra tio n re su lts in in ju ry w h e n tissues are cooled b e lo w a c ritical tem p eratu re o r if th ey are h e ld fro zen fo r a p ro lo n g ed perio d o f tim e at a te m p e ra tu re slightly w a rm e r th a n th e k illin g te m p e ra tu re. T his m ay resu lt in a fo rm o f d ro u g h t stress, a p h y sical im p a irm e n t o f cell m e m b ran es, red u ctio n o f c e llu la r v o lu m e b e lo w a criti­ cal size, o r c o n c e n tra tio n o f th e cell sap to resu lt in th e d é n a tu ra tio n o f p ro tein s. E arly w o rk e rs b eliev ed th e p rin cip al cause o f freezin g in ju ry w as p h y sic a l dam ­ age cau se d b y ice c ry stals. T h e v o lu m e increase asso ciated w ith th e fre e z in g o f w a­ ter w as th o u g h t to ru p tu re p la n t cells, thus d estro y in g th e ir c e llu la r stru ctu re. This early e x p la n a tio n w as reje c te d w h en it w as observ ed th at c e lls w ere c o lla p se d (not ex p a n d e d ) d u rin g fre e z in g and d id not rupture w hile in a fro zen state. It is g e n e ra lly acc e p ted th a t in tracellu lar freezin g a lw ay s re su lts in cell death, p ro b ab ly d u e to m e c h a n ic al d e stru ctio n o f m em b ran e sy ste m s asso c ia te d w ith ice g row th in th e p ro to p la sm . S o m e c e lls survive in tracellu lar fre e z in g if fre e z in g is so rap id th at a q u e o u s g lasses an d very fine ice cry stals are fo rm ed . In th ese cases, th aw in g m u s t also be very rap id to p rev en t the very sm all ice c ry sta ls fro m g ro w in g d u rin g th e th a w in g p ro cess to a size that results in in ju ry . T h e ex tre m e ly rapid c o o lin g an d th a w in g ra te s u sed in so m e cry o p re se rv atio n p ro c e d u re s (above 1,000°C p er m in u te ) are g o o d ex a m p le s o f this. T h e n a tu re o f fro st-in d u c e d d a m ag e to the p lasm a m e m b ra n e is still op en to q u estio n . S te p o n k u s e t al. (1 9 9 0 ) stressed the im p o rtan ce o f sp e cific lip id c o m p o ­ nents in th e p la s m a m e m b ra n e th at m ay p rev en t o r red u ce in ju ry d u rin g freezing. P o ssib le c a u se s o f m e m b ra n e in ju ry d u rin g freezing m ay b e th e a c tiv a tio n o f phosp h o lip ase D (Y o sh id a and S ak ai, 1974), a la m ellar-to -h ex ag o n al II p h ase tran sitio n (G o rd o n -K a m m an d S tep o n k u s, 1984), and irrev ersib le e n d o c y to tic v é sic u la tio n o f th e p la sm a m e m b ra n e (S te p o n k u s e t al., 1982). P a lta ’s g ro u p , h o w e v e r, em p h asizes the im p o rta n c e o f in ju ry to m e m b ra n e A T P ases cau sed d ire c tly o r in d irectly by the free z e-th a w p ro c e ss (Isw ari an d P alta, 1989).

Metastable Water and Plant Hardiness S u p e rc o o lin g d isc u sse d th ro u g h o u t this b o o k is o nly o n e o f th e sev eral m eta­ stab le states o f w a te r im p o rta n t in p la n t stress responses. T h e fo llo w in g are th e four p rin cip al m e ta sta b le states fo r p la n t stress responses: 1. S u p erco o led (som etim es ca lled undercooled) tissue a n d cell solutions w here the solutions are betw een the equilibrium freezin g p o in t a n d the hom o g en eo u s ice nucleation tem perature (usually betw een -1 a n d -4 1 °C ). T h is m e ta sta b le c o n d i­ tion is te rm in a te d by h e te ro g e n e o u s o r h o m o g en eo u s ice n u c le a tio n an d su b se­ q u en t ice g ro w th (B u rk e e t al., 1976)

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2. Sup ersa tu ra ted cell solu tio n s where the concentration o f one solute (usually a sugar) is above the crystallization concentration. A n e x am p le o ccu rs d u rin g the d eh y d ra tio n o f seed s w h en cell so lu tio n s pass th ro u g h the eu tectic p o in t and re a c h su g a r c o n c e n tra tio n s g re a te r th an the su g a r c ry stallizatio n point. T h e m e ­ ta sta b le c o n d itio n is te rm in a te d u p o n the fo rm a tio n o f the first su g ar cry stal, w h ich then g ro w s and p re c ip ita te s th e su g ar fro m solution. S u p ersatu ratio n is th e first c ritical ste p in g lass fo rm atio n in th e cell so lu tio n s o f seeds (L eo p o ld et al., 1992). 3. Tension stressed (hydrostatic) cell a n d tissue solutions resulting fro m stretching, cohesion, a n d tensile strength o f the solutions. F o r tall trees, it has lo n g been p ro p o se d th at w a te r in x y lem v essels has a ten sile stren g th su fficien t to p ull w a ­ ter to th e to p s o f th e trees. S u ch ten sile stren g th lead s to h y d ro static ten sio n s o f up to 0.5 M P a an d a llo w s a su ffic ie n t free en erg y lo w erin g to pull w ater up to th e to p o f the tree. It h as also b een p ro p o sed th at w ater in d eep su p erco o led ray p a re n c h y m a c e lls is re ta in e d in the cells d u e to h y d ro static ten sio n s as larg e as 4 0 M P a. T h e m e ta sta b le c o n d itio n is te rm in ated w hen a cav itatio n o ccu rs, c a u s­ ing a w ater v a p o r b u b b le to fo rm an d releasin g th e ten sio n (G eo rg e and B u rk e, 1977). 4. G lassed cell solutions. T h is is a u n iq u e m e ta sta b le c o n d itio n th at can n o t be te r­ m in ated b elo w th e glass tra n sitio n tem p eratu re. A q u e o u s g lasses are ex trem ely v isco u s so lu tio n s w ith th e v isco sity b ro u g h t a b o u t by high so lu te (su g ar) c o n ­ c e n tratio n s at a su ffic ie n tly low tem p eratu re. G lasses h av e b een re p o rted to form at tem p e ra tu res as h ig h as 30 °C in seeds (W illia m s and L eo p o ld , 1989; B runi and L e o p o ld , 1991, 1992). In ex trem ely cold h ard y w o o d y stem s, glasses are re ­ p o rte d to fo rm b e lo w - 2 0 ° C (H irsh et al., 1985). G lasses in such sy stem s m ay be su p e rc o o le d , su p e rsa tu ra te d , and u n d er h y d ro sta tic ten sio n ; h o w ev er, they are n o t su b je c t to ice n u c le a tio n (h e te ro g en eo u s o r h o m o g e n e o u s), so lu te c ry sta lli­ z atio n , o r w a te r v a p o r c a v ita tio n so lo n g as th e so lu tio n rem ain s b elow the glass tran sitio n tem p e ra tu re. In d e e d the g lass is e x tre m e ly stable. C learly , glass fo rm a tio n can be a hig h ly d esirab le m etastab le state for co n d itio n s o f low te m p e ra tu re an d e x tre m e d esiccatio n . G lasses in liv in g tissu es are u su ally a sso ciated w ith h ig h su g a r co n c e n tra tio n s; su cro se and o lig o sacch arid es (raffin o se and sta c h y o se) a re c o m m o n in plants, and tre h a lo se is c o m m o n in an im als and fu n g i (L eo p o ld e t al., 1992). G lasses are fo rm ed fro m su g ars th a t p ro tect m a c ro ­ m o lecu les and m e m b ra n e s an d fu n ctio n as im p o rta n t sto rag e m aterials (C ro w e et al., 1984a,b). G la sse s fill sp ace an d sto p fu rth er c e ll co llap se o r c h an g es in so lu te c o n c e n tratio n , p H , o r d e h y d ra tio n . T h ey are ex tre m e ly visco u s and p re v e n t all p h y sical and c h e m ic a l re a c tio n s th at req u ire m o le c u la r d iffu sio n in clu d in g ice n u ­ c leatio n , so lu te c ry sta l n u c le a tio n , and v ap o r c a v ita tio n (B u rk e, 1986). F o r glasses to fo rm in m o st p la n t sy stem s, th e so lu tio n s m u st u sually be d e h y ­ d rated to a p o in t w h e re a su g a r is su p ersatu rated , an d so m etim es such so lu tio n s are su p erco o led a n d /o r u n d e r h y d ro sta tic tension. A n e x a m p le w o u ld be in the d e h y ­ d ratio n o f a c o rn e m b ry o a t ro o m te m p eratu re w h e re su cro se b eco m es su p e rsa tu ­ rated. K o ster (1 9 9 1 ) su g g e ste d th at a low co n c e n tra tio n o f ra ffin o se in these seeds e ffectiv ely p o iso n s th e in itia tio n o f su cro se cry stal fo rm atio n and th erefo re p re ­ vents p re c ip ita tio n o f su cro se fro m so lution. H irsh has d e m o n strated th at tw ig cells o f P opulus spp. d e h y d ra te w h e n fro z e n slow ly an d co n c e n tra te p ro tein s and su g ars (p re d o m in a n t su g a rs are su c ro se , raffin o se, an d sta c h y o se) un til a su g ar-p ro tein w ater g lass tra n sitio n is re a c h e d a t a b o u t - 2 0 ° C (H irsh , 1987; H irsh et al., 1985).

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H irs h ( 1 9 8 7 ) p r o v id e s fu r th e r e v id e n c e fo r tw o a d d itio n a l g la s s tr a n s itio n s a t lo w e r t e m p e r a t u r e s in t w i g s o f

Populus. H y d r o p h i l i c p r o t e i n s , a s f i r s t d e s c r i b e d b y S im i-

n o v i t c h a n d B r i g g s ( 1 9 5 3 ) a n d d i s c u s s e d l a t e r in t h i s c h a p t e r , m a y a l s o p l a y a k e y r o l e in g l a s s f o r m a t i o n . H y d r o p h i l i c p r o t e i n s a t s u f f i c i e n t l y h i g h c o n c e n t r a t i o n s m a y a d d t o t h e s o l u t i o n v i s c o s i t y ; th e y w o u l d f ill s p a c e a n d m i g h t a l s o p r e v e n t c r y s ta lliz a tio n o f c r itic a l s o lu te s . M a n y t r e e s u s e d e e p s u p e r c o o l i n g o f c e l l s in w o o d y t i s s u e s to s u r v i v e t o t h e i r h o m o g e n e o u s ic e n u c le a tio n te m p e r a tu re , u s u a lly a b o u t - 4 1 ° C . P rin c ip a l c e lls o f th i s t y p e a r e r a y p a r e n c h y m a a n d p i t h c e l l s in t h e x y l e m . A t lo w t e m p e r a t u r e s , th e s e s u p e r c o o lin g c e lls d o n o t c o lla p s e a n d a p p e a r fu lly tu r g id ( M a lo n e a n d A s h ­ w o r t h , 1 9 9 1 ) . M o s t i n t e r e s t i n g in M a l o n e a n d A s h w o r t h ’s w o r k is t h a t t h e v e r y h a r d y s p e c ie s w ith ra y p a r e n c h y m a a n d p ith c e lls th a t s u r v iv e - 1 9 6 ° C a ls o d o n o t c o lla p s e a n d a p p e a r tu r g id a t lo w te m p e r a tu r e s . O n e m ig h t s p e c u la te th a t th e s e v e ry h a rd y s p e c ie s h a v e g la s s f o r m a tio n a s o u tlin e d b y H irs h ( 1 9 8 7 ) a n d th a t th e g la s s e s o c c u r in r a y p a r e n c h y m a a n d p i t h c e l l s d u e t o h i g h s u g a r c o n c e n t r a t i o n s . O n ly s l i g h t d e h y d r a t i o n a n d c e l l c o l l a p s e m i g h t l e a d to a g l a s s t r a n s i t i o n , w h i c h p r o t e c t s t h e c e l l s f r o m f u r t h e r d e h y d r a t i o n , i c e n u c l e a t i o n ( to 0 ° K ) , v a p o r c a v i t a t i o n , a n d s o lu te c ry s ta lliz a tio n . I n d e e d , m e t a s t a b l e w a t e r is v e r y i m p o r t a n t f o r o u r c o n s i d e r a t i o n s o f p l a n t s u r ­ v iv a l o f e x t r e m e c o n d i t i o n s . M o s t o f t h e m e t a s t a b l e s o l u t i o n s a r e s u b j e c t t o t e r m i ­ n a tio n b y ic e n u c le a tio n , v a p o r c a v ita tio n , a n d /o r s o lu te c r y s ta lliz a tio n . H o w e v e r , if t h e s o l u t i o n g l a s s e s , it b e c o m e s r e s i s t a n t to t h e s e o c c u r r e n c e s .

Changes During Plant Cold Acclimation A l t h o u g h i t is g e n e r a l l y t h o u g h t t h a t a c t i v e l y g r o w i n g p l a n t s h a v e a l i m i t e d a b i l ­ ity to t o l e r a t e ic e f o r m a t i o n in t h e i r ti s s u e s , m a n y p l a n t s t o l e r a t e - 5 to - 1 0 ° C d u r ­ in g t h e g r o w i n g s e a s o n . F o r e x a m p l e , w i n t e r r y e ( Secale

cereale ‘P u m a ’) g r o w i n g

in t h e f ie l d in m i d - J u n e a t d a y t e m p e r a t u r e s o f 3 0 ° C c a n t o l e r a t e - 1 0 ° C . P l a n t s g r o w n in a n e n v i r o n m e n t a l c h a m b e r m a i n t a i n e d a t 3 0 ° C , h o w e v e r , a r e k i l l e d a t 3 ° C (L . V . G u s t a ,

unpublished). T h e r e a s o n f o r s u c h a d i f f e r e n c e in f r e e z i n g t o l e r ­

a n c e is c u r r e n t l y u n k n o w n . T h e L T 5I) ( t e m p e r a t u r e a t w h i c h 5 0 % k ille d ) o f s o m e

o f c e lls w e re

Solanum s p e c i e s g r o w i n g a t w a r m t e m p e r a t u r e s c a n b e a s lo w a s - 5

t o - 6 ° C ( R a j a s h e k a r e t a l ., 1 9 8 3 ) . U p o n e x p o s u r e to h a r d e n i n g c o n d i t i o n s , s o m e o f t h e s e s p e c i e s c a n s u r v i v e - 8 to —1 2 ° C . C h e n e t a l. ( 1 9 7 6 ) s t u d i e d t h e f r e e z i n g c h a r a c t e r i s t i c s o f h a r d y a n d n o n h a r d y S o ­ lanum s p e c i e s a n d c o n c l u d e d t h a t t i s s u e s o f h a r d y s p e c i e s t o l e r a t e m o r e f r o s t i n d u c e d d e h y d r a t i o n t h a n d o t i s s u e s o f n o n h a r d y s p e c i e s . Solanum s p e c i e s h a v e b e e n d i v i d e d i n t o f i v e h a r d i n e s s c a t e g o r i e s : G r o u p 1, f r o s t r e s i s t a n t a n d a b l e to c o l d h a r d e n ; G r o u p 2 , f r o s t r e s is ta n t a n d u n a b le to c o ld h a r d e n ; G ro u p 3 , f ro s t s e n s itiv e a n d a b l e to c o l d h a r d e n ; G r o u p 4 , f r o s t s e n s i t i v e a n d u n a b l e to c o l d h a r d e n ; a n d G r o u p 5 , c h i l l i n g s e n s i t i v e ( C h e n a n d L i, 1 9 8 0 b ) . R e c e n t l y , S t o n e e t al. ( 1 9 9 3 ) p e r ­ f o r m e d a n i n t e r s p e c i f i c c r o s s b e t w e e n a G r o u p 1 s p e c i e s ( Solanum a n d a G ro u p 4 s p e c ie s

com m ersonii ), (S. cardiophyllum ). I n th e s e g r e g a t i n g g e n e r a t i o n s , f r o s t t o l ­

e r a n c e a n d a b i l i t y t o c o l d h a r d e n w e r e s e p a r a t e l y i n h e r i t e d , i m p l y i n g t h a t t h e s e tw o tr a i t s w e r e c o n t r o l l e d b y d i f f e r e n t l o c i , n o t c l o s e l y li n k e d . L o w g r o w in g te m p e r a tu r e s a re n o t th e o n ly s tim u lu s in d u c in g fre e z in g to le r ­ a n c e . S h o r t d a y s ( C h e n a n d L i, 1 9 7 8 ) , d e h y d r a t i o n o r d r o u g h t ( C h e n a n d L i, 1 9 7 8 ; C l o u t i e r a n d A n d r e w s , 1 9 8 4 ; S i m i n o v i t c h a n d C l o u t i e r , 1 9 8 2 ) , a n d a b s c i s i c a c id

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(A B A ) tre a tm e n t (C h en e t al., 1983; C h en an d G u sta, 1983; K eith and M cK ersie, 1986; L an g e t al., 1989) also in d u c e freezin g to le ra n c e . It is u n k n o w n if the v ario u s ex tern al stim u li in d u c e fre e z in g to leran ce v ia a c o m m o n m etab o lic p ath w ay o r by a lte rn ativ e p ath w ay s.

Extracellular Polysaccharides O lien e t al. (1 9 6 5 ) id e n tifie d a g ro u p o f c o m p o u n d s term ed freezin g in h ib ito rs th at are d isp e rse d in th e liq u id w a te r o f the in te rc e llu la r spaces. T h e in h ib ito rs are large p o ly sa c c h a rid e p o ly m e rs, g e n erally w ith a m o le c u la r w eig h t o f several h u n ­ d red th o u san d D a lto n s. T h e in teractio n o f th ese p o ly m e rs w ith ice is v ariable; so m e bind o r are c a p tu re d in th e g ro w in g ice cry stals w ith little effe c t on crystal g ro w th rate o r cry stal stru c tu re . O th ers fo rm a c o h esiv e film a t th e ice-liq u id in terface and slo w th e rate o f ice cry sta l g ro w th d u rin g freezin g . T h e se e ffectiv e in h ib ito rs in flu ­ en ce th e path o f ice g ro w th th ro u g h th e p la n t an d th e su b se q u e n t size and sh ap e o f ice crystals. In su sp e n sio n -c u ltu re d p e a r cells, co ld a c clim atio n is acco m p an ied by ch an g es in so lu b le e x tra c e llu la r p o ly sa c c h a rid es (W alln er e t al., 1986). S p ecifically , th ere is an in crease in th e re le a se o f a relativ ely sm all n eu tral p o ly sacch arid e into the c u l­ ture m ed iu m , a d e c re a se in p ro d u c tio n o f a large m o le c u la r w eig h t pectic p o ly sa c ­ c h arid e, and a d e p o sitio n o f c a llo se at th e cell su rfa c e (W alln er et al., 1986). T h e lo calizatio n o f c a llo se a t th e p la sm a m e m b ra n e -c e ll w all in terface in p ear cells m ay stab ilize this re g io n fro m the fre e z in g stress.

Cell Wall T h ere is g ro w in g e v id e n c e th at th e cell w all p lay s an im p o rtan t role in freezin g injury an d re sista n c e (B arto lo an d W alln er, 1986; G riffith e t al., 1985; O lien and S m ith, 1977; P a ro sc h y e t al., 1980; R a jash ek ar an d B u rk e, 1982). N eg ativ e tu rg o r m easu rem en ts (R a ja rsh e k a r and B u rk e, 1982) h a v e p ro m p ted so m e re search ers to su g g est th at e n h a n c e d rig id ity o f cell w alls in creases th e co ld hard in ess o f plan ts by d e creasin g th e se v e rity o f cell v o lu m e red u ctio n d u rin g e x tra c e llu lar freezing. C ell w all a u g m e n ta tio n h as o ften been o b se rv e d as a p la n t resp o n se to low te m ­ p eratu re. F o r e x a m p le , p e a ep ic o ty l cell w all w eig h t in creases by 4 0 % d u rin g co ld acclim atio n (W e ise r e t al., 1990). A h ard y p o ta to sp ecies, S. acaule, has a th ick er cell w all th an a fro st-se n sitiv e sp ecies, S. tuberosum (C h en e t al., 1977). T h e e p i­ derm al an d m e so to m e sh e a th cell w alls o f h a rd e n e d P u m a ry e leav es are sig n ifi­ cantly th ick er th an th o se o f n o n h ard en ed leav es (G riffith et al., 1985). U ltra stru c tu ra l stu d ie s o f b ro m e g ra ss su sp en sio n c u ltu re cells in d icated that A B A treated cells h av e m a rk e d ly th ic k e r cell w alls th an d o u n treated cells (T an in o e t al., 1991). In c o ld -a c c lim atin g p e a seed lin g s, cell w all w eig h t n o t only in creases b u t also sh o w s c o m p o sitio n a l ch an g es: arab in o sy l c o n te n t in creases by 100% , cell w all g ly co sy l re sid u e s and cellu lo se increase by a p p ro x im ately 20% , and hyd ro x y p ro lin e c o n te n t in c reases by 80% (W eiser e t al., 1990). M o st b io c h e m ic a l stu d ies o n th e co ld a c clim atio n o f cells h av e co n c e n tra ted on the so lu b le ra th e r th an th e in so lu b le dry m atter fractio n s (L evitt, 1980). C a rb o h y ­ drates a c c o u n t fo r a sig n ific a n t p ro p o rtio n o f th e dry m atter accu m u latio n d u rin g co ld a c c lim a tio n (C h en an d L i, 1980a; L evitt, 1980). A lth o u g h carb o h y d ra te re ­ serv es are im p o rta n t as an en e rg y so u rce fo r w in te r su rv iv al (L ev itt, 1980), stru c ­ tu ral c a rb o h y d ra tes m ay also be clo sely a sso ciated w ith h ard in ess. In b ro m eg rass cells, T a n in o e t al. (1 9 9 0 ) o b se rv e d th at A B A c a u se s a sig n ifican t a c c u m u latio n o f

122

C h e n , B u r k e , a n d G u s ta

dry m atter, p a rtic u la rly in the 85% eth an o l-in so lu b le fractio n , o f w h ich cell walls re p resen t th e m o st a b u n d a n t c o m p o n en t. F ro m l4C -su c ro se la b e lin g stu d ies, it was d e m o n stra te d th at a high p e rc e n ta g e o f the ex o g en o u s su c ro se is in c o rp o ra te d into the cell w all fractio n . W e ise r e t al. (1990) ob serv ed th at in c re a se d fre e z in g toler­ ance is a sso c ia te d w ith e le v a te d levels o f extensin, a cell w all g ly c o p ro te in that c ro ss-lin k s c e llu lo se m icro fib rils, thereby ad d in g rig id ity an d stre n g th to cell w alls. N o rth e rn b lo t an a ly sis in d ic a te d th at the level o f sp ecific e x te n sin tra n sc rip ts in­ creases d u rin g c o ld a c c lim a tio n (W eiser e t al., 1990). P e rh ap s an in c re a se in the rig id ity o f th e cell w all in c reases the resistan ce o f the cell to c o lla p se d u rin g freez­ in g -in d u ced d e h y d ra tio n . It has b e e n p o stu la te d th a t m em b ran e d am ag e is the re su lt o f p h y sical tearing th at o c c u rs w h e n th e cell w all sep arates from the p lasm a m e m b ra n e d u rin g freezing (T an in o e t al., 1991). A g re a te r cell w all-to -p lasm a m e m b ra n e ad h e sio n has been su g g ested to in c re a se th e re sista n c e to cell co llap se and d e h y d ra tio n stress (B artolo and W a lln e r, 1986). T h e n u m b e r o f m em b ran e a ttach m en ts to th e cell w all in­ creases d u rin g co ld a c c lim a tio n in alfalfa cell cu ltu res (Jo h n so n -F la n a g a n and Singh, 1986). T h e ch a n g e s o b se rv e d in cell w all and m e m b ra n e c o m p o sitio n d u ring co ld a c c lim a tio n m ay alter th e attach m en t ch aracteristics an d th e re fo re m ay serve to in crease p la s m a m e m b ra n e -to -ce ll w all adhesion.

Plasma Mem brane S in ce th e p la sm a m e m b ra n e is co n sid ered the p rim ary site o f fre e z in g injury, any fre e z in g to le ra n c e m e c h a n ism should acco u n t for p ro te c tio n o f th e m em b ran e from fre e z in g stress. D u rin g c o ld acclim atio n , ch an g es to lip id (S te p o n k u s e t al., 1988; U e m u ra an d Y o sh id a, 1984; Y o sh id a and U em u ra, 1984) and protein (U em u ra an d Y o sh id a , 1984; Y o sh id a and U em ura, 1984) c o n stitu e n ts, as w ell as altered b io p h y sic a l p ro p e rtie s (S tep o n k u s et al., 1990; Y o sh id a, 1 9 8 4 a,b ) o f m em ­ b ranes h av e b e e n re p o rte d . S u g a w a ra and Sakai (1978) o b se rv e d th a t th e n u m b e r o f in n er m e m b ra n e p a rtic le s o f th e p la sm a m em b ran e p rim arily on th e fra c tu re surface facing th e c e ll w all (face E ), w as m arkedly red u ced in h a rd e n e d Je ru sa le m arti­ c h o k e tu b e r ca lli b u t w as re sto re d to initial levels fo llo w in g d e h a rd e n in g . S im ilar resu lts w ere a lso o b se rv e d in S. acaule (T o iv io -K in n u can e t al., 1981) an d w heat (P earce, 1985) d u rin g h a rd e n in g an d d ehardening. L ip id a n a ly sis o f p u rified p la sm a m em b ran e fractio n s h av e d e m o n stra te d either d ram atic o r s lig h t ch a n g e s in th e lip id co m p o sitio n o f the p la sm a m e m b ra n e d u ring the d e v e lo p m e n t o f co ld h a rd in e ss (U em u ra and Y o sh id a, 1984; Y o sh id a and U em ura, 1984). In p u rified p la sm a m em b ran es o f co ld -a c c lim ate d w in te r rye seed ­ lings, U e m u ra an d Y o sh id a (1 9 8 4 ) rep o rted 1) slight ch a n g e s in th e d eg re e o f fatty acid u n s a tu ra tio n an d p ro p o rtio n s o f p h o sp h o lip id s; 2) a sm all c h a n g e in sterol co m p o sitio n c o n sistin g o f in c re a se d b eta-sito stero l and d e c re a se d cam p e stro l and stig m astero l le v e ls; 3) an in c re a se in the p h o sp h o lip id -to -p ro tein ratio ; an d 4 ) a d e­ clin e in th e ste ro l-to -p h o sp h o lip id ratio. S te p o n k u s e t al. (1 9 9 0 ) also d e m o n strated the fo llo w in g c h a n g e s in th e p ro p e r­ ties o f p la s m a m e m b ra n e : 1) th e cry o b io lo g ical b e h av io r o f h a rd y and no n h ard y rye are v ery d iffe re n t; 2) fu sio n o f lip o so m es prepared fro m h a rd y p la n ts to n o n ­ hardy rye p ro to p la sts in c re a se d th e freezin g to leran ce o f th e n o n h a rd y p ro to p lasts (S te p o n k u s e t al., 1988). T h e se stu d ies p ro v id ed e v id en ce th at c h a n g e s in th e lipid co m p o sitio n o f th e p la sm a m e m b ra n e are related to co ld h ard in ess.

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Y o sh id a (1 9 8 4 b ) n o ted sig n ific a n t ch an g es in th e p la sm a m em b ran e p ro tein p a t­ tern o f m u lb erry b a rk c e lls d u rin g a c clim atio n to co ld . A n altered g ly co p ro tein fraction co in c id e d w ith th e d e v e lo p m e n t o f fre e z in g to leran ce. U em u ra and Y o sh id a (1 9 8 4 ) o b se rv e d sev eral p ro tein s p re se n t in the p lasm a m em b ran e o f h a rd ­ en ed w in te r ry e see d lin g s th at w ere n o t p re se n t in n o n h ard en ed tissue. In w in ter can o la p lan ts, J o h n so n -F la n a g a n and S in g h (1 9 8 7 ) re p o rted the id en tificatio n o f a m em b ran e p ro te in a sso c ia te d w ith th e in d u ctio n o f fre e z in g tolerance.

Cytoplasmic Changes Sugars A ll m a jo r c la sse s o f o rg a n ic co m p o u n d s lo cated in th e cy to p lasm , o f w hich su g ­ ars are th e m o st a b u n d a n t, h av e b een im p licated in th e h ard e n in g resp o n se o f plan ts (L ev itt, 1980; S ak ai an d L a rc h e r, 1987). M ajo r c h a n g e s in total o sm o tic p o ten tial, w hich a c c o m p a n y se a so n a l ch a n g e s in freezin g to le ra n c e , are related to c h an g es in the c o n c e n tra tio n s o f su g a rs an d p o ly h y d ric a lc o h o ls (S ak ai and L arch er, 1987). In h erb aceo u s and w o o d y p lan ts, so lu b le c a rb o h y d ra tes in crease from fall to w in te r and d ec re a se in th e sp rin g d u rin g d e h a rd e n in g (S ak ai and L arch er, 1987). C h an g es in su g a r c o n te n t d u rin g a rtificial h ard e n in g fo llo w a v ery sim ilar resp o n se p a ttern (S akai and L a rc h e r, 1987). In ad d itio n to q u a n tita tiv e c h an g es in the su g a r c o n te n t o f p lan t cells, q u a litativ e ch an g es also o c c u r d u rin g co ld h ard en in g . F o r e x a m p le , P ark er (1 9 5 9 ) o b serv ed th at lev els o f ra ffin o se an d sta ch y o se increased m a rk e d ly in the b ark and leav es o f six co n ife r sp e c ie s d u rin g late fall; co n c e n tra tio n s o f su cro se and so m etim es g lu ­ cose an d fru c to se also in creased . T h e p ro p o rtio n o f th e v ario u s sugars d iffered in 18 w o o d y p la n t sp ecies, b u t the a c c u m u latio n o f a sp ecific su g ar w as n o t c o n sis­ ten tly c o rre la ted w ith h a rd in e ss (S ak ai, 1962). T h e ty p e o f c a rb o h y d rate th at a c c u ­ m u lates d u rin g h a rd e n in g is d e p e n d e n t o n a sp e cies-sp ecific pattern o f carb o h y d ra te m e ta b o lism . In sev eral sp ecies, p o ly h y d ric alco h o ls, such as sorbitol or m an n ito l, a c c o u n t fo r ap p ro x im a te ly 4 0 % o f th e to tal so lu b le carb o h y d ra te c o n ­ ten t and m ay th e re fo re c o n trib u te to h ard en in g (Ich ik i an d Y am ay a, 1982; R aese et al., 1977). S u g a rs a c c u m u la te in th e ch lo ro p la sts o f a c clim atin g cab b ag e, sp in ach (K rau se e t al., 1978), an d w h e a t (T ru n o v a an d Z v e re v a , 1974). S u g a r fe e d in g trials h a v e b een u sed to d e m o n stra te the ro le o f su g ars in fro st h ard in ess. F o r e x a m p le , th e m ax im u m hard in ess p o ten tial o f w in te r cereals an d c a l­ lus tissu es o f w o o d y p lan ts c o u ld n ot be attain ed u n less, in ad d itio n to ex p o su re to low te m p e ra tu re, th e p la n ts and tissu es w ere fed su c ro se (S akai, 1962; O g o lev ets, 1976). In th e d a rk , C hlorella spp. h a rd en ed o n ly m a rg in ally , b u t th e ad d itio n o f g lu co se cau sed a d ra m a tic in c re a se in fro st re sista n c e (H atan o , 1978). T h e re is a m p le e v id e n c e o f a clo se c o rre la tio n b etw een the a c c u m u latio n o f so lu b le c a rb o h y d ra te s an d fre e z in g to leran ce. S u g g e ste d p o ssib ilities as to ho w su g ars are in v o lv e d in p ro te c tio n a g a in st freezin g in ju ry in clu d e the follow ing: 1. O sm otic effect. S u g ars d ec re a se c ry stallizatio n o f w ater and therefo re red u c e fre e z e-in d u c e d d e h y d ra tio n . 2. M etabolic effect. M e ta b o liz a tio n o f su g ars in th e c y to p lasm d u rin g acclim atio n p ro d u c e s o th e r p ro te c tiv e su b sta n c e s or m e ta b o lic en erg y . 3. C ryoprotective effect. S u g ars m ay p ro te c t c e llu la r c o n stitu en ts in clu d in g m e m ­ b ran es d u rin g fre e z e /th a w c y c le (S ak ai an d L a rc h e r, 1987). 4. G lass effect. H ig h su g a r co n c e n tra tio n s m ay sto p all b io ch em ical an d m o st p h y sic a l activ ity (d e h y d ra tio n ) w h en they fo rm a solid glass (B runi an d L eo -

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p o ld , 1991; W illia m s and L e o p o ld , 1989; H irsh e t al., 1985). It is a lso p o ssib le th at sp e c ific in teractio n s b etw een su g ars a n d cell stru ctu res p ro tect o r a m e lio ra te th e d e le te rio u s effects o f freezing.

Proteins P ro tein a c c u m u la tio n d u rin g co ld acclim atio n has b een fre q u e n tly o b se rv e d in n u m ero u s h e rb a c e o u s p lan t sp e c ie s (C hen and Li, 1980a; G u y , 1990; Joh n so n F lan ag an an d S in g h , 1988; L e e an d C hen, 1993a) u n d er both n a tu ra l and artificial c o n d itio n s. In m o st in stan ces, th ere is an increase in b o th to tal p ro tein co n ten t (L evitt, 19 8 0 ) an d in sp e cific p o ly p e p tid e species (G ilm o u r e t al., 1988; G uy e t al., 1985; G u y an d H ask ell, 1987; L an g et al., 1989; L ee e t al., 1990; L ee et al., 1992; P erras an d S a rh a n , 1989; R o b e rtso n et al., 1987). T he re q u ire m e n t fo r p ro tein sy n ­ thesis d u rin g c o ld a c c lim a tio n has been d em o n strated in w h eat (T ru n o v a , 1982), w in ter c a n o la (K a c p e rsk a -P a la c z e t al., 1977), Chlorella (H atan o , 1978), an d S ola­ rium sp e c ie s (C h e n e t al., 1983). A high co rrelatio n b etw een th e in c re a se in p rotein c o n te n t an d c o ld h ard in ess w as o b serv ed in all o f these sp ecies. I f ap p lied p rio r to co ld a c c lim a tio n , c y c lo h e x im id e , a p rotein syn th esis in h ib ito r, su p p re sse d th e h a rd ­ en in g p ro c e ss (C h en et al., 1983), th u s in d icatin g the need fo r p ro te in sy n th esis. G uy (1 9 9 0 ) p ro p o se d th at c h a n g e s d u rin g cold a c clim atio n in v o lv e b oth th e in­ d u ctio n o f fre e z in g to le ra n c e an d m etab o lic ad ju stm en t to c o n stra in ts im p o sed by low te m p e ra tu re. T h is m ay in v o lv e structural pro tein s as w ell as en z y m e s. T h e im ­ p licatio n is th a t p ro tein c h a n g e s can in clu d e eith er th e m o d ific a tio n o f ex istin g p o ly p e p tid e s to g e n e ra te d iffe re n t isoform s, o r the p ro d u c tio n o r e n h a n c e d p ro d u c ­ tion o f n ew p o ly p e p tid e s w ith u n iq u e p ro p erties, or the cre a tio n o f a n ew m etabolic p ath w ay . O n th e b asis o f th ese assu m p tio n s, it is n ot su rp risin g to o b se rv e ch an g es to m any e n z y m e s d u rin g co ld a c clim atio n (G uy, 1990; L ev itt, 1980). A sid e fro m p ro te in s w ith c a ta ly tic o r stru ctu ral p ro p erties, p ro te in s m ay h av e d i­ rect c ry o p ro te c tiv e ro les. F o r ex a m p le , V o lg er and H e b e r (1 9 7 5 ) iso la te d a p rotein fraction fro m fro st-h a rd y sp in a c h leaves th at p ro tected m e m b ra n e s in v itro from freezin g in ju ry . R e c e n tly , H in c h a e t al. (19 8 9 , 1990) id e n tifie d p ro te in fractio n s from c o ld -a c c lim a te d , fro st-h a rd y cab b ag e and spinach leav es th a t p ro te c te d n o n ­ h ard y sp in a c h th y la k o id s a g a in st a freeze-th aw stress. O n a p e r m o le c u le basis, the p ro te c tiv e p ro te in s are a b o u t 2 0 ,0 0 0 to 4 0 ,0 0 0 tim es m o re e ffe c tiv e th an su cro se in p re v e n tin g fre e z e -th a w ru p tu re to isolated th y lak o id m e m b ra n e s (H in c h a e t al., 1989). A c o rre la tio n b e tw een h y d ro p h ilic p ro tein s and h ard in e ss in b la c k lo cu st w as o b se rv e d by S im in o v itc h an d B riggs (1953). It w as c o n c lu d e d th a t h y d ro p h ilic p ro tein s im p ro v e th e w ate r-b in d in g capacity o f the c y to p la sm ic flu id an d th erefo re red u ce the fre e z in g stress. R ec e n tly , m any co ld -in d u ced m R N A s e n c o d in g h y d ro ­ ph ilic p ro te in s h a v e b een c lo n e d , su g g estin g th at these ty p es o f p ro te in s m ay p ro ­ tect the c y to p la sm o r m e m b ra n e s from freeze-in d u ced d e h y d ra tio n stress. S o m e o f the c o ld -re g u la te d g en es o f A rabidopsis en co d e for h eat-stab le p ro te in s (L in e t al., 1990). A lth o u g h th e e x a c t ro le o f th ese h eat-stab le p ro tein s is n o t k n o w n , th ey h av e a ran d o m c o il stru c tu re and m ay re p re se n t a u n iq u e gro u p o f p ro te in s th a t so m eh o w reduce a fre e z in g stress. R ecen tly , se v e ra l c D N A c lo n e s from S. com m ersonii cell cu ltu re s h av e been iso ­ lated d u rin g th e in d u c tio n o f c o ld h ard in ess by low te m p eratu re o r A B A treatm en t (Z hu e t al, 199 3 ). T h re e o f th e c D N A clo n es h av e high h o m o lo g y to to b a c c o osm o tin , w h ic h a c c u m u la te s in to b a c c o cells u n d erg o in g g rad u al o sm o tic ad ju stm en t to N aC l stre ss (S in g h e t al., 1989).

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In A B A -h a rd e n e d b ro m e g ra ss cell cu ltu res, L e e an d C h en (1 9 9 3 b ) iso lated an A B A -re sp o n siv e c D N A clo n e th at sh ares 82% n u c le ic acid seq u en ce sim ilarity to b arley d eh y d rin (C lo se e t al., 1989). In b arley , d e h y d rin m R N A a ccu m u lates d u r­ in g th e p ro g ra m m e d d e h y d ra tio n p h a se o f em b ry o d e v e lo p m e n t and is also in d u ced by a d e sic c a tio n stress o r A B A treatm en t. A s d isc u sse d ab o v e, freezin g is also a fo rm o f d e sic c a tio n stress. T h e en h a n c e d ex p re ssio n o f o sm o tin - o r d e h y d rin -lik e g en es d u rin g th e d e v e lo p m e n t o f freezin g to le ra n c e in p la n t cells in d icates a re ­ q u ire m e n t fo r to le ra n c e to the d e sic c a tio n stress m a n ife ste d by the freezin g stress.

Ice Nucleation and Freezing Tolerance in Plants Ice fo rm a tio n in v o lv e s th e initial ice n u c le a tio n an d th e co n se q u e n t c ry sta lliz a ­ tion o f w ater m o le c u le s. In co n tra st to the c o m m o n b e lie f that aq u eo u s so lu tio n s freeze a t th e m e ltin g p o in t o f th e solid p h ase (ice), cell so lu tio n s rarely freeze at th eir m eltin g p o in t. L iq u id w ater, su p erco o led sev eral d eg rees below its freezin g point, w ill freeze only if spontaneous ice form ation occurs (usually only below -3 8 ° C ) o r if an e x trin sic ice n u c le a to r is p re se n t th at acts as a ca ta ly st for the liq u id -so lid p h ase tran sitio n . T h e re are tw o g e n eral types o f ice n u clei in b io lo g ical system s: h o m o g e n e o u s an d h e te ro g e n e o u s. In h o m o g e n e o u s ice n u cleatio n , the n u clei form sp o n tan eo u sly in th e liq u id (u su ally b elo w - 3 8 ° C ) . In h etero g en eo u s ice nu cleatio n , n u c le a tio n o c c u rs as a re su lt o f e x trin sic n u clei, such as ice. P o ten t b io ­ lo g ical h e te ro g e n e o u s n u c le a to rs are b acteria, fu n g i, p lan ts, and insects (see o th er ch ap ters in th e b o o k ). E x trin sic ice n uclei are th e m a jo r cau se o f ice fo rm atio n in te n d e r g ro w in g p la n ts (see C h a p te r 2). It is g e n erally b e lie v e d th at m o st p la n t cells d o n ot co n tain intrinsic ice n u c le a ­ tors; h o w ev er, th e re are e x c e p tio n s (see C h a p te r 8). K ro g et al. (1 9 7 9 ) re p o rted flo w er tissu es o f th e A fro -a lp in e p lan t Lobelia telekii co n tain a slightly v isco u s flu id in th e c e n tral p a rt o f th e in flo rescen ce. F re e z in g o f this cen tral flu id o ccu rs n ear 0 °C and re su lts in a slo w and steady re le a se o f heat, w hich m ain tain s th e p la n t’s te m p e ra tu re at a b o u t 0 °C d u rin g a frost. A n a ly sis o f th e ch em ical c o m p o si­ tio n o f th is c e n tra l flu id re v e a le d th at the n u c le a tin g ag en ts are carb o h y d ra te, p ro b a b ly h igh m o le c u la r w eig h t p o ly sa c c h a rid es. T h u s, this p la n t has e v o lv ed a u n iq u e w ay o f su rv iv in g a fro st in th e a lp in e zo n e o f M o u n t K enya. V a rio u s stu d ie s h a v e p ro v id e d ev id e n c e fo r e ffe c tiv e ice n u cleato rs w ithin p lan t tissu es., A n d re w s e t al. (1 9 8 6 ) m easu red th e ice n u cleatio n tem p eratu res o f peach and sw eet ch e rry flo w e rs, fru its, an d stem s. T h e m ean ice n u cleatio n tem p eratu re o f flo w ers w as b e tw e e n - 4 an d - 6 ° C , w h ile th o se o f 0 .5 -c m stem seg m en ts w ere b e­ tw een - 6 and - 9 ° C . T h e stem tissu e c o n tain ed a lo w e r co n c e n tra tio n o f ice n u c le a ­ to rs a c tiv e a b o v e - 5 ° C th an d id the flo ral tissu e. H o m o g en izatio n o f th e tissu e re d u c e d th e n u c le a tio n tem p e ra tu re, w h ich su g g e sts th at tissu e stru ctu ral in teg rity is n ecessary fo r th e m a n ife sta tio n o f op tim al ice n u c le a tio n activity. In o th e r stu d ies, w o o d y tissu e o f P runus spp. n u cleated at - 2 ° C , and the n u c le a ­ tor w as c o n sid e re d to be n o t o f b acterial o rig in fo r th e fo llo w in g reasons: F irst, p each stem s n u c le a te d a t - 2 ° C ev en th o u g h IN A b a c te ria co u ld n o t be d etected . S eco n d , su p p re ssio n o f IN A b ac te ria in o rc h a rd s w ith eith e r b actericid es o r a n ta g o ­ n istic b a c te ria fa ile d to p re v e n t ex te n siv e su p e rc o o lin g (P ro eb stin g and G ro ss, 1988). T h ird , b a se d o n se a so n a l m o n ito rin g o f activ ity , b a cteria ice n u clei d id n o t rem ain activ e fo r e x te n d e d p e rio d s in n atu re (C o d y et al., 1987). A sh w o rth and D av is (1 9 8 4 ) c o n d u c te d th e fo llo w in g stu d ies o n p e a c h to estab lish th at th e ice nu-

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I clei w ere o f p lant origin. A utoclaved shoots were supercooled to approxim ately -4 ° C , w h ich w as 1.5 to 2 .0 °C lo w e r th an the ice n u cleatio n te m p e ra tu re o f untreated peach sh o o ts. S o a k in g 5 -cm p ea c h stem sections in w ater fo r 4 h r lo w ered the m ain ice n u c le a tio n te m p e ra tu re to b elo w - 4 ° C ; how ever, ice n u c le a tio n activ ity w as fully re sto re d b y a ir-d ry in g w o o d y stem sections for a few h o u rs. T h e ice n uclei in w o o d y tissu e s w ere in a c tiv a te d b e tw een 4 0 and 50°C b u t u n a ffe c ted by treatm en t w ith b a c te ria l ice n u c le a tio n in h ib ito rs (i.e., N aO C l, ta rta ric acid , trito n X Q S -2 0 ), su lfh y d ry l re a g e n ts (i.e., p -h y d ro x y m e rc u rib e n z o a te an d io d in e), an d p ro n ase. Ice n uclei c o u ld n o t be d islo d g e d fro m stem s by so n icatio n . T h ey w e re sh o w n to be d istrib u ted u n ifo rm ly in bu d an d in tern o d al stem tissue. T h e ice n u c le a tio n activity in o u ter an d in n e r stem tissu es w as also in d istin g u ish ab le. T h e d e v e lo p m e n t o f ice nuclei in im m a tu re p each an d sw eet ch erry stem did n o t o c c u r un til m id su m m er, w as c o m p le te b y A u g u st, an d w as u n affected by seaso n al ch a n g e s an d ad d itio n al grow th.

M olecular Biology of Cold Hardiness Development G en etic a sp e c ts o f co ld h ard in e ss have been rev iew ed (L im in an d F o w ler, 1983; T h o m a sh o w , 1990) and w ill n o t be reiterated here. T h e c o m p le x g e n e tic basis o f cold to le ra n c e h as tra d itio n a lly b een stu d ied using te c h n iq u es o f q u a n tita tiv e an aly ­ sis (E u n u s e t al., 1962) su p p o rte d by cy to g en etic ev id en ce. M o n o so m ic analysis and su b stitu tio n lin es h av e b e e n u sed in w h eat to lo cate c o ld to le ra n c e g en es on 15 o f the 21 c h ro m o so m e s (C a h a la n an d L aw , 1979; S utka, 1981; S u tk a an d V eisz, 1988). S im ila r stu d ie s in o th e r sp ecies indicate that cold h ard in e ss is c o n tro lled by m any gen es.

Molecular Biology of Plant Cold Acclimation In th e p a s t few y ears, ra p id p ro g re ss has been m ad e in th e c h a ra c teriz a tio n o f m o le c u la r c h a n g e s asso c ia te d w ith fro st tolerance. In all th e sy ste m s stu d ied , the in d u ctio n o f fre e z in g to le ra n c e is g en erally acco m p an ied b y th e a p p e a ra n c e o f new m R N A s an d n ew p o ly p e p tid e s. cD N A s o f co ld -reg u lated g en es h av e b een clo n ed fro m A ra b id o p sis thaliana (G ilm o u r e t al., 1992; H ajela e t al., 1990; K u rk e la and F ran ck , 1990, 1992; N o rd in e t al., 1991), B rom us inerm is (L ee an d C h en , 1993b), B rassica n a p u s (W e re tiln y k e t al., 1993), H ordeum vulgare (D u n n e t al., 1990; H u g h es e t al., 1992; C a ttiv elli and B artels, 1990), M edicago spp. (L u o , e t al., 1992; M o h a p a tra e t al., 1989), S. com m ersonii (Z hu e t al., 1993), an d Triticum aestivum (G uo e t al., 1992; H o u d e e t al., 1992). F o r a th o ro u g h rev iew o n m o le c u la r b iology o f co ld a c c lim a tio n , th e re a d e r is d irected to review s by G u y (1 9 9 0 ), L e e an d C hen (1 9 9 3 a), an d T h o m a sh o w (1 9 9 0 ). T he n u c le o tid e se q u e n c e s fo r so m e o f these cD N A s h av e been p u b lish e d . T he search o f th e G e n B a n k o r E M B L d a ta bases has id en tified h o m o lo g y w ith rep o rted seq u en ces o f k n o w n D N A s an d p ro te in s (T able 1). T he e x p re ssio n o f so m e o f these g enes is also in d u c e d by A B A , a p lan t h o rm o n e know n fo r its ab ility to induce freezin g to le ra n c e in p lan ts (C h en e t al., 1983; C hen and G u sta, 1983). It a p p e a rs th a t p lan ts h a v e e v o lv e d so p h isticated m ech an ism s th a t p ro v id e p ro ­ tectio n to all m a jo r fre e z in g -sen sitiv e sites o f p lan t cells. T h e re are few e x a m p le s in w hich th e p u ta tiv e fu n c tio n s o f th e g en e pro d u cts h av e b een p ro p o se d (T ab le 2). T he p u ta tiv e fu n c tio n s o f th ese c o ld -reg u lated p ro tein s in c lu d e 1) cry o p ro te c tiv e p ro p erties (L in an d T h o m a sh o w , 1992), 2) altered lipid m e ta b o lism (H u g h e s et al.,

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T e m p e ra tu re (°C) F ig u re 2. C u m u lativ e ice n u cleatio n sp ectra o f lichens (K ieft, 1988). A , L ichens show ing ice n u cleatio n activ ity at tem p eratu res ab o v e -3 ° C : Rhizoplaca chrysoleuca ( ■ ), Xanthoparm elia sp. ( • ) , X anthoria elegans ( A ) , all fro m n e a r Jem ez Springs, N M , and Rhizoplaca chrysoleuca ( O ) from the M ag d alen a M o u n tain s, N M . B , L ich en s w ith interm ediate ice n u cleatio n activ ity : Platism atia sp. ( • ) , Acarospora sp., from near Je m ez S prings, NM (■ ), Acarospora sp.. P latoro, C O (A), A carospora sp. from near S o co rro , N M (O ), L eptogium sp. ( □ ) . (C) L ichens w ith lo w levels o f ice nucleation activity: P sora decipiens ( • ) , Xanthoparm elia sp., M agdalena M o u n tain s (■ ), Xanthoparem lia sp. from n ear S ocorro, N M ( A ) , L etharia sp. (O ), Peltigera sp. ( □ ) , Usnea sp ( A ) .

Ice Nucleation Activity Associated with Plants and Fungi

151

Ice Nucleation Activity Associated with Fungi W arm -tem perature IN A has been found within m em bers of the fungal kingdom as well as in the plant kingdom . In som e species, the fungal ice nucleation sites are as potent as those produced by the m ost active bacterial ice nucleators. Kieft (1988) reported a wide range o f cum ulative ice nucleation spectra for a variety o f ho­ mogenized lichens. Threshold tem peratures for IN A (i.e., the warm est tem perature at which IN A is detected) ranged from less than -8 ° C for the least active lichens to -2 .3 °C for the m ost active nucleators (Fig. 2). O ne of the lichen partnerships with the highest threshold nucleation tem peratures was Rhizoplaca chrysoleuca. As part of the same study, Kieft reported that an axenic culture of the lichen fungus R. chrysoleuca initiated ice form ation in supercooled w ater at tem peratures as warm as -2 ° C and that the cum ulative num ber of nucleation sites active at -5 ° C or w arm er was approxim ately 107 per gram dry weight of fungus (Fig. 3). M ore re­ cently, Pouleur et al. (1992) and Tsum uki et al. (1992) reported INA in species of the free-living fungus Fusarium. To date, reports o f w arm -tem perature INA (i.e., maxim um threshold tem peratures m ore than -5 ° C or Type 1 INA as described by Phelps et al. (1986)) in fungi, have been limited to lichen fungi and to the genus Fusarium. The fungal ice nucleation sites have a num ber of sim ilarities to, but also differences from, the m ore thoroughly studied bacterial nucleators. These differ­ ences and sim ilarities have im plications for the evolution of biological ice nuclea­ tion and may shed light on the intrinsic properties of biological ice nucleation sites. The unique properties o f fungal ice nucleators also suggest a num ber of potential applications.

Lichens and Lichen Fungi Lichens are sym biotic associations of fungi with algae and/or cyanobacteria. The association is generally an obligate one for the fungus (the m ycobiont); how ­ ever, m any o f the photosynthetic partners (the photobionts) can also be found in natural environm ents w ithout the m ycobionts. The lichen association is designated by the taxonom ic nam e of the fungus. Nearly all lichen fungi are A scom ycetes; the photobionts com prise a few genera o f green algae, with the genus Trebouxia being the m ost com m on, plus som e species of cyanobacteria, e.g., Nostoc (Ahm adjian, 1967). C yanobacteria can be the sole photobiont or in some cases, they are a third symbiont. The advantage o f the sym biosis to the fungus is fairly obvious: the pho­ tobiont releases photosynthate in the form of sugars or sugar alcohols for heterotrophic m etabolism by the fungus. W hen cyanobacteria are present, they confer the added advantage of nitrogen fixation. The fungi, in return, provide a protective structure for the photobiont and may also add to the nutrient budget of the partner­ ship by solubilizing nutrients from the mineral substrata through the action of car­ bonic and organic acids. The fungal structures and physiology may also function in tolerance o f environm ental extrem es (especially desiccation) experienced by many lichens. Epilithic lichens occur as pioneer organism s on mineral surfaces and are thus im portant agents in the initial stages of pedogenesis. O ther lichens are epi­ phytic, i.e., the w oody structures o f perennial plants form the substrata for these species. Lichens are fam ous for their ability to survive harsh environm ental condi­ tions that include desiccation, heat, and freezing. Lichens are dependent on the at­ m osphere for m oisture, either as precipitation or as water vapor. Many lichens can remain viable in a quiescent state while severely desiccated for prolonged periods;

Ashworth and Kieft

152

d e s e r t lic h e n s c a n r e m a in v ia b le f o r lo n g e r th a n a y e a r a t m o is tu re c o n te n ts b e lo w 1% b y w e ig h t ( R u n d e l, 1 9 8 8 ). W h e n m o is tu r e b e c o m e s a v a ila b le , p h o to s y n th e s is c o m m e n c e s w i t h i n m i n u t e s . M a n y s p e c i e s o f l i c h e n s s u r v i v e p r o l o n g e d e x p o s u r e to t e m p e r a t u r e s in e x c e s s o f 7 0 ° C ( K a p p e n , 1 9 7 3 ) ; a n d l i c h e n s h a v e b e e n s h o w n to s u r v i v e f r e e z i n g t o a t l e a s t - 5 0 ° C ( N a s h e t a l. 1 9 8 7 a ,b ) . I c e n u c l e a t i o n a c t i v i t y in li c h e n s m a y b e a n a d a p t a t i o n f o r i n c r e a s e d u p t a k e o f a t m o s p h e r i c w a t e r v a p o r . T h e o r i g i n a l s u r v e y o f I N A in l i c h e n s ( K i e f t , 1 9 8 8 ; F ig . 2 ) s h o w e d a n u m b e r o f tre n d s . O v e ra ll, th e th r e s h o ld te m p e r a tu r e s fo r fre e z in g w e re h ig h e r f o r th e e p ilith ic lic h e n s th a n f o r th e e p ip h y tic lic h e n s . A ls o , th e o n e lic h e n w ith a c y a n o b a c te r ia l p h o to b io n t,

P eltigera s p ., w a s a r e l a t i v e l y p o o r n u c l e a t o r . E x t e n s i v e e f f o r t s to i s o -

Table 4. I c e n u c l e a t i o n a c t i v i t i e s ( I N A ) d e t e c t e d i n p u r e c u l t u r e s o f f r e e - l i v i n g f u n g i , l i c h e n f u n g i ( m y c o b io n ts ) , a n d lic h e n a lg a e a n d c y a n o b a c te r ia ( p h o t o b io n t s )

N u c le i p e r g r a m O rg a n is m

at -5 ° C

W a rm e s t IN A d e te c te d (° C )

R e fe re n c e

F re e -liv in g fu n g i

Fusarium avenaceum F. acu m in a tu m F usarium s p . f r o m in s e c t la r v a l g u t

1 .0 X I 0 9 NDa ND

- 2 .5

P o u l e u r e t a l ., 1 9 9 2

> - 5 .0

P o u l e u r e t a l., 1992

- 5 .5

T s u m u k i e t a l ., 1 9 9 2

L ic h e n m y c o b io n ts

A carospora fu sc a ta

0

- 9 .1

K ie ft a n d A h m a d jia n , 1989

A n ta r c tic e n d o lith ic lic h e n f u n g u s

0 0

- 5 .9

K ie ft a n d A h m a d jia n , 1989 K ie ft a n d A h m a d jia n , 198 9

C ladonia bellidiflora C. boryi C. cariosa C. chlorophaea 1 4 2 9 C. cristalella 113 C . p leu ro ta C. rangiferina C. subcariosa Lecanora dispersa Pertusaria fla vica n s Phaeographis leucophelia R hizoplaca chrysoleuca

- 8 .3

8 .3 X 1 0 3

- 5 .0

K ie ft a n d A h m a d jia n , 198 9

0

- 6 .3

K ie ft a n d A h m a d jia n , 198 9

0

- 1 0 .0

K ie ft a n d A h m a d jia n , 198 9

0

- 6 .3

K ie ft a n d A h m a d jia n , 19 89

0

- 9 .1

K ie ft a n d A h m a d jia n , 19 89

5 .7 X 1 0 '

- 5 .0

K ie f t a n d A h m a d j i a n , 1 9 8 9

0

- 6 .2

K ie ft a n d A h m a d jia n , 1989

2 .0 X 1 0 4

- 4 .2

K ie f t a n d A h m a d j i a n , 1 9 8 9

3 .1 X 1 0 4

- 4 .1

K ie ft a n d A h m a d jia n , 1989

0

- 8 .3 - 1 .9

K ie ft a n d A h m a d jia n , 1989

1.3 X 10®

K ie f t, 1988; K ie ft a n d A h m a d jia n , 1989

L ic h e n p h o t o b i o n t s

C occom yxa peltigera v a r . variolosa N o sto c f r o m Peltigera canina f r o m P. rufescens f r o m P. spuria T rebouxia erici f r o m C ladonia cristatella T rebouxia incrustata f r o m L ecanora dispersa T rebouxia f r o m A carospora fu sc a ta f r o m A spicilia calcarea f r o m C ladonia coniocraea f r o m L ecidea tum ida f r o m Parmelia tin cto ru m f r o m P arm eliopsis hyp ero p ia f r o m Stereocaulon saxatile aN o d a ta .

0

- 8 .3

K ie ft a n d A h m a d jia n , 1989

0

- 9 .1

K ie ft a n d A h m a d jia n , 19 89

0

- 7 .5

K ie ft a n d A h m a d jia n , 19 89

0

- 9 .1

K ie ft a n d A h m a d jia n , 1989

0

- 9 .2

K ie ft a n d A h m a d jia n , 198 9

0

- 9 .1

K ie ft a n d A h m a d jia n , 1989

0

- 5 .1

K ie ft a n d A h m a d jia n , 19 89

0

- 6 .3

K ie ft a n d A h m a d jia n , 19 89

0

- 1 1 .3

K ie f t a n d A h m a d j i a n , 1 9 8 9

0

- 6 .2

K ie ft a n d A h m a d jia n , 1989

0

- 1 6 .0

K ie ft a n d A h m a d jia n , 198 9

0

- 6 .3

K ie ft a n d A h m a d jia n , 198 9

0

- 8 .8

K ie ft a n d A h m a d jia n , 198 9

Ice Nucléation Activity Associated with Plants and Fungi

153

late bacteria with IN A from the lichens were unsuccessful, suggesting that the li­ chens them selves m ight be the source of the nucleation sites. The finding o f warmtem perature INA in a pure culture of the m ycobiont R. chrysoleuca supported the hypothesis that lichen m ycobionts synthesize intrinsic ice nucleation sites. This finding led to a survey of a greater num ber of lichen sym bionts for INA. M any of the individual sym bionts o f lichens have been cultured axenically in vitro (A hm adjian et al., 1980). K ieft and Ahm adjian tested many o f A hm adjian’s pure cultures of sym bionts and reported that w arm -tem perature ice nucleation, when it occurred, was restricted to the m ycobionts; threshold tem peratures in the photobionts were all below -5 ° C (K ieft and A hm adjian, 1989) (Table 4). The INA data for the m ycobionts also corroborated the results for the hom ogenized lichens (Kieft, 1988). For instance, pure cultures o f the fungus R. chrysoleuca and the closely re­ lated Lecanora dispersa were am ong those with the warm est threshold tem pera­ tures. Lichens were first tested for IN A (Kieft, 1988) using the droplet-freezing assay (Vali, 1971). M ore recently, A shw orth and Kieft (1992) tested INA in whole lichen thalli by therm al analysis. W ith therm ocouple probes attached to lichen thalli, freezing was detected by the release of the latent heat o f fusion (the exotherm ) as the tem perature of the lichen was slowly decreased. By this method of analysis, it was found that the range o f threshold temperatures for freezing in lichens is much narrow er than that detected by the droplet-freezing assay. Freezing was detected at tem peratures above -5 ° C in all lichens tested, even epiphytic lichens like Usnea that had appeared to be poor nucleators in the droplet-freezing assay. This discrep­ ancy in results from the two m ethods can be explained by the differences in con­

M e a s u re d by • d ro p le tfre e z in g a ssa y

M e a s u re d by • th e rm a l a n a ly s is 0

-5

-1 0

-1 5

-2 0

Temperature (°C) F ig u re 3. Id ealized cu m u lativ e ice n u cleatio n spectra for a strong fungal ice n u cleato r, e.g., Rhizoplaca chrysoleuca, an d a w eak fu n g al ice nucleator, e.g., Usnea sp., show ing the ranges o f d ata co llected by the d ro p let-fre ezin g assay an d th erm al analysis m ethods.

154

Ashworth and Kieft

centrations o f w arm -tem perature ice nucleation sites am ong the various species of lichens and by the differences in sensitivities of the two tests (Fig. 3). The thermal analysis m ethod can detect a single ice nucleus in a relatively large sample, whereas the low er limit o f detection for the droplet-freezing assay is about 103 nu­ cleation sites per gram. It appears that some lichens previously thought not to have w arm -tem perature ice nucleation sites actually possess them , albeit in low num ­ bers. W hen freezing is initiated at a single site, ice form ation is propagated throughout the lichen thallus. Thus, INA appears to be even m ore widespread am ong lichens than was previously known.

Free-living Fungi: Fusarium spp. In the years since Schnell and Vali (1972) first reported the presence o f warmtem perature IN A in decaying leaf litter, many free-living fungi have been screened for INA, especially as part o f tests for INA among epiphytic m icroorganism s and m icrobes from the guts of insects (Lindow et al., 1978a; T sum uki, 1992; Pouleur et al., 1992; S. Lindow , personal communication). However, it has only very recently been dem onstrated that the nonlichenized fungi identified that have w arm -tem pera­ ture IN A are all m em bers o f the genus Fusarium. Pouleur et al. (1992) screened 145 strains com prising 20 genera of fungi for their ability to nucleate ice at —5°C. O f these, 16 strains o f Fusarium avenaceum and 5 strains o f Fusarium accuminatum were positive. All others were negative at this tem perature, including 12 other species o f Fusarium. The threshold tem perature for nucleation in F. avenaceum was —2.5°C, and the num ber of nucleation sites per gram o f fungus was approxim ately 109 (Table 4). IN A could be detected in m acroconidia that were physically separated from the m ycelium (Pouleur et al., 1992). F. avenaceum and F. accuminatum are pathogens o f plants, causing root rot in a variety of plants, in­ cluding alfalfa, especially in cold climates (Pouleur et al., 1992). Tsum uki et al. (1992) screened bacteria from the guts of insect larvae (the rice stem borer Chilo suppressalis) for INA at -1 0 °C . O f the approximately 600 colonies tested (75% bacterial, 25% fungal), only one fungus, an unidentified strain o f Fusarium, was positive. The threshold tem perature of freezing for this strain was -5 .5 °C (Table 4). As part of the same study, it was shown that inoculation o f plants and insect larvae with this Fusarium strain raised the tem perature o f freezing by several de­ grees. As noted by Pouleur et al. (1992), the fungi that display w arm -tem perature INA are phylogenetically related. N early all lichenized fungi are m em bers of the Ascomycetes; Fusarium spp. are in the form-class D euterom ycetes (the Fungi Imperfecti) but w ould belong to the A scom ycetes if they had know n perfect stages. This suggests a possible com m on evolutionary origin for the ice nucleation trait within these phylogenetically related fungi. Characteristics of Fungal Ice Nucleation Sites Currently, the lichen ice nucleators are not nearly as well characterized as their bacterial counterparts. As w ith the bacterial nucleators, characterization o f fungal ice nucleation sites has been ham pered by the difficulties o f isolating them in large num bers. W arm -tem perature ice nucleation sites are generally produced in low num bers, usually few er than one per cell. Consequently, characterization of fungal ice nucleation sites has thus far been limited to indirect m eans. Activities have been m easured after various physical and chemical treatments to gain insights regarding

Ice Nucléation Activity Associated with Plants and Fungi

155

their structure and function. In lichens, R. chrysoleuca has been the source of nucleators for study. A few sim ilar studies have also been perform ed on F. avenaceum. As dem onstrated for bacteria, the lichen fungal ice nucléation sites appear to be proteinaceous. For exam ple, lichen IN A is reduced follow ing treatm ent with the proteases pronase E and papain (K ieft and Ruscetti, 1990). Further support for the proteinaceous nature o f lichen ice nuclei was shown in their inactivation by the de­ naturing agents guanidine hydrochloride and urea. The effect o f these dénaturants also suggests that, as with the bacterial ice nucléation proteins, w arm -tem perature ice nucléation sites m ay form by the aggregation o f m any sm aller protein subunits. Theories of physical chem istry indicate that w arm -tem perature INA requires m ole­ cules o f large size (Fletcher, 1958; Rogers et al., 1987; Burke and Lindow, 1990). Govindarajan and L indow (1988a) showed by gam m a irradiation inactivation analysis that a direct relationship exists between the num ber o f aggregated bacterial ice nucléation proteins and the tem perature of freezing. Using the same approach, Kieft and Ruscetti (1992) found a sim ilar pattern in R. chrysoleuca (Fig. 4). The first clue that fungal ice nucleators differed from those of bacteria appeared when it was dem onstrated that lichen ice nucléation sites are relatively temperature-stable. Fungal ice nuclei m aintained activity even after exposure to tem pera­ tures o f 60°C. As stated earlier, epilithic lichens are frequently subjected to these tem peratures in w arm desert and sem iarid environm ents, so it is not surprising to find tem perature stability in substances produced by these organisms. Bacterial nu­ cleators are inactivated relatively quickly at 40°C (Maki and W illoughby, 1978).

re

Q

o

E o> o

Temperature (°C) F ig u re 4. M o lecu lar m asses o f the ice n u cleatio n sites in Pseudom onas syringae strain C it7 bacterial cells; field -co llected , h o m o g en ized R hizoplaca chrysoleuca lichen; cells o f the lich en fungus R. chrysoleuca cu ltu red ax en ically ; and a cru d e ex tract o f the lichen R. chrysoleuca, all d eterm in ed by g am m a-irrad iatio n -in activ atio n analysis (R edraw n from K ieft and R uscetti, 1992).

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T h e l i c h e n n u c l é a t i o n s i t e s a l s o d i f f e r f r o m t h o s e o f b a c t e r i a in b e i n g a c t i v e o v e r a n e x t r a o r d i n a r i l y b r o a d p H r a n g e . T h r e s h o l d t e m p e r a t u r e s in a n e x t r a c t o f ic e n u c l é a ­ ti o n s i t e s f r o m

R. chrysoleuca w e r e u n c h a n g e d o v e r a p H r a n g e f r o m 2 to 11 ( K i e f t

a n d R u s c e tti, 1 9 9 0 ), a p r o p e r ty th a t m ig h t b e e x p la in e d b y a h ig h p r o p o rtio n o f u n ­ c h a r g e d a m i n o a c i d r e s i d u e s in t h e ic e n u c l é a t i o n p r o t e i n . B a c t e r i a l I N A is d i m i n ­ i s h e d b e l o w p H 4 ( K o z l o f f e t a l ., 1 9 8 3 ) . T h e g r e a t e s t d i f f e r e n c e b e t w e e n t h e l i c h e n a n d b a c t e r i a l ic e n u c l é a t i o n r e l a t e s to t h e i n v o l v e m e n t o f m e m b r a n e l i p i d s . B a c t e r i a l i c e n u c l é a t i o n p r o t e i n s o c c u r in th e o u t e r m e m b r a n e o f g r a m - n e g a t i v e b a c t e r i a ( L i n d o w e t a l ., 1 9 8 9 ) , a n d I N A r e q u i r e s th e p r e s e n c e o f lip id m a te r ia l ( G o v in d a r a ja n a n d L in d o w , 1 9 8 8 b ). T h e n u c lé a tio n s ite s o f th e lic h e n

R. chrysoleuca d i f f e r in t h a t t h e y c a n s ti ll n u c l e a t e ic e a t w a r m

t e m p e r a t u r e s in a c r u d e e x t r a c t c o m p l e t e l y d e l i p i d a t e d w ith a n o n p o l a r s o l v e n t s u c h as c h lo ro fo rm

( K ie f t a n d R u s c e tti, 1 9 9 0 ). T h e c r u d e e x tr a c t c a n a ls o b e f ilte re d

t h r o u g h a 0 . 2 2 - |i m p o r e s i z e f i l t e r , a n d a c t i v i t y is r e t a i n e d , i n d i c a t i n g t h e p r e s e n c e o f c e llfre e n u c le i (K ie ft a n d R u s c e tti, 1 9 9 0 ). The

F usarium ic e n u c l e a t o r s h a v e p r o p e r t i e s in c o m m o n w ith th e l i c h e n ic e n u ­

c l é a t i o n s i t e s . T h e s e i n c l u d e a c t i v i t y o v e r a b r o a d p H r a n g e ( p H 1 to 1 3 ) a n d t e m ­ p e ra tu re

s ta b ility

to

60°C

(P o u le u r

et

a l .,

1 9 9 2 ).

The

ic e

n u c le a tio n - a c tiv e

Fusarium s p p . a l s o a p p e a r to g e n e r a t e c e l l f r e e n u c l é a t i o n s i t e s , s i n c e f i l t r a t e s f r o m th e f u n g i th a t p a s s th r o u g h ( P o u l e u r e t a l .,

a 0 . 2 2 - |i m

filte r re ta in

w a rm - te m p e ra tu re

1 9 9 2 ). T h e s e s im ila ritie s b e tw e e n IN A

in

a c tiv ity

Fusarium a n d l i c h e n

fu n g i a ls o s u g g e s t a c o m m o n g e n e tic o rig in . A t t h e p r e s e n t t i m e , t h e s t r u c t u r e o f t h e f u n g a l ic e n u c l é a t i o n p r o t e i n ( s ) is u n ­ k n o w n , a n d it is a l s o n o t k n o w n w h e t h e r o t h e r m o l e c u l e s , e . g ., c a r b o h y d r a t e s , i n ­ o r g a n ic

c o f a c to rs , e tc .,

are

in v o lv e d

in

th e

n u c lé a tio n

p ro ce ss.

S im ila rly , th e

g e n e t i c b a s i s o f f u n g a l I N A is l a r g e l y u n e x p l o r e d . I n o n e p r e l i m i n a r y s t u d y , C . O rse r

(personal com m unication) f o u n d n o s e q u e n c e h o m o l o g y b e t w e e n a b a c t e r i a l

I N A g e n e p r o b e a n d D N A f r o m a l i c h e n f u n g u s . I N A in l i c h e n s m a y h a v e r e s u l t e d f r o m c o n v e r g e n t e v o l u t i o n , i.e ., t h e g e n e ( s ) c o d i n g f o r I N A in f u n g i m a y h a v e a r i s e n i n d e p e n d e n t l y f r o m t h o s e in b a c t e r i a . H o w e v e r , t h e f u n g a l ic e n u c l e a t o r s m a y s h a re in tr in s ic p r o p e rtie s r e q u ir e d fo r IN A

w ith t h o s e in o t h e r t a x o n o m i c

g r o u p s . T h e s e m a y i n c l u d e l a r g e m o l e c u l a r s iz e a n d p r o t e i n s w i t h r e p e a t i n g s e ­ q u e n c e s o f h y d r o p h ilic a m in o a c id s .

Selective Advantages of Fungal INA A l t h o u g h t h e s e l e c t i v e a d v a n t a g e o f l i c h e n ic e n u c l e i h a s n o t b e e n f i r m l y e s t a b ­ lis h e d , a n u m b e r o f e x p la n a tio n s h a v e b e e n p r o p o s e d . T h e m o s t p la u s ib le h a s to d o w ith t h e m o i s t u r e r e l a t i o n s o f l i c h e n s . A s s t a t e d a b o v e , l i c h e n s a r e d e p e n d e n t o n th e a tm o s p h e r e f o r w a te r a n d th e y a r e a ls o e x tr e m e ly f re e z e - to le r a n t. M a n y lic h e n s o c c u r in h a b i t a t s t h a t u n d e r g o d a i l y f r e e z e - t h a w c y c l e s . B e c a u s e t h e v a p o r p r e s s u r e o f w a t e r is

lo w e r o v e r

ic e

th a n

over

s u p e rc o o le d

w a te r, d e p o s itio n

fre e z in g

( fr e e z in g o f w a te r v a p o r o n ic e s u r fa c e s ) o n lic h e n s c o u ld b e e n h a n c e d b y w a rm te m p e r a tu r e IN A . I c e n u c le a tio n - a c tiv e lic h e n s m a y g a in e x tr a m o is tu r e th r o u g h d e p o s i t i o n f r e e z i n g a t n i g h t a n d t h e n a b s o r b t h e m o i s t u r e a s w a t e r w h e n it m e l t s th e f o llo w in g d a y . T h is m e c h a n is m o f m o is tu re u p ta k e w o u ld b e p a rtic u la rly b e n e fic ia l u n d e r c o n d i t i o n s in w h i c h n i g h t t e m p e r a t u r e s d r o p b e l o w 0 ° C b u t n o t lo w e n o u g h f o r n o n b i o l o g i c a l ic e n u c l e i t o b e a c t i v e . A s a n e c d o t a l e v i d e n c e in s u p p o r t o f th i s th e o r y , t h e w a r m e s t t h r e s h o l d t e m p e r a t u r e s a n d th e h i g h e s t d e n s i t i e s o f ic e n u c l é a ­ ti o n s i t e s in f i e l d - c o l l e c t e d

R. chrysoleuca a r e f o u n d in t h e m o u n t a i n s o f N e w M e x -

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ico during the fall and spring, w hen nighttime low tem peratures are typically approxim ately -5 ° C and conditions are generally dry. During midwinter and m id­ sum m er at the same sites, when nighttim e low tem peratures are below -5 ° C and above 0°C, respectively, and precipitation is m ore com m on, R. chrysoleuca sam ­ ples are less active as ice nucleators. A second possible explanation for lichen INA relates to freeze tolerance. W hen freezing is initiated at w arm er tem peratures, the rate of ice crystal grow th and cellular dehydration is slow er than when freezing ini­ tiates at colder tem peratures. Therefore, prom oting ice nucleation may enhance freeze tolerance. T hese tw o hypotheses are not mutually exclusive; INA may bene­ fit lichens by enhancing both m oisture uptake and freeze tolerance. The selective advantage of INA in Fusarium may be sim ilar to that proposed for bacteria, i.e., the ability to initiate ice formation at relatively warm tem perature ap­ pears to make these fungi conditional pathogens of plants. Infection of alfalfa roots by Fusarium spp. is enhanced by freezing (Richard et al., 1982, 1985; Pouleur et al., 1992). As with ice nucleating bacteria on leaf surfaces, fungi associated with plant roots may dam age plant tissue by initiating freezing o f supercooled w ater at relatively warm tem peratures, thereby gaining access to plant nutrients and also facilitating invasion o f plant root tissue. On the other hand, pathogenic fungi, in­ cluding Fusarium spp., are norm ally not dependent on wounds or other openings for entry into plants as are pathogenic bacteria like P. syringae. This suggests that INA in F. acuminatum and F. avenaceum may not be essential for invasion of plant tissue. Fungal ice nucleators may contribute, along with other biogenic ice nucleators, to heterogeneous ice nucleation in the atm osphere, thereby influencing hydrologie cycles. They may contribute to IN A on the surface o f plants, as well. For instance, lichens may function as ice nucleators on the surface of woody plants. Fungal nu­ cleators may also initiate freezing at warm tem peratures in insects (Duman and M ontgom ery, 1991; Tsum uki et al., 1992).

Potential Applications of Fungal Ice Nucleators The potential applications o f ice nuclei have been reviewed elsewhere and in­ clude uses in snow m aking, cloud seeding, frozen food additives, and as signal transducers in im m unological tests (W arren, 1987; M argaritas and Bassi, 1991). To date, these developm ents have been pursued exclusively with bacterial ice nuclea­ tion proteins; how ever, fungal ice nucleators may also gain favor. The unique properties of fungal ice nucleators (tem perature stability, activity over a broad pH range, activity in the absence of lipids, etc.) may make these substances especially attractive for biotechnological applications requiring w arm -tem perature ice nuclea­ tion. For instance, the tem perature stability would simplify storage and lengthen the shelf-life o f ice nucleation products. The fact that lichen nucleators do not require lipid material for activity may m ake them especially suitable for the food industry and for im m unological applications. Using lichen nuclei would avoid the use of bacterial outer m em branes (w hich contain bacterial endotoxin). A purified prepara­ tion of lichen nuclei m ight also pose fewer nonspecific binding problem s than lipid-associated bacterial ice nucleation-active proteins when used in im m unologi­ cal assays. U nfortunately, know ledge of the genetics o f the fungal systems lags far behind that of the bacteria. A dditionally, because lichens grow very slowly both in nature and in the laboratory, the com m ercial use o f lichen nuclei would require that the gene(s) coding for this trait be cloned and expressed in a faster-grow ing organ­

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ism. U nfortunately, the genetic studies of lichen fungi are only in the beginning stages (A hm adjian, 1991). Because Fusarium is much m ore am enable than the li­ chens to grow th in the laboratory, progress in fungal INA studies may proceed more rapidly w ith this organism .

Concluding Remarks Extracellular freezing in plant and fungal cells is initiated by heterogeneous ice nucleation and generally begins following only a few degrees o f supercooling. Al­ though m uch o f the IN A associated with plant and fungal tissues can often be as­ cribed to Ina+ bacteria, there is significant evidence that plant and fungal tissues also contain intrinsic ice nucleation sites. Unfortunately, the ice nucleation-active com ponents w ithin these tissues have been only partially characterized. Progress in identifying these intrinsic ice nucleation-active sites has been ham pered by their apparent low concentration w ithin tissues and the difficulty o f studying ice form a­ tion in tissues under conditions sim ilar to those in nature.

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Pseudomonas syringae a n d Escherichia coli.

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M a k i, L . R ., a n d W i l l o u g h b y , K . J. 1 9 7 8 . B a c t e r i a a s b i o g e n i c s o u r c e s o f f r e e z i n g n u c l e i . J. A p p l. M e t e o r o l. 1 7 :1 0 4 9 - 1 0 5 3 . M a k i, L . R „ G a l y a n , E . L ., C h a n g - C h ie n , M „ a n d C a ld w e l l, D . R . 1 9 7 4 . I c e n u c l e a t i o n in d u c e d b y

Pseudomonas syringae. A p p l. M i c r o b i o l . 2 8 : 4 5 6 - 4 5 9 . M a l o n e , S . R ., a n d A s h w o r t h , E . N . 1 9 9 1 . F r e e z i n g s tr e s s r e s p o n s e in w o o d y t i s s u e s o b s e r v e d u s in g l o w - t e m p e r a t u r e s c a n n i n g e l e c t r o n m ic r o s c o p y a n d f r e e z e s u b s t i t u t i o n t e c h n i q u e s . P la n t P h y s io l. 9 5 :8 7 1 -8 8 1 . M a r c e ll o s , H ., a n d S in g l e , W . V . 1 9 7 6 . I c e n u c l e a t i o n o n w h e a t. A g r ic . M e t e o r o l . 1 6 : 1 2 5 - 1 2 9 . M a r c e ll o s , H „ a n d S in g l e , W . V . 1 9 7 9 . S u p e r c o o l i n g a n d h e te r o g e n e o u s n u c l e a t i o n o f f r e e z i n g in t i s ­ s u e s o f t e n d e r p l a n t s . C r y o b i o l o g y 1 6 :7 4 - 7 7 . M a r e n t e s , E ., G r i f f i t h , M ., M l y n a r z , A ., a n d B r u s h , R . A . 1 9 9 3 . P r o t e in s a c c u m u l a t e in th e a p o p l a s t o f w i n t e r r y e le a v e s d u r i n g c o l d a c c l i m a t i o n . P h y s i o l . P la n t. 8 7 : 4 9 9 - 5 0 7 . M a r g a r i t a s , A ., a n d B a s s i , A . S . 1 9 9 1 . P r i n c ip a ls a n d b i o t e c h n o l o g i c a l a p p l i c a t i o n s o f b a c t e r i a l ic e n u c l e a t i o n . C r it. R e v . B i o t e c h n o l . 1 1 :2 7 7 - 2 9 5 . M a rs h a ll, D . 198 8 . A r e la tio n s h ip b e tw e e n ic e - n u c le a tio n - a c tiv e b a c te ria , fre e z e d a m a g e , a n d g e n o ty p e i n o a ts . P h y t o p a t h o l o g y 7 8 : 9 5 2 - 9 5 7 . M o d l i b o w s k a , I. 1 9 6 2 . S o m e f a c t o r s a f f e c t in g s u p e r c o o l i n g o f f r u it b lo s s o m s . J.

H o r t. S c i. 3 7 :2 4 9 -

261. M o r r is , G . J ., a n d M c G r a t h , J . J. 1 9 8 1 . I n t r a c e ll u l a r ic e n u c l e a t i o n a n d g a s b u b b l e f o r m a t i o n in

gyra. C r y o L e t t. 2 : 3 4 1 - 3 5 2 .

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N ash, T. H., K appen, L., L ö sch , R., L arso n , D. W ., and M athes-S ears, U. 1987a. C old resistance o f lichens w ith T rentepohlia o r Trebouxia p hotobionts from the N orth A m erican W est C oast. Flora 179:241-251. N ash, T. H ., K appen, L., L ö sch , R., M a th es-S ears, U., and L arson, D. W 1987b. C old resistance o f lichens. P rogress and P ro b le m s in L ich en o lo g y in the E ighties. B ibl. L ichenol. 25:313-317. N ath, J., and Fisher, T. C . 1971. A n ato m ical study o f freezing injury in hardy and nonhardy alfalfa varieties treated w ith c y to sin e and gu an in e. C ryobiology 8:420-430. O ’B rien, R. D ., and L in d o w , S. E. 1988. E ffect o f plant sp ecies and environm ental conditions on ice n u cleatio n activity o f P seudom onas syringae on leaves. A ppl. E nviron. M icrobiol. 54:2281-2286. P earce, R. S. 1988. E x tra c e llu la r ice and cell shape in frost-stressed cereal leaves: A low -tem perature sc an n in g -electro n -m icro sco p y study. P lan ta 175:313-324. P earce, R. S., and A sh w o rth , E. N. 1992. L o calizatio n o f ice and cell shape in leaves o f overw intering w heat during frost stress in the field. P lan ta 188:324-331. Phelps, P., G iddings, T. H ., P ro ch o d a, M ., an d Fall, R.. 1986. R elease o f cell-free nuclei by Erwinia herbicola. J. B acteriol. 167:496-502. Pouleur, S., Richard, C ., M artin , J.-G ., an d A ntoun, H. 1992. Ice nucleation activity in Fusarium acum inatum and F usarium avenaceum . A ppl. E nviron. M icrobiol. 58:2960-2964. P roebsting, E. L., Jr., A n d rew s, P. K., an d G ross, D. C. 1982. S u p erco o lin g you n g developing fruit and floral buds in decid u o u s orch ard s. H o rtS cien ce 17:67-68. Proebsting, E. L „ Jr., and G ro ss, D. C. 1988. F ield evaluations o f frost injury to deciduous fruit trees as influenced by ice n u cleatio n -activ e Pseudom onas syringae. J. A m . Soc. H ort. Sei. 103:57-61. P ruppacher, H. R. 1967a. In terp retatio n o f ex perim entally d eterm in ed grow th rates o f ice crystals in sup erco o led w ater. J. C h e m . Phys. 47:18 0 7 -1 8 1 3 . P ruppacher, H. R. 1967b. S o m e relatio n s b etw een the stru ctu re o f the ice-solution interface and the free g row th rate o f ice cry stals in su p e rco o led aqueous so lu tio n s. J. C olloid Interface Sei. 25:285294. Q uam m e, H. A. 1978. M e ch an ism o f su p e rco o lin g in o v erw in terin g peach flow er buds. J. Am. Soc. Hort. Sei. 103:57-61. R ajashekar, C. B , Li, P. H ., an d C arter, J. V . 1983. F rost injury and h eterogeneous ice n u cleatio n in leaves o f tu b er-b earin g Solanum species. P lan t Physiol. 71:749-755. R ichard, C ., M ichaud, R ., W illem o t, C ., B em ier-C ardo u , M ., an d G agnon, C. 1985. E ffect o f frost on Fusarium root rot o f a lfa lfa and p o ssib ility o f double trait selection. Pages 209-211 in: E cology and M anagem ent o f S o ilb o m e P lant P ath o g en s. C. A. Parker, A. D. R ovira, K. J. M oore, P. T. W . W ong, and J. F. K ollm orgen, eds. A m erican P hyto p ath o lo g ical S ociety, St. Paul, MN. Richard, C „ W illem ot, C ., M ich au d , R., B em ier-C ardou, M „ and G agnon, C. 1982. L ow -tem perature interactions in F usarium w ilt and ro o t ro t o f alfalfa. P hyto p ath o lo g y 72:293-297. R ogers, J. S., Stall, R. E., an d B urke, M . J. 1987. Low tem p eratu re co n d itio n in g o f the ice nucleationactive b acterium E rw inia herbicola. C ry o b io lo g y 24:270-279. R ogers, R. R., and Y au, M . K . 1989. A S h o rt C o u rse in C loud Physics. Pergam on Press, N ew Y ork. R undel, P. W . 1988. W a te r relatio n s. P ag es 17-36 in: C R C H an d b o o k o f L ichenology, vol. 2. M. G alun, ed. C R C P ress, B o c a R aton, FL. Sakai, A. 1979. F reezin g a v o id an ce m ech an ism o f prim ordial shoots o f co n ifer buds. P lant C ell Physiol. 20:1381-1390. Sakai, A. 1982. E x trao rg an freezin g o f p rim o rd ial shoots o f w in ter buds o f conifer. Pages 199-209 in: P lant C old H ardiness an d F reezin g S tress, vol 2. P. H. Li, A. S akai, eds. A cadem ic Press, N ew Y ork. Salt, R. W ., and K aku, S. 1967. Ice n u cleatio n and p ro p ag atio n in spruce needles. C an. J. Bot. 45:1335-1346. Schnell, R. C., and V ali, G . 1972. A tm o sp h eric ice n uclei from deco m p o sin g vegetation. N ature (L ondon) 2 36:163-165. S im inovitch, D., and S carth, G . W . 1938. A study o f the m ech an ism o f frost injury to plants. C an. J. Res. C 16:467-481. Single, W. V ., and M arcello s, H . 1981. Ice form ation and freezing injury in actively grow ing cereals. P ages 17-33 in: A n aly sis and Im p ro v em en t o f P lant C old H ardiness. C. R. O lein and M . N. Sm ith, eds. C R C Press, B oca R a to n , FL. Steffens, K. L., A rora, R ., and Palta, J. P. 1989. R elative sen sitiv ity o f p h otosynthesis and respiration to freeze-thaw stress in h erb aceo u s sp ecies. P lant Physiol. 89:1372-1379. S torey, J. M ., and Storey, K . B. 1985. T rig g erin g o f cry o p ro tectan t synthesis by the initiation o f ice nucleation in the freeze to le ra n t frog, R ana sylvatica. J. C om p. Physiol. B 156:191-195. T hom as, D. A ., and B arb er, H . N. 1974. S tudies on le a f ch aracteristics o f a cline o f Eucalyptus u m igera from M o u n t W ellin g to n , T asm an ia. I. W ater rep ellen cy and the freezing o f leaves. A ust. J.

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Bot. 2 2 :501-512. T su m u k i, H ., H aru y o sh i, K., M aed a, T., and O kam oto, T. 1992. An ice-n u cleatin g active fungus iso ­ lated from the gu t o f the rice stem b orer, Chilo suppressalis W alk er (L ep id o p tera: P yraldae). J. In­ sect P hysiol. 3 8 :119-125. V ali, G . 1971. Q u an titativ e e v alu atio n o f experim ental results on the h etero g en eo u s freezing nuclea­ tion o f su p erco o led liquids. J. A tm o s. Sci. 28:402-409. V ali, G ., and S tan sb u ry , E. J. 1966. T im e-d ep en d en t characteristics o f th e h etero g en eo u s nucleation o f ice. C an. J. P hys. 44:477-502. W arren, G . J. 1987. B acterial ice n u cleatio n : M olecular biology and ap p licatio n s. B io tech n o l. & Gen. Eng. Rev. 5 :107-135. W iegand, K. M . 1906. S om e stu d ies reg ard in g the biology o f b uds and tw igs in w inter. Bot. Gaz. 4 1 :3 7 3 -4 2 4 . Y elen o sk y , G. 1983. Ice n u cleatio n activ e (IN A ) agents in freezing o f y o u n g citrus trees. J. Am. Soc. H ort. Sci. 108:1030-1034. Z ach ariassen , K. E ., and H am m el, H. T. 1988. The effect o f ice-n u cleatin g agents on ice-nucleating activ ity . C ry o b io lo g y 2 5 :1 4 3 -1 4 7 .

CHAPTER 9

Deep Supercooling in Woody Plants and the Role of Cell Wall Structure Michael Wisniewski

Deep supercooling o f xylem parenchym a cells is a m echanism of freeze avoid­ ance and can be defined as the ability of a population of cells to retain cellular w a­ ter in a liquid phase at low, subfreezing tem peratures by rem aining free from internal, heterogeneous ice nuclei and isolated from the nucleating effect of extra­ cellular ice (Burke, 1979). Supercooled water is in a m etastable condition and will form intracellular ice in response to a heterogeneous nucleation event or when the hom ogeneous nucleation tem perature of water (—40°C) is reached (Rassmussen and M ackenzie, 1972). A dditionally, colligative properties of the cell sap may further depress the freezing point beyond the hom ogeneous nucleation point by several m ore degrees (G eorge and Burke, 1977; Rassm ussen and M ackenzie, 1972). W hen freezing does occur, intracellular ice is formed, w hich results in lysis of the cell. Deep supercooling has also been referred to as deep undercooling. The term s are synonym ous, and although the latter term may represent m ore appropriate term i­ nology, the term deep supercooling has found w idespread and common use in the literature. Due to the intrinsic tem perature constraints for deep supercooling, which limit its effectiveness as a strategy for freeze protection, it has been implicated in determ ining species distribution on both a latitudinal and elevational gradient (George et al., 1974; B ecw ar et al., 1981). O f the many aspects o f biological ice nucleation and cold hardiness o f plants, deep supercooling is perhaps the m ost enigmatic. The ability of some plants to maintain sym plastic w ater in an unfrozen condition and without m ovem ent of the water into the apoplast is a rem arkable adaptation that has not failed to im press both biophysicists and plant physiologists. A lthough the ability of woody plant tis­ sues to avoid freezing by deep supercooling was first suggested in the 1960s (Tum anov and K rasavtsev, 1962; Tum anov et al., 1969) and 1970s (K rasavtsev, 1970; Quam m e et al., 1972, 1973), the mechanism that allows small dom ains of water to avoid freezing, despite the presence of extracellular ice, rem ains little un­ derstood (Franks, 1985). This is partly due to the fact that the properties which al­ low deep supercooling to occur apparently rely on the structural organization of the tissue or organ. This feature has m ade it very difficult to m anipulate plant material 163

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in a way to discover the fundam ental mechanism and/or properties that allow deep supercooling to occur. D espite this lim itation, evidence indicates that the structure of the cell wall plays an integral role in determ ining whether or not a species will exhibit deep super­ cooling. The purpose o f this chapter is to provide an overview o f the topic of deep supercooling, to discuss the role o f cell wall structure in regulating the ability of plants to exhibit this trait, and to pose some possible areas o f future research. The review and discussion are lim ited to deep supercooling o f xylem tissues of woody plants (see also A shw orth, 1993; W isniewski et al., 1993). A lthough deep super­ cooling o f overw intering floral buds also occurs, the response o f these tissues is unique, and the reader should refer to Chapter 10 of this volum e for a detailed elu­ cidation o f this topic.

Characterization of Freezing Events in Xylem Tissues The freezing response o f xylem tissues (or other m aterial) can be monitored us­ ing differential therm al analysis (DTA) as outlined by Q uam m e et al. (1972) and since m odified to take advantage of recent advances in digital data-acquisition equipm ent and m icrocom puters (see references in W isniew ski et al., 1990). Ther­ m ocouples are used to detect the latent heat released by w ater in the samples as it undergoes a liquid to solid phase change. C oncom itantly, m elting curves can also be obtained with this technique. Sample temperatures are com pared to a reference (freeze-dried tissue) undergoing the same rate o f cooling. This produces a flat base­ line on the therm ogram until the water in the sample undergoes a phase change, resulting in a difference in tem perature between the sam ple and the reference. The sam ple/reference differential is recorded as a peak on the therm ogram and repre­ sents an exotherm during freezing or an endotherm during melting. Differential scanning calorim etry and nuclear magnetic resonance have also been used to obtain invaluable inform ation about deep supercooling in plant tissues (George and Burke, 1977; Q uam m e et al., 1982). U sing DTA, two exotherm s have been observed during the controlled freezing of xylem tissues o f many tem perate hardwood and softw ood trees (Q uam m e et al., 1972; G eorge et al., 1974; Q uam m e, 1976; George and Burke, 1977; Becwar et al., 1981; A shw orth et al., 1983; Rajashekar and Reid, 1989). Exam ples o f therm o­ gram s are illustrated in Figure 1. The initial “high-tem perature exotherm ” (HTE) represents the freezing o f bulk w ater within the lumens o f tracheary elem ents and extracellular spaces, w hereas the subsequent, “low -tem perature exotherm ” (LTE), which often occurs near -4 0 ° C , represents the freezing o f w ater within living cells (Fig. 1). Evidence for the existence of a large fraction o f deep supercooled water has also been obtained from studies utilizing nuclear m agnetic resonance in which a sharp change in the freezing curve is observed within the sam e tem perature range o f the LTE (G eorge and Burke, 1977; Quamme et ai., 1982). Providing further evi­ dence for deep supercooling o f xylem tissue, A shworth et al. (1988) and Malone and A shw orth (1991) used low -tem perature- or conventional scanning electron mi­ croscopy of freeze-substituted specimens to observe the response of ray cells to freezing. They noted, in xylem tissues from several species o f trees which exhibit deep supercooling, that the cell walls of ray cells rem ained rigid during freezing. There was no evidence o f cytorrhysis as had been observed in bark tissues, which exhibited extracellular freezing and a concomitant loss o f cellular water to sites of extracellular ice.

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In contrast to therm ogram s obtained from species that exhibit deep supercool­ ing, only a single H T E is exhibited in species, such as Salix babylonica, that lack the ability to deep supercool (Fig. 1). In these species, survival of living xylem tis­ sues depends upon m echanism s associated with freeze tolerance rather than freeze avoidance (see C hapter 7). A lthough xylem tissues that deep supercool are often referred to as having a single G aussian-shaped LTE, it should be noted that in some species, the shape of the LTE, as well as the num ber, can be quite variable and com plex (Figs. 1 and 2). The basis for the com plex shape o f the exotherm s, w hich is often accentuated dur­ ing periods of acclim ation and deacclim ation (A shw orth, 1993), is not understood. It appears the xylem tissue in som e species does not freeze in a hom ogeneous m an­ ner but instead, as indicated by the com plex shape o f the LTE, responds as a het­ erogeneous population o f cells that freeze over a wide tem perature range. In apple (.Malus pumila), K etchie and K am m ereck (1987) observed at least three exotherm s (Fig. 2) and indicated that they represent the freezing of different tissues of the stem (i.e., pith, prim ary xylem , secondary xylem). The shape of the LTE and the tem perature at w hich it is initiated is also subject to seasonal changes (Q uam m e et al., 1972; George and Burke, 1977; Ashworth et al., 1983; Ketchie and K am ­ mereck, 1987; W isniew ski and A shw orth, 1986; A rora et al., 1992) and in general follows a pattern sim ilar to those o f other processes involved in cold acclimation: increasing in the fall, reaching a m axim um in m idw inter, and then decreasing in the spring.

TE M P E R A T U R E

°C

F ig u re 1. F reezin g resp o n se o f d eb ark ed , in tem o d al tw ig sectio n s o f peach (Prunus persica), flow ering dog w o o d (C o m u s flo rid a ), an d w illow (Salix babylonica). T he H T E (high-tem perature exotherm ) represents th e freezin g o f in tercellu lar w ater and w ater w ithin the lum en o f non liv in g tracheary elem ents. T h e L T E (lo w -tem p eratu re exotherm ) rep resen ts the freezing o f intracellular, d eep supercooled w ater, w ith in liv in g ray cells. N o te the ab sen ce o f any L T E in w illow , w hich lack s the ability to d eep supercool.

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f

T ISSU E TEM PERATURE C C ) F ig u re 2. T y p ical ex o th erm p attern s o b serv ed in basal in tem o d es o f cu rre n t-seaso n lateral shoots o f apple cv. G o ld en D elicio u s p rio r to v eg etativ e m aturity (A ugust), d u rin g acclim atio n (O ctober), at m axim um co ld resistan ce (February), and during deacclim ation (A pril). X represents a high-tem perature ex o th erm w h ereas P, H, and L T E all represent low -tem perature ex o th erm s. L T E in this study cor­ related w ith injury o n ly at the sta g e o f m axim um cold resistance. T h e n u m b er asso ciated w ith the ex o th erm rep resen ts p ro p o rtio n o f to tal tissue w ater, in percent. (R ep rin ted , by perm ission, from K etchie and K am m ereck , 1987.)

Deep Supercooling and Tissue Injury The initial research on deep supercooling of xylem tissues established a correla­ tion betw een the tem perature range of the LTE and tissue injury (Q uam m e et al., 1972; G eorge et al., 1974; G eorge and Burke, 1977; H ong et al., 1980). This was a significant discovery in regards to the biology and survival o f woody plants at freezing tem peratures and has led to many practical applications in evaluating woody plants for cold hardiness (Stushnoff, 1972; Q uam m e, 1976; Quam m e and Stushnoff, 1983). In particular, D TA of stem tissues has played an im portant role in evaluating germ plasm of deciduous fruit and nut trees for cold hardiness (Quamme, 1976; R ajashekar et al., 1982; A shw orth et al., 1983; R ajashekar and Reid, 1989). It is believed that the correlation of the LTE with tissue injury is due to the for­ mation o f intracellular ice w ithin living cells, which results in lysis o f the cell (Burke and Stushnoff, 1979). H ong and Sucoff (1980), in a study of eight species of woody plants, observed a linear relationship between the num ber of dead xylem ray cells and the am ount o f supercooled water that had frozen. This work con­ firm ed and extended earlier observations made by G eorge and B urke (1977) on shagbark hickory (Carya ovata). Using differential scanning calorim etry, the latter authors dem onstrated that the bell-shaped LTE is actually com posed o f m any inde­ pendent freezing events (Fig. 3) and, based on low -tem perature m icroscopy, indi­ cated that the intact ray was the unit of freezing. H ong and Sucoff (1980) later

Deep Supercooling in Woody Plants

Temperature

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(C)

F ig u re 3. E xotherm fine stru ctu re as rev ealed by calorim etric analysis. A large peak roughly equals 100 ray p aren ch y m a cells free zin g sim u ltan eo u sly . (R eprinted, by perm ission, from G eorge and B urke, 1977.)

reported individual or sm all groups o f cells were the units o f freezing and that ice spread very slowly, if at all, from the initial freezing units. W isniewski and Ash­ worth (1985), using electron microscopy in combination with freeze-fixation, also confirmed the units o f freezing to be individual or small groups of cells within a ray. It appears likely that cells that freeze near the hom ogeneous ice nucleation tem ­ perature o f water do so due to spontaneous intracellular nucleation. N evertheless, the cause o f ice nucleation is by no means clear when cells freeze over a very broad tem perature range, as indicated by an irregularly shaped, broad LTE. As Ashworth (1993) has correctly indicated, freezing at these tem peratures could be due to the presence o f an internal, heterogeneous ice-nucleating agent or due to external seed­ ing by extracellular ice. T hese two alternatives have im portant implications regard­ ing the m echanism o f deep supercooling. In the first, seasonal changes in the tem perature at which ice is initiated would be due to the activity of an intracellular ice-nucleating agent, w hereas the latter would be due to some structural m odifica­ tion in the barrier that prevents extracellular ice from propagating, contacting the ray parenchym a cell, and either puncturing the plasm a m em brane or acting as a seeding agent. As Pitt (1992) indicated, the m olecular basis o f seeding is not well understood. Either ice is able to grow through aqueous channels in the plasm a m em brane (M azur, 1965, 1977), or the extracellular ice interacts with the plasm a m em brane in such a m anner as to cause intracellular nucleation on the m em brane surface (Toner et al., 1990). The reader is referred to Pitt (1992) for a detailed dis­ cussion on the therm odynam ics o f intracellular ice form ation. In situations w here the LTE is quite broad or exhibits several peaks, it is also unclear w hether or not the entire freezing curve is associated with tissue injury. Ketchie and K am m ereck (1987) observed multiple LTEs in apple and found that only the peaks at the w arm er tem peratures (peaks P and H, Fig. 2) were associated with injury. The peak that occurred near the tem perature o f the hom ogeneous nu­ cleation point of w ater w as only associated with injury at m idwinter, when all peaks becam e superim posed, and occurred in the range o f -3 5 to -40°C .

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Deep Supercooling and the Distribution of Woody Plants An im portant extension that developed from the recognition o f deep supercool­ ing o f xylem as a freeze-avoidance mechanism was its im plication for defining the distribution o f w oody plants (G eorge et al., 1974; Becw ar et al., 1981). If the ob­ served LTEs were due to sim ple supercooling, then the lim its o f this freeze avoid­ ance should occur in a tem perature range near the hom ogeneous nucleation point o f w ater (—38°C). G eorge et al. (1974), in a survey of 49 species o f woody plants native to N orth A m erica, found a good correlation betw een the tem perature at which an L TE occurred and the minim um tem peratures at the boundary of the northern range o f that species. B ased on this correlation, they proposed that deep supercooling played a m ajor role in defining the northern lim it o f the distribution o f m any native species o f hardw oods. In addition to defining the northern latitu­ dinal lim its o f a species, B ecw ar et al. (1981) later extended this hypothesis to the distribution o f w oody plants on an elevational gradient. A gain, regarding the distri­ bution o f a species, there was a good correlation between the extent of deep super­ cooling and the m inim um tem perature occurring at the elevational lim it of the species. C om prehensive lists o f species that exhibit deep supercooling and those that do not have been published (G eorge et al., 1974, 1982; B ecw ar et al., 1981) In attem pting to associate anatom ical features o f xylem tissues with the ability to deep supercool, G eorge et al. (1974) reported that large LTEs, and deep super­ cooling in general, predom inate in species with ring porous xylem . It should be noted that species with ring porous xylem have very wide diam eter vessel elements that are very efficient in conducting water com pared with species with diffuse po­ rous xylem (Zim m erm an, 1983). These large-diam eter vessel elem ents, however, are also subject to cavitation during freezing, resulting from the separation of dif­ fused gases from w ater contained in vessel elements. This occurs during the initial freezing of bulk water, recorded as an HTE. Because o f the large size o f the air bubble that form s in vessel elem ents o f ring porous xylem, the air cannot be redis­ solved when the ice melts at the return of warm tem peratures, and therefore the xy­ lem elem ent rem ains nonfunctional in regard to w ater transport (Zimmerman, 1983; E llm ore and Ewers, 1985). W hether or not this characteristic is related to deep supercooling in a causal m anner is not known but bears further consideration (W isniew ski et al., 1987b). G usta et al. (1983) reported several exceptions to the —40°C isotherm limit pro­ posed by G eorge et al. (1974). A m ajor finding of this report was that, in a number o f woody species, LTEs could occur at much lower tem peratures than had been previously reported, allow ing these species to survive in areas w here w inter tem ­ peratures o f - 4 5 ° C are not uncom m on. Furthermore, in som e species (e.g., red ash, Fraxinus pennsylvanica), LTEs observable in early w inter com pletely disappeared during prolonged exposure of the tissue to subzero temperatures approaching -38°C . Based on their observations, the authors concluded that, in som e species, xylem ray cells w ere able to gradually lose w ater to extracellular ice, resulting in a four- to fivefold concentration o f the dissolved cell solutes (i.e., an increase from 0.5-1.0 osM to 4 .0 -5 .0 osM ). This intriguing data indicated that not only was cell wall structure an im portant com ponent o f the mechanism of deep supercooling, but that properties o f the plasm a m em brane were also a critical elem ent. Earlier work by Hong and Sucoff (1982) reported that rapid shifts in deep supercooling to lower tem peratures could occur in apple during a 10-h exposure to —5°C. Dehydration of

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the living cells, how ever, w as not responsible for this shift, and no causal m echa­ nism was suggested. M ore recently, W isniewski et al. (1991 c) noted that contact of the plasm a m em brane w ith the cell wall was essential for maximum supercooling to occur. Again, the causal basis for this observation was not known, but collec­ tively these reports indicate that the relationship betw een cell wall structure, the plasm a m em brane, and the ability to deep supercool needs a great deal of further investigation.

Cell Wall Structure and Deep Supercooling In order for deep supercooling to occur, a tissue m ust exhibit several features (G eorge and Burke, 1977): 1) cells m ust be free o f heterogeneous nucleating sub­ stances active at “w arm ” subzero tem peratures; 2) a barrier m ust be present that excludes the grow th o f ice crystals into a cell; concom itantly 3) a barrier to water movem ent m ust exist that prevents a “rapid” loss o f cellular water to extracellular ice in the presence o f a strong vapor pressure gradient; and 4) cell walls must have sufficient tensile strength to counteract the negative hydrostatic pressures that result from the large vapor pressure deficit. Current opinion holds that physical properties o f the apoplast (i.e., cell wall structure) rather than the sym plast (i.e., protoplast) largely account for the ability to deep supercool (G eorge and Burke, 1977; George, 1983; Ashworth and Abeles, 1984). In this regard, porosity of the cell wall w ould play an essential role, al­ though as noted by W isniew ski et al. (1987a), perm eability should also be consid­ ered. George and B urke (1977) found that although the xylem o f shagbark hickory exhibited an LTE around -4 0 ° C in m idwinter, there was no substantial barrier to water exchange betw een the xylem ray parenchym a cells and neighboring vessel elements. They suggested the w ater potential o f xylem ray cells at subfreezing temperatures was reduced by some means such that ice outside the ray cells and cellular water could be in therm odynam ic equilibrium . They proposed a sim ple sur­ face chem ical effect, the “ ink bottle” pore effect, to explain their observations. As illustrated in Figure 4, the ink bottle represents the w ater contained within a group of ray cells. The pore represents cell wall m icrocapillaries of ray cells containing vapor-phase water that is in equilibrium with ice present in neighboring vessel elements. The stability o f the liquid to desiccation depends on the radius of curva­ ture of the meniscus (liquid-vapor) according to the Kelvin equation. As illustrated, a unit of w ater separated from its external environm ent by a pore with a diam eter of 100 A would, due to the highly curved meniscus at point A, be in equilibrium at 80% relative hum idity. A ny further drop in relative hum idity, however, would cause evaporation, resulting in a low ering of the m eniscus to point B and subse­ quent evaporation o f the rem aining water. Utilizing this concept to explain the iso­ lation and desiccation resistance o f water within deep supercooled tissues, the water potential in supercooled cells would have to be low ered to about -4 0 0 bars in order for the cells to be in vapor equilibrium with ice at -4 0 °C . How the cells could withstand so great a hydrostatic tension rem ains an open question. Subsequent work by A shw orth and Abeles (1984), using controlled-pore-size glass particles and polycarbonate filters, also docum ented that pore size could have an effect on the tem perature at w hich water within the pores would freeze. They found water within the pores supercooled and that the extent o f supercooling in­ creased as the diam eter o f the pores decreased. W ithin different sized pores, the

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observed m elting point o f w ater (Fig. 5) agreed very closely with predicted values generated using equations developed by M azur (1965) and H om shaw (1980), al­ though glass particles with a pore size of less than 7.5 nm (75 A) could not be ob­ tained. The freezing point of water within these smallest pores occurred at -1 0 °C . As G eorge and B urke (1977) had used the pore effect to explain observations related to desiccation resistance, A shw orth and Abeles (1984) proposed that ice could not propagate through a cell wall m icrocapillary until the tem perature dropped below the m elting point of the w ater within the pore, which in turn w ould be determined

F ig u re 4. “Ink b o ttle ” pores h av in g vo lu m es approxim ately equal to 100 ray p aren ch y m a cells. The relative h u m id ity calcu lated for th e m en iscu s at point A is 80% and at p o in t B is slig h tly below 100%. Such pores w o u ld n o t deh y d rate sig n ifican tly until the relative h u m id ity w as d ro p p ed to below 80%. (R eprinted, by p erm issio n , from G eo rg e and Burke, 1977.)

z o- 20

PREDI CTED VALUES

CL

AT= 30

- 300 COS 6

0. 1 r

AT = - ^ , HOMSHAW 1980

h_l Ü J- 40

*

OBSERVED

Z

50, 0

20

40

60

80

100

120

PORE DI AMETER,

140

00C

nm

F ig u re 5. R e la tio n sh ip betw een p o re d iam eter and m elting point. E x p erim en tal values obtain ed using co n tro lled pore g lass an d p o ly carb o n ate filters w ere com pared to v alu es p red icted by eq u atio n s d ev el­ oped b y M a zu r (1 9 6 5 ) and H o m sh aw (1980). (R eprinted, by perm ission, from A shw orth and Abeles. 1984.)

Deep Supercooling in Woody Plants

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by the diam eter of the pore. There is a large discrepancy in the effect of pore size on freezing point depression in these tw o reports, w hich may be explained by the fact that w ater m ovem ent is assum ed to occur in the vapor phase in George and Burke (1977), while the system of A shw orth and A beles (1984) utilized a continu­ ous colum n of water. A n im portant corollary of both hypotheses, however, is that freezing or evaporative loss o f w ater contained in the deep supercooled cells would be determ ined by the diam eter of the largest size pores. The data obtained from theoretical calculations and model systems clearly indi­ cate that pore size should have a profound effect on the m ovem ent and freezing point o f water within supercooled tissues. How these constraints on pore size relate to actual cell wall structure and tissue anatomy, how ever, is not clear. These hy­ potheses do not indicate w hether the entire prim ary and/or secondary wall or ju st specific sites within the cell wall need exhibit restricted pore size. It is also unclear w hether all cells or ju st living cells o f xylem tissues need possess this structure. Furtherm ore, hypotheses on cell wall structure m ust also account for seasonal changes in the extent o f deep supercooling, as well as differences in wood structure between species that exhibit deep supercooling and those that do not. O ver the past several years, W isniew ski and co-w orkers (W isniew ski et al., 1987a,b, 1991a,b; W isniewski and Davis, 1989) have focused on trying to elucidate the aspects o f cell wall structure that prom ote deep supercooling and regulate seasonal variation in the extent of deep supercooling. Their data, as presented in the following sections, support a m ajor role for the structure o f the pit m em brane of xylem parenchym a and further indicate that pectins may determ ine the porosity and/or perm eability of this region of the cell wall.

Characterization of Cell Wall Porosity and Permeability Lanthanum nitrate has been extensively used as a free-space m arker at the ultrastructural level in both plant and anim al tissues, and is believed to penetrate spaces as small as 20 A (see citations in W isniew ski, 1987a). It is administered as an ionic solution, and the lanthanum is precipitated when tissue is immersed in a fixative with a neutral to basic pH . The lanthanum is seen as black, electron-dense deposits when tissue sections are observed with a transm ission electron microscope. Utilizing this apoplastic tracer, the distribution of lanthanum deposits in stem tissues o f several species o f woody plants was characterized (W isniewski et al., 1987a,b). Intergeneric and intrageneric com parisons were made between species that exhibit deep supercooling ( Prunus persica, Cornus florida, Betula lenta) and those that do not (Salix babylonica, Betula papyrifera). The distribution of lantha­ num was generally the sam e in all species. A lthough som e differences were noted, they were not associated with the freezing response (i.e., supercooling vs. non­ supercooling) of the tissue. W hereas the primary cell walls of cortical cells were freely perm eable to the lanthanum solution, both the prim ary and secondary cell walls o f xylem parenchym a cells w ere im perm eable (Fig. 6). This indicates either an extrem ely small pore size or low accessibility (i.e., porous but low perm eabil­ ity). In contrast, the pit m em brane and am orphous (protective) layer of xylem pa­ renchym a cells were very perm eable. The absence of lanthanum deposits in secondary cell walls o f ray cells, in all species exam ined, indicates that if pore size in the cell wall is integral to defining the freezing response o f xylem tissue, then the structure and perm eability o f the pit m em brane (and associated am orphous

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l a y e r ) r a t h e r t h a n t h e s e c o n d a r y c e l l w a ll , s h o u l d p la y a n i n t e g r a l r o l e . T h i s is d u e to t h e f a c t t h a t t h e w i d e s t c a p i l l a r i e s l i m i t t h e a b i l i t y o f a c e l l t o r e t a i n w a t e r a g a i n s t a s t r o n g v a p o r - p r e s s u r e g r a d i e n t a n d p r e v e n t t h e i n t r u s i v e g r o w t h o f e x t e r n a l ic e c r y s ta ls . E v i d e n c e o f p o r e d i a m e t e r s in t h e r a n g e o f 5 to 1 7 0 n m ( 5 0 - 1 , 7 0 0 m e m b r a n e s a n d 1 0 to 1 3 0 n m ( 1 0 0 - 1 3 0 0

A)

A)

f o r p it

fo r x y le m c e ll w a lls o f h a rd w o o d s

h a v e b e e n r e p o r t e d ( S i a u , 1 9 8 4 ) . H o w e v e r , a s n o t e d b y P r e s t o n ( 1 9 7 4 ) , t h e te r m p o r o s i t y c a n o n l y b e u s e d l o o s e l y in t h a t t h e p a t h t h r o u g h t h e c e l l w a ll r e p r e s e n t s a v e r y t o r t u o u s s e r i e s o f c a n a l s , a n g u l a r in c r o s s - s e c t i o n a n d m a i n l y a l i g n e d p a r a l l e l to t h e m i c r o f i b r i l s . W h e r e a s t h e “ p o r e ” s y s t e m in p i t m e m b r a n e s m a y c o m e c l o s e to r e p r e s e n t i n g t r u e c a p i l l a r i e s , t h e “ p o r e ” s y s t e m in t h e s e c o n d a r y c e ll w a l l m u s t b e m o r e c o m p l e x d u e t o t h e l a m i n a t e c o n s t r u c t i o n o f t h e c e ll w a ll w ith o p p o s i n g m i c r o f i b r i l o r i e n t a t i o n s in t h e

S 2, a n d S 3 la y e r s . T r a n s i t i o n l a m e l l a e h a v e a l s o

b e e n r e p o r t e d ( V i a n e t a l., 1 9 8 6 ) , w h i c h w o u l d f u r t h e r c o m p l i c a t e t h e o r g a n i z a t i o n o f a n y p o r e s y s t e m in t h e s e c o n d a r y c e ll w a ll . T h i s f e a t u r e , a l o n g w ith t h e d e m o n -

Figure 6. D i s t r i b u t i o n o f l a n t h a n u m ( b l a c k , c r y s t a l l i n e d e p o s i ts ) in A, b a r k a n d B -D , r a y c e lls . N o te d i s t r i b u t i o n o f l a n t h a n u m w i t h i n th e p r i m a r y c e ll w a l l s ( p w ) o f b a r k c e lls . In x y l e m r a y c e lls , l a n t h a ­ n u m w a s a b s e n t f r o m th e s e c o n d a r y c e ll w a l l s ( s w ) b u t p r e s e n t in l a r g e a m o u n t s in th e p i t m e m b r a n e a n d u n d e r l y i n g a m o r p h o u s l a y e r ( a l) . ( S e e F i g u r e 7 f o r d i a g r a m m a t i c i l l u s t r a t i o n o f x y l e m r a y p a r e n ­

Prunus persica ( A a n d B ) , Salix babylonica ( C ) , C o m u s flo rid a ( D ). v = v e s s e l e l e m e n t , r p = x y l e m r a y p a r e n c h y m a c e ll. ( R e p r i n t e d , b y c h y m a c e ll a n d c e ll w a ll l a y e r s c o m p o s i n g th e p i t m e m b r a n e .)

p e r m i s s io n , f r o m W i s n ie w s k i e t a l., 1 9 8 7 a .)

Deep Supercooling in Woody Plants

173

strated perm eability o f the pit m em brane to lanthanum , adds credibility to the idea that differences in the structure of pit m em branes may be responsible for determ in­ ing w hether or not a cell or tissue will deep supercool. The studies utilizing lanthanum as an apoplastic tracer clearly revealed that the pit m em brane was the m ost perm eable portion o f the xylem ray cell. Therefore, if differences in cell wall structure exist between species that supercool and those that do not, and which directly im pact on freezing response, they should be present in the pit m em brane portion o f the cell wall. Based on the hypotheses o f George and Burke (1977) and A shw orth and A beles (1984), pores in the size range o f 6 0 -1 0 0 A or less would be expected to have an impact on freezing response. Lanthanum can penetrate voids as sm all as 20 A. Therefore, apoplastic tracers of a larger size would be necessary to conduct exclusion studies to reveal m ore details about the pore structure o f the pit m em brane in supercooling vs. non-supercooling species. Research on the m ovem ent o f solutions through xylem tissues has been reported in the wood technology literature (Siau, 1984) and supports the prem ise that, although cell wall capillaries exist in secondary cell walls, the m ajor pathway of both longi­ tudinal and lateral m ovem ent o f solutions is via the pit system into the lum ens of cells and then into the cell wall (K ininm onth, 1971, 1972; M urmanis and Chudnoff, 1979).

Pit Membrane Structure and Deep Supercooling A lthough the structure o f the pit m em brane o f ray cells of hardwoods can be variable depending on the type of cell it interfaces (Esau, 1977), the structure o f a typical pit m em brane o f a ray cell bordering a vessel elem ent (such ray cells are designated as contact cells) is diagram m atically represented in Figure 7. As seen with an electron m icroscope, it is com posed of an outer layer of electron-dense material (black cap), a m iddle layer o f primary cell wall derived from both the ray cell and adjoining vessel elem ent, and an inner layer (am orphous or protective layer) that lines the inside o f the secondary cell wall but is thickened in the vicinity of the pit m em brane. T he function of this inner wall layer is not fully understood; VESSEL

F ig u re 7. D iagram m atic rep re sen tatio n o f a x y lem ray cell (tran sv erse section) ad jacen t to a vessel elem ent. T he pit m em brane co n sists o f three layers: an o u term o st b lack cap (B C); a prim ary w all (PW ), derived from b o th th e ray cell and v essel elem ent, an d an am orphous lay er (A L), w h ich lies interior to the secondary cell w all (SW ) o f th e ray cell. T he ch an n els are m eant to diagram m atically illustrate cell w all m icro cap illaries. (R ep rin ted , by perm ission, from W isniew ski et al., 1991b.)

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Wisniewski

hence, am biguous term inology has arisen to describe it (Schaffer and W isniewski, 1989). It should be noted that only a single pit m em brane is illustrated; in reality, a ray cell will have m any o f these pits interfacing with its neighboring cells. W isniew ski et al. (1991a) found that when stem segm ents o f peach and flow er­ ing dogw ood were treated with m acerase (an enzym e m ixture rich in pectinase), T a b le 1. C o m p o u n d s o r tr e a tm e n ts th a t m o d ify th e s tru c tu re o f th e p it m e m b ra n e o f xylem p aren ch y m a* C om pound or tre a tm e n t

P o rtio n o f p it m e m b ra n e affe c te d 11

P ectin ase C ellu lase H em icellu lase O x alic acid

BC , P W , A L BC BC BC , P W , A L

EGTAC W a te r s o a k (3 -1 0 d ay s)

BC, PW BC , P W , A L

S o d iu m p h o s p h a te b u ffer

BC (slight)

P rin c ip a l effect o n lo w -te m p e ra tu re e x o th e rm F la tte n in g S h ift to w a rm e r te m p e ra tu re S h ift to w a rm e r te m p e ra tu re F la tte n in g a n d slig h t shift to w a rm e r te m p e ra tu re S h ift to w a rm e r te m p e ra tu re F la tte n in g a n d slig h t shift to w a rm e r te m p e ra tu re S h ift to w a rm e r te m p e ra tu re

'S o u r c e : W isn ie w sk i et al. (1991); used by p erm issio n . bBC = b la c k c a p o r to ru s-lik e co v e rin g o n vessel side o f p it m e m b ra n e ; P W = p rim a ry w all p o r tio n o f p it m e m b ra n e ; a n d A L = a m o rp h o u s lay er o f p it m e m b ra n e . cE th y len e g ly c o l-b is (/3 -am in o -eth y l e th e r)-M W '-tetraacetic acid.

F ig u re 8. E ffect o f m acerase on freezin g response and pit m em brane stu c tu re o f peach. A , Control tis­ sue. N ote L T E (lo w -tem p eratu re ex o th erm ) and intact structure o f th e p it m em brane (larg e arrow ). B, M acerase-treated tissue. N ote flatten in g o f L TE and disruption o f p it m em brane structure (large arrow ), v = v essel elem en t, sw = seco n d ary cell w all, pw = prim ary cell w all, b e - black cap, al = am o rp h o u s layer, rp = xy lem ray p aren ch y m a cell.

Deep Supercooling in Woody Plants

175

the enzym e had a profound effect on both pit m em brane structure and the ability to deep supercool. In contrast, treatm ents with cellulase, hemicellulase, or phosphate buffer had relatively m inor effects on the character o f the LTE and pit m em brane structure (Table 1). As illustrated in Figure 8, the m acerase treatm ent resulted in a flattening (i.e., disappearance) o f the LTE. This was associated with an alm ost com plete digestion o f the outer two layers of the pit m em brane and a partial diges­ tion o f the inner am orphous layer. T he other enzym e and buffer treatm ents ap­ peared to affect only the outer layer (black cap) and m ainly resulted in a shift of the LTE to w arm er tem peratures. No alterations were observed in the structure of the secondary cell wall or prim ary cell wall (outside o f the pit m embrane). Based on these observations, it was concluded that treatm ents (cellulase, hem icellulase, buffer) that resulted in only a slight m odification in pit m em brane structure (via partial dissolution o f cell wall polysaccharides) altered the “barrier” properties of the cell wall. This caused a shift of the LTE to w arm er tem peratures. In com pari­ son, the pectinase treatm ent, which substantially altered the structure of the pit m em brane, nearly elim inated this “barrier” property, thus elim inating the ability of the tissue to deep supercool. These observations supported a previous report (W isniewski and D avis, 1989) in w hich self-induced alterations in pit m em brane structure in peach w ere accom panied by a dram atic shift o f the LTE to w arm er

TEMPERATURE °C F ig u re 9. D ifferential th erm al an aly sis profiles o f ‘L oring’ peach xy lem tissue before and after e x p o ­ sure to 5 .0 -5 0 .0 m M o x alic acid for 24 h. N ote th e gradual flatten in g o f the lo w -tem perature ex o th erm (LTE) w ith in creasin g c o n cen tratio n o f o x alic acid. (R eprinted, by perm ission, from W isniew ski e t al„ 1991b.)

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Wisniewski

F ig u re 10. Pit m em b ran e o f ray cells o f p each (Primus persica) after ex p o su re to o x alic acid for 24 h. A , L ow co n cen tratio n (5 m M ) resu lted in loosening o f the black cap (be, cu rv ed arrow ). B, High co n ­ cen tratio n (5 0 m M ) resu lted in sev ere deg rad atio n o f the black cap (be, arrow s) and u nderlying layers o f the p it m em b ran e, v = vessel elem en t, sw = secondary cell w all, b e = b lack cap, pw = prim ary cell wall, rp = xylem ray parenchym a cell, al = am orphous layer. (Reprinted, by perm ission, from W isniew ski et al., 1991b.)

tem peratures. The alterations were brought about by prolonged soaking of twigs in water. B oth the alterations and the shift in the LTE were prevented by soaking the twigs in cyclohexim ide. Subsequent w ork with calcium chelating agents (W isniew ski et al., 1991b) pro­ vided further evidence that the pit m em brane of xylem ray cells is rich in pectin. A sum m ary o f the effects of com pounds or treatments w hich m odify the structure of the pit m em brane and cause an alteration of the LTE profile is presented in Table 1. Besides the pectinase treatm ent, oxalic acid in particular had a significant effect on both the character of the L TE and the structure of the pit m em brane (Figs. 9 and 10, respectively). M ore recently, utilizing m onoclonal antibodies (Knox et al., 1990) to esterified (JIM 7) and nonesterified (JIM 5) pectin, W isniewski and Davis (unpublished) ob­ served that in pit m em branes o f peach ray cells, nonesterified epitopes of pectin were confined to the outer layer o f the pit m em brane, w hereas esterified epitopes predom inated in the m iddle (prim ary wall) and inner layer (am orphous layer) (Fig. 11). Labeling o f the pit m em brane by either antibody (JIM 5 and JIM 7) was absent when tissues were first treated with pectinase. The am orphous layer was also abun­ dant in epitopes o f arabm ogalactan-rich glycoprotein recognized by the m ono­ clonal antibody JIM 14 developed by Knox et al. (1989, 1991). An increase in extensin, a cell wall glycoprotein, in epicotyl tissue of pea (Pisum sativum) during cold acclim ation has been previously reported (W eiser et al., 1990). C ollectively, the data on cell wall structure and deep supercooling indicate that the type, am ount , and degree o f cross-linking of pectin w ithin the pit membrane may determ ine the size of the pores and/or their perm eability to water (som e pectin polym ers m ay increase hydrophobicity and therefore decrease perm eability). The

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F ig u re 11. D istribution o f m o n o clo n al a n tib o d ies to esterified o r nonesterified epitopes o f pectin (antibodies term ed JIM 7 an d JIM 5) and to ep ito p es o f arab in o g alactan -rich glycoprotein (JIM 14) in pit m em brane o f ray cells o f p each (Prunus persica). A , D istrib u tio n o f esterified ep ito p es o f p ectin recognized by JIM 7. B , D istrib u tio n o f ep ito p es o f nonesterified p ectin recognized by JIM 5. C , D ras­ tic red u ctio n o f lab elin g w ith JIM 7 after treatm en t w ith p ectinase. D , D istribution o f ep ito p es reco g ­ nized by JIM 14. v = vessel elem en t, rp = x y lem ray parenchym a, sw = secondary cell w all, be = black cap, pw = prim ary cell w all, al = am o rp h o u s layer. (See F ig u re 7 for diagram m atic illustration o f pit m em brane structure.)

structure o f the pit m em brane, along with the tensile strength imparted by the sec­ ondary cell wall, could account for the ability to deep supercool. Baron-Epel et al. (1988) also suggested that pectins determ ine pore size in cell walls of soybean sus­ pension cells. Pectin-m ediated regulation of porosity/perm eability of the pit m em ­ brane is an attractive hypothesis because it provides a plausible basis to explain the seasonal shifts that occur in the extent o f deep supercooling. Loosening o f cell wall structure (i.e., increased porosity/perm eability) could occur in the spring via disruption of metal ion bridges (prim arily Ca2+) or breakage of covalent bonds by intrinsic pectinases. Such m odifications do occur, for exam ple, during tylose for­ mation when the cell w all within the pit m em brane loosens considerably to allow for growth and extension o f the living ray cell into a neighboring vessel elem ent (Beckm an, 1971).

Summary and Future Research As previously stated, deep supercooling of plant tissues is perhaps one o f the more enigm atic aspects o f biological ice nucleation. B ecause the full expression of

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f this trait is dependent on the existence of intact tissue, it has been difficult to dis­ cern the fundam ental m echanism and/or properties that regulate deep supercooling. A lthough the im portance o f cell wall porosity has been im plied via the use o f theo­ retical calculations (G eorge and Burke, 1977) and m odel system s (Ashworth and Abeles, 1984), how these constraints translate to actual cell wall structure and tis­ sue anatom y is not well understood. As presented, the com bined evidence indicates an im portant role for pit m em brane structure in regulating deep supercooling and implicates pectin and the interaction of pectin with other cell wall constituents as playing a role in defining the porosity and/or perm eability o f this region o f the cell wall. This evidence, how ever, is only correlative or inferential at this stage. A lthough pectin-m ediated regulation of deep supercooling is an attractive hy­ pothesis because it can account for many of the observations presented, there are many questions that m ust be resolved before a definitive know ledge of the underly­ ing m echanism of deep supercooling of xylem tissues is developed. Perhaps up­ perm ost is the fact that com ponents of cell wall structure that specifically dictate freezing response and differ betw een species that supercool and those that do not have yet to be docum ented. This is true in regards to both dem onstrated porosity (W isniew ski et al., 1987a,b) and appearance during exposure to subzero tem pera­ tures (A shw orth et al., 1988, M alone and Ashworth, 1991). Furtherm ore, in apple (K etchie and K am m ereck, 1987), peach (W isniewski and D avis, 1989), and some other species (A shw orth, 1993), it is clear that the xylem tissue does not exhibit a hom ogeneous freezing response. This is evidenced by m ultiple LTEs in apple and bim odal peaks in peach. How the complex freezing response of these tissues is regulated is not understood. The work o f G usta et al. (1983) also indicates that, despite exhibiting deep su­ percooling, som e species also have the ability to slowly dehydrate at very low tem ­ peratures ( - 3 0 to -4 0 °C ) resulting in a com plete disappearance of the LTE. The viability o f these tissues after exposure to cold tem peratures w ould be dependent on variables associated with deep supercooling as well as additional cellular prop­ erties associated with cold acclim ation. How these m echanism s o f cold adaptation and ice nucleation are regulated is, again, not understood. Due to the lim its im posed on the cold hardiness o f tem perate fruit trees (and other econom ically im portant woody plants) by the tem perature constraints associ­ ated with deep supercooling of xylem tissue and floral buds (see C hapter 10), selection to elim inate this trait in favor of other m echanism s o f cold hardiness has been proposed (B urke and Stushnoff, 1979). The inheritance and genetic regulation of deep supercooling, how ever, have not been investigated. A lthough there is a dis­ tinct seasonality in the expression of deep supercooling o f xylem tissues, which implies a controlled sequence o f active processes, the biochem ical and structural changes responsible for this seasonal regulation remain to be elucidated. As scien­ tific evidence for the control and regulation of deep supercooling becom es avail­ able, determ ination o f w hether or not this trait can be m anipulated using traditional m ethods o f plant breeding or m odern techniques o f m olecular biology will be forthcom ing. It is hoped that a better understanding of deep supercooling o f xylem tissues will directly or indirectly result in new strategies having the potential to prevent freeze injury in econom ically im portant horticultural plants.

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in mure a n d o n is o l a t e d m o l e c u l e s . P r o t o ­

p l a s m a 1 3 1 :1 8 5 - 1 8 9 . W e i s e r , R . L ., W a l l n e r , S . J ., a n d W a d e l l , J. W . 1 9 9 0 . C e ll w a ll a n d e x t e n s i n m R N A c h a n g e s d u r i n g c o ld a c c lim a tio n o f p e a s e e d lin g s . P la n t P h y sio l. 9 3 :1 0 2 1 -1 0 2 6 . W i s n ie w s k i , M ., A r o r a , R ., a n d D a v is , G . 1 9 9 1 c . R o le o f th e p r o t o p la s t in d e e p s u p e r c o o l i n g o f x y le m tis s u e . H o r t S c i e n c e 2 6 : 7 2 7 . W i s n ie w s k i , M ., a n d A s h w o r t h , E . N . 1 9 8 5 . C h a n g e s in th e u l t r a s t r u c t u r e o f x y l e m p a r e n c h y m a c e lls o f peach

(Prunus persica) a n d r e d o a k (Quercus rubra) in r e s p o n s e to a f r e e z i n g s tr e s s . A m . J. B o t.

7 2 :1 3 6 4 - 1 3 7 6 . W i s n ie w s k i , M ., a n d A s h w o r t h , E . N . 1 9 8 6 . S e a s o n a l v a r i a t i o n in d e e p s u p e r c o o l i n g a n d d e h y d r a t i v e re s is ta n c e . H o rtS c ie n c e 2 1 :5 0 3 -5 0 5 . W i s n ie w s k i , M ., a n d D a v i s , G . 1 9 8 9 . E v id e n c e f o r th e i n v o l v e m e n t o f a s p e c i f i c c e l l - w a l l l a y e r in

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reg u latio n o f deep su p e rco o lin g o f xylem p arenchym a. P lant P hysiol. 91:151-156. W isniew ski, M ., D avis, G ., a n d A ro ra, R. 1991b. E ffect o f m acerase, o x alic acid, and EG TA on deep su p erco o lin g and p it m em b ran e structure o f x y lem p aren ch y m a o f peach. P lant Physiol. 96:13541359. W isniew ski, M „ D avis, G ., and A ro ra, R. 1993. T h e role o f pit m em brane structure in deep su p e rco o l­ ing o f xy lem p arenchym a. P ag es 215 -2 2 8 in: A dvances in P lant C old H ardiness. P. H. Li and L. C hristersson, eds. C R C P ress, B o c a R ato n , FL. W isniew ski, M ., D avis, G ., and Schaffer, K. 1991a. M ediation o f d eep supercooling in Prunus and Cornus v ia en zy m atic m o d ifica tio n s in cell w all structure. P lan ta 184:254-260. W isniew ski, M ., L ig h tn er, G ., D av is, G ., an d Schiavone, M . 1990. System configuration for m icro ­ com p u ter-co n tro lled , lo w -tem p eratu re, d ifferen tial therm al analysis. C om p. E lec. A gric. 5:223-230. W isniew ski, M ., A shw orth, E . N „ an d Schaffer, K. 1987a. T he use o f lanthanum to characterize cell w all p erm eab ility in relatio n to d eep su p e rco o lin g and ex tracellu lar freezing in w oody plants. I. In ­ tergeneric co m p ariso n s b etw een Prunus, Cornus, and Salix. P ro to p lasm a 139:105-116. W isniew ski, M „ A shw orth, E. N „ an d S ch affer, K. 1987b. T he u se o f lanthanum to characterize cell w all p erm eab ility in relatio n to d eep su p e rco o lin g and ex tracellu lar freezing in w oody plants. II. In­ trageneric co m parisons b etw een B etula lenta an d Betula papyrifera. P rotoplasm a 141:160-168. Z im m erm an, M. H. 1983. X y lem S tru ctu re and the A scent o f Sap. S p rin g er V erlag, Berlin, H eidelberg, New York.

CHAPTER 10

Deep Supercooling in Buds of Woody Plants H. A. Quamme

Introduction Ice form ation at certain preferred sites of freezing within overwintering leaf and flower buds was first observed in w oody plants by W eigand (1906). Dorsey (1934) suggested that in dorm ant peach flow er buds, water withdrawal to preferred sites of freezing within the bud scales and bud axis protects the flower by lowering its freezing point. The release o f latent heat of fusion associated with the freezing of supercooled water w as first detected in cherry flow er buds by Tum anov et al. (1969) using heat calorim etry, but the full significance of this finding to flow er bud survival was not understood until therm al analysis studies perform ed on Rhodo­ dendron mollis and R. mollis x R. roseum flower buds by Graham (1971). G raham was able to show that w ater in the flow ers of the azalea flower buds supercools and freezes at the tem perature that corresponds to the killing point. The m aintenance of supercooled water in certain tissues below the freezing point of surrounding tissue has since been term ed deep supercooling (Burke et al., 1976). Since G raham ’s re­ port (1971), deep supercooling has been observed in the flower buds of a num ber of species, vegetative buds o f conifers, and mixed buds of grape (Table 1), and the m echanism o f supercooling has been elucidated.

Expression of Deep Supercooling in Buds Therm al analysis of dorm ant buds o f most woody plants usually reveals a hightem perature exotherm (H TE) ju st below the freezing point (-5 to -1 0 °C ) which results from extracellular ice form ation. The HTE may be followed by one or m ore low-tem perature exotherm s (LTEs) that arise from the freezing of supercooled water within the flow er(s) or the shoot prim ordium o f the bud. Typical thermal profiles are shown for peach, R. japonicum, Abies firm a, and grape (Fig. 1). The expression of supercooling in dorm ant buds cooled at rates that correspond to environm ental rates (

r ° C/3
c/i

S S' *2 §» Ä3 ?§ I» . 'S 2.

î

Î O^ S §--8 H 8 3 c-°-= m-S" §. 11 "S '

OT3 Û .n j

3

O

o

CD

—î

C

i—h

—\ CD

CD

rs

3

CD

CD

O

CD 13

—1

CD *> CD

Exothermic Response

< -1 0 - 1 9 to - 2 4 - 1 2 to - 3 2 — 12 to —45 < -1 1 - 2 0 to - 3 0 -2 8 < -1 1 < -1 1 < -1 3 - 1 5 to - 2 7

F F F F F F F F F F F S a p a lta ch erry S o u r ch erry E u ro p e a n plu m D w a rf flo w erin g ch erry M u ck le p lu m P in ch erry P each

-2 0 -2 8 -2 5 -2 4 -2 1 -2 3

to to to to to to W ild plu m A p rico t Sw eet ch erry W estern sa n d ch erry

W ild ro sem ary

F lo w erin g d o g w o o d F o rsy th ia

K a tsu ra tree

-2 2 -2 5 -2 5 -2 4 -2 6 -2 3

to to to to to to

-1 8 -1 8 -1 8 -1 9 -2 1 -1 5 -2 3 -1 2 -2 4 -1 0 -2 2 -1 2 -1 0

B ud type*

R a n g e in te m p e ra tu re of LTE (°C )

F F F F F F F F F F F F F B og ro sem ary

C o m m o n n am e

aF = flo w er b u d , V = v eg etativ e b u d , an d M = m ixed bud.

besseyi X P. salicina cerasus dom estica ja p o n ica nigra X P. lenella pen nsylvanica persica

A n g io sp e rm s A n d ro m e d a po lifia A rcterica nana Cassiope lycopodioides Cerciciphyllum ja p o n ica C ham aedaphne calyculata Cornus flo rid a F orsythia spp. koreana suspensa viridissim a L ed u m p a lustre P hyllodoce sp. Pieris ja po nica P runus am ericana arm eniaca avium besseyi

S pecies in w hich d eep su p e rc o o lin g has b een d etected in flo w er b u d s

G en u s a n d species

Table 1. O ccu rren ce o f deep su p e rc o o lin g in b u d s o f w o o d y p la n t species

(co n tin u ed o n n e x t p a g e)

B u rk e a n d S tu sh n o ff, 1978 A s h w o rth et al., 1981; Q u am m e, 1974 A n d rew s et al., 1983b; Q u am m e, 1974 B urke a n d S tu sh n o ff, 1978; Q u a m m e et al., 1982 B urke a n d S tu sh n o ff, 1978 B urke a n d S tu sh n o ff, 1978 Q u a m m e , 1974 B urke a n d S tu sh n o ff, 1978 B urke a n d S tu sh n o ff, 1978 B urke an d S tu sh n o ff, 1978 P ro e b stin g a n d S a k a i, 1979; Q u am m e, 1974

Ish ik a w a a n d S a k a i, 1982 Ish ik a w a a n d S a k a i, 1982 Ish ik a w a a n d S a k a i, 1982 Ish ik a w a an d S a k a i, 1982 Ish ik a w a a n d S a k a i, 1982 S a k a i, 1979a N u s et al., 1981 Ish ik a w a a n d S a k a i, 1982 Ish ik a w a a n d S a k a i, 1982 A s h w o rth et al., 1992 Ish ik a w a an d S a k a i, 1982 Ish ik aw a a n d S a k a i, 1982 Ish ik a w a an d S a k a i, 1982

R eferen ce

P runus (c o n tin u e d ) salicina salicina X m u nso n ia na salicina X (P. salicina X P. am ericana) R ibes nigrum sa tivu m R h o d o d en d ro n brachycarpum dauricum dilatatu m keiskei ja p o n ica o b tu su m tsch o n o skii X m ollis X m ollis X R. roseum ko steria n um R u b us ideas spp. T su siophyllum tanakae U lmus d avidiana p u m ila Vaccinium co rym b o su m sm allii vitis-idea

G en u s a n d species

T ab le 1. ( c o n tin u e d )

- 1 9 to - 3 7 - 1 6 to - 2 8 - 2 0 to - 2 4 - 2 5 to - 3 3 - 2 1 to - 2 7

F F F F F F F F F F F F F F F F F F F

B lack c u rra n t R e d c u rra n t R h o d o d e n d ro n R h o d o d e n d ro n R h o d o d e n d ro n R h o d o d e n d ro n R h o d o d e n d ro n R h o d o d e n d ro n R h o d o d e n d ro n A zalea A zalea A zalea R ed ra sp b e rry B lack b erry

M a n c h u ria n elm B lu eb erry B lueberry

to to to to to to to to to to

-2 8 -3 1 -2 5 —17 -3 4 -2 9 -2 4 -2 2 -3 8 -2 7

< -2 3 - 1 2 to - 2 0 - 1 5 to - 2 9

-2 2 -2 7 -1 6 -1 1 -1 4 -1 9 -2 0 -1 6 -1 7 -1 2

- 1 2 to - 3 5 - 1 0 to - 3 1

< -1 7

F

P ip esto n e p lu m

-2 4 < -7

F F

B ud ty p e '

Ja p a n e s e p lum R ed g lo w plu m

C o m m o n n am e

R a n g e in te m p e ra tu re of LTE (°C )

1982 1982 1981 1981 1982 1981 1981 !9 7 6 a,b 1976a,b

B ierm an n et al., 1979 Ish ik a w a a n d S a k a i, 1981 Ish ik a w a a n d S a k a i, 1981

Ish ik a w a an d S a k a i, 1982 Ish ik a w a an d S a k a i, 1982

W a rm u n d a n d G eo rg e, 1990 W a rm u n d et al., 1988 Ish ik a w a an d S a k a i, 1982

Ish ik a w a an d S a k a i, Ish ik a w a an d S a k a i, Ish ik a w a a n d S a k a i, Ish ik a w a a n d S a k a i, Ish ik a w a a n d S a k a i, Ish ik a w a a n d S a k a i, Ish ik a w a a n d S a k a i, G ra h a m a n d M u llin , G ra h a m a n d M u llin , G eo rg e et al., 1974

W a rm u n d et al., 1991 W a rm u n d et al., 1991

B u rk e an d S tu sh n o ff, 1978

Q u a m m e , 1974 B u rk e an d S tu sh n o ff, 1978

R eferen ce

186 Quamme

S pecies in w h ich d eep su p e rco o lin g o f th e b u d s is ab sen t A n g io sp e rm s A eseu lus turbenata Betula pla typ h ylla Cornus stolonifera Gauteria a d in o th ix migueliana M alus dom estica P opulus spp. Prunus m a aki pa d u s virginiana Pyrus co m m u n is G y m n o sp e rm s Picea spp.

S a k a i, 1978

E u ro p e a n b ird ch erry C h o k e ch erry P ear S p ru c e

S tu sh n o ff, 1978 S tu sh n o ff, 1978 S tu sh n o ff, 1978 1976

B u rk e a n d B u rk e a n d B u rk e a n d Q u am m e,

A p p le P o p u la r

S a k a i, 1978, 1979 S a k a i, 1978, 1979 S a k a i, 1978, 1979 S a k a i, 1978, 1979 S a k a i, 1978, 1979 S a k a i, 1978, 1979 G eo rg e, 1982

Ish ik a w a an d S a k a i, 1982 Ish ik a w a a n d S a k a i, 1982 Q u a m m e , 1976 Ish ik a w a a n d S a k a i, 1982

« -3 5 -3 0 -3 2 = —30 “ -3 0 -3 0 -4 0

Ish ik a w a a n d S a k a i, 1982 Ish ik a w a a n d S a k a i, 1982 Ish ik a w a a n d S a k a i, 1982

V V V V V V F

P ie rq u e t e t al., 1977 A n d rew s et al., 1984; Q u a m m e , 1986 W o lf an d P o o l, 1987 A n d rew s et al., 1984; Q u a m m e , 1986 W o lf a n d P o o l, 1987

C h e stn u t Birch R e d -o sier d o g w o o d

F ir F ir F ir F ir F ir L arch J u n ip e r

- 2 2 to - 2 8 M

G rap e

spp. G y m n o sp e rm s A bies balsam a ftr m a h o m o lep is sachalinensis veitchii L a rix leptolepis Ju niperus V irginia

- 2 7 to - 4 2 - 2 2 to - 2 5 F M

G rap e G rap e

Vitis riparia vinifera

Deep Supercooling in Buds

187

188

Quamme

(Proebsting and Sakai, 1979; Quam m e, 1974) and m ale cone o f juniper (George, 1982) produce a single LTE that corresponds to a single flow er. A single LTE is also produced by the vegetative buds of fir that corresponds to freezing of the shoot prim ordium (Sakai, 1978, 1979b). In larch, a single shoot prim ordium produces several LTEs that correspond to leaf prim ordia (Sakai, 1978). Racem ose flower buds o f blueberry (B ierm ann et al., 1979), flowering dogw ood (Sakai, 1979a), Prunus spp. (B urke and Stushnoff, 1978), and Rhododendron spp. (G eorge et al., 1974a; G raham and M ullin, 1976a; Kaku et al., 1980; Ishikaw a and Sakai, 1981) produce m ultiple exotherm s. The num ber of LTEs produced in Rhododendron spp. corresponds to the num ber o f florets (Graham and M ullin, 1976a; Kaku et al., 1980), but the num ber o f LTEs in sweet cherry (A ndrew s et al., 1983b), black and red currant (W arm und et al., 1991), black raspberry (W arm und et al., 1988), and red raspberry (W arm und and G eorge, 1990) is less than the num ber of florets. The exact cause for the discrepancy is unknown, but it may result from an absence of freezing in som e o f the florets, simultaneous freezing o f florets, or an inability to sense LTE o f all florets. The inflorescence of Prunus serotina , which is racemose, freezes as a unit and produces a single LTE (Kader and Proebsting, 1992). One or two broad LTEs are present on the differential thermal analysis (DTA) profiles of black and red currant flow er buds in addition to sharp LTEs. The broad LTEs cor­ respond to the freezing o f w ater in the outer nonliving region of the cane periderm tissue attached to the flow er bud, whereas the sharp LTEs correspond to the freez­ ing o f the flow ers (W arm und et al., 1991). G rape has a com plex bud containing primary, secondary, and tertiary buds, each of which may contain an apical m eristem and flowers. Up to three LTEs, thought to correspond to the freezing o f each bud type, are detected below the HTE (Andrews et al., 1984; Q uam m e, 1986; Pierquet and Stushnoff, 1980; W olf and Pool, 1987). The LTEs o f each bud type occur at a progressively low er tem perature and are progressively sm aller in size. The size o f the LTEs is probably related to bud size. The LTE o f m any buds including those of many conifers (Sakai, 1978, 1979b), Prunus spp. (A shw orth, 1982; B urke and Stushnoff, 1978; K ader and Proebsting, 1992; Q uam m e, 1983; Q uam m e et al., 1982; Proebsting and Sakai, 1979), and Rhododendron spp. (Kaku et al., 1980), occurs at a w arm er tem perature with in­ creasing cooling rate. The response is unlike that of the xylem of many of these same plant species, in w hich LTEs usually vary little with cooling rate (Burke and Stushnoff, 1978). The exception is grape, in which the LTEs o f buds remain at constant tem perature irrespective of cooling rate (Quam m e, 1986). Deep supercooling appears to be a true avoidance m echanism for low-temperature survival for flow er buds of certain genera. Flowers o f both peach (Quamme, 1983) and Rhododendron spp. (Graham and M ullin, 1976b; Kaku et al., 1981) re­ main supercooled throughout the winter. Excised flow ers of R. japonicum (Ishikaw a and Sakai, 1981) and peach (Quamme, unpublished) freeze and are killed at tem peratures well above the LTE tem perature o f the intact flower bud when inoculated with ice. Thus, the floral tissue of these tw o genera is inherently nonhardy and survives only by supercooling. A voidance o f freezing injury by supercooling appears to be transitory in some species and m ay be im portant to survival in these species only under certain envi­ ronm ental conditions. The LTEs o f blueberry (Bierm ann et al., 1979), Prunus besseyi, and Prunus pennsylvanica (Burke and Stushnoff, 1978) buds shift rapidly to low tem perature and eventually disappear when exposed to freezing tem pera­

Deep Supercooling in Buds

189

tures above the LTE. In m idw inter, LTEs are often absent in the buds o f these plants. The flowers o f these species survive the disappearance of LTEs.

Freezing Pattern in Dormant Buds A lthough few attem pts have been made to observe intracellular ice in buds that exhibit deep supercooling, the supposition is that the freezing is intracellular be­ cause o f the sudden occurrence o f the LTE and the invariably lethal effects on the tissue. Ice crystals have been observed with a light m icroscope within the tissue subtending the flow er in frozen R. japonicum (Ishikaw a and Sakai, 1981), peach (Dorsey, 1934; Q uam m e, 1974), and plum (D orsey and Strausbaugh, 1923), but not in the flow er itself. A t subzero tem peratures, the shoot prim ordium of fir (Sakai, 1979b) and flow er buds o f peach (Quamme, 1978) appear greenish and pli­ able but suddenly turn w hite and stiff when nucleated with ice crystals. The pres­ ence o f intercellular ice in peach flow ers has been verified by rapidly cooling frozen flow ers in supercooled liquid nitrogen, fracturing the flower, and exam ining the surfaces of the fractures in a cryostage with a scanning electron m icroscope (Quam m e et al., unpublished). Extracellular ice is absent in dorm ant peach flow ers but is present in the pedun­ cle and in the base o f the peach flow er during the late stages of deacclim ation (Ashworth et al., 1989). It is also present in the peduncle and base of dorm ant forsythia flowers before deacclim ation (Ashworth et al., 1992). The presence of intra­ cellular ice was not determ ined in either deacclim ated peach flowers or dorm ant forsythia, but the upper regions o f these flowers are believed to deep supercool be­ cause LTEs are present. The tissue subtending the supercooled flow er in the flower bud, including the scales and flow er bud axis, appears to freeze extracellularly. Ice accum ulates in preferred sites at the base o f the scales and in the flow er bud axis ju st below the flower in peach (D orsey, 1934; Q uam m e, 1978) and plum (Dorsey and Straus­ baugh, 1923) and in the scales o f R. japonicum (Ishikawa and Sakai, 1981). In vegetative buds o f fir, ice accum ulates in a cavity beneath the shoot prim ordium and in the scales (Sakai, 1979b). Ice crystals that form by extracellular freezing dis­ rupt and produce voids in the tissue (Ashworth et al., 1989, 1992; Ishikaw a and Sakai, 1981). A lthough cell dam age occurs near the voids, bud survival is not re­ duced. The initiation o f freezing in w oody plant buds appears to result from the propa­ gation of ice from the shoot. Freezing within several species spreads throughout the tree from a few intrinsic nucleation sites (A nderson and Smith, 1989; Ashworth and Davis, 1984; A shw orth et al., 1985; A ndrew s et al., 1983b). In peach, the ex­ cised flow ers and bud scales do not differ appreciably in their nucleation tem pera­ tures. N ucleation o f the flow er bud occurs at w arm er tem peratures as the am ount of tissue attached to the bud increases, which im plies that nucleation occurs outside the bud (Q uam m e and G usta, 1987).

Mechanism of Deep Supercooling in Dormant Buds M elting points o f w ater have not been determ ined in the buds of many plants with LTEs. H ow ever, it is presum ed that the LTE is not a eutectic point, because the LTE tem perature is usually m uch lower than the freezing point depression,

190

Quamme

w h i c h s e l d o m e x c e e d s - 4 ° C in p l a n t s ( L e v i t t , 1 9 7 2 ) . T h e m e l t i n g p o i n t o f t h e w a ­ te r a s s o c ia te d w ith f r e e z in g o f

R. kosterianum ( G e o r g e e t a l ., 1 9 7 4 a ) a n d p e a c h

f l o w e r s ( Q u a m m e , 1 9 7 8 ) is a p p r o x i m a t e l y - 2 ° C , w h e r e a s t h e f r e e z i n g p o i n t o f th e f l o w e r s is b e l o w —1 8 ° C . N u c l e a r m a g n e t i c r e s o n a n c e m e a s u r e m e n t s m a d e o n R. kosterianum ( G e o r g e e t a l ., 1 9 7 4 a ) a n d p e a c h ( R a j a s h e k a r , 1 9 8 9 ) c o n f i r m t h a t i n ­ j u r y to t h e f l o w e r f o l l o w s t h e s u d d e n f r e e z i n g o f a f r a c t i o n o f s u p e r c o o l e d w a te r . I c e f o r m a t i o n in p e a c h f l o w e r s ( Q u a m m e , 1 9 7 8 ) a n d f ir s h o o t p r i m o r d i a ( S a k a i , 1 9 7 9 b ) , p r o b e d w i t h a n ic e c r y s t a l u n d e r a m i c r o s c o p e in a c o l d c h a m b e r , a p p e a r e d to s p r e a d f r o m t h e p o i n t o f i n o c u l a t i o n . T h e p r e s e n c e o f t h e i n o c u l a t i n g ic e c r y s t a l a l l o w s a l o w e r e d a c t i v a t i o n e n e r g y f o r t h e s u b s e q u e n t r a p i d ic e c r y s t a l g r o w t h . A l t h o u g h s u p e r c o o l i n g o f b u d s h a s b e e n s t u d i e d in a n u m b e r o f s p e c i e s , i t is n o t k n o w n h o w t h e s u p e r c o o l e d s t a t e o f t h e t i s s u e is m a i n t a i n e d o r h o w f r e e z i n g o f s u ­ p e r c o o l e d t i s s u e is i n i t i a t e d . O n e o f t h e r e q u i r e m e n t s f o r s u p e r c o o l i n g o f t i s s u e is a lo w n u c l e a t i o n t e m p e r a t u r e o f th e c e l l u l a r w a t e r s o l u t i o n . I t is k n o w n t h a t in th e a b s e n c e o f n u c l e a t o r s , p u r e w a t e r s u p e r c o o l s to t e m p e r a t u r e s a s lo w

as -3 8 °C

(h o m o g e n e o u s n u c le a tio n p o in t) (F le tc h e r, 1 9 7 0 ). T h e p r e s e n c e o f s o lu te s fu rth e r l o w e r s t h e h o m o g e n e o u s n u c l e a t i o n p o i n t b y a v a l u e t h a t is p r o p o r t i o n a l t o th e f r e e z i n g p o i n t d e p r e s s i o n ( R a s m u s s e n a n d M a c K e n z i e , 1 9 7 2 ) . Y e a s t c e l l s in o il s u s p e n s i o n s s u p e r c o o l to n e a r t h e h o m o g e n e o u s n u c l e a t i o n p o i n t ( R a s m u s s e n e t al., 1 9 7 5 ) . T h u s , in y e a s t c e l l s , n u c l e a t o r s a p p e a r to b e e i t h e r a b s e n t o r to h a v e lo w a c ­ tiv ity . In p la n t c e lls th a t s u p e r c o o l, n u c le a to rs m a y a ls o b e e ith e r a b s e n t o r h a v e lo w a c t i v i t y . A s e c o n d r e q u i r e m e n t f o r d e e p s u p e r c o o l i n g is t h e p r e v e n t i o n o f ic e p r o p a g a t i o n i n to t h e s u p e r c o o l e d f l o w e r s o r a p i c a l m e r i s t e m f r o m t h e b u d a x i s . B a r r i e r s to ic e p r o p a g a t i o n m a y e x i s t t h a t i n v o l v e t h e p o r e s t r u c t u r e o f t h e tis s u e . M e l t i n g p o i n t a n d v a p o r p r e s s u r e a r e d e c r e a s e d in s t r u c t u r e s w i t h f i n e p o r e s b y t h e e f f e c t s o f i n ­ c re a s e d s u rfa c e te n s io n . M a z u r (1 9 6 5 ) a n d H o m s h a w (1 9 8 0 ) d e v e lo p e d e q u a tio n s t h a t p r e d i c t t h e d e c r e a s e s in t h e m e l t i n g p o i n t o f w a t e r w i t h p o r e s iz e . T h e s e e q u a ­ t i o n s s h o w t h a t m e l t i n g p o i n t d e c r e a s e s f r o m 2 ° C in p o r e s o f 2 0 n m d i a m e t e r to b e l o w - 4 0 ° C in p o r e s o f 1 n m d i a m e t e r . U s i n g g l a s s p a r t i c l e s w ith a r a n g e in i n t e r ­ n a l p o r e s iz e , A s h w o rth a n d A b e le s ( 1 9 8 4 ) d e m o n s tr a te d th a t m e ltin g p o in t d e ­ p r e s s io n fits th e e q u a tio n s o f M a z u r ( 1 9 6 5 ) a n d

H om shaw

(1 9 8 0 ). T h e y

a ls o

d e m o n s tra te d th a t th e p re s e n c e o f s o lu te s fu rth e r s u p p re s s e s th e m e ltin g p o in t o f w a t e r in p o r e s ( A s h w o r t h

a n d A b e le s ,

1 9 8 4 ) . In t i s s u e t h a t l a c k s e x t r a c e l l u l a r

s p a c e s , ic e p r o p a g a t i o n w o u l d b e r e s t r i c t e d a n d s u p e r c o o l i n g f a c i l i t a t e d b y th e p r e s e n c e o f w a t e r in c e l l w a ll m i c r o c a p i l l a r i e s . T h e p r e s e n c e o f s o l u t e s in w a t e r o f t h e c e l l w a l l m i c r o c a p i l l a r i e s w o u l d f u r t h e r r e s t r i c t ic e p r o p a g a t i o n . T h e f r e e z i n g b e h a v i o r o f w a t e r in s m a l l p o r e s m a y e x p l a i n t h e r e s t r i c t e d ic e p r o p a g a t i o n a n d its a c c u m u l a t i o n a t c e r t a i n p r e f e r r e d s i t e s in b u d s . T h e f l o w e r s o f b o th

peach

(A s h w o rth ,

1982; Q uam m e

e t a l .,

unpublished) a n d R. ja p o n icu m

(I s h ik a w a a n d S a k a i, 1 9 8 1 ) a n d s h o o t p r im o r d ia o f fir (S a k a i, 1 9 7 9 b ) d o n o t c o n ­ t a i n e x t r a c e l l u l a r s p a c e s t h a t a r e p r e s e n t in t h e b u d a x i s . R e s t r i c t e d p o r e s i z e is p r o b a b l y a n i m p o r t a n t f a c t o r p r e v e n t i n g ic e s p r e a d i n t o t h e f l o w e r . T h e e x c l u s i o n o f a z o s u l f a m i d e ( A s h w o r t h , 1 9 8 2 ) a n d a c i d f u c h s i n d y e ( Q u a m m e , 1 9 7 8 ) f r o m th e f l o w e r s in p e a c h b u d s is a n i n d i c a t i o n o f t h e s m a l l p o r e s i z e w it h i n t h e f l o w e r . In w i n t e r b u d s o f f i r a n d la r c h , a d e n s e z o n e o f c o l l e n c h y m a c e l l s is p r e s e n t a t t h e b a s e o f t h e s h o o t p r i m o r d i u m . T h i s a n a t o m i c a l s t r u c t u r e p r e v e n t s ic e p r o p a g a ­ ti o n i n t o t h e s h o o t p r i m o r d i u m f r o m t h e s u b t e n d i n g t i s s u e . R e m o v a l o f th e b a s a l tis s u e fro m e x c is e d s h o o t p r im o r d iu m o f f ir r a is e s th e L T E fro m - 2 5

to - 1 5 ° C

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( S a k a i, 1 9 7 8 ). D o r m a n t p e a c h f lo w e r s a ls o h a v e a b a s a l z o n e o f c e lls th a t la c k s in ­ te r c e llu la r s p a c e s ( Q u a m m e e t al., u n p u b lis h e d ). T h e f lo w e r c a n b e in d u c e d to f r e e z e b y ic e i n o c u la tio n a b o v e th e b a s a l z o n e b u t n o t b e lo w it. S im ila r a n a to m ic a l b a r r ie r s th a t r e s tr ic t th e s p r e a d o f ic e a re a ls o p r e s e n t in w h e a t f lo w e r s ( M a r c e llo s a n d S in g le , 1 9 7 6 ) a n d s te m s ( O lie n , 1 9 6 7 ). L o s s o f s u p e r c o o lin g in p e a c h d u r in g d e a c c lim a t io n is a s s o c ia te d w ith th e d e ­ v e lo p m e n t o f f u n c tio n a l x y le m ( A s h w o r th , 1 9 8 4 ). V a s c u la r tr a c e s a r e p r e s e n t in th e b a s e o f th e f o r s y th ia f lo w e r , in w h ic h e x tr a c e llu la r ic e f o rm s , b u t n o t in th e p e ta ls , f ila m e n ts , a n d p is til t h a t s u p e r c o o l ( A s h w o r th e t a l., 1 9 9 2 ). T h e x y le m o f P r u n u s v ir g in ia n a a n d P. p a d u s f lo w e r s , w h ic h d o e s n o t s u p e r c o o l, is m o r e d if f e r e n tia te d th a n th a t o f P. s e r o tin a , w h ic h d o e s s u p e r c o o l ( K a d e r a n d P r o e b s tin g , 1 9 9 2 ). T h e r e f o r e , fu lly d if f e r e n t i a t e d x y le m m a y a c t a s a c o n d u it f o r ic e p r o p a g a tio n in to th e flo w e r. T h e p o s s ib ility t h a t a n t in u c le a tin g s u b s ta n c e s m a y r e s tr ic t ic e p r o p a g a tio n in s u ­ p e r c o o le d f lo w e r s w a s f ir s t p r o p o s e d b y G e o r g e e t a l. ( 1 9 7 4 a ) b u t to d a te h a s n o t b e e n c o n f ir m e d . A n t i f r e e z e p r o te in s s im ila r to th o s e th a t e x is t in w in te r c e r e a ls ( G r if f ith e t a l., 1 9 9 2 ) m a y b e p r e s e n t in f lo w e r s o f w o o d y p la n ts , b u t th is h a s y e t to b e d e te r m in e d . P o r e s tr u c tu r e o f t h e tis s u e a ls o a f f e c ts th e e x tr a c e llu la r f r e e z in g p a tte r n . I c e fir s t f o r m s in th e la r g e r p o r e s o f s tr u c tu r e s h a v in g ir r e g u la r p o r o s ity . W a te r m o v e s f r o m th e p o r e s to f r e e z e a t th e in te r f a c e o f th e ic e c r y s ta l b e c a u s e o f th e d if f e r e n c e in w a te r p o te n tia l. T h e r e s u l t is c o n tin u e d g r o w th o f th e ic e c r y s ta l w h e r e it fir s t f o r m e d . T h e ic e c r y s ta l g r o w s a s lo n g a s th e s tr u c tu r a l m a te r ia l e x p a n d s to a c c o m ­ m o d a te th e v o lu m e c h a n g e , u n til th e s tr u c tu r a l f o r c e s b a la n c e th e w a te r p o te n tia l, o r u n til th e s tr u c tu r e b r e a k s ( E v e r e tt, 1 9 6 1 ; J a c k s o n a n d C h a lm e r s , 1 9 5 8 ). M ig r a tio n o f w a te r to p r e f e r r e d s ite s o f f r e e z in g a p p e a r s to b e e s s e n tia l to th e s u p e r c o o lin g o f p e a c h f lo w e r b u d s . D is r u p tio n o f th e f lo w e r b u d a x is b y c u ttin g th e f lo w e r b u d j u s t b e lo w th e f lo w e r p r e v e n ts s u p e r c o o lin g ( A s h w o r th , 1 9 8 2 ; Q u a m m e , 1 9 7 8 ). S c a le r e m o v a l s tu d ie s s h o w th a t th e f ir s t tw o p r o x im a l s c a le s s u b te n d in g th e p e a c h f l o w e r a re n e c e s s a r y f o r s u p e r c o o lin g to o c c u r b e lo w - 1 0 ° C ( Q u a m m e , u n p u b lis h e d ) . R a p id c o o lin g ( > 1 5 ° C /h ) o f p e a c h f lo w e r b u d s , w h ic h d is r u p ts w a te r m ig r a tio n to p r e f e r r e d s ite s , e lim in a te s s u p e r c o o lin g ( A s h w o r th , 1 9 8 2 ; Q u a m m e , 1 9 8 3 ). I c e p r o p a g a tio n th r o u g h th e tis s u e c a n b e p r e v e n te d b y d is c o n tin u itie s in w a te r flo w to w a r d s g r o w in g ic e c r y s ta ls . U s in g a m o d e l b a s e d o n th e in itia l s o lu te c o n ­ c e n tr a tio n o f th e f l o w e r p a r ts a n d h e a t a n d w a te r f lo w d u r in g f r e e z in g , C a r y ( 1 9 8 5 ) d e m o n s tr a te d th e p o s s ib ility o f a d is c o n tin u ity in th e liq u id p h a s e th a t a llo w s th e o v a r ie s o f fu lly d e v e lo p e d p e a c h a n d p lu m f lo w e r s to s u p e r c o o l. Ic e f o r m a tio n is r e s tr ic te d to th e s te m a n d r e c e p ta c le b e c a u s e a d r y la y e r f o r m s in th e r e c e p ta c le d u r in g th e in itia l s ta g e s o f c o o lin g . I f a d is c o n tin u ity in th e liq u id p h a s e o c c u r s , w a te r c a n o n ly m ig r a te f r o m th e s u p e r c o o le d tis s u e to th e s u r f a c e o f a d ja c e n t ic e c r y s ta ls th r o u g h th e v a p o r p h a s e . S u c h a d is c o n tin u ity m a y a ls o o c c u r in d o r m a n t w in te r b u d s . T h e w a te r a n d o s m o tic p o te n tia l o f th e p e a c h f lo w e r a re h ig h e r th a n th a t o f th e s u b te n d in g tis s u e , w h ic h m a y p r o m o te d e v e lo p m e n t o f a w a te r d is c o n ti­ n u ity ( Q u a m m e a n d G u s ta , 1 9 8 7 ). A p o s s ib ility e x is ts t h a t a d is c o n tin u ity in th e liq u id p h a s e c a n o c c u r a t th e p la s m a m e m b r a n e . A le th a l f r e e z e ( A s h w o r th , 1 9 8 2 ; Q u a m m e , 1 9 7 8 ) a n d h e a t tr e a tm e n t ( A s h w o r th , 1 9 8 2 ), b o th o f w h ic h d is r u p t th e p la s m a m e m b r a n e o f th e f lo w e r , r a is e th e L T E te m p e r a tu r e b u t d o n o t e lim in a te th e L T E . N u c le a tio n o f th e

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p e a c h f lo w e r c a n b e in d u c e d a t te m p e r a tu r e s b e lo w - 1 0 ° C b y p r o b in g th e f lo w e r th r o u g h th e s c a le s w ith ic e ( Q u a m m e , 1 9 7 8 ). P r e s u m a b ly , o n c e ic e p r o p a g a tio n is in itia te d in s o m e f lo w e r c e lls , it c a n s p re a d e a s ily f r o m c e ll to c e ll th r o u g h th e tis ­ s u e . I n d e a c c lim a t in g p e a c h ( A s h w o r th e t a l., 1 9 9 2 ) a n d d o r m a n t f o r s y th ia flo w e rs ( A s h w o r th e t a l., 1 9 8 9 ), th e d is c o n tin u ity a p p e a r s to o c c u r a m o n g d if f e r e n t p a rts o f th e flo w e r . A l th o u g h a d is c o n tin u ity in th e liq u id p h a s e a t th e b a s e o f th e f lo w e r o r th e s h o o t p r i m o r d i u m p r e v e n ts th e s p r e a d o f ic e , it d o e s n o t p r e v e n t w a te r m ig r a tio n . G r a v im e tr ic d e te r m in a tio n s o f th e w a te r c o n te n t o f f lo w e r s o f d o g w o o d (S a k a i, 1 9 7 9 a ), p e a c h ( Q u a m m e , 1 9 8 3 ), R h o d o d e n d r o n s p p . ( G r a h a m a n d M u llin , 1 9 7 6 a ; K a k u e t a l., 1 9 8 1 ) a n d s h o o t p r im o r d ia o f fir (S a k a i, 1 9 7 9 b ) c o n f ir m th a t w a te r m i­ g r a te s f r o m th e f lo w e r o r b u d a p ic a l m e r is te m d u r in g f r e e z in g . In b u d s o f b lu e b e r r y ( B ie r m a n n e t a l., 1 9 7 9 ), j u n i p e r ( G e o r g e , 1 9 8 2 ), a n d s e v e r a l P r u n u s s p p . (B u r k e a n d S tu s h n o f f , 1 9 7 8 ; Q u a m m e e t a l., 1 9 8 2 ), th e s iz e o f th e L T E , w h ic h is a n in d i­ c a tio n o f th e a m o u n t o f s u p e r c o o le d w a te r, d e c r e a s e s w ith a s lo w c o o lin g r a te ( > 5 ° C /h ) o r in c r e a s e d le n g th o f s to r a g e tim e a t s u b z e r o te m p e r a tu r e s . W a te r e q u ili­ b r a tio n b e tw e e n d if f e r e n t p a r ts o f th e p e a c h f lo w e r b u d , h o w e v e r , is n o t u n if o r m ( Q u a m m e , 1 9 8 3 ). T h e f lo w e r lo s e s w a te r m o r e s lo w ly th a n th e b u d a x is a t th e f lo w e r b a s e . T h e w a te r c o n t e n t o f th e f lo w e r b u d a x is n e a r th e f lo w e r b a s e d r o p s q u ic k ly a f te r f r e e z in g , w h e r e a s th a t o f th e f lo w e r d r o p s s lo w ly a f te r p r o lo n g e d s to r a g e a t s u b z e r o te m p e r a tu r e s . I f th e w a te r c o n te n t o f th e f lo w e r d e c r e a s e s , th e n th e L T E o c c u r s a t a c o ld e r t e m p e r a tu r e . T h is h a s b e e n d e m o n s tr a te d in th e f o llo w in g s p e c ie s : b lu e b e r r y ( B ie r m a n n e t a l., 1 9 7 9 ), s w e e t c h e r r y ( A n d r e w s a n d P r o e b s tin g , 1 9 8 7 ), flo w e r in g d o g w o o d ( I s h ik a w a a n d S a k a i, 1 9 8 5 ), p e a c h ( Q u a m m e , 1 9 8 3 ), a n d R h o d o d e n d r o n s p p . ( G r a h a m a n d M u llin , 1 9 7 6 b ; K a k u e t a l., 1 9 8 1 ; G e o r g e e t a l., 1 9 7 4 a ). T h e r e ­ la tio n s h ip b e tw e e n w a te r c o n te n t o f th e e x c is e d f lo w e r a n d L T E is lin e a r in p e a c h ( Q u a m m e , 1 9 8 3 ), R. m o llis , a n d R. m o llis x R. r o s e u m ( G r a h a m a n d M u llin , 1 9 7 6 b ). I t is n o t k n o w n e x a c tly h o w d e h y d r a tio n d e c r e a s e s th e te m p e r a tu r e o f th e L T E , b u t I s h ik a w a a n d S a k a i ( 1 9 8 1 ) o b s e r v e d th a t a f te r d r y in g , th e f r e e z in g p o in t o f s a p e x p r e s s e d f r o m f lo w e r s o f R . ja p o n ic u m d e c r e a s e d . P r e s u m a b ly , c e ll s o lu te s w e r e c o n c e n tr a te d b y d r y in g , a n d th is d e c r e a s e d th e n u c le a tio n te m p e r a tu r e o f w a ­ te r w ith in th e p o r e s tr u c tu r e . S a k a i ( 1 9 7 9 a ) te r m e d th e s e g r e g a tio n o f ic e w ith in p l a n t tis s u e s “e x tr a o r g a n ” f r e e z in g . A s a c o n s e q u e n c e o f e x tr a o r g a n fr e e z in g , w a te r is w ith d r a w n f r o m o n e tis s u e to f r e e z e in a n o th e r w h e r e it is le s s in ju r io u s . E x tr a o r g a n f r e e z in g a ls o o c c u rs in s e e d s o f C e la s tr u s a r b r ic u la tu s a n d le ttu c e ( I s h ik a w a a n d S a k a i, 1 9 8 2 ). T h e s tr u c tu r e o f b u d s a p p e a r s to b e a d a p te d to a c c o m m o d a te ic e a t p r e f e r r e d site s . F o llo w in g f r e e z in g a n d th a w in g , v o id s a re p r o d u c e d in th e tis s u e o f d o r m a n t f lo w e r b u d s o f p e a c h ( A s h w o r th e t a l., 1 9 8 9 ) a n d f o r s y th ia ( A s h w o r th e t a l., 1 9 9 2 ). It is u n k n o w n i f th e r e is a p r e d is p o s itio n f o r v o id f o r m a tio n a t p r e f e r r e d s ite s o f f r e e z in g , b u t c e ll w a lls m a y b e m o r e s u s c e p tib le to s e p a r a tio n a n d d e f o r m a tio n a t th e s e s ite s th a n in s u r r o u n d in g tis s u e . In p e a c h , th e c e lls a t p r e f e r r e d s ite s a re r e la ­ tiv e ly la r g e a n d h a v e th in n e r w a lls o f f r e e z in g th a n th e c e lls in s u r r o u n d in g tis s u e ( Q u a m m e , u n p u b lis h e d ). A lth o u g h ic e a p p e a r s to p r o p a g a te s u d d e n ly f r o m a s in g le p o in t in b u d tis s u e , it is u n k n o w n w h e t h e r ic e p r o p a g a te s f r o m c e ll to c e ll a lo n g th e c e ll w a ll o r th r o u g h th e c y to p la s m . P o s s ib ly , th e p la s m o d e s m a ta c o u ld a llo w ic e p r o p a g a tio n fro m c e ll to c e ll. I n x y le m r a y p a r e n c h y m a , s in g le c e lls o r s m a ll g r o u p s o f c e lls f r e e z e o v e r a

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b r o a d te m p e r a tu r e r a n g e ( H o n g a n d S u c o f f , 1 9 8 2 ). T h e p it s tr u c tu r e o f x y le m ra y p a r e n c h y m a a p p e a r s to b e a n i m p o r ta n t c o n s tr a in t to w a te r p e r m e a b ility a n d ic e p r o p a g a tio n . A “ p r o t e c t i v e la y e r ” w ith lo w p o r o s ity u n d e r lie s th e p it m e m b r a n e in th e x y le m o f s p e c ie s th a t d e e p s u p e r c o o l, a n d th is m a y p r e v e n t c e ll- to - c e ll ic e p r o p a g a tio n ( W is n ie w s k i a n d A s h w o r th , 1 9 8 6 ; W is n ie w s k i e t a l., 1 9 8 7 ). T h e s u d ­ d e n s p r e a d o f ic e th a t o c c u r s in b u d s th a t d e e p s u p e r c o o l s u g g e s ts th a t n o s u c h b a r ­ r ie r to ic e p r o p a g a tio n is p r e s e n t a m o n g c e lls . T h e la c k o f a p r o te c tiv e la y e r in th e p it s tr u c tu r e o f b u d s w o u ld a ls o e x p la in th e ir m o r e r a p id w a te r lo s s a n d d e c lin e in th e L T E te m p e r a tu r e d u r i n g c o o lin g c o m p a r e d to th e x y le m . A n o th e r d if f e r e n c e in d e e p s u p e r c o o lin g b e tw e e n b u d s a n d th e x y le m ra y p a r e n ­ c h y m a is th a t th e r a y s a r e in te r s p e r s e d a m o n g lig n if ie d e le m e n ts s u c h a s f ib e r s a n d v e s s e ls . R ig id ity o f th e tis s u e m a y r e s tr ic t a d e c r e a s e in c e ll v o lu m e , w h ic h m a y e x p la in th e s lo w e q u i lib r a tio n b e tw e e n s u p e r c o o le d w a te r a n d ic e w ith in th e x y le m ( G e o r g e a n d B u r k e , 1 9 7 7 ; Q u a m m e e t a l., 1 9 7 3 ). T h e s u p e r c o o le d w a te r w ith in b u d s o f m a n y s p e c ie s a p p e a r s to e q u ilib r a te r a p id ly w ith ic e in a d ja c e n t tis s u e , w h e r e a s th a t o f th e x y le m d o e s n o t ( B ie r m a n n e t a l., 1 9 7 9 ; B u r k e a n d S tu s h n o f f , 1 9 7 8 ). T h e d e e p s u p e r c o o le d c e lls o f b u d s m a y h a v e c e ll w a lls th a t a re m o r e e la s tic th a n th o s e o f x y le m r a y p a r e n c h y m a c e lls . T h e p o s s ib ility a ls o e x is ts th a t f lo w e r s o f s o m e s p e c ie s , s u c h a s p e a c h a n d R . j a p o n ic u m , w h ic h a r e s u b j e c t to ic e n u c le a tio n f r o m th e s u r f a c e , m a y b e in o c u la te d b y f r e e z in g r a in o r d e w o r b y h o a r f r o s t. I c e is p r o b a b ly e x c lu d e d fro m th e s u r f a c e o f th e f lo w e r s o f t h e s e s p e c ie s b y th e b u d s c a le s . I c e f o r m s w ith in th e s c a le s s u r ­ r o u n d in g th e f lo w e r , b u t n o t a t th e s u r f a c e o f th e b u d s c a le . T h u s , ic e w ith in th e f lo w e r b u d , w h ic h is c a p a b l e o f n u c le a tin g th e f lo w e r , d o e s n o t a p p e a r to b e in c o n ta c t w ith th e f lo w e r s u r f a c e ( M o n e t a n d B a s ta r d , 1 9 8 0 ; Q u a m m e , 1 9 7 8 ; I s h i ­ k a w a a n d S a k a i, 1 9 8 1 ).

Acclimation Cycle of Buds That Deep Supercool T h e s e a s o n a l c h a n g e s in s u p e r c o o lin g o f b u d s h a v e b e e n e x te n s iv e ly s tu d ie d in p e a c h ( Q u a m m e , 1 9 7 4 a n d 1 9 8 3 ), R h o d o d e n d r o n s p p . ( G r a h a m a n d M u llin , 1 9 7 6 b ; K a k u e t a l., 1 9 8 1 ), a n d s w e e t c h e r r y ( A n d r e w s a n d P r o e b s tin g , 1 9 8 7 ). In th e s e s p e c ie s , L T E te m p e r a tu r e s g e n e r a lly d e c r e a s e w ith th e o n s e t o f w in te r a n d in c r e a s e w ith b u d d e v e lo p m e n t in th e s p r in g . D e p e n d in g o n s ta g e o f d o r m a n c y a n d a ir t e m ­ p e r a tu r e , f lu c tu a tio n in L T E te m p e r a tu r e o c c u r s th r o u g h o u t th e w in te r u n til b u d d e v e lo p m e n t. P e a c h a n d c h e r r y f l o w e r b u d s a tta in a m in im u m h a r d in e s s le v e l in e a r ly a u tu m n a n d r e m a in a t th is le v e l th r o u g h o u t th e r e s t p e r io d i f c o ld w e a th e r d o e s n o t o c c u r . U p o n e x p o s u r e to p r o l o n g e d p e r io d s o f s u b z e r o te m p e r a tu r e s , th e f lo w e r b u d s in ­ c r e a s e in h a r d in e s s . A f t e r th e c o m p le tio n o f re s t, w h ic h c o in c id e s w ith m ic r o s p o r e m e io s is , th e r e is a p r o g r e s s iv e lo s s in h a r d in e s s a b o v e th e m in im u m le v e l, a n d h a r d in e s s le v e ls f lu c tu a te m o r e w id e ly w ith a ir te m p e r a tu r e ( P r o e b s tin g , 1 9 7 0 ). L T E s a re p r e s e n t in f l o w e r b u d s o f th e s e s p e c ie s a t a ll s ta g e s o f d o r m a n c y u n til b u d s w e ll ( A n d r e w a n d P r o e b s tin g , 1 9 8 7 ; Q u a m m e , 1 9 8 3 ), b u t th e n a tu r e o f th e c h a n g e s in b u d d e v e lo p m e n t th a t a f f e c t th e e x p r e s s io n o f s u p e r c o o lin g is u n c le a r . A t b u d s w e ll, h a r d in e s s d e c r e a s e s r a p id ly , a n d L T E s o c c u r a t w a r m e r te m p e r a ­ tu r e s a n d e v e n tu a lly d i s a p p e a r . A lth o u g h th e L T E s d is a p p e a r , s w o lle n f lo w e r b u d s c a n s till to le r a te s o m e f r e e z in g . T h e d is a p p e a r a n c e o f th e L T E s c o in c id e s w ith v a s ­ c u la r d if f e r e n tia tio n in th e f lo w e r ( A s h w o r th , 1 9 8 4 ). A f te r th e p e ta l tip s e m e r g e

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th r o u g h th e c a ly x , to le r a n c e is d e p e n d e n t o n th e H T E te m p e r a tu r e . T h e f lo w e r s to l­ e r a te a lm o s t n o f r e e z in g a f te r p e ta l e m e r g e n c e ( A n d r e w s e t a l., 1 9 8 2 ; K a k u e t a l., 1 9 8 1 ). C h a n g e s in L T E te m p e r a tu r e o f th e f lo w e r b u d s o f p e a c h ( Q u a m m e , 1 9 8 3 ), s w e e t c h e r r y ( A n d r e w s e t a l., 1 9 8 3 b ; A n d r e w s a n d P r o e b s tin g , 1 9 8 7 ), a n d s e v e ra l R h o d o d e n d r o n s p e c ie s ( G r a h a m a n d M u llin , 1 9 7 6 b ; K a k u e t a l., 1 9 8 1 ) d u r in g th e a c c lim a tio n c y c le a r e s ig n if ic a n tly c o r r e la te d w ith c h a n g e s in m in im u m a ir te m ­ p e r a tu r e a n d w a te r c o n te n t o f th e f lo w e r b u d . In p e a c h , th e w a te r c o n te n t o f th e w h o le f lo w e r b u d a n d L T E te m p e r a tu r e a re d e p e n d e n t u p o n th e a ir te m p e r a tu r e ( Q u a m m e , 1 9 8 3 ). P e a c h a n d c h e r r y f lo w e r b u d s e x h ib it g r e a te r h a r d in e s s i f th e a ir te m p e r a tu r e is b e lo w - 2 ° C ( a p p r o x im a te ly , th e m e ltin g p o in t) th a n i f th e a ir te m ­ p e r a tu r e is a b o v e th is te m p e r a tu r e . T h e e x c e p tio n is th a t b u d s d o n o t lo s e h a r d in e s s a t a ir t e m p e r a tu r e s a b o v e - 2 ° C w h e n th e y a re a t th e m in im u m h a r d in e s s le v e l d u r ­ in g r e s t ( P r o e b s tin g a n d M ills , 1 9 7 2 ). E x p o s u r e to c o n tr o lle d te m p e r a tu r e s c o n ­ f ir m s th a t th e L T E te m p e r a tu r e o f s w e e t c h e r r y f lo w e r d e c r e a s e s w h e n th e a ir te m p e r a tu r e is b e lo w th e m e ltin g p o in t o f th e s u b te n d in g tis s u e a n d in c r e a s e s w h e n it is a b o v e th is p o i n t ( A n d r e w s a n d P r o e b s tin g , 1 9 8 7 ). In c o n tr o lle d e n v ir o n m e n t c h a m b e r s , th e d r o p in th e L T E is a s s o c ia te d w ith a c h a n g e in in te r n a l w a te r b a l­ a n c e , b u t in th e o r c h a r d e x te r n a l w a te r lo s s a ls o o c c u r s ( Q u a m m e , 1 9 8 3 ). J o h n s to n (1 9 2 5 ) , w h o f ir s t o b s e r v e d th e in v e r s e r e la tio n s h ip b e tw e e n w a te r c o n te n t a n d f lo w e r b u d h a r d in e s s in p e a c h , d e te r m in e d th a t in th e o r c h a r d , f lo w e r b u d s lo s e w a te r w h e n f r o z e n a n d r e g a in w a te r f ro m o th e r p a r ts o f th e tr e e d u r in g th a w in g . I s h ik a w a a n d S a k a i ( 1 9 8 1 ) a ls o d e m o n s tr a te d th a t d e h y d r a tio n b y w in d e n h a n c e s th e s u p e r c o o lin g a b ility o f R . ja p o n ic u m f lo w e r b u d s . C h a n g e s in f lo w e r b u d h a r ­ d in e s s , t h e r e f o r e , d e p e n d o n c h a n g e s in w a te r d is tr ib u tio n w ith in th e f lo w e r b u d th a t a f f e c t s u p e r c o o lin g . T h e f lo w e r b u d c a n u n d e r g o c h a n g e s in w a te r d is tr ib u tio n as a r e s u lt o f in te r n a l w a te r m ig r a tio n d u r in g f r e e z in g o r th a w in g , o r a s a r e s u lt o f d e h y d r a tio n o f th e b u d a n d u p ta k e o f w a te r f ro m th e o th e r p a r ts o f th e tre e . S to n e f r u it o r c h a r d s a r e o f te n p r o te c te d f r o m s p r in g f r o s t in ju r y b y w in d m a ­ c h in e s a n d /o r o r c h a r d h e a te r s . P r o te c tio n o f f lo w e r b u d s b e lo w - 2 0 ° C in m id w in te r is p o s s ib le b u t r e q u ir e s a c c u r a te d e te r m in a tio n o f f lo w e r b u d h a r d in e s s . U n f o r tu ­ n a te ly , th e le n g th o f tim e r e q u ir e d to m e a s u r e f lo w e r b u d h a r d in e s s m a k e s p r o te c ­ tio n p r o g r a m s im p r a c tic a l. T o o v e r c o m e th is p r o b le m , A n d r e w s e t al. ( 1 9 8 7 ) d e v e lo p e d a m o d e l th a t p r e d ic ts th e h o u r ly m e d ia n L T E o f c h e r r y b a s e d o n c h a n g e s in a ir te m p e r a tu r e a n d c h ill u n it a c c u m u la tio n , s ta r tin g w ith a n in itia l L T E m e a s ­ u r e m e n t. T h e i r m o d e l, u s in g in itia l a n d u p d a te d L T E s , s u c c e s s f u lly p r e d ic te d d a ily c h a n g e s in h a r d in e s s o f s w e e t c h e r r y f lo w e r b u d s d u r in g tw o w in te r p e r io d s . T h e p r e d ic te d v a lu e s a r e a c c u r a te e n o u g h to p e r m it th e p r a c tic e o f w in te r f r e e z e p r o te c ­ tio n o f c h e r r y o r c h a r d s .

Relationship of Deep Supercooling in Buds to Plant Distribution T h e n a tu r a l d is tr ib u tio n o f m a n y w o o d y p la n ts is lim ite d b y th e d e e p s u p e r c o o l­ in g c a p a c ity o f th e x y le m r a y p a r e n c h y m a ( G e o r g e e t a l., 1 9 7 4 b ). T h e p r o d u c tio n a r e a o f m a n y c u ltiv a te d f r u it c r o p s is a ls o lim ite d b y th e s u p e r c o o lin g c a p a c ity o f th e x y le m ( Q u a m m e , 1 9 7 6 ; Q u a m m e e t a l., 1 9 8 2 ). In th e s e s p e c ie s , th e te m p e r a tu r e o f th e L T E c lo s e ly c o in c id e s w ith th e a v e r a g e a n n u a l m in im u m te m p e r a tu r e a t th e m o s t n o r th e r n lim it o f n a tu r a l d is tr ib u tio n o r c u ltiv a tio n . S p e c ie s th a t a r e fo u n d in

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c o ld e r r e g io n s th a n th o s e w h e r e d e e p s u p e r c o o lin g s p e c ie s g r o w d o n o t d e e p s u p e r ­ c o o l a n d a re c a p a b le o f h a r d e n in g to lo w e r te m p e r a tu r e s , e .g ., r e d o s ie r d o g w o o d , p o p la r , a n d w illo w ( G e o r g e e t a l., 1 9 7 4 b ). T h e r e la tio n s h ip o f d e e p s u p e r c o o lin g c a p a c ity o f b u d s to s p e c ie s d is tr ib u tio n is le s s c le a r . P r u n u s m a a k i, P . p a d u s , P. p e n n s y lv a n ic a , a n d P . v ir g in ia n a , w h ic h d o n o t s u p e r c o o l, s u r v iv e f u r t h e r n o r th th a n P r u n u s s p e c ie s th a t d o s u p e r c o o l ( B u r k e a n d S tu s h n o f f , 1 9 7 8 ). A m o n g s p e c ie s w ith b u d s th a t d e e p s u p e r c o o l, th e n o r th e r n lim it o f d is tr ib u tio n o f r ip a r ia n g r a p e c o in c id e s c lo s e ly w ith th e a v e r a g e a n n u a l w in te r m in im u m te m p e r a t u r e s a n d th e L T E te m p e r a tu r e s o f b o th b u d s a n d x y le m (P ie r q u e t e t a l., 1 9 7 7 ). O n th e o th e r h a n d , th e L T E te m p e r a tu r e s ( - 2 2 to —2 6 ° C ) o f p r im a r y b u d s o f c u ltiv a te d g r a p e ( A n d r e w s e t a l., 1 9 8 4 ; Q u a m m e , 1 9 8 6 ; W o l f a n d P o o l, 1 9 8 7 ) c o in c id e a p p r o x im a te ly w ith w in te r a v e r a g e a n n u a l m in im u m te m p e r a ­ tu re s fo u n d a t th e n o r th e r n lim it o f c u ltiv a tio n ( - 2 1 to - 2 4 ° C ) . T h is a ls o a p p e a r s to b e tr u e o f s w e e t c h e r r y , p e a c h , a n d a p r ic o t, in w h ic h c r o p p r o d u c tio n is d e p e n d e n t o n f lo w e r b u d s u r v iv a l ( Q u a m m e e t a l., 1 9 8 2 ). T h e b u d s o f f ir a n d la rc h d e e p s u ­ p e r c o o l. T h e s e tw o s p e c ie s a re n e ith e r a s h a r d y n o r a s w e ll d is tr ib u te d in to c o ld c lim a te s a s o th e r c o n i f e r s p e c ie s th a t d o n o t d e e p s u p e r c o o l ( S a k a i, 1 9 7 8 ). O th e r s p e c ie s th a t d e e p s u p e r c o o l e ith e r d o n o t h a v e L T E s in b u d s , e .g ., a p p le a n d p e a r ( Q u a m m e , 1 9 7 6 ), o r th e L T E s o f th e b u d o c c u r a t a lo w e r te m p e r a tu r e th a n th a t o f th e x y le m L T E ( B u r k e a n d S tu s h n o f f , 1 9 7 8 ). It is u n c le a r w h a t a d a p tiv e a d v a n ta g e , i f a n y , d e e p s u p e r c o o lin g h a s f o r th e p la n t. N o t a ll s p e c ie s e x h i b i t d e e p s u p e r c o o lin g , a n d c o m p a r e d w ith s p e c ie s th a t d o n o t s u p e r c o o l, th e le v e l o f p r o te c tio n f r o m f r e e z in g is lim ite d . P e r h a p s s u p e r c o o l­ in g c o n f e r s s o m e a d v a n t a g e to th e p la n t o th e r th a n w in te r s u r v iv a l. In th e f lo w e r s o f s o m e s p e c ie s , e .g ., a p r ic o t, p e a c h , a n d s w e e t c h e r r y , f lo w e r b u d d e v e lo p m e n t o c c u r s e a r ly in th e s p r in g . S p e c u la tio n is th a t b u d s u r v iv a l b y d e e p s u p e r c o o lin g m a y n o t r e q u ir e c h a n g e s in c e llu la r s tr u c tu r e n e c e s s a r y to w ith s ta n d th e d e h y d r a ­ tio n o f e x tr a c e llu la r f r e e z in g . T h is m a y a llo w c e ll d iv is io n r e q u ir e d fo r f lo w e r d e ­ v e lo p m e n t to ta k e p la c e in la te w in te r o r e a r ly s p r in g . In tu rn , e a r ly f lo w e r in g m a y b e r e q u ir e d f o r e a r ly f r u it r ip e n in g a n d /o r la r g e f r u it s iz e . O th e r P r u n u s s p p . th a t d o n o t d e e p s u p e r c o o l o r h a v e te m p o r a r y d e e p s u p e r c o o lin g p r o d u c e f r u it n e ith e r a s la r g e n o r a s e a r ly in th e s e a s o n a s a p r ic o t, c h e r r y , a n d p e a c h . O n e c a n a ls o s p e c u ­ la te th a t s e le c tio n p r e s s u r e f o r th e s e c h a r a c te r is tic s , a n d th u s s e le c tio n f o r h ig h s u ­ p e r c o o lin g t e m p e r a tu r e s in th e s e c r o p s , is in p a r t d u e to d o m e s tic a tio n a n d b r e e d in g . T h e im p r o v e m e n t o f la r g e , e a r ly f r u ite d c u ltiv a r s o f P r u n u s s p p . b y b r e e d in g m a y h a v e b e e n a t th e e x p e n s e o f c o ld h a r d in e s s ( Q u a m m e , 1 9 9 1 ).

Measurement of Bud Hardiness with Thermal Analysis T h e r m a l a n a ly s is is a n id e a l m e th o d o f d e te r m in in g th e h a r d in e s s le v e ls o f s p e ­ c ie s th a t s u p e r c o o l, b e c a u s e th e b u d s a re k ille d a t th e m o m e n t o f ic e f o r m a tio n . T h e r m a l a n a ly s is m e a s u r e m e n ts a r e m o r e e a s ily a u to m a te d a n d r e q u ir e little tim e a n d la b o r c o m p a r e d w ith o th e r v ia b ility te s ts s u c h a s b r o w n in g , re g r o w th , a n d c o n ­ d u c tiv ity . S a m p le s iz e is s m a ll, b e c a u s e a d e f in ite k illin g te m p e r a tu r e c a n b e d e ­ te r m in e d b y th e L T E , a n d it is n o t n e c e s s a r y to f r e e z e b u d s o v e r a r a n g e o f te s t te m p e r a tu r e s to o b ta in th e k illin g te m p e r a tu r e . T h e L T E o f b u d s c a n b e d e te c te d d ir e c tly w ith e ith e r th e r m o c o u p le s o r t h e r m is ­ to rs ( Q u a m m e , 1 9 7 4 ; G r a h a m a n d M u llin , 1 9 7 6 a ), o r b y D T A u s in g s in g le s e n s o r s ( G e o r g e e t a l., 1 9 7 4 a ), o r w ith a th e r m o p ile ( A n d r e w s e t a l., 1 9 8 3 a ). S c a n n in g d i f ­

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f e r e n tia l c a lo r im e te r s c a n a ls o b e u s e d f o r m e a s u r in g th e L T E b u t a re n o t p r a c tic a l f o r r o u tin e h a r d in e s s d e te r m in a tio n s . T h e u s e o f a t h e r m o c o u p le o r th e r m is to r is th e s im p le s t m e th o d to m e a s u r e th e t e m p e r a tu r e o f th e L T E o f b u d s . I c e o r s ilic o n e g r e a s e is u s e d to e n s u r e g o o d th e r ­ m a l c o n ta c t b e tw e e n th e s e n s o r a n d th e b u d (Q u a m m e , 1 9 9 1 ). T h e L T E c a n r e a d ily b e d e te c te d o n th e t i m e - te m p e r a tu r e p r o f ile i f th e L T E is a la r g e s h a r p , d is c r e te e v e n t, b u t is d if f ic u lt to d e t e c t i f th e L T E is s m a ll o r s p r e a d o v e r a w id e te m p e r a ­ tu r e r a n g e . N u s e t al. ( 1 9 8 1 ) a n d G e o r g e ( 1 9 8 2 ) d e s c r ib e m e th o d s f o r a m p lif y in g th e L T E in b u d s u s in g D T A w ith th e r m is to r s to im p r o v e th e s e n s itiv ity o f d e te c ­ tio n . R o u tin e d e te r m in a tio n o f b u d h a r d in e s s o f te n r e q u ir e s a la r g e n u m b e r o f m e a s ­ u r e m e n ts o f L T E . T h e n u m b e r o f L T E m e a s u r e m e n ts m a y b e in c r e a s e d b y p la c in g m o r e th a n o n e b u d o n a s in g le s e n s o r ( P r o e b s tin g a n d S a k a i, 1 9 7 9 ) o r b y c o n n e c t­ in g th e r m o c o u p le s in s e r ie s to m o n ito r m o r e th a n o n e b u d p e r c h a n n e l ( Q u a m m e e t a l., 1 9 7 5 ). P r o e b s tin g a n d S a k a i ( 1 9 7 9 ) fo u n d th a t th e y ie ld o f L T E s in p e a c h b u d s d e c r e a s e s a s th e n u m b e r o f b u d s p la c e d o n th e t h e r m o c o u p le in c r e a s e s , a n d at m o s t, 2 5 p e a c h f lo w e r b u d s c o u ld b e p la c e d o n a s in g le c h a n n e l a t a l° C / h c o o lin g r a te w ith o u t a p p r e c ia b ly a f f e c tin g th e y ie ld o f L T E s . T h e r m o p ile s c a n b e u s e d to m o n ito r s e v e ra l b u d s a t a tim e a n d h a v e th e a d v a n ­ ta g e th a t th e s ig n a l is a m p lif ie d . A th e r m o e le c tr ic m o d u le ( M e lc o r M a te r ia ls E le c ­ tr o n ic P r o d u c ts C o r p ., T r e n to n , N .J .) , w h ic h w a s d e s ig n e d a s a c o o lin g p la te , h a s b e e n a d a p te d to m e a s u r e s e v e r a l b u d s s im u lta n e o u s ly ( A n d r e w s e t a l., 1 9 8 3 a ). A th e r m a l c o n d u c tin g p a s te ( O m e g a th e r m O m e g a E n g in e e r in g , In c ., S ta m f o r d , C o n n .) o r s ilic o n e g r e a s e im p r o v e s th e th e rm a l c o n ta c t b e tw e e n th e b u d s a n d th e p la te . M o r e L T E s a re d e te c te d f r o m a g iv e n n u m b e r o f p e a c h f lo w e r b u d s w ith a th e r m o e le c tr i c m o d u le th a n w ith a th e r m o c o u p le . I n m e a s u r in g th e h a r d in e s s le v e l o f b u d s w ith th e r m a l a n a ly s is , th e p o in t o f b u d e x c is io n a n d c o o lin g r a te a r e im p o r ta n t. In b o th p e a c h ( P r o e b s tin g a n d S a k a i, 1 9 7 9 ; Q u a m m e , 1 9 7 8 ) a n d g r a p e ( Q u a m m e , 1 9 8 6 ; W o l f a n d P o o l, 1 9 8 7 ), i f th e b u d is c u t to o c lo s e to th e s h o o t, th e L T E o c c u r s a t a h ig h e r te m p e r a tu r e th a n i f it is n o t. R a p id c o o lin g ( > 1 5 ° C /h ) a ls o r a is e s th e s u p e r c o o lin g p o in t o f p e a c h f lo w e r b u d s ( A s h w o r th , 1 9 8 2 ; P r o e b s tin g a n d S a k a i, 1 9 7 9 ; Q u a m m e , 1 9 8 3 ). C lo s e a g r e e m e n t h a s b e e n f o u n d b e tw e e n th e L T E m e a s u r e m e n ts a n d b u d in ju ry a s m e a s u r e d b y b r o w n in g e v a lu a tio n . T h e d is tr ib u tio n o f L T E s o f p e a c h f lo w e r b u d s is v e r y c lo s e to th e s ta n d a r d s k e w e d m o r ta lity c u r v e f o r f lo w e r b u d s e v a lu a te d b y th e tis s u e b r o w n in g m e th o d ( P r o e b s tin g a n d S a k a i, 1 9 7 9 ). T h e L T E te m p e r a tu r e v a lu e s a g r e e w ith t e m p e r a tu r e s a t w h ic h 5 0 % o f th e b u d s a r e k ille d f o r s e v e ra l p e a c h ( Q u a m m e e t a l., 1 9 7 5 ) a n d a p r ic o t c u ltiv a r s ( A s h w o r th e t a l., 1 9 8 1 ). T h e m e a n L T E t e m p e r a tu r e o f g r a p e b u d s f a lls w ith in 1 °C o f th e te m p e r a tu r e th a t k ills 5 0 % o f th e b u d s ( A n d r e w s e t a l., 1 9 8 4 ; Q u a m m e , 1 9 8 6 ).

Summary T h e a v o id a n c e o f f r e e z in g in b u d s b y d e e p s u p e r c o o lin g r e p r e s e n ts a u n iq u e s u r v iv a l a d a p ta tio n in m a n y w o o d y p la n t s p e c ie s . In b u d s th a t d e e p s u p e r c o o l, ic e is s e g r e g a te d in p r e f e r r e d s ite s o f f r e e z in g . T h e m e c h a n is m o f th is s e g r e g a tio n is n o t f u lly u n d e r s to o d b u t a p p e a r s to in v o lv e tis s u e s tr u c tu r e , c e ll w a ll p o r o s ity , a n d d i f f e r e n c e s in w a te r p o te n tia l. S e a s o n a l c h a n g e s in b u d h a r d in e s s a r e r e la te d to c h a n g e s in th e s u p e r c o o lin g p o in t th a t c a n b e p r e d ic te d f r o m c h a n g e s in te m p e r a ­

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tu r e e x p o s u r e . T h e s u p e r c o o lin g c a p a c ity o f b u d s lim its th e n o r th e r n d is tr ib u tio n o f s e v e r a l w o o d y p la n t s p e c ie s . T h e a d a p tiv e v a lu e o f th e d e e p s u p e r c o o lin g m e c h a ­ n is m in b u d s is u n c le a r , b u t m e a s u r e m e n t o f th e s u p e r c o o lin g p o in t b y th e r m a l a n a ly s is c a n b e u s e d to d e te r m in e b u d h a r d in e s s .

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A n e x o th e r m s e n s o r f o r m e a s u rin g th e c o ld h a rd in e s s o f d e e p s u p e r c o o le d f lo w e r b u d s b y d if f e r e n tia l th e rm a l a n a ly s is . H o rtS c ie n c e 1 8 :7 7 78. A n d re w s , P . K „ P r o e b s tin g , E . L ., a n d G ro s s , D . C . 1 9 8 3 b . D iffe re n tia l th e rm a l a n a ly s is a n d fre e z in g in ju r y o f d e a c c lim a tin g p e a c h a n d s w e e t c h e r r y re p ro d u c tiv e o rg a n s . J. A m . S o c . H o rt. S ci. 1 0 8 :7 5 5 759. A n d re w s , P. K ., P r o e b s tin g , E . L ., Jr., a n d S w e e t L e e , C . 1 98 7 . A c o n c e p tu a l m o d el o f th e c h a n g e s in d e e p s u p e r c o o lin g o f d o r m a n t s w e e t c h e r r y f lo w e r b u d s . J. A m . S o c . H o rt. S c i. 1 1 2 :3 2 0 -3 2 4 . A n d re w s , P . K ., S a n d r id g e , C . R ., I ll, a n d T o y a m a , T . K . 1 9 8 4 . 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T h e r e la tio n s h ip b e tw e e n v a s c u la r d if f e r e n ­ tia tio n a n d d is tr ib u tio n o f ic e w ith in f o r s y th ia f lo w e r b u d s . P la n t C e ll E n v iro n .. 1 5 :6 0 7 -6 1 2 . B ie rm a n n , J., S tu s h n o ff, C ., a n d B u rk e , M . J. 1 9 7 9 . D iff e r e n tia l th e rm a l a n a ly s is a n d fre e z in g in ju ry in c o ld h a rd y b lu e b e rry f lo w e r b u d s . J. A m . S o c . H o rt. S c i. 1 0 4 :4 4 4 -4 4 9 . B u rk e , M . J ., a n d S tu s h n o f f , C . 1 97 8 . F r o s t h a rd in e s s : A d is c u s s io n o f p o s s ib le m o le c u la r c a u s e s o f in ju r y w ith p a r tic u la r r e f e r e n c e to d e e p s u p e r c o o lin g o f w a te r. P a g e s 1 9 7 -2 2 5 in: S tre s s P h y s io lo g y in C r o p P la n ts . H . M u s s e ll a n d R . S ta p le s , e d s . W ile y & S o n s , N e w Y o rk . B u rk e , M . J., G u s ta , L . V ., Q u a m m e , H . A ., W e is e r, C . J ., a n d L i, P . H . 1 9 7 6 . 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t h e s is . U n i v e r s i t y o f M i n n e s o t a , S t. P a u l. G r a h a m , P . R ., a n d M u l l i n , R . 1 9 7 6 a . T h e d e t e r m i n a t i o n o f l e th a l f r e e z i n g te m p e r a t u r e s in b u d s a n d s t e m s o f d e c i d u o u s a z a l e a b y a f r e e z i n g c u r v e m e th o d . J . A m . S o c . H o r t. S e i. 1 0 1 :3 - 7 . G r a h a m , P . R ., a n d M u l l i n , R . 1 9 7 6 b . A s t u d y o f f l o w e r b u d h a r d i n e s s in a z a l e a . J. A m . S o c . H o r t. S e i. 1 0 1 :7 - 1 0 . G r i f f i t h , M ., A la , P ., Y a n g , D . S . C ., H o u , W ., a n d M o f f a tt, B . A . 1 9 9 2 . A n ti f r e e z e p r o t e i n p r o d u c e d e n d o g e n o u s l y in w i n t e r r y e le a v e s . P l a n t P h y s i o l . 1 0 0 :5 9 3 - 5 9 6 . H o m s h a w , L . G . 1 9 8 0 . F r e e z i n g a n d m e l t i n g t e m p e r a t u r e h y s t e r e s i s o f w a t e r in p o r o u s m a t e r ia ls : A p ­ p l i c a t i o n to s t u d y o f p o r e f o r m . J. S o il S e i. 3 1 : 3 9 9 - 4 1 4 . H o n g , S ., a n d S u c o f f , E . 1 9 8 2 . R a p id i n c r e a s e in d e e p s u p e r c o o l i n g o f x y l e m p a r e n c h y m a . P la n t P h y s io l. 6 9 :6 9 7 -7 0 0 . I s h i k a w a , M „ a n d S a k a i , A . 1 9 8 1 . F r e e z i n g a v o i d a n c e m e c h a n i s m s b y s u p e r c o o l i n g in s o m e

R hodo­

dendron f l o w e r b u d s w i t h r e f e r e n c e to w a t e r r e la t i o n s . P l a n t C e ll P h y s io l. 2 2 :9 5 3 - 9 6 7 . I s h i k a w a , M ., a n d S a k a i , A . 1 9 8 2 . C h a r a c t e r i s t i c s o f f r e e z i n g a v o i d a n c e in c o m p a r i s o n w i t h f r e e z in g t o l e r a n c e : A d e m o n s t r a t i o n o f e x t r a o r g a n f r e e z i n g . P a g e s 3 2 5 - 3 4 0 in : P la n t C o l d H a r d i n e s s a n d F r e e z i n g S tr e s s : M e c h a n i s m s a n d C r o p I m p li c a ti o n s . V o l. 1. P . H . L i a n d A . S a k a i, e d s . A c a d e m ic P re ss, N e w Y o rk . I s h i k a w a , M ., a n d S a k a i , A . 1 9 8 5 . E x t r a o r g a n f r e e z i n g in w i n t e r i n g f l o w e r b u d s o f

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Prunus, s u b g e n u s Padus, f l o w e r b u d s .

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Rhododendron f l o w e r b u d s in

r e l a t i o n to c o o l i n g r a t e a n d c o l d h a r d i n e s s . P l a n t C e ll P h y s i o l . 2 1 :1 2 0 5 - 1 2 1 6 . K a k u , S ., T a w a y a , M „ a n d J e o n , F . B . 1 9 8 1 . S u p e r c o o l i n g a b i l i t y , w a t e r c o n t e n t , a n d h a r d i n e s s o f

R hododendron f l o w e r b u d s d u r i n g c o l d a c c l i m a t i o n a n d d e a c c li m a t i o n . P la n t C e ll P h y s i o l . 2 2 : 1 5 6 1 1569. L e v i tt , J . 1 9 7 2 . T h e h a r d i n e s s o f p l a n t s . A c a d e m i c P r e s s , N e w Y o r k . M a r c e ll o s , H ., a n d S i n g l e , W . V . 1 9 7 6 . I c e n u c l e a t i o n o n w h e a t . A g r ic . M e t e r o r o l . 1 6 :1 2 5 - 1 2 9 . M a z u r , P . 1 9 6 5 . T h e r o l e o f c e l l m e m b r a n e s in f r e e z i n g o f s i n g l e c e l l s . A n n . N e w Y o r k A c a d . S e i. 1 2 5 :6 5 8 - 6 7 6 . M o n e t, R ., a n d B a s t a r d , Y . 1 9 8 0 . E tu d e d u m é c a n i s m e d u g e l s u r d e s b o u r g e o n s f l o r a u x e t f l e u r s d u P ê c h e r , p a r a n a l y s e th e r m iq u e . C . R . A c a d . S e i. P a r i s , S e r . D 2 9 1 : 1 1 3 - 1 1 6 . N u s , N . L ., W e i g l e , J. L ., a n d S c h o r a d l e , J. J . 1 9 8 1 . S u p e r i m p o s e d a m p l i f i e d e x o t h e r m d i f f e r e n t i a l a n a l y s i s s y s t e m . H o r t S c i e n c e 1 6 :7 5 3 - 7 5 4 . O l i e n , C . R . 1 9 6 7 . F r e e z i n g s t r e s s e s a n d s u r v i v a l . A n n u . R e v . P l a n t P h y s io l. 1 8 : 3 8 7 - 4 0 8 . P i e r q u e t , P ., a n d S t u s h n o f f , C . 1 9 8 0 . R e la ti o n s h i p s o f lo w t e m p e r a t u r e e x o t h e r m to c o l d i n j u r y in

Vitis

riparia M i c h x . A m . J . E n o l. V itic . 3 1 :1 - 6 . P i e r q u e t , P ., S t u s h n o f f , C ., a n d B u r k e , M . J. 1 9 7 7 . L o w t e m p e r a t u r e e x o t h e r m s in s te m a n d b u d ti s s u e s of

Vitis riparia M i c h x . J . A m . S o c . H o r t. S e i. 9 7 : 6 0 8 - 6 1 3 .

P r o e b s t i n g , E . L ., J r ., 1 9 7 0 . R e la ti o n o f f a ll a n d w i n t e r t e m p e r a t u r e s to f l o w e r b u d b e h a v i o r a n d w o o d h a r d i n e s s o f d e c i d u o u s f r u i t tr e e s . H o r t S c i e n c e 5 :4 2 2 - 4 2 4 . P r o e b s t i n g , E . L ., J r ., a n d M i l l s , H . H . 1 9 7 2 . A c o m p a r i s o n o f h a r d i n e s s r e s p o n s e s in f r u i t b u d s o f ‘B i n g ’ c h e r r y a n d ‘E l b e r t a ’ p e a c h . J . A m . S o c . H o r t. S e i. 9 7 : 8 0 2 - 8 0 6 . P r o e b s t i n g , E . L ., J r ., a n d S a k a i , A . 1 9 7 9 . D e t e r m i n i n g T S(I o f p e a c h f l o w e r b u d s w i t h e x o t h e r m a n a l y ­ s is . H o r t S c i e n c e 1 4 :5 9 7 - 5 9 8 . Q u a m m e , H . A . 1 9 7 4 . A n e x o t h e r m i c p r o c e s s in v o l v e d in f r e e z i n g i n j u r y to f l o w e r b u d s o f s e v e r a l P r u n u s s p e c i e s . J. A m . S o c . H o r t. S e i. 9 9 : 3 1 5 - 3 1 7 . Q u a m m e , H . A . 1 9 7 6 . R e l a t i o n s h i p o f th e lo w t e m p e r a t u r e e x o t h e r m to a p p l e a n d p e a r p r o d u c t i o n in N o r t h A m e r i c a . C a n . J. P l a n t S e i. 5 6 : 4 9 3 - 5 0 0 . Q u a m m e , H . A . 1 9 7 8 . M e c h a n i s m o f s u p e r c o o l i n g in o v e r w i n t e r i n g p e a c h f l o w e r b u d s . J . A m . S o c .

Deep Supercooling in Buds

199

H o rt. S c i. 1 0 3 :5 7 - 6 1 . Q u a m m e , H . A . 1 9 8 3 . R e la t io n s h ip o f a ir te m p e ra tu r e to w a te r c o n te n t a n d s u p e r c o o lin g o f o v e r w in te r ­ in g p e a c h f lo w e r b u d s . J . A m . S o c . H o rt. S c i. 1 0 8 :6 9 7 -7 0 1 . Q u a m m e , H . A . 1 9 8 6 . U s e o f th e r m a l a n a ly s e s to m e a s u re th e fr e e z in g re s is ta n c e o f g ra p e b u d s . C a n . J. P la n t S c i. 6 6 :9 4 5 -9 5 2 . Q u a m m e , H . A . 1 9 9 1 . A p p lic a tio n o f th e rm a l a n a ly s is to b r e e d in g f ru it c ro p s f o r in c re a s e d c o ld h a r d i­ n e s s . H o rtS c ie n c e 2 6 :5 1 3 - 5 1 7 . Q u a m m e , H . A ., a n d G u s ta , L . V . 1 9 8 7 . R e la tio n s h ip o f ic e n u c le a tio n a n d w a te r s ta tu s to f re e z in g p a tte rn s in d o rm a n t p e a c h f lo w e r b u d s . H o r tS c ie n c e 2 2 :4 6 5 -4 6 7 . Q u a m m e , H . A ., L a y n e , R . E . C ., J a c k s o n , H . O ., a n d S p e a rm a n , G . A . 1 9 7 5 . A n im p ro v e d e x o th e rm m e th o d f o r m e a s u r in g c o ld h a r d in e s s o f p e a c h f lo w e r b u d s . H o r tS c ie n c e 1 0 :5 2 1 -5 2 3 . Q u a m m e , H . A ., L a y n e , R . E . C ., a n d R o n a ld , W . G . 1 98 2 . R e la tio n s h ip o f s u p e r c o o lin g to c o ld h a r d i­ n e s s a n d n o rth e rn d i s t r ib u t i o n o f s e v e ra l c u ltiv a te d a n d n a tiv e P ru n u s s p e c ie s a n d h y b rid s . C a n . J. P la n t S c i. 6 2 :1 3 7 -1 4 8 . Q u a m m e , H . A ., W e is e r, C . J ., a n d S tu s h n o ff, C . 1 9 7 3 . T h e m e c h a n is m o f f re e z in g in ju ry in x y le m o f w in te r a p p le tw ig s . P la n t P h y s io l. 5 1 :2 7 3 -2 7 7 . R a ja s h e k a r , C . B . 1 9 8 9 . S u p e r c o o lin g c h a r a c te ris tic s o f is o la te d p e a c h f lo w e r b u d p rim o rd ia . P la n t P h y s io l. 8 9 :1 0 3 1 -1 0 3 4 . R a s m u s s e n , D . H ., a n d M a c K e n z ie , A . P. 1 9 7 2 . E ffe c t o f s o lu te o n ic e - s o lu tio n in te rfa c ia l fre e e n e rg y , c a lc u la tio n fro m m e a s u r e d h o m o g e n e o u s n u c le a tio n te m p e ra tu re s . P a g e s 1 2 6 -1 4 5 in : W a te r S tr u c ­ tu re a t th e W a te r P o ly m e r In te rfa c e . H . H . G . J e llin e c k , ed . P le n e u m P re ss , N e w Y o rk . R a s m u s s e n , D . H ., M a c a u le y , M . N ., a n d M a c K e n z ie , A . P. 1 9 7 5 . S u p e r c o o lin g a n d n u c le a tio n in ic e in s in g le c e lls. C ry o b io l. 1 2 :3 2 8 -3 3 9 . S a k a i, A . 1 9 7 8 . L o w te m p e r a tu r e e x o th e r m s o f w in te r b u d s o f h a rd y c o n if e rs . P la n t C e ll P h y s io l. 1 9 :1 4 3 9 -1 4 4 6 . S a k a i, A . 1 9 7 9 a. D e e p s u p e r c o o lin g o f w in te r f lo w e r b u d s o f C o m u s flo r id a . L . H o rtS c ie n c e 1 4 :6 9 -7 0 . S a k a i, A . 1 9 7 9 b . F r e e z in g a v o id a n c e m e c h a n is m o f p r im o r d ia l s h o o ts o f c o n if e r b u d s . P la n t C e ll P h y s io l. 2 0 :1 3 8 1 . T u m a n o v , I. I., K r a s a v ts e v , O . A ., a n d T r u n o v a , T . I. 1 9 6 9 . In v e s tig a tio n o f ic e fo rm a tio n p ro c e s s in p la n ts b y m e a s u rin g h e a t e v o lu tio n . S o v ie t P la n t P h y s io l. 1 6 :7 5 4 -7 6 0 . W a rm u n d , M . R ., a n d G e o r g e , M . F . 1 9 9 0 . F r e e z in g s u r v iv a l a n d s u p e r c o o lin g in p rim a ry a n d s e c o n ­ d a ry b u d s o f R u b u s s p p . C a n . J. P la n t S c i. 7 0 :8 9 3 -9 0 4 . W a rm u n d , M . R ., G e o r g e , M . F ., a n d C u m b ie , B . G . 1 9 8 8 . S u p e r c o o lin g in ‘D a r r o w ’ b la c k b e r r y b u d s . J. A m . S o c . H o rt. S c i. 1 1 3 :4 1 8 -4 2 2 . W a rm u n d , M . R ., G e o r g e , M ., a n d T a k e d a , F . 1 9 9 1 . S u p e r c o o lin g in flo ra l b u d s o f ‘D a n k a ’ b l a c k a n d ‘R e d L a k e ’ r e d c u rra n ts . J. A m . S o c . H o rt. S c i. 1 1 6 :1 0 3 0 -1 0 3 4 . W e ig a n d , K . M . 1 9 0 6 . S o m e s tu d ie s r e g a r d in g th e b io lo g y o f b u d s a n d tw ig s in w in te r. B o t. G a z. 1 0 3 :3 7 3 -4 2 4 . W is n ie w s k i, M ., a n d A s h w o r th , E . N . 1 9 8 6 . A c o m p a r is o n o f s e a s o n a l u ltr a s tru c tu re c h a n g e s in s te m tis s u e s o f p e a c h (P r u n u s p e r s ic a ) th a t e x h ib it c o n tr a s tin g m e c h a n is m s o f c o ld h a rd in e s s . B o t. G a z. 1 4 7 :4 0 7 -4 1 7 . W is n ie w s k i, M ., A s h w o r th , E . N ., a n d S c h a f fe r , K . 1 98 7 . T h e u s e o f la n th a n u m to c h a r a c te riz e cell w a ll p e r m e a b ility in r e la tio n to d e e p s u p e r c o o lin g a n d e x tr a c e llu la r f re e z in g in w o o d y p la n ts . I. I n ­ te r g e n e r ic c o m p a r is o n s b e tw e e n P ru n u s, C o m u s a n d S a lix . P r o to p la s m a 1 3 9 :1 0 5 -1 1 6 . W o lf , T . K ., a n d P o o l, R . M . 1 9 8 7 . F a c to r s a f fe c tin g e x o th e r m d e te c tio n in th e d if f e r e n tia l th e rm a l a n a ly s is o f g r a p e v in e d o r m a n t b u d s . J. A m . S o c. H o rt. S ci. 1 1 2 :5 2 0 -5 2 5 .

CHAPTER 11

The Roles of Ice Nucleators in Cold Tolerant Invertebrates John G. Duman, T. Mark Olsen, King Lun Yeung, and Fred Jerva

I c e n u c le a to r s i n itia te h e te r o g e n e o u s n u c lé a tio n b y o r g a n iz in g w a te r m o le c u le s in to e m b r y o c r y s ta ls o f c r itic a l s iz e a t te m p e r a tu r e s a b o v e th o s e a t w h ic h h o m o g e ­ n e o u s n u c lé a tio n w o u ld o c c u r . In f r e e z e - a v o id in g a n im a ls , th e p r e v e n tio n o f i n o c u ­ la tiv e f r e e z in g a n d e x te n s io n o f s u p e r c o o lin g a b ilitie s to te m p e r a tu r e s b e y o n d th o s e lik e ly to b e e x p e r ie n c e d a r e o f o b v io u s im p o r ta n c e . C o n s e q u e n tly , e lim in a tio n o f ic e n u c le a to r s , e ith e r o v e r e v o lu tio n a r y o r s h o r te r (i.e ., s e a s o n a l) tim e f r a m e s , a n d /o r m a s k in g o r i n h ib itio n o f ic e n u c lé a tio n a c tiv ity b y a n tif r e e z e s in w in te r b e ­ c o m e s c r u c ia l. In c o n tr a s t, in m a n y f r e e z e - to le r a n t a n im a ls , th e r e a p p e a r s to b e a r e q u ir e m e n t th a t ic e f o r m a tio n b e in itia te d a t “ f a ir ly h ig h ” s u b z e r o te m p e r a tu r e s , th u s a p p a r e n tly p r o v i d i n g s e le c tio n p r e s s u r e f o r th e e v o lu tio n o f e x tr a c e llu la r ic e n u c le a to r s f o r th is p u r p o s e . H o w e v e r , n u c lé a tio n te m p e r a tu r e s o f f r e e z e - to le r a n t s p e c ie s v a ry w id e ly , a n d m a n y s p e c ie s s e e m n o t to h a v e th is r e q u ir e m e n t. T h u s , th e a b s e n c e o r p r e s e n c e o f ic e n u c le a to r s is c r itic a l to th e o v e r w in te r in g s u c c e s s o f b o th f r e e z e - a v o i d i n g a n d f r e e z e - to le r a n t s p e c ie s . F r e e z e - a v o id in g s p e ­ c ie s m u s t e lim in a te o r m a s k ic e n u c le a to r s , w h e r e a s m a n y f r e e z e - to le r a n t s p e c ie s h a v e a p p a r e n tly s e le c te d f o r p o te n t ic e n u c le a to r s . W h ile th is tr e n d a p p lie s to v e r ­ te b r a te s ( s e e C h a p te r 1 2 ) a s w e ll a s in v e r te b r a te s , th is r e v ie w w ill c o n c e r n th e la t­ ter.

Freeze-A voiding Species W h ile te m p e r a tu r e s a b o v e th o s e a t w h ic h f r e e z in g o c c u r s m a y in itia te le th a l c h ill, c o m a , e tc ., f r e e z e - a v o id in g o r g a n is m s d ie if ic e f o r m s in th e ir tis s u e s . T h e r e ­ fo re , a d a p ta tio n s th a t l o w e r th e t e m p e r a tu r e a t w h ic h b o d y f lu id s r e m a in liq u id c o n f e r s ig n if ic a n t a d v a n ta g e s to th e s e o r g a n is m s . A n u m b e r o f p r o c e s s e s m a y c o n ­ tr ib u te to a v o id a n c e o f f r e e z in g . S o m e o f th e s e a re a n tif r e e z e p r o d u c tio n , ic e n u c le a to r r e m o v a l, p r e v e n tio n o f in o c u la tiv e f r e e z in g , m ic r o h a b ita t s e le c tio n , d e s ic c a tio n , a n d , o f c o u r s e , a p p r o p r ia te a c c lim a tiz a tio n p r o c e s s e s to c o n tr o l th e tim in g o f th e s e s e a s o n a l a d a p ta tio n s ( F o r r e v ie w s s e e S o m m e , 1 9 8 2 ; B a u s t a n d

201

202

Duman, Olsen, Yeung, and Jerva

R o ja s , 1 9 8 5 ; Z a c h a r ia s s e n , 1 9 8 5 ; B a le , 1 9 8 7 ; B lo c k , 1 9 9 0 ; L e e a n d D e n lin g e r, 1 9 9 1 ; D u m a n e t a l., 1 9 9 1 a ) . T h e s e a d a p ta tio n s a re n o t m u tu a lly e x c lu s iv e , a n d a n i­ m a ls e x h ib it v a r io u s c o m b in a tio n s o f th e s e p ro c e s s e s . T h is s e c tio n w ill c o n c e n tr a te o n th e b a s ic p h y s io lo g ic a l m e c h a n is m s fo r p r o m o tio n o f s u p e r c o o lin g , n a m e ly , r e ­ m o v a l o f ic e n u c le a to r s , a n tif r e e z e p r o d u c tio n to o v e r c o m e ic e n u c le a to r a c tiv ity , a n d th e in te r a c tio n s o f th e s e tw o a d a p ta tio n s . T h e m a g n itu d e o f th e s u p e r c o o lin g p r o d u c e d b y a n tif r e e z e d e p e n d s g r e a tly o n th e p r e s e n c e o r a b s e n c e o f ic e n u c le a to r s a n d o n th e a c tiv ity o f ic e n u c le a to r s i f th e y a re p r e s e n t. H o w e v e r , u n le s s th e re a re m e c h a n is m s in p la c e to p r e v e n t in o c u la tiv e fre e z in g , s e e d in g a c r o s s th e b o d y s u r ­ fa c e m a y p r e c lu d e th e c h a n c e to s u p e r c o o l. T h e r e f o r e , it is n e c e s s a r y to b rie fly d is ­ c u s s in o c u la tiv e fre e z in g .

Inoculative Freezing P r e v e n tio n o f in o c u la tio n f r o m e x te r n a l ic e b y a p h y s ic a l s u r f a c e b a r r ie r (i.e ., th e w a x - c o a te d c u tic le o f in s e c ts o r th e c o r n if ie d e p ith e liu m o f s o m e v e r te b r a te s ) is o f p a r a m o u n t im p o r ta n c e to a f r e e z e - a v o id in g a n im a l. T h e a b s e n c e o f s u c h a b a r ­ r ie r r e q u ir e s p r o d u c tio n o f s u f f ic ie n t c o n c e n tr a tio n s o f a n tif r e e z e s to lo w e r th e f r e e z in g p o in t o f th e b o d y f lu id s b e lo w e n v ir o n m e n ta l te m p e r a tu r e s . T h is m a y b e e n e r g e tic a lly c o s tly , i f n o t im p o s s ib le , w h e n m ic r o h a b ita t te m p e r a tu r e s a re v e ry lo w . A n tif r e e z e p r o te in s , e v e n th e m o s t a c tiv e o n e s a t h ig h c o n c e n tr a tio n s , a re c a ­ p a b le o f d e p r e s s in g th e h y s te r e tic f r e e z in g p o in t o f w a te r b y o n ly ~ 6 ° C ( D u m a n e t a l., 1 9 9 3 ). A ls o , s in c e th e m o la l f r e e z in g p o in t c o n s ta n t f o r w a te r is 1 .8 6 ° C , e v e n a 5 M c o n c e n tr a tio n o f c o llig a tiv e a n tif r e e z e lo w e r s th e e q u ilib r iu m f r e e z in g p o in t b y o n ly ~ 9 .3 ° C . C o n s e q u e n tly , th e p r e s e n c e o f a p h y s ic a l b a r r ie r to in o c u la tio n is c r u ­ c ia l. T h e d i f f e r e n c e in th e lim ite d s u p e r c o o lin g a b ilitie s o f e a r th w o r m s a f te r h a tc h ­ in g c o m p a r e d to th e p o te n tia lly e x te n d e d c a p a b ilitie s o f in s e c ts , w ith th e ir w a x c o a te d , c h itin o u s c u tic le , illu s tr a te s th is p o in t. In s o m e in s e c ts , h o w e v e r , it is b e c o m in g a p p a r e n t th a t th e r e a r e s e a s o n a l a d a p ta tio n s to in h ib it in o c u la tio n . S o m e s p e c ie s th a t a r e i m m u n e to in o c u la tiv e f r e e z in g in w in te r a r e r e a d ily in o c u la te d a t t e m p e r a tu r e s o n ly s lig h tly b e lo w th e b o d y flu id f r e e z in g p o in t in s u m m e r (R o ja s e t a l., 1 9 9 2 ). T h is s u g g e s ts s e a s o n a l v a r ia tio n in w a x e s a n d /o r o th e r c u tic u la r m o d if i­ c a tio n s . G e h r k e n ( 1 9 9 2 ) s h o w e d th a t in Ip s a c u m in a tu s b e e tle s , h e m o ly m p h a n ti­ f r e e z e p r o te in s p r o te c t a g a in s t in o c u la tiv e f r e e z in g to a g r e a te r e x te n t th a n e x p e c te d b a s e d o n th e m e a s u r e d th e r m a l h y s te r e s is o f th e h e m o ly m p h . F r e e z e - a v o id in g s p e c ie s in w h ic h th e b o d y s u r f a c e h a s lim ite d a b ilitie s to p r e ­ v e n t i n o c u la tiv e f r e e z in g m a y r e ly u p o n c o c o o n s to p r o v id e s o m e b a r r ie r to in o c u ­ la tio n . T h is is th e c a s e in th e n o r th e r n lu m b r ic id e a r th w o r m D e n d r o b a e n a o c ta e d r a . P o s te m b r y o n ic in d iv id u a ls a re re a d ily in o c u la te d , b u t th e c o c o o n s (e g g c a p s u le s ) p r o v id e p r o te c tio n f o r th is f r e e z e - s e n s itiv e s p e c ie s ( H o lm s tr u p , 1 9 9 2 ).

Adaptations That Promote Supercooling I f a n a n im a l is a b le to s to p in o c u la tiv e f r e e z in g , th e n m e c h a n is m s th a t p r o m o te s u p e r c o o lin g a n d d e p r e s s th e ic e n u c le a tio n te m p e r a tu r e b e c o m e c r itic a l, p o te n ­ tia lly a llo w in g th e o r g a n is m to s u r v iv e in e n v ir o n m e n ts w ith m u c h lo w e r te m p e r a ­ tu re s . R e m o v a l o f ic e n u c le a to r s f r o m b o d y flu id s a n d /o r p r o d u c tio n o f a n tif r e e z e s a re c o m m o n a d a p ta tio n s .

Colligative Antifreezes P r o d u c tio n o f h ig h c o n c e n tr a tio n s o f c o m p a tib le s o lu te s , u s u a lly p o ly o ls , w h ic h f u n c tio n a s c o llig a tiv e a n tif r e e z e s h a s lo n g b e e n r e c o g n iz e d a s a n im p o r ta n t a d a p ta ­

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tio n in f r e e z e a v o id a n c e . I n h is c la s s ic s tu d ie s o f p a r a s itic la r v a e o f th e w h e a t s te m s a w f ly , B r a c o n c e p h i, w h ic h s u p e r c o o l to - 4 5 ° C , S a lt ( 1 9 5 9 ) d e m o n s tr a te d th a t g ly c e r o l c o n c e n tr a tio n s a s h ig h a s 5 M a re a c c u m u la te d . H o w e v e r , o n e m u s t b e c a u tio u s in a ttr ib u tin g a ll o f a n o b s e r v e d w in te r s u p e r c o o lin g p o in t d e p r e s s io n to c o llig a tiv e a n tif r e e z e , s in c e in s tu d ie s o f in ta c t o r g a n is m s , o r e v e n o f th e ir h e m o ­ ly m p h , it c a n n o t b e a s c e r ta in e d w h e th e r a d d itio n a l f a c to r s (i.e ., r e m o v a l o f ic e n u ­ c le a to r s , p r e s e n c e o f a n tif r e e z e p r o te in s ) m a y a ls o b e r e s p o n s ib le fo r a p o r tio n o f th e o b s e r v e d s e a s o n a l e x t e n s io n o f th e c a p a c ity to s u p e r c o o l. In f r e e z e - a v o id in g s p e c ie s , it is a d v a n ta g e o u s to m a x im iz e s u p e r c o o lin g p o in t d e p r e s s io n r e s u ltin g f r o m a c c u m u la tio n o f c o llig a tiv e a n tif r e e z e s . Y e t, w h ile in m o s t f r e e z e - to le r a n t s p e c ie s h ig h c o n c e n tr a tio n s o f c r y o p r o te c ta n ts a re c o m m o n , s u p e r c o o lin g m u s t o f te n b e in h ib ite d b y e x tr a c e llu la r ic e n u c le a to r s ( Z a c h a r ia s s e n a n d H a m m e l, 1 9 7 6 ; Z a c h a r ia s s e n 1 9 8 2 , 1 9 9 2 ). I n c o n tr a s t to th e s itu a tio n in f r e e z e a v o id in g o r g a n is m s , e x te n s io n o f s u p e r c o o lin g b y c r y o p r o te c ta n ts is c o u n te r p r o ­ d u c tiv e in f r e e z e - to le r a n t a n im a ls . T h e r e f o r e , q u e s tio n s a r is e a s to w h e th e r f r e e z e a v o id in g s p e c ie s h a v e e v o lv e d m e c h a n is m s to in c r e a s e th e e f f e c ts o f c o llig a tiv e a n tif r e e z e s o n s u p e r c o o lin g b e y o n d th o s e in f r e e z e - to le r a n t s p e c ie s , o r w h e th e r f r e e z e - to le r a n t s p e c ie s h a v e e v o lv e d m e c h a n is m s to lim it th e e f f e c ts o f c o llig a tiv e a n tif r e e z e s o n s u p e r c o o lin g . T h e a p p a r e n t a n s w e r s to th e a b o v e q u e s tio n s a r e y e s , b u t th e e f f e c t o f th e c o lli­ g a tiv e a n tif r e e z e s a p p e a r s to d e p e n d u p o n w h e th e r p o te n t ic e n u c le a to r a c tiv ity is p r e s e n t. S tu d ie s o n w e ll- d e f in e d s y s te m s , in w h ic h c o m p o n e n ts o th e r th a n w a te r a n d a d d e d a n tif r e e z e s a r e id e n tif ie d , a re lim ite d . M a c K e n z ie ( 1 9 7 7 ) d e m o n s tr a te d th a t c o llig a tiv e a n tif r e e z e s d e p r e s s th e h o m o g e n e o u s n u c le a tio n te m p e r a tu r e o f w a ­ te r (A N T ) b y a p p r o x im a te ly tw ic e th e m a g n itu d e o f th e c o llig a tiv e m e ltin g p o in t d e p r e s s io n (A M P ), A N T /A M P = 2 :1 . T h e e f f e c t w a s in d e p e n d e n t o f th e n a tu r e o f th e a d d e d s o lu te ( g lu c o s e , g ly c e r o l, s u c r o s e , N a C l, N H 4F , u re a , e th y le n e g ly c o l) , e x c e p t f o r p o ly m e r s ( P E G , P V P ) , w h ic h h a d a s ig n if ic a n tly g r e a te r e f f e c t (A N T /A M P = - 5 : 1 ) . F r a n k s ( 1 9 8 1 ) p r e s e n te d a th e o r e tic a l a r g u m e n t th a t in d ic a te d th a t th e d e p r e s s io n o f h o m o g e n e o u s n u c le a tio n te m p e r a tu r e p r o d u c e d b y a g iv e n s o lu te is d ir e c tly p r o p o r tio n a l to m e ltin g p o in t d e p r e s s io n . T h e s e d a ta o n e f f e c ts o f s o lu te s o n h o m o g e n e o u s n u c le a tio n a r e p o te n tia lly a p p lic a b le to s p e c ie s th a t la c k ic e n u c le a to r s in w in te r a n d th a t p r o d u c e s ig n if ic a n t c o n c e n tr a tio n s o f c o llig a tiv e a n tif r e e z e s ( M ille r , 1 9 8 2 ; R in g , 1 9 8 2 ; M ille r a n d W e r n e r , 1 9 8 7 ). L ik e w is e , B lo c k a n d Y o u n g ( 1 9 7 9 ) s h o w e d th a t a d d itio n o f g ly c e r o l to d r o p le ts o f w a te r r e s u lte d in A N T /A M P = - 2 : 1 . S in c e th e n u c le a tio n te m p e r a tu r e o f th e w a te r d r o p le ts w ith o u t g ly c e r o l w a s a p p r o x im a te ly —2 1 ° C ( r a n g e o f - 1 5 to —3 0 ° C ), th e a u th o r s c o r r e c tly c o n s id e r e d th is r e s u lt to illu s tr a te th e e f f e c t o f g ly c e r o l o n h e te r o g e n e o u s n u c le a ­ tio n , a lth o u g h th e ic e n u c le a to r s p r e s e n t in th e w a te r w e r e n o t p o te n t o n e s . R e id e t al. ( 1 9 8 5 ) o b ta in e d s im ila r r e s u lts in v e s tig a tin g e f f e c ts o f s o lu te s ( s u c r o s e , g ly c e r o l, e th y le n e g ly c o l, a n d d i m e th y ls u lf o x id e ) o n h e te r o g e n e o u s n u c le a tio n in “ p o lis h e d ” w a te r d r o p le ts , A N T /A M P = - 5 : 3 to 2 :1 , e x c e p t th a t th e r a tio fo r g ly c e r o l w a s - 2 : 3 . In th e p r e s e n c e o f m o r e a c tiv e ic e n u c le a to r s , c o llig a tiv e a n tif r e e z e s a p p e a r to lo w e r n u c le a tio n t e m p e r a tu r e b y a n a m o u n t e q u a l to th e c o llig a tiv e d e p r e s s io n o f th e m e ltin g p o in t. L u s e n a ( 1 9 5 5 ) s h o w e d th a t g ly c e r o l o r N a C l d e p r e s s e s th e h e t­ e r o g e n e o u s n u c le a tio n te m p e r a tu r e in itia te d b y s ilv e r io d id e c r y s ta ls b y a n a m o u n t e q u a l to th e m e ltin g p o i n t d e p r e s s io n . T h is is a t o d d s w ith R e id e t al. ( 1 9 8 5 ) , w h o s h o w e d th a t in th e p r e s e n c e o f g ly c e r o l, e th y le n e g ly c o l, o r D M S O , th e A N T /A M P o b ta in e d u s in g s ilv e r io d id e to in d u c e h e te r o g e n e o u s n u c le a tio n w a s - 5 : 3 , w h e r e a s

204

Duman, Olsen, Yeung, and Jerva

s u c r o s e r e s u lte d in a r a tio o f - 1 : 2 . T h e re a s o n f o r th is d is c r e p a n c y b e tw e e n th e tw o s tu d ie s is n o t o b v io u s . T o o u r k n o w le d g e , th e e f f e c t o f c o llig a tiv e a n tif r e e z e o n h e t e r o g e n e o u s n u c le a tio n in a n a q u e o u s s o lu tio n o f a p u r if ie d b io lo g ic a l (p ro te in ) ic e n u c le a to r h a s n o t b e e n d e te r m in e d . H o w e v e r , s tu d ie s o n in s e c t h e m o ly m p h c o n ­ ta in in g p o te n t ic e n u c le a to r s d e m o n s tr a te th a t n u c le a tio n te m p e r a tu r e is d e p r e s s e d b y a d d itio n o f c o llig a tiv e a n tif r e e z e b y a n a m o u n t e q u a l to m e ltin g p o in t d e p r e s ­ s io n . L e e e t al. ( 1 9 8 1 ) e x te n d e d a n e a r lie r s tu d y b y Z a c h a r ia s s e n a n d H a m m e l ( 1 9 7 6 ) b y d e m o n s tr a tin g t h a t a d d itio n o f a n y o f 11 s o lu te s ( g lu c o s e , g ly c e r o l, s u ­ c r o s e , la c ta te , f r u c to s e , N a C l, tr e h a lo s e , p r o lin e , e t h y le n e g ly c o l, m a n n ito l, a n d P V P ) to h e m o ly m p h o f th e f r e e z e - to le r a n t b e e tle E le o d e s b la n c h a r d i (w h ic h c o n ­ ta in s p o te n t ic e n u c le a to r s ) r e s u lte d in A N T /A M P = - 1 : 1 . T h e a b o v e s u g g e s t th a t a d d itio n o f s o lu te s in th e a b s e n c e o f ic e n u c le a to r s o r in th e p r e s e n c e o f ic e n u c le a to r s th a t a re n o t e s p e c ia lly p o te n t w ill p r o d u c e A N T /A M P r a tio s o f - 2 : 1 . I n c o n tr a s t, w ith th e e x c e p tio n o f th e s tu d y b y R e id e t al. (1 9 8 5 ) , it a p p e a r s th a t a d d itio n o f s o lu te s to a q u e o u s s o lu tio n s c o n ta in in g p o te n t ic e n u c le a ­ to r s r e s u lts in A N T /A M P r a tio s o f - 1 : 1 . N u m e r o u s s tu d ie s h a v e c o r r e la te d m e ltin g p o in t d e p r e s s io n w ith s u p e r c o o lin g p o in t d e p r e s s io n in in ta c t in s e c ts . N o ta b le e a r ly s tu d ie s a r e th o s e o f S a lt ( 1 9 5 9 ) o n B r a c o n c e p h i ( A N T /A M P r a tio o f - 1 : 1 ) a n d S o m m e ( 1 9 6 7 ) o n s e v e r a l s p e c ie s (A N T /A M P r a tio o f - 3 . 7 : 1 ) . B lo c k a n d Y o u n g ( 1 9 7 9 ) f o u n d a r a tio s im ila r to th o s e o f S o m m e ( 1 9 6 7 ) in th e f r e e z e - a v o id in g m ite A la s k o z e te s a n ta r c tic u s . A p a tte r n h a s e m e r g e d th a t in d ic a te s th a t c o llig a tiv e a n tif r e e z e s in f r e e z e - a v o id in g in s e c ts u s u a lly d e p r e s s n u c le a tio n te m p e r a tu r e m o r e th a n m e ltin g p o in t ( r a tio s u s u a lly 2:1 to 3 :1 ). In f r e e z e - to le r a n t s p e c ie s , d e p r e s s io n o f n u c le a tio n te m p e r a tu r e is g e n e ra lly e q u a l to m e ltin g p o in t d e p r e s s io n (S o m m e 1 9 8 2 ; Z a c h a r ia s s e n 1 9 8 5 , 1 9 9 2 ; B lo c k 1 9 9 0 ). T h u s , f r e e z e - a v o id in g s p e c ie s a p p e a r to m a x im iz e e f f e c ts o f c o llig a tiv e a n ti­ f r e e z e s o n d e p r e s s io n o f n u c le a tio n te m p e r a tu r e s , w h ile f r e e z e - to le r a n t s p e c ie s s e e m to m in im iz e th e e f f e c ts . B a s e d o n th e s im p lif ie d in v itr o s y s te m s d e s c r ib e d e a r lie r , th e k e y to w h e th e r s u p e r c o o lin g e f f e c ts a re m a x im iz e d (A N T /A M P o f 2:1 to 3 :1 ) o r m in im iz e d ( A N T /A M P o f - 1 : 1 ) a p p e a r s to d e p e n d u p o n w h e th e r p o te n t ic e n u c le a to r s a r e p r e s e n t ( m o s t f r e e z e - to le r a n t in s e c ts ) o r n o t ( m o s t f r e e z e - a v o id in g s p e c ie s in w in te r ) . I n a d d itio n to th e ir f u n c tio n in f r e e z e - to le r a n t s p e c ie s o f r a is in g th e a b s o lu te n u c le a tio n t e m p e r a tu r e , p o te n t ic e n u c le a to r s a ls o a p p e a r to lim it th e s u p e r c o o lin g e f f e c t o f c r y o p r o te c ta n ts s o th a t A N T /A M P r a tio s a r e - 1 : 1 . In f r e e z e a v o id in g s p e c ie s , r e m o v a l o f p o te n t ic e n u c le a to r s f r o m g u t a n d h e m o ly m p h m a y b e r e q u ir e d to a c h ie v e th e m a x im u m s u p e r c o o lin g e f f e c t f r o m c o llig a tiv e a n ti­ freezes.

Antifreeze Proteins A n tif r e e z e p r o te in s lo w e r th e n o n e q u ilib r iu m f r e e z in g p o in t o f w a te r , b u t n o t th e m e ltin g p o in t, th e r e b y p r o d u c in g a d if f e r e n c e b e tw e e n th e f r e e z in g a n d m e ltin g p o in ts , te r m e d th e r m a l h y s te r e s is . A lth o u g h a n tif r e e z e p r o te in s w e r e f ir s t d is c o v ­ e r e d in p o la r m a r in e f is h ( D e V r ie s , 1 9 6 8 , 1 9 7 1 ), th e y h a v e a ls o b e e n id e n tif ie d in c e r ta in a r th r o p o d s in c lu d in g in s e c ts (D u m a n 1 9 7 7 a ; D u m a n e t a l., 1 9 9 1 b , 1 9 9 3 ), s p id e r s ( D u m a n , 1 9 7 9 ), m ite s ( B lo c k a n d D u m a n , 1 9 8 9 ), a n d c e n tip e d e s ( T u r s m a n e t a l., 1 9 9 4 ). T h e e d e e t a l. ( 1 9 7 6 ) r e p o r te d th e r m a l h y s te r e s is a c tiv ity in th e in te r ­ tid a l m u s s e l M y tilu s e d u lis f r o m E u r o p e ; h o w e v e r , M . e d u lis c o lle c te d in J a n u a r y o n C a p e C o d d id n o t e x h i b i t th e r m a l h y s te r e s is a c tiv ity a n d la c k e d th e r e c r y s ta lli­ z a tio n in h ib itio n a c tiv ity a s s o c ia te d w ith a n tif r e e z e p r o te in s ( D u m a n a n d K n ig h t,

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u n p u b lis h e d ). M o s t in v e r te b r a te s th a t p r o d u c e th e r m a l h y s te r e s is p r o te in s a re f r e e z e a v o id in g , a n d p r o d u c tio n o f th e p r o te in s is s e a s o n a l. C o r r e la tio n s b e tw e e n in c r e a s e d th e r m a l h y s te r e s is a c tiv ity a n d lo w e r e d s u p e r c o o lin g p o in ts a re g e n e r a lly o b v io u s in m o s t s tu d ie s o f a n tif r e e z e p r o te in s in te r r e s tr ia l a r th r o p o d s , w h ic h le d to s p e c u la tio n th a t a n tif r e e z e p r o te in s w e r e r e s p o n s ib le f o r e x te n d e d s u p e r c o o lin g . H o w e v e r , c o r r e la tio n s b e tw e e n a n tif r e e z e a c c u m u la tio n a n d e x te n d e d s u p e r c o o lin g d o n o t p r o v e a c a u s e a n d e f f e c t, a n d th e m a g n itu d e o f th e e f f e c t a ttr ib u ta b le to a n ti­ f r e e z e m a y b e d if f ic u lt to a s c e r ta in . In s p ite o f th is c a u tio n , s u c h c o r r e la tio n s b e ­ tw e e n a n tif r e e z e p r o te in s a n d e x te n d e d s u p e r c o o lin g w e r e p a r tic u la r ly e n tic in g in th o s e a n tif r e e z e - p r o te in - p r o d u c in g s p e c ie s th a t d o n o t a c c u m u la te p o ly o ls in w in te r ( D u m a n 1 9 7 7 a ; P a tte r s o n a n d D u m a n , 1 9 7 8 ). T h e f ir s t e x p e r im e n ta l e v id e n c e th a t a n tif r e e z e p r o te in s m ig h t e x te n d s u p e r c o o l­ in g w a s p r o v id e d b y Z a c h a r ia s s e n a n d H u s b y ( 1 9 8 2 ) , w h o d e m o n s tr a te d a r e la tio n ­ s h ip b e tw e e n a m o u n t o f t h e r m a l h y s te r e s is m e a s u r e d a n d s iz e o f th e s e e d c r y s ta l u s e d in th e m e a s u r e m e n t. T h e s e d a ta w e r e e x tr a p o la te d to e m b r y o c r y s ta l d i m e n ­ s io n s to s u g g e s t th a t a n tif r e e z e p r o te in s c o u ld e x p la in th e le v e l o f s u p e r c o o lin g s e e n in h e m o ly m p h s a m p le s ( a p p r o x im a te ly - 2 0 ° C ) . T h e f ir s t d ir e c t e v id e n c e th a t a n tif r e e z e p r o te in s c o u ld in h i b i t ic e n u c le a to r s w a s p r o v id e d b y P a r o d y - M o r r e a le e t al. ( 1 9 8 8 ) , w h o s h o w e d th a t a d d itio n o f g ly c o p r o te in a n tif r e e z e f r o m A n ta r c tic fis h s h if te d th e ic e n u c le a tio n s p e c tr u m o f w a te r c o n ta in in g ic e n u c le a tin g b a c te r ia to lo w e r te m p e r a tu r e s . S im ila r ly , p u r if ie d a n tif r e e z e p r o te in s fro m la rv a e o f th e b e e tle D e n d r o id e s c a n a d e n s is in h ib it th e a c tiv ity o f c e r ta in h e m o ly m p h ic e n u c le a tin g p r o te in s o f th is s a m e s p e c ie s ( D u m a n e t a l., 1 9 9 1 b , 1 9 9 2 ). T h e s e s a m e a n tif r e e z e p r o te in s , h o w e v e r , d id n o t i n h ib it th e a c tiv ity o f h e m o ly m p h lip o p r o te in ic e n u c le a ­ to rs f r o m f r e e z e - to le r a n t la r v a e o f th e c r a n e f ly T ip u la tr iv itta ta (W u a n d D u m a n , 1 9 9 1 ). B a u s t a n d Z a c h a r ia s s e n ( 1 9 8 3 ) f o u n d th a t th e r m a l h y s te r e s is p r o te in s f r o m R h a g iu m in q u is ito r b e e tle s d id n o t in h ib it ic e n u c le a to r s f r o m th e s a m e s p e c ie s . S im ila r ly , a n tif r e e z e p r o te in s f r o m th e b e e tle Ip h th im u s la e v is s im u s d id n o t in h ib it h e m o ly m p h ic e n u c le a to r s f r o m f r e e z e - to le r a n t E le o d e s b la n c h a r d i b e e tle s . T h u s , a n tif r e e z e p r o te in s c a n in h i b i t s o m e ic e n u c le a to r p r o te in s b u t n o t o th e rs .

Removal of Ice Nucleators T h e o r e tic a lly , i f a n o r g a n is m r e m o v e d a ll ic e n u c le a to r s a n d p r e v e n te d in o c u la ­ tiv e f r e e z in g , f r e e z in g c o u l d b e a v o id e d d o w n to h o m o g e n e o u s n u c le a tio n t e m p e r a ­ tu re s . I n a d d itio n , c o llig a tiv e a n tif r e e z e s m ig h t f u r th e r e x te n d s u p e r c o o lin g b e y o n d th e h o m o g e n e o u s n u c le a tio n te m p e r a tu r e . I f p o te n t ic e n u c le a to r s a re r e m o v e d , c o l ­ lig a tiv e a n tif r e e z e s c a n lo w e r th e h e te r o g e n e o u s n u c le a tio n te m p e r a tu r e b y a p ­ p r o x im a te ly tw ic e th e m e ltin g p o in t d e p r e s s io n . A ls o , a n tif r e e z e p r o te in s a p p e a r to b e c a p a b le o f in h ib itin g s o m e ic e n u c le a to r s , b u t n o t o th e r s , a n d th e r e f o r e th e r e ­ m o v a l o f u n a f f e c te d ic e n u c le a to r s m a y b e c r itic a l. C o n s e q u e n tly , r e m o v a l o f ic e n u c le a to r s , e ith e r o n a s e a s o n a l o r e v o lu tio n a r y tim e s c a le , a p p e a r s to b e a n im p o r ­ ta n t a d a p ta tio n in f r e e z e - a v o id in g s p e c ie s . It h a s lo n g b e e n r e c o g n iz e d ( W a lla c e a n d B e a r d , 1 9 4 3 ; S a lt, 1 9 5 3 ) th a t in s e c ts m a y in g e s t ic e n u c le a to r s . T h e r e f o r e , c e s s a tio n o f f e e d in g a n d c le a r in g th e g u t a re im p o r ta n t a d a p ta tio n s in m a n y f r e e z e - a v o id in g s p e c ie s . K ru n ic a n d R a d o v ic ( 1 9 7 4 ) s u g g e s te d th a t a 2 0 ° C d e p r e s s io n in n u c le a tio n te m p e r a tu r e o f th e s o lita r y b e e M e g a c h ile r o tu n d a ta w a s p r im a r ily d u e to g u t e v a c u a tio n . W h ile th e r e m a y b e e x ­ c e p tio n s to th is ( B a u s t a n d R o ja s , 1 9 8 5 ), a n d w h ile th e p e r c e n ta g e o f th e o b s e r v e d s u p e r c o o lin g e n h a n c e m e n t a ttr ib u ta b le to a n y g iv e n a d a p ta tio n is d if f ic u lt to d e ­

206

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te r m in e in a n in t a c t o r g a n is m , th e e v id e n c e is o v e r w h e lm in g th a t, in m a n y f r e e z e a v o id in g s p e c ie s , r e m o v a l o f ic e n u c le a to r s f r o m th e g u t p r i o r to th e o n s e t o f c o ld te m p e r a tu r e s is c r itic a l. ( S e e th e r e v ie w s b y S o m m e , 1 9 8 2 ; Z a c h a r ia s s e n , 1 9 8 5 ; C a n n o n a n d B lo c k , 1 9 8 8 ). P e r h a p s th e m o s t c o n v in c in g e v id e n c e c o m e s f r o m e x ­ p e r im e n ts in w h ic h th e o r g a n is m is fe d p o te n t ic e n u c le a to r s w h ic h in d u c e a s ig ­ n if ic a n t in c r e a s e in n u c le a tio n te m p e r a tu r e . F o r e x a m p le , s ta r v a tio n o f fie ld c o lle c te d C o lle m b o la , C r y p to p y g u s a n ta r c tic u s , d u r in g th e A n ta r c tic s u m m e r lo w ­ e re d th e ir s u p e r c o o lin g p o in t. S u b s e q u e n t f e e d in g o f a n h o m o g e n a te o f m o s s tu r f c o n ta in in g n u c le a to r s r a is e d s u p e r c o o lin g p o in ts , w h ile f e e d in g o n p u r if ie d g re e n a lg a e la c k in g ic e n u c le a to r s d id n o t (S o m m e a n d B lo c k , 1 9 8 2 ). R e c e n t s tu d ie s , w h ic h h a v e im p lic a te d ic e n u c le a tin g b a c te r ia in g u t f lu id n u c le a tio n , h a v e r e in ­ f o r c e d th is p o i n t ( s e e C h a p te r 1 4 ). S u p e r c o o lin g p o in ts o f la d y b e e tle s , H ip p o d a m ia c o n v e r g e n s , f e d w a te r c o n ta in in g ic e n u c le a tin g b a c te r ia (P s e u d o m o n a s s y r in g a e o r E r w in ia h e r b ic o la ) in c r e a s e d f r o m - 1 6 t o - 4 ° C ( S tr o n g - G u n d e r s o n e t a l., 1 9 9 0 ). I c e n u c le a tin g b a c te r ia ( E n te r o b a c te r a g g lo m e r a n s a n d E . ta y lo r a e ) w e re is o la te d f r o m g u ts o f s u m m e r - c o lle c te d b e e tle s , H . c o n v e r g e n s a n d C e r a to m a tr ifu r c a ta ( L e e e t a l., 1 9 9 1 ). F e e d in g th e s e to H. c o n v e r g e n s c a u s e d s u p e r c o o lin g p o in ts o f th e in s e c ts to in c r e a s e f r o m - 1 6 to - 3 ° C . T h e s e r e s u lts s u g g e s t th a t ic e n u c le a tin g b a c te r ia m u s t b e r e m o v e d o r m a s k e d in f r e e z e - a v o id in g in s e c ts p r io r to w in te r. L a r v a e o f th e b e e tle D e n d r o id e s c a n a d e n s is h a v e ic e n u c le a tin g b a c te r ia in th e g u t in s u m m e r a n d t h r o u g h p a r t o f th e a u tu m n . B y w in te r , th e g u t h a s b e e n e v a c u a te d a n d ic e n u c le a tin g b a c te r ia c a n n o lo n g e r b e is o la te d f r o m th e g u t (O ls e n a n d D u m a n , 1 9 9 2 ). A ls o , r e c e n t s tu d ie s h a v e s h o w n th a t p u r if ie d D . c a n a d e n s is a n tif r e e z e p r o te in in h ib its th e a c tiv ity o f a n ic e n u c le a tin g a c tiv e is o la te o f P s e u ­ d o m o n a s f l u o r e s c e n s f r o m D . c a n a d e n s is g u t (O ls e n a n d D u m a n , u n p u b lis h e d ). S in c e a n tif r e e z e p r o te in s a r e a c c u m u la te d in th e g u t o f D . c a n a d e n s is b y la te S e p ­ te m b e r ( D u m a n , 1 9 8 4 ), it a p p e a r s th a t a c o m b in a tio n o f a n tif r e e z e p r o te in p lu s g u t e v a c u a tio n to r e m o v e ic e n u c le a to r s p r o te c ts g u t flu id . A p p lic a tio n o f f r e e z e - d r ie d P. s y r in g a e to s to r e d w h e a t o r c o r n c o n ta in in g in s e c t p e s ts r e s u lte d in in c r e a s e s in s u p e r c o o lin g p o in ts o f 4 .7 to 1 1 .9 ° C in v a r io u s in s e c t s p e c ie s , s u g g e s tin g th a t b a c ­ te r ia m ig h t b e u s e d a s b io lo g ic a l in s e c tic id e s to k ill o v e r w in te r in g s to r e d g ra in in ­ s e c t p e s ts ( L e e e t a l., 1 9 9 3 a ). In c o n tr a s t to th e s e a s o n a l r e m o v a l o f g u t ic e n u c le a to r s , w h ic h h a s b e e n in v e s ­ tig a te d f o r m a n y y e a r s , r e m o v a l o f ic e n u c le a to r s f r o m c o m p a r tm e n ts o th e r th a n g u t h a s o n ly r e c e n tly b e e n r e c o g n iz e d . Z a c h a r ia s s e n ( 1 9 8 2 ) d e m o n s tr a te d th a t th e b e e tle B o lito p h a g u s r e tic u la tu s r e m o v e s ic e n u c le a to r s , t h o u g h t to b e in tr a c e llu la r , in w in te r . W i n t e r b e e tle s th a t w e r e w a r m a c c lim a te d , w ith o u t b e in g fe d , r a p id ly ( 3 4 d a y s ) i n c r e a s e d n u c le a tio n te m p e r a tu r e fro m - 3 0 to —1 0 ° C , in d ic a tin g in d u c tio n o f e n d o g e n o u s ic e n u c le a to r s . A ls o , B a k k e n ( 1 9 8 5 ) s h o w e d th a t n u c le a tio n te m ­ p e r a tu r e s d e c r e a s e d s ig n if ic a n tly in tw o a lp in e b e e tle s , w ith o u t a n tif r e e z e p r o d u c ­ tio n , v ia r e m o v a l o f u n id e n tif ie d ic e n u c le a to r s . A s im ila r s itu a tio n o c c u r s in f r e e z e - a v o id in g la r v a e o f th e s ta g b e e tle C e r u c h u s p ic e u s , w h ic h lo w e r e d s u p e r ­ c o o lin g p o in ts f r o m - 7 ° C in s u m m e r to b e lo w - 2 0 ° C in w in te r w ith o u t a n tif r e e z e p r o d u c tio n . T h e s e la r v a e b o th c le a r th e g u t a n d r e m o v e h e m o ly m p h lip o p r o te in s w ith ic e n u c le a to r a c tiv ity ( N e v e n e t a l., 1 9 8 6 ). S in c e m e ta b o lis m is g re a tly r e ­ d u c e d in w in te r , th e n o r m a l lip id s h u ttle f u n c tio n o f h e m o ly m p h lip o p r o te in s is a p ­ p a r e n tly n o t e s s e n tia l a t th is tim e , a n d th e lip o p r o te in c a n b e r e m o v e d . T h e h e m o ly m p h le v e ls o f th is lip o p r o te in ic e n u c le a to r a re c o n tr o lle d b y h o r m o n e s , in ­ c lu d in g a d ip o k in e tic h o r m o n e a n d p r o b a b ly j u v e n ile h o r m o n e (X u e t a l., 1 9 9 0 ). It

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is lik e ly th a t th e e n d o g e n o u s ic e n u c le a to r s r e m o v e d s e a s o n a lly in o th e r fr e e z e a v o id in g s p e c ie s m a y a ls o b e p r o te in s . H o w e v e r , th e r e a re o th e r p o s s ib ilitie s . L e e e t al. ( 1 9 9 2 ) id e n tif ie d c r y s ta llo id s p h e r e s o f tr ib a s ic c a lc iu m p h o s p h a te w ith ic e n u c le a tin g a c tiv ity in M a l p ig h ia n tu b u le s o f o v e r w in te r in g la r v a e o f E u r o s ta so lid a g in is .

Examples of Freeze-Avoiding Strategies W h a te v e r th e lo c a tio n o f ic e n u c le a to r s , it s e e m s c r itic a l th a t th e y b e r e m o v e d o r th e ir a c tiv ity m a s k e d in w in te r . T h e in a b ility to d o s o r e q u ir e s th a t a f r e e z e - a v o id in g o r g a n is m a c c u m u la te v e r y h ig h c o n c e n tr a tio n s o f a n tif r e e z e if it is to s u r v iv e e v e n m o d e r a te ly lo w w i n t e r te m p e r a tu r e s . T a b le 1 illu s tr a te s th is w ith th r e e fr e e z e a v o id in g s p e c ie s o f b e e tle s th a t w e h a v e s tu d ie d . A ll a re c o m m o n in n o r th e r n I n d i­ a n a , o v e r w in te r in s im ila r m ic r o h a b ita ts , a n d a re e x p o s e d to s im ila r te m p e r a tu r e s . Y e t th e ir a d a p ta tio n s a r e q u ite d iv e r s e a n d a p p a r e n tly d e p e n d e n t u p o n th e a b ility , o r la c k th e r e o f , to r e m o v e ic e n u c le a to r s . A ll c le a r th e g u t in w in te r , th e r e b y r e ­ m o v in g ic e n u c le a to r s . A s n o te d e a r lie r , in w in te r , C e r u c h u s p ic e u s la r v a e r e m o v e a h e m o ly m p h lip o p r o te in ic e n u c le a to r ( N e v e n e t a l., 1 9 8 6 ). A n tif r e e z e p r o te in s a re n o t p r o d u c e d , a n d o n ly s m a ll a m o u n ts o f p o ly o ls ( s o r b ito l) a re a c c u m u la te d . Y e t th e le v e l o f s u p e r c o o lin g ( b e lo w th e f r e e z in g p o in t) is ~ 2 5 ° C , a ttr ib u ta b le p r im a r ily to r e m o v a l o f ic e n u c le a to r s . D e n d r o id e s c a n a d e n s is la r v a e p r o d u c e b o th a n tif r e e z e p r o te in a n d p o ly o ls ( g ly c e r o l a n d s o r b ito l) th a t p r o m o te s u p e r c o o lin g . R e c a ll th a t th e D . c a n a d e n s is a n tif r e e z e p r o te in s in h ib it h e m o ly m p h ic e n u c le a to r s ( D u m a n e t a l., 1 9 9 1 b , 1 9 9 2 ). In a d d itio n , th e c o n c e n tr a tio n o f h e m o ly m p h ic e n u c le a tin g p r o ­ te in s a ls o d e c r e a s e s in w in te r ( O ls e n a n d D u m a n , 1 9 9 2 ). In c o n tr a s t, U lo m a im ­ p r e s s a a c c u m u la te s b o th a n tif r e e z e p r o te in s a n d v e r y h ig h c o n c e n tr a tio n s o f c o llig a tiv e a n tif r e e z e s ( g ly c e r o l a n d s o r b ito l) b u t r e ta in s a c tiv e ic e n u c le a to rs . N o te

Table 1. E x a m p l e s o f f r e e z e - a v o id i n g i n s e c ts w ith d i f f e r e n t s t r a te g i e s f o r d e p r e s s in g th e s u p e r c o o l i n g p o i n t in w in te r

Species Season C eruchus p ic e u s b S um m er W in te r D e n d r o id e s c a n a d e n s is c Sum m er W in te r U lo m a im p r e s s a d Sum m er W in te r R h a b d o p h a g a s tr o b ilo id e s ' Sum m er W in t e r

Melting point

Supercooling point (°C)

Supercooling capacity below the freezing point (°C)

(°C )

Freezing point (°C)

-0 .5 - 1 .1

- 0 .5 - 1 .1

0 0

-7 -2 6

6 25

- 0 .6 - 2 .5

- 1 .7 - 5 .7

1.1 3 .2

-9 -2 7

7 21

- 0 .9 - 9 .9

- 2 .0 -1 4 .7

1.1 4 .8

-6 -2 1

4 6

Thermal hysteresis*

- 1 .2 - 1 9 .3

“T h e r m a l h y s te r e s is i n d ic a te s t h e p r e s e n c e o f a n tif r e e z e p r o t e i n . b D a t a f r o m N e v e n e t al. (1 9 8 6 ). ‘ D a t a f r o m D u m a n (1 9 8 4 ). " D a t a f r o m D u m a n (1 9 7 9 ). 'D a t a f r o m M ille r (1 9 8 2 ).

- 2 6 .5 -5 6

25 37

208

Duman, Olsen, Yeung, and Jerva

th a t th e h e m o ly m p h m e ltin g p o in t is a p p ro x im a te ly - 1 0 ° C a n d th e h y s te r e tic fre e z in g p o in t —1 5 ° C in w in te r . Y e t th e b e e tle s s u p e r c o o l j u s t 6 ° C b e lo w th e h y s te r e tic f r e e z in g p o in t. E v id e n tly t h e s e b e e tle s c o n ta in v e r y a c tiv e ic e n u c le a to r s , w h ic h a re n o t r e m o v e d in w in te r a n d w h ic h a n tif r e e z e p r o te in c a n n o t in h ib it. T h e r e f o r e , a c ­ c u m u la tio n o f v e r y h ig h le v e ls o f a n tif r e e z e s is r e q u ir e d to lo w e r th e f r e e z in g p o in t t o —1 5 ° C . A n e x a m p le o f th e e f f ic a c y o f c o m b in in g r e m o v a l o f ic e n u c le a to r s w ith e x te n ­ s iv e a n tif r e e z e a c c u m u la tio n is p r o v id e d b y w illo w c o n e g a ll la r v a e , R h a b d o p h a g a s tr o b ilo id e s f r o m A la s k a ( M ille r , 1 9 8 2 ). T h e s e u n d e r c o o l b y ~ 2 5 ° C in s u m m e r , in d ic a tin g a la c k o f p o te n t ic e n u c le a to r s ( T a b le 1). In w in te r , th e h e m o ly m p h m e lt­ in g p o in t is d e c r e a s e d to - 1 9 . 3 ° C , a t le a s t p a r tly d u e to e x te n s iv e p o ly o l a c c u m u la ­ tio n ( g ly c e r o l - 4 . 8 M ). T h e la r v a e u n d e r c o o l b y ~ 3 7 ° C , a n d th e s u p e rc o o lin g p o in t o f th e s e e x c e p tio n a lly fre e z e -a v o id in g in sec ts is re d u c e d to - 5 6 ° C .

An Evolutionary Perspective S in c e e n d o g e n o u s ic e n u c le a to r s p r e s e n t p r o b le m s f o r f r e e z e - a v o id in g s p e c ie s , th e q u e s tio n a r is e s a s to w h y s u c h n e g a tiv e s e le c tio n p r e s s u r e d id n o t r e s u lt in r e ­ m o v a l o f th e s e n u c le a to r s , o r a t le a s t o f th e a c tiv ity , o v e r e v o lu tio n a r y tim e . A p p a r ­ e n tly th e ic e n u c le a to r a c tiv ity e v o lv e d w h e n th is n e g a tiv e s e le c tio n p r e s s u r e d id n o t e x is t. T h e p r o te in s ( o r p e r h a p s o th e r ty p e s o f n u c le a to r s ) a s s u m e d c e r ta in s tr u c ­ tu re s , r e q u ir e d b y th e ir p a r tic u la r f u n c tio n s , w h ic h b y c h a n c e h a p p e n e d to r e s u lt in s u r f a c e w a te r b e in g s tr u c tu r e d in a n ic e lik e f a s h io n . T h is s h o u ld n o t b e s u r p r is in g s in c e , a s D a r w in r e c o g n iz e d in th e O rig in o f S p e c ie s , “ A n o r g a n b u ilt u n d e r th e in ­ f lu e n c e o f s e le c tio n f o r a s p e c if ic r o le m a y b e a b le a s a c o n s e q u e n c e o f i t ’s s tr u c ­ tu r e , to p e r f o r m m a n y o th e r , u n s e le c te d f u n c tio n s , a s w e ll” ( D a r w in , 1 8 5 9 ). S u b s titu te p r o te in f o r o r g a n , a n d th e s ta te m e n t m a y a p p ly to ic e n u c le a to r p r o te in s . In s o m e c a s e s , th e p r o te in f u n c tio n m a y r e q u ir e th is e m b r y o c r y s ta l p r o m o tin g s tr u c tu r e a n d th u s it c a n n o t n o w b e e lim in a te d . W h ile th is is a p la u s ib le s c e n a rio , th e r e d o s e e m to b e e x a m p le s o f in v e r te b r a te s th a t la c k e n d o g e n o u s ic e n u c le a tin g a c tiv ity , th u s s h o w in g t h a t ic e n u c le a tin g s tr u c tu r e s , a t le a s t n o t h ig h ly a c tiv e o n e s , a re n o t r e q u ir e d f o r n o r m a l a c tiv ity . A p h id s a s a g r o u p g e n e r a lly a re c a p a b le o f f a ir ly e x te n s iv e s u p e r c o o lin g e v e n in s u m m e r a n d a p p a r e n tly la c k a c tiv e ic e n u ­ c le a to r s ( O ’D o h e r ty a n d B a le , 1 9 8 5 ; K n ig h t a n d B a le , 1 9 8 6 ; O ’D o h e r ty a n d R in g , 1 9 8 7 ). M i l l e r a n d W e r n e r ( 1 9 8 7 ) d e s c r ib e d th e o v e r w in te r in g a d a p ta tio n o f th re e s p e c ie s o f f r e e z e - a v o id in g w illo w g a ll in s e c ts f r o m A la s k a , in c lu d in g R . stro b ilo id es d is c u s s e d a b o v e . M e a n s u p e rc o o lin g p o in ts in s u m m e r w e re - 2 5 to - 3 0 ° C , in d ic a tin g th e a b s e n c e o f p o t e n t ic e n u c le a to r s . H o w e v e r , in w in te r , s u p e r c o o lin g p o in ts d e ­ c r e a s e d to —5 8 ° C . G ly c e r o l a c c u m u la te d to 4 - 6 M a n d c a n a c c o u n t f o r a m a jo r p o r ­ tio n o f th e i n c r e a s e d s u p e r c o o lin g ( b a s e d o n A N T /A M P = 2 :1 to 3 :1 in th e a b s e n c e o f a c tiv e ic e n u c le a to r s ) , b u t it is lik e ly th a t r e m o v a l o f e v e n th e m in im a lly a c tiv e ic e n u c le a to r s p r e s e n t in s u m m e r m a y a ls o o c c u r . O th e r A la s k a n (M ille r, 1 9 8 2) an d a lp in e (R in g , 1 9 8 2 ) s p e c ie s th a t s u p e rc o o l to te m p e ra tu re s o f - 5 0 to - 6 0 ° C h a v e b e e n r e p o r te d . I n te r e s tin g ly , s o m e o f th e s e c a n s u r v iv e f r e e z in g a t th e s e lo w n u c le a tio n te m p e r a tu r e s , a lth o u g h it is lik e ly th a t s u c h te m p e r a tu r e s w o u ld o n ly r a r e ly b e e n ­ c o u n te r e d . I t s e e m s th a t i f s e le c tio n p r e s s u r e is s u f f ic ie n t (i.e ., th e e x tr e m e c o ld o f th e n o r th e r n R o c k ie s o r c e n tr a l A la s k a ) , e n d o g e n o u s ic e n u c le a tin g a c tiv ity c a n b e e lim in a te d o v e r e v o lu tio n a r y tim e .

Cold Tolerant Invertebrates

209

Freeze-Tolerant Species T h e r e a r e a n u m b e r o f a d a p ta tio n s r e c o g n iz e d a s c o n tr ib u tin g to th e a b ility o f a n im a ls to s u r v iv e f r e e z in g o f th e ir b o d y f lu id s ( s e e R in g , 1 9 8 0 ; Z a c h a r ia s s e n , 1 9 8 5 ; B a u s t a n d R o ja s , 1 9 8 5 ; S to r e y a n d S to r e y , 1 9 8 8 ; C a n n o n a n d B lo c k , 1 9 8 8 ; D u m a n e t a l., 1 9 9 1 a ; L e e a n d D e n lin g e r , 1 99 1 f o r v a r io u s p e r s p e c tiv e s ) . H o w e v e r , o n e o f th e b a s ic te n e ts o f f r e e z in g to le r a n c e is th a t in tr a c e llu la r f r e e z in g is le th a l. W ith a f e w n o ta b le e x c e p tio n s , s u c h a s fa t b o d y c e lls o f th e g o ld e n r o d g a ll fly E u r o s ta s o lid a g itiis ( S a lt, 1 9 5 9 ; L e e e t a l., 1 9 9 3 b ), th is c o n c e p t a p p e a r s to h o ld . E x te n s iv e s u p e r c o o lin g , f o llo w e d b y r a p id f r e e z in g , c a n r e s u lt in in tr a c e llu la r ic e f o r m a tio n ( M a z u r , 1 9 7 7 , 1 9 8 4 ) a n d in d a m a g e f r o m o s m o tic s tr e s s ( Z a c h a r ia s s e n , 1 9 9 2 ). C o n s e q u e n tly , in h ib itio n o f e x te n s iv e s u p e r c o o lin g is a n im p o r ta n t a d a p ta ­ tio n in m a n y f r e e z e - to le r a n t in v e r te b r a te s .

Advantage of Freezing at a High Temperature O n e o f th e m o s t i n te r e s tin g d is c o v e r ie s c o n c e r n in g n a tu r a l f r e e z e to le r a n c e a d ­ a p ta tio n s w a s th a t b y Z a c h a r ia s s e n a n d H a m m e l ( 1 9 7 6 ) d e s c r ib in g h e m o ly m p h ic e n u c le a to r s in f r e e z e - to le r a n t b e e tle s th a t in itia te f r e e z in g a t r e la tiv e ly h ig h t e m p e r a ­ tu re s . W ith s u p e r c o o lin g c o n tr o lle d a t j u s t a f e w d e g r e e s b e lo w th e h e m o ly m p h f r e e z in g p o in t, th e p la s m a m e m b r a n e is g e n e r a lly a b le to p r e v e n t in o c u la tiv e f r e e z ­ in g o f c y to p la s m . A s s o lu te s a r e e x c lu d e d f r o m e x tr a c e llu la r ic e , th e o s m o tic c o n ­ c e n tr a tio n o f u n f r o z e n e x tr a c e l l u l a r w a te r in c r e a s e s , th u s g e n e r a tin g an o s m o tic o u tf lu x o f w a te r f r o m c e lls , w h ic h lo w e r s f r e e z in g a n d s u p e r c o o lin g p o in ts o f in ­ tr a c e llu la r w a te r a n d th e r e b y d e c r e a s in g th e c h a n c e f o r in tr a c e llu la r f r e e z in g . A ls o , f r e e z in g a t h ig h e r te m p e r a tu r e s le s s e n s o s m o tic im b a la n c e s b e tw e e n in tr a c e llu la r a n d e x tr a c e llu la r c o m p a r tm e n ts , m in im iz in g th e p o te n tia l f o r o s m o tic s h o c k ( Z a c h a r ia s s e n , 1 9 9 2 ). A n o th e r p o s s ib le a d v a n ta g e o f f r e e z in g a t h ig h e r te m p e r a tu r e s is e n e r g y s a v in g s . S c h o la n d e r e t al. ( 1 9 5 3 ) d e m o n s tr a te d th a t f r o z e n in s e c ts h a d m u c h r e d u c e d m e ta ­ b o lic r a te s c o m p a r e d w ith u n f r o z e n s u p e r c o o le d in d iv id u a ls a t th e s a m e t e m p e r a ­ tu re s . M o r e tim e s p e n t f r o z e n o v e r th e w in te r r e s u lts in le s s e n e r g y s to r e s b e in g u tiliz e d ; th e r e f o r e , m o r e e n e r g y is a v a ila b le f o r g r o w th a n d /o r r e p r o d u c tio n in s p rin g . A n a d d itio n a l a d v a n ta g e o f f r e e z in g a t h ig h e r te m p e r a tu r e s m a y b e w a te r c o n ­ s e r v a tio n . S in c e o v e r w in te r in g te r r e s tr ia l in v e r te b r a te s m a y n o t e a t o r d r in k , n e g a ­ tiv e w a te r b a la n c e c o u ld r e s u l t in s e r io u s d e s ic c a tio n . R e s p ir a to r y w a te r lo s s v a r ie s d ir e c tly w ith m e ta b o lic r a te . S in c e f r e e z in g r e s u lts in r e d u c e d m e ta b o lis m , f r e e z in g a t h ig h e r t e m p e r a tu r e s r e d u c e s r e s p ir a to r y w a te r lo s s . A ls o , th e b o d y f lu id s o f a s u ­ p e r c o o le d o r g a n is m in a h ib e r n a c u lu m c o n ta in in g ic e h a v e a h ig h e r v a p o r p r e s s u r e th a n ic e . C o n s e q u e n tly , th e o r g a n is m w ill lo s e w a te r . I n c o n tr a s t, i f th e o r g a n is m is fr o z e n ( r e s u ltin g in v a p o r p r e s s u r e e q u ilib r iu m ) , w a te r w ill b e c o n s e r v e d ( Z a c h a r ia s s e n , 1 9 9 2 ). S o m e o r a ll o f t h e s e p o te n tia l a d v a n ta g e s o f e x tr a c e llu la r ic e n u c le a to r s a r e u n ­ d o u b te d ly im p o r ta n t to th e o v e r w in te r in g s u c c e s s o f f r e e z e - to le r a n t s p e c ie s . In a d ­ d itio n , e a r ly in th e e v o lu tio n o f c o ld to le r a n c e in a p a r tic u la r s p e c ie s , e n d o g e n o u s ic e n u c le a to r s m a y h a v e b e e n p r e s e n t, a n d it m a y h a v e b e e n im p o s s ib le , o r e n e r ­ g e tic a lly c o s tly , to r e m o v e o r m a s k th e s e . C o n s e q u e n tly , e v o lu tio n o f f r e e z e to l e r ­ a n c e a n d p e r h a p s f u r th e r d e v e l o p m e n t o f in c r e a s e d ic e n u c le a to r a c tiv ity , r a th e r th a n f r e e z e a v o id a n c e , m a y h a v e r e s u lte d . In s o m e f r e e z e - to le r a n t in s e c ts , ic e n u -

210

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c le a to r a c tiv ity in h e m o ly m p h in c r e a s e s in w in te r , in d ic a tin g th a t ic e n u c le a tio n is t h e ir p r im a r y f u n c tio n ( Z a c h a r ia s s e n , 1 9 8 0 , 1 9 9 2 ). H o w e v e r , th is is n o t a lw a y s th e c a s e . F o r e x a m p le , th e h e m o ly m p h lip o p r o te in ic e n u c le a to r in f r e e z e - to le r a n t la r ­ v a e o f th e c r a n e f ly T ip u la tr iv itta ta is th e m a jo r h e m o ly m p h lip o p r o te in d u r in g all s e a s o n s , a n d t h e r e f o r e a lm o s t c e r ta in ly f u n c tio n s to s h u ttle lip id ( N e v e n e t al., 1 9 8 9 ).

Inoculative Freezing in Freeze-Tolerant Organisms T h e p r e c e d in g a r g u m e n ts f o r h e m o ly m p h ic e n u c le a to r s in f r e e z e - to le r a n t o r ­ g a n is m s c o u ld b e a p p lie d to in o c u la tiv e f r e e z in g o f e x tr a c e llu la r f lu id s b y e x te r n a l ic e , th e u ltim a te ic e n u c le a to r . T h e r e c o u ld e v e n b e a d d itio n a l a d v a n ta g e s s u c h as n o t h a v in g to m a in ta in e n d o g e n o u s ic e n u c le a to r s a n d p o te n tia lly to e lim in a te a n y s u p e r c o o lin g w h a ts o e v e r . R e c e n tly , a fe w in s e c ts ( T a n n o , 1 9 7 7 ; F ie ld s a n d M c N e il, 1 9 8 6 ; S h im a d a a n d R iih im a a , 1 9 8 8 ; G e h r k e n e t a l., 1 9 9 1 ) a n d a c e n tip e d e ( T u r s m a n e t a l., 1 9 9 4 ) w e r e d e s c r ib e d th a t s u r v iv e f r e e z in g o n ly i f in o c u la tiv e fre e z in g o c c u rs . O v e rw in te rin g la rv a e o f th e m o th C is s e p s fu lv ic o llis s u p e rc o o l to - 1 2 to - 1 7 ° C u n d e r d r y c o n d itio n s ; h o w e v e r , th e y d o n o t s u r v iv e th is f r e e z in g . In c o n tr a s t, i f c o o le d w h ile in c o n ta c t w ith e x te r n a l ic e , t h e y f r e e z e a b o v e - 4 ° C b u t s u r v iv e ( F ie ld s a n d M c N e il, 1 9 8 6 ). A s im ila r s itu a tio n p e r ta in s in o v e rw in te rin g a d u lt B o lito p h a g u s re tic u la tu s b e e tle s, w h ic h c a n s u p e rc o o l to - 3 0 ° C b u t th e n d ie if f r o z e n . H o w e v e r , i f in c o n t a c t w ith ic e , th e b e e tle s f r e e z e in o c u la tiv e ly a b o v e - 6 ° C a n d s u r v iv e f r e e z in g ( G e h r k e n e t a l., 1 9 9 1 ). O v e r w in te r in g L ith o b iu s fo r fic a tu s c e n tip e d e s h a v e p o t e n t h e m o ly m p h ic e n u c le a to r s , w h ic h in itia te n u c le a tio n a t a p ­ p r o x im a te ly - 3 ° C . H o w e v e r , th e c e n tip e d e s d o n o t s u r v iv e e v e n th is m in im a l s u ­ p e r c o o lin g . I f f r o z e n in c o n ta c t w ith e x te r io r ic e , f r e e z in g o c c u r s a t th e h e m o ly m p h f r e e z in g p o in t ( a p p r o x im a te ly - 1 ° C ) , a n d th e c e n tip e d e s s u r v iv e ( T u r s m a n e t al., 1 9 9 4 ). C e n tip e d e s la c k th e w a x c o a tin g o f th e c u tic le o f in s e c ts , a n d th e r e f o r e th e c u tic le a p p a r e n tly d o e s n o t p r e s e n t a b a r r ie r to i n o c u la tiv e f r e e z in g . T h e o v e r w in ­ te r in g m i c r o h a b ita ts o f t h e s e in s e c ts a n d c e n tip e d e s a r e g e n e r a lly d a m p , a n d c o n s e ­ q u e n tly w in te r ic e f o r m a tio n in th e h ib e r n a c u lu m is c o m m o n . T h e r e f o r e , it is lik e ly th a t th e y a r e f r o z e n f o r p e r i o d s o f tim e e a c h w in te r.

Other Sites of Nucleation In a d d itio n to i n o c u la tiv e f r e e z in g , o th e r n o n h e m o ly m p h s ite s o f n u c le a tio n in i­ tia te f r e e z in g in s o m e f r e e z e - to le r a n t s p e c ie s . W in te r a c c lim a te d , f r e e z e - to le r a n t la r v a e o f th e a r c tic m o th G y n a e p h o r a g r o e n la n d ic a h a v e a h e m o ly m p h s u p e r c o o l­ in g p o i n t o f - 2 0 ° C . H o w e v e r , in ta c t la r v a e n u c le a te a t - 7 ° C ; th e r e f o r e , it is o b v i­ o u s th a t h e m o ly m p h n u c le a to r s d o n o t in itia te n u c le a tio n ( K u k a l e t a l., 1 9 8 8 ). Ic e n u c le a tin g b a c te r ia in th e g u t o f f r e e z e - to le r a n t o r g a n is m s a re p o s s ib le s ite s o f n u ­ c le a tio n . S o m e f r e e z e - to le r a n t p o p u la tio n s o f th e g a ll fly E u r o s ta s o lid a g in is a p p a r e n tly h a v e h e m o ly m p h ic e n u c le a to r s (S o m m e , 1 9 7 8 ; L e e e t a l., 1 9 8 1 ; Z a c h a r ia s s e n e t a l., 1 9 8 2 ). H o w e v e r , B a le e t a l. ( 1 9 8 9 ) r e p o r te d th a t a N e w Y o r k p o p u la tio n la c k e d h e m o ly m p h ic e n u c le a to r s a n d p r e s e n te d e v id e n c e th a t f r a s s in th e g a ll h a s p o te n t ic e n u c le a to r a c tiv ity a n d in itia te s f r e e z in g . A ls o , a s m e n tio n e d e a r lie r , L e e e t al. ( 1 9 9 2 ) s h o w e d th a t E . s o lid a g in is h e m o ly m p h s u p e r c o o le d to - 1 8 ° C , b u t c ry s ta ls o f tr ib a s ic c a lc iu m p h o s p h a te in th e M a lp ig h ia n tu b u le s in itia te n u c le a tio n a t te m ­ p e r a tu r e s a s h ig h a s - 7 . 8 ° C .

Cold Tolerant Invertebrates

211

Table 2. E x a m p l e s o f v a r ia b il i ty in t h e a m o u n t o f s u p e r c o o l i n g b e lo w t h e h e m o l y m p h f r e e z in g p o i n t s e e n in v a r io u s f r e e z e - t o l e r a n t t e r r e s t r i a l a rth ro p o d s 3

Species L ith o b iu s f o r f i c a t u s b E le o d e s b la n c h a r d i c T ip u la tr i v i t a tt a d A n th e r e a p o l y p h e m u s ' B ra c o n c e p h i1 P y th o d e p la n a ta s g P. a m e r ic a n u s 8

Organismal freezing temperature (°C)

Supercooling

-I - 6 .3 - 6 .5 -2 1 .1 -4 7 -5 4 -6

0 - 9 to -1 5 - 9 to - 1 5 - 9 to - 1 5

3 -5 3 2 -5 3 6 2 -6 9 9 8 -9 9

M o r ta lity (% ) T re a te d 3 -7 9 8 -9 9 98 100 100

“D a t a f r o m F ie ld s (1993).

fo r 24 h o u rs ev e n w h e n n o t tre a te d w ith b a c te ria , tre a tm e n t w ith e ith e r 100 o r 1,0 0 0 p p m o f P. syrin g a e in w h e a t c a u se d a sig n ific a n t, a n d a d o s e -d e p e n d e n t re d u c tio n in su rv iv a l fo r all s p e c ie s te s te d (T a b le 3). W h e n th e te m p e ra tu re o f e x p o s u re w as d e ­ c re a se d to —8°C , th is tre n d c o n tin u e d a n d su rv iv a l ra te s d e c re a s e d still fu rth e r (R .E . L ee et al., 1992b). F ie ld s (1 9 9 3 ) also e x a m in e d th e e ffic a c y o f P. syrin g a e in re d u c in g th e c o ld to l­ e ra n c e o f C. fe r r u g in e u s u n d e r fie ld c o n d itio n s in M a n ito b a g ran a rie s. G ro u p s o f c o ld -a c c lim a te d ru s ty g ra in b e e tle a d u lts w ere tre a te d w ith 1,0 0 0 p p m o f P. sy rin ­ gae an d p la c e d in w h e a t g ra n a rie s in e a rly D e c e m b e r. O n d a y s 15 an d 2 2 tre a te d b e e tle s h ad sig n ific a n tly lo w e r su rv iv a l ra te s th a n u n tre a te d g ro u p s (T a b le 4). H o w ­ ev e r, by d ay 3 0 n e a rly all in d iv id u a ls in b o th tre a te d a n d u n tre a te d g ro u p s h ad died . F ro m th e se in itia l s tu d ie s w ith s to re d p ro d u c t p e s ts, it is c le a r th a t In a + b a c te ria m ay b e u se d to d e c r e a s e th e s u p e rc o o lin g c a p a c ity an d c o ld -h a rd in e ss e v e n o f c o ld a c c lim a te d in se cts. In a n a tte m p t to sta n d a rd iz e p ro to c o ls fo r fu tu re stu d ies o f s u r­ v iv a l o f s to re d p ro d u c t in s e c ts at e x tre m e te m p e ra tu re s, F ie ld s (1 9 9 2 ) re c o m m e n d e d th e fo llo w in g : 1) te sts s h o u ld b e c o n d u c te d w ith in s e c t strain s th a t h a v e b ee n in th e la b o ra to ry n o m o re th a n 2 y e a rs, 2 ) th e m o st te m p e ra tu re -re s is ta n t d e v e lo p m e n ta l sta g e o f th e p e s t s h o u ld b e u se d , 3) te m p e ra tu re -a c c lim a te d in se cts sh o u ld b e u sed , 4 ) a ra n g e o f e x tre m e te m p e ra tu re s sh o u ld be te s te d so th a t d a ta m ay b e a n a ly z e d u sin g p ro b it an a ly sis a n d th a t fid u c ia l lim its m a y b e re p o rte d , an d 5 ) th e re su lts o f la b o ra to ry stu d ie s s h o u ld b e c o n firm e d w ith field te sts. F u tu re in v e stig a tio n s u sin g In a + m ic ro o rg a n is m s f o r th e c o n tro l o f s to re d p ro d u c t p e s ts s h o u ld fo llo w th e se c r i­ teria.

P r o s p e c ts fo r th e B io lo g ic a l C o n tr o l o f t h e C o lo r a d o P o t a t o B e e t le T h e C o lo ra d o p o ta to b e e tle , L ep tin o ta rsa d ecem lin ea ta (S ay ), is th e m o s t s e ri­ o u s d e fo lia tin g p e s t o f p o ta to e s , S o lan um tu b ero sum L ., in N o rth A m eric a. T h is s p e c ie s o v e rw in te rs b y b u rro w in g in to the soil in la te su m m e r o r e a rly au tu m n . W h e n o v e rw in te rin g a d u lts e m e rg e fro m d o rm a n c y , th e y can sig n ific a n tly re d u c e y ie ld s b y d e fo lia tin g th e e a rly g ro w th sta g es o f th e p o ta to p la n ts (S h ie ld s a n d W y ­ m an, 1984). T h is p e s t is k n o w n for th e w id e ran g e o f p esticid es, in clu d in g synthetic p y reth ro id s, to w h ich it h as rap id ly d ev e lo p e d resistan c e (C asag ran d e, 1987). T h e c u rre n t a g ric u ltu ra l p r a c tic e o f p la n tin g e x te n siv e m o n o c u ltu re s o f p o ta to e s fu rth e r p ro m o te s th e c u m u la tiv e b u ild u p o f C o lo ra d o p o ta to b e e tle p o p u la tio n s fro m y e a r to y ea r. B e c a u s e o f th e s e fa c to rs , c u rre n t re s e a rc h e ffo rts h a v e in c re a sin g ly b e g u n to

264

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fo cu s on a lte r n a tiv e fo rm s o f c o n tro l. O n e n o v e l a p p r o a c h to m a n a g e m e n t o f the p est u se s c u ltu ra l m e th o d s d e s ig n e d to e x p o s e C o lo ra d o p o ta to b e e tle s to leth al low te m p e ra tu re s d u rin g th e w in ter (M iln e r e t al., 1 9 9 2 ; K u n g e t al., 1992). T ra p c ro p s o f p o ta to e s are p la n te d in m id ­ su m m er, a n d b e c a u s e th e se y o u n g e r p la n ts are m o re a ttra c tiv e to th e a d u lts, the b e e tle s c o n c e n tra te in th e se a re as. In late su m m er, tra p p la n ts are m u lc h e d to e n ­ c o u ra g e th e b e e tle s to re m a in a t th e se sites o v e r w in te r r a th e r th a n d is p e rs in g fro m th e field s. In s e c t m o rta lity is in d u c e d by re m o v in g th e m u lc h in m id w in te r im m e­ d ia te ly p rio r to a c o ld fro n t, w h ich c a u se s the soil te m p e ra tu re s to d ro p ra p id ly . M u lc h in g m a y a ls o r e d u c e th e d e p th to w h ich the b e e tle s w ill b u rro w d u rin g the w in te r, in c re a s in g fu rth e r th e ir s u sc e p tib ility to the lo w e rin g o f so il te m p e ra tu re . T h e fe a s ib ility o f th is a p p ro a c h w as re c e n tly s u p p o rte d b y th e w o rk o f M iln e r e t al. (1 9 9 2 ). T h e y fo u n d th a t a d u lt b e e tle su rv iv al w as sig n ific a n tly lo w e r at site s w here m u lc h w a s re m o v e d in m id w in ter. In c o lla b o r a tio n w ith J e ffre y W y m a n an d P h il K a u fm a n , U n iv e rsity o f W is c o n ­ sin, w e h a v e b e g u n in v e s tig a tin g th e u se o f In a + m ic ro o rg a n is m s to in c re a s e th e su s­ c e p tib ility o f th e o v e rw in te rin g b e e tle s to lo w te m p e ra tu re s (L e e e t al., 1994). O u r ra tio n a le is to d e c r e a s e th e c o ld -h a rd in e s s o f the b e e tle s u sin g th e se b io lo g ic a l nuc le a to rs w h en a p p lie d in c o n ju n c tio n w ith the cu ltu ral c o n tro l a p p ro a c h (M iln e r et al., 1992). In o u r in itia l stu d y , w e d e te rm in e d th a t th e C o lo ra d o p o ta to b e e tle is a fre ez ein to le ra n t s p e c ie s th a t d ie s w h en c o o le d to its s u p e rc o o lin g p o in t (L e e e t al., 1994). H o w e v e r, th e o v e rw in te rin g b e e tle s su rv iv e to te m p e ra tu re s im m e d ia te ly ab o v e th e ir s u p e rc o o lin g p o in t, in d ic a tin g th a t d e a th is d u e to th e o n s e t o f in te rn a l ice fo r­ m a tio n , a n d n o t lo w te m p e ra tu re p e r se. T h is re su lt a lso in d ic a te s th a t th e s u p e r­ c o o lin g p o in t m a y b e u se d as a m e a su re o f the leth al lo w te m p e ra tu re , at le a st d u r­ in g sh o rt-te rm e x p o s u re to co ld . C o n s id e rin g th e re la tiv e ly h ig h su p e rc o o lin g p o in t o f a p p ro x im a te ly - 7 ° C for o v e rw in te rin g a d u lts , it is o b v io u s th a t th is sp e cie s la ck s e x c e p tio n a l c o ld to le ran ce . T h e ir lim ite d c a p a c ity fo r s u p e rc o o lin g is n o t su rp risin g , h o w e v e r, c o n s id e rin g th eir th e rm a lly p r o te c te d o v e rw in te rin g site in the so il (L e e, 1 9 9 1 ). N o n e th e le ss, an e le ­ v atio n in th e s u p e rc o o lin g p o in t o f as little as 2 to 4 d e g re e s in th e le th al lo w te m ­ p e ra tu re w o u ld b e o f m a jo r s ig n ific a n c e in d e c re a s in g th e p r o p o rtio n o f b ee tle s s u rv iv in g th e w in te r. C o lo ra d o p o ta to b e e tle s e x p o s e d to - 4 ° C had a su rv iv a l ra te o f 5 4 .8 % , w h e re a s o n ly 6 .2 % o f th o se e x p o s e d to - 6 ° C s u rv iv e d (K u n g e t al., 1992). T o s im u la te o v e rw in te rin g c o n d itio n s in th e la b o ra to ry , w e te ste d w h e th e r su ­ p e rc o o lin g p o in ts in c re a s e d in b e e tle s th a t w ere e x p o s e d to a c o n c e n tra te d , freezed rie d a n d k ille d p re p a ra tio n o f P. syrin g a e m ix ed w ith s o il (L e e et al., 1994). M ean s u p e rc o o lin g p o in ts o f b e e tle s tre a te d w ith P. syringa e in c o n c e n tra tio n s ran g in g fro m 0 to 1 ,0 0 0 p p m w ere d e te rm in e d (F ig. 2A ). In b o th 1991 an d 1992, th e s u p e r­ c o o lin g p o in t m e a n s in c re a se d s ig n ific a n tly w hen b e e tle s w e re e x p o s e d to in c re a s ­ ing c o n c e n tra tio n s o f P. sy ringa e, ra n g in g fro m - 7 .6 ± 0 .2 ° C (u n tre a te d ) to - 3 .7 ± 0.1 °C (1 ,0 0 0 p p m ). In th e 1992 te sts, as little as 1 p p m re s u lte d in a su p e rc o o lin g p o in t th a t w as s ta tis tic a lly h ig h e r th a n th a t o f the u n tre a te d c o n tro l. T h e s e resu lts in d ic a te th a t th e e f f e c t o f In a + m ic ro o rg a n ism s on th e s u p e rc o o lin g p o in t is d o sed e p e n d e n t as h a s b e e n r e p o rte d p re v io u sly in o th e r in s e c ts (F ie ld s, 1990; R .E . L ee e t al., 1992b). T h e c u m u la tiv e fre e z in g d istrib u tio n s , c o m p a ra b le to th e ice n u c le a tio n sp e c tra th a t are ty p ic a lly u s e d to d e s c rib e th e ic e -n u c le a tin g a c tiv ity o f In a + b a c te ria , w ere

B io lo g ic a l C o n tr o l o f In s e c t P e s ts

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also d e te rm in e d fo r th e s e b e e tle s (F ig. IB ). T h e s e c u rv e s are a u se fu l fo rm o f d a ta p re s e n ta tio n b e c a u s e th e y sh o w a p ro file o f th e th e o re tic a l le th al lo w te m p e ra tu re fo r a p o p u la tio n o f b e e tle s tre a te d w ith v a rio u s c o n c e n tra tio n s o f P. syringae (L e e e t al., 1994). F o r e x a m p le , if b e e tle s w ere e x p o s e d to —5 °C , 80% o f th o se tre a te d w ith 100 p p m o f P. sy rin g a e w o u ld b e e x p e c te d to fre e z e an d d ie ; in co n tra st, n o n e o r v ery few o f th e u n tre a te d c o n tro l b e e tle s w o u ld b e e x p e c te d to fre e z e at th is te m ­ p e ra tu re . T h e s im ila rity o f th e 100 an d 1,000 p p m c u rv e s fu rth e r su g g e sts th a t, u n ­ d e r th e se c o n d itio n s , th e e le v a tio n o f th e s u p e rc o o lin g p o in t re a c h e s a m a x im u m n e a r 100 pp m .

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F i g u r e 2. A , E ffe c t o f P seu d o m o n a s syringae o n th e m e a n (± sta n d a rd erro r) su p e rc o o lin g p o in t o f d ia p a u s in g a d u lts o f th e C o lo r a d o p o ta to b e e tle . B e e tle s w e re e x p o s e d to v a rio u s c o n c e n tra tio n s ( 0 1 ,0 0 0 p p m ) o f P. syrin g a e in s o il fo r 48 h o u rs a t 4 °C . In 1991, sa m p le siz es w ere n = 1 0 -1 1 , a n d in 1992, n = 44—58. B , C u m u la tiv e f re e z in g p ro file fo r b e e tle s e x p o s e d to v a rio u s co n c e n tra tio n s o f P. syringae in 1 9 9 2 . (A d a p te d f ro m L e e e t al., 1994.)

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A n o th e r c ritic a l fa c to r in th e d e v e lo p m e n t o f a b io lo g ic a l co n tro l stra te g y fo r this sp e c ie s is th e d u r a tio n o f th e su p e rc o o lin g p o in t e le v a tio n a fte r a p p lic a tio n o f m i­ c ro o rg a n is m s . T h e s u p e rc o o lin g p o in t o f b e e tle s tre a te d w ith P. sy rin g a e w as e le ­ v a te d s ig n ific a n tly fo r 7 d a y s afte r a p p lic a tio n at 4 °C b u t w as s im ila r to th at o f u n tre a te d b e e tle s b y d ay 14 (L e e e t al., 1994). P re v io u s stu d ie s h a v e re p o rte d a loss o f b a c te ria l ic e -n u c le a tin g ac tiv ity w ith tim e at te m p e ra tu re s a b o v e 0 °C (G o o d n o w et al., 1 9 90). T re a tm e n t te m p e ra tu re also affec ted s u p e rc o o lin g p o in t e le v a tio n o f b e e tle s fo llo w in g tre a tm e n t w ith P. syringae. A lth o u g h a f te r 7 d a y s th e m e an su ­ p e rc o o lin g p o in ts fo r u n tre a te d b e e tle s in c u b a te d at 4 o r 1 0°C w ere sim ila r, the v a l­ u es fo r th o se tr e a te d w ith P. syrin g a e rem a in ed s ig n ific a n tly h ig h e r a t 4 th a n at 10°C . T h e s e d a ta su g g e st th a t ic e -n u c le a tin g ac tiv ity w a s b e tte r re ta in e d d u rin g in ­ c u b a tio n at th e lo w e r te m p e ra tu re . In c o n tra st, F ie ld s e t al. (1 9 9 3 ) re p o rte d lo n g ­ te rm sta b ility o f a P. syrin g a e p re p a ra tio n h eld at 3 0 °C fo r 8 w ee k s. O b v io u sly , a d d itio n a l s tu d y is n e e d e d re g a rd in g th e effec ts o f te m p e ra tu re a n d d u ra tio n o f e x ­ p o s u re o n s u p e rc o o lin g p o in t e le v a tio n b y P. syringae. A t th is tim e it is e n v is io n e d th a t n o rth ern p o p u la tio n s o f the C o lo ra d o p o ta to b e e tle w o u ld b e tre a te d w ith In a + b a c te ria in late A u g u s t o r ea rly S e p te m b e r, w hen a d u lts h a v e b e e n a ttra c te d to fee d on tra p c ro p s on th e e d g e s o f fie ld s, b u t b efo re th e y h a v e b e g u n to b u rro w in to th e so il to o v erw in te r. S in c e a m b ie n t te m p e ra tu re s a re still r e la tiv e ly h ig h at th is tim e o f the y ear, th e ic e -n u c le a tin g a c tiv ity o f the b a c te ria m ay b e lo s t b e fo re e n v iro n m e n ta l te m p e ra tu re s d ro p lo w e n o u g h (even a fte r a p p ly in g th e c u ltu ra l m a n ip u la tio n s o f M iln e r e t al. [1 9 9 2 ]) to k ill th e b ee tle s. O n e a lte rn a tiv e a p p r o a c h to th is p ro b le m w o u ld b e to fin d w ay s to m a in ta in th e icen u c le a tin g a c tiv ity o f m ic ro o rg a n is m s in th e g u t o r on th e su rfa c e o f th e in se c t o r to a p p ly o th e r I n a + m ic ro o rg a n is m s th a t w o u ld b e re ta in e d b y th e b e e tle s u n til e n v i­ ro n m e n ta l te m p e r a tu re s in th e ir m ic ro h a b ita t d e c re a s e to le th al le v els. C o n s e ­ q u en tly , w e h a v e b e g u n to te st th e e ffic a c y o f se v eral d if fe r e n t sp e c ie s o f liv in g In a + b a c te ria a n d fu n g i fo r th e ir e ffe c t o n th e b e e tle ’s s u p e rc o o lin g p o in t. W e h av e also te ste d th e e f f e c t o f su sp e n sio n s o f liv in g In a+ b a c te ria o n th e s u p e rc o o lin g p o in t. S u sp e n s io n s o f liv in g P. flu o resc en s, P. syringae, an d P. p u tid a s p ra y e d o n to th e ad u lts all c a u s e d a sig n ific a n t in c re a se in th e s u p e rc o o lin g p o in t, in d ic a tin g th a t liv in g b a c te ria l c e lls m a y also b e u se d fo r su p e rc o o lin g p o in t m a n ip u la tio n . A n o th e r m o d ific a tio n th a t m a y b e o f v alu e in th e d e v e lo p m e n t o f b io lo g ica l c o n tro l m e th o d s is th e u se o f su rfa c ta n ts in c o m b in a tio n w ith In a + m ic ro o rg a n ism s (L e e e t al., 1 9 9 3 ). T h e a d d itio n o f T w e e n 80 to F. a cu m in a tu m su s p e n s io n s sig n ifi­ c a n tly in c re a s e d th e a m o u n t o f su p e rc o o lin g p o in t e le v a tio n in th e b e e tle , H. convergens, c o m p a r e d w ith u se o f th is In a + fu n g u s alo n e (M .R . L e e e t al., 1992b). In th e c a s e o f th e C o lo ra d o p o ta to b e e tle , the a p p lic a tio n o f th e In a + b a c te ria w o u ld b e fa c ilita te d b y th e u se o f tra p c ro p p in g , w h ich c o n c e n tra te s th e b e e tle s in a n a rro w p o rtio n o f th e fie ld an d , th e re b y , w o u ld re d u c e th e am o u n t a n d c o s t o f b io ­ lo g ic al n u c le a to rs th a t m u st b e a p p lie d . T h e su c ce ssfu l in te g ra tio n o f th e se b io lo g i­ cal ic e n u c le a to rs w ith th e c u ltu ra l co n tro l strateg y o f M iln e r et al. (1 9 9 2 ) also w o u ld a llo w u s e o f th is c u ltu ra l c o n tro l ap p ro a c h in a re a s o f th e c o u n try w h ere it o th e rw is e c o u ld n o t b e u se d b e c a u s e w in te r soil te m p e ra tu re s w o u ld b e to o m ild.

C o n c lu d in g R e m a r k s U se o f I n a + m ic ro o rg a n is m s fo r b io lo g ic a l co n tro l h a s b o th a d v a n ta g e s an d d is ­ a d v a n ta g e s. F irs t, sin c e it a p p e a rs to b e effec tiv e a g a in s t a d iv e rs e ra n g e o f in sects,

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co n s id e ra tio n m u st b e g iv e n to a v o id in g d e trim e n ta l e ffe c ts on b e n e fic ia l in se cts. T h is m ay b e a c c o m p lis h e d if a p p lic a tio n s can b e ta rg e te d to sp e c ific are a s w h ere few n o n ta rg e t in se c ts w o u ld b e e x p o s e d , su ch as in th e tra p c ro p s fo r C o lo ra d o p o ­ ta to b e e tle s o r s to ra g e p r o d u c t sites. I f fie ld iso la te s o f In a + m ic ro o rg a n ism s ca n be u se d fo r c o n tro l, it w o u ld c irc u m v e n t p ro b le m s a s s o c ia te d w ith th e re le a se o f g e ­ n e tic a lly e n g in e e re d o rg a n is m s in to the e n v iro n m e n t. C o n s id e rin g th e a p p a re n t e a se a n d ra p id ity w ith w h ich I n a + b a c te ria c a u s e an in c re a se in th e su p e rc o o lin g p o in t, it m a y b e re la tiv e ly d if fic u lt fo r in se c ts to d e v e lo p re s is ta n c e to th is co n tro l m e asu re, sin c e it w o u ld re q u ire b lo c k in g a n y an d all a v e n u e s o f c o n ta c t b etw e en the In a + o r ­ g an ism s an d in te rn a l w a te r. T h e d e v e lo p m e n t o f r e s is ta n c e to tra n sc u tic u la r n u ­ c le a tio n is u n d o u b te d ly m o re c o m p le x a n d is e x p e c te d to be an u n lik e ly o r at le a st slo w e r p ro c e s s th a n c o m m o n m e c h a n ism s o f re s is ta n c e , su c h as a lte ra tio n in the stru c tu ra l ta rg e t fo r a to x in o r th e p ro d u c tio n o f to x in -d e s tro y in g en z y m e s. O n the o th e r h an d , i f in se c ts m o v e to a re a s (e .g ., b u rro w m o re d e e p ly in to th e so il) s u ffi­ cie n tly w arm to re m a in a b o v e th e s u p e rc o o lin g p o in t, th is a p p ro a c h w o u ld be in e f­ fec tiv e . A n o th e r a d v a n ta g e o f th is tre a tm e n t is th e b io d e g ra d a b ility o f th e se p re p a ra tio n s , w h ic h are u n lik e ly to le av e b e h in d c o n ta m in a tin g re sid u e s. L astly , th is a p p ro a c h is fu lly c o m p a tib le w ith o th e r co n tro l m e a s u re s th a t m ig h t b e u sed c o n ­ c o m ita n tly fo r in te g ra te d p e s t m a n a g e m e n t o f a g iv e n sp e cie s. A lth o u g h th e in itial s tu d ie s r e la te d to th e p o te n tia l u se o f ic e -n u c le a tin g m ic ro ­ o rg a n ism s are e n c o u ra g in g , c o n s id e ra b ly m o re w o rk is n e e d e d to d e te rm in e w h eth e r th is a p p ro a c h w ill p ro v e u s e fu l fo r b io lo g ic a l c o n tro l.

A c k n o w le d g m e n ts W e th a n k P au l F ield s fo r g e n e ro u sly p ro v id in g us w ith an a d v a n c e co p y o f a m a n u sc rip t in p ress an d V alerie B e n n e tt, C h u c k B u rk s, a n d P au l F ield s fo r th eir c o m m en ts on th e m a n u scrip t. T h is resea rch w as su p p o rte d by th e C o o p e ra tiv e S ta te R e se a rc h S erv ice (U S D A ) g ra n t # 9 3 -3 7 3 0 2 -9 0 0 3 , N S F g ran t IB N 9 3 0 5 8 0 9 , a n d G e n e n c o r In tl., S a n F ra n c isc o .

L it e r a t u r e C ite d B ale, J. S. 1987. R ev iew . In se c t co ld h ard in ess: F reezin g an d su p e rc o o lin g -a n eco p h y sio lo g ic al p e r­ sp ectiv e. J. In sect P h y sio l. 1 2 :8 9 9 -9 0 8 . B a u st, J. G ., an d R o ja s, R. R . 1 9 8 5 . R e v ie w — In se c t co ld h a rd in e ss: F acts an d fan cy . J. In sect P h y sio l. 3 1 :7 5 5 -7 5 9 . C a n n o n , R. J. C ., a n d B lo ck , W . 1988. C o ld to leran ce o f m ic ro a rth ro p o d s. B iol. R ev. 6 3 :2 3 -7 7 . C a sa g ra n d e , R. A . 1987. T h e C o lo ra d o p o ta to beetle: 125 years o f m ism an ag e m e n t. B ull. E n to m o l. Soc. A m . 3 3 :1 4 2 -1 5 0 . D u m an , J. G ., W u , D. W ., X u , L ., T u rsm a n , D ., an d O lsen , T . M . 1991. A d ap tatio n s o f in se cts to s u b ­ z ero tem p eratu res. Q . R ev. B io l. 6 6 :3 8 7 -4 1 0 . F ield s, P. G . 1990. T h e c o ld -h a rd in e s s o f C ryptolestes fe rru g in e u s an d th e u se o f ice n u c le a tio n -a c tiv e b a c te ria as a co ld -sy n e rg ist. P a g e s 1183-1191 in: Proc. Int. W o rk in g C o n f. o n S to re d -P ro d u c t P ro ­ tectio n , 5 th . V ol. 2. F. F le u ra t-L e ss a rd an d P. D u co n , eds. B o rd e a u x , F rance. F ield s, P. G . 1992. T h e c o n tro l o f sto re d -p ro d u c t in se cts an d m ites w ith ex tre m e tem p eratu res. J. S to re d P rod. R es. 2 8 :8 9 -1 1 8 . F ield s, P. G . 1993. R e d u c tio n o f c o ld to leran ce o f sto re d -p ro d u c t in se cts by ic e -n u c le a tin g -a c tiv e b a c ­ teria. E n v iro n . E n to m o l. 2 2 :4 7 0 -4 7 6 . F ield s, P. G ., a n d M c N eil, J. N . 1986. P o ssib le d u al c o ld -h a rd in e ss strateg ies in C isseps fu lv ic o llis (L ep id o p tera: A rctiid ae). C an . E n to m o l. 1 1 8 :1 3 0 9 -1 3 1 1 . F ield s, P .G ., P o u leu r, S., a n d R ic h a rd , C . 1993. S tab ility o f ic e -n u c le a tin g b a c te ria a n d fu n g u s as a m ean s o f re d u c in g th e c o ld -h a rd in e s s o f in se c t p ests. C ry o b io lo g y 30:623. G o o d n o w , R. A ., H a rriso n , M . D ., M o rris, J. D „ S w eetin g , K . B ., an d L aD u ca, R. J. 1990. F ate o f ic e n u c le a tio n -a c tiv e P seu d o m o n a s sy ringae stra in s in alp in e so ils an d w aters in sy n th e tic snow s a m ­ ples. A p p l. E n v iro n . M ic ro b io l. 5 6 :2 2 2 3 -2 2 2 7 .

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H ag stru m , D . W „ a n d F lin n , P. W . 1992. In teg rated pest m a n g e m e n t o f sto red -g ra in in se cts. P ages 5 355 6 2 in: S to ra g e o f C e re a l G rain s a n d T h e ir P ro d u cts, 4 th ed. D. B. S au er, ed. A m e ric a n A sso ciatio n o f C e re a l C h e m is ts, St. P au l, M N . Jo h n sto n , S. L., a n d L ee, R. E. 1990. R e g u la tio n o f su p erco o lin g a n d n u c le a tio n in a freeze in to leran t b e etle ( Tenebrio m o lito r). C ry o b io lo g y 2 7 :5 6 2 -5 6 8 . K an ek o , J., K ita, K ., a n d T an n o , K. 1989. B a cteria in th e g u t d e te rm in e s th e su p e rc o o lin g p o in t o f the d ia m o n d b a c k m o th , P lu tella xylo stella , p u p a e reared on g e rm in a tin g ra d ish se ed s (R aphanus satiuus L. var. a ca n th ifo rm is M a k in o ). Jp n . J. A p p l. E n to m o l. Z ool. 3 3 :8 2 -9 1 . K an ek o , J „ K ita, K ., a n d T an n o , K. 1991a. Ice n u cleatin g activ e b a c te ria iso lated fro m th e d ia m o n d b a c k m o th , P lu tella x y lo ste lla L. p u p a e (L ep id o p tera: Y p o n o n eu tid ae). Jp n . J. A ppl. E n to m o l. Z ool. 3 5:711 .

K an ek o , J., Y o s h id a , T ., O w ad a, T ., K ita, K ., an d T an n o , K. 1991b. E rw in ia h e rb ic o la : Ice n u cleatio n a c tiv e b a c te ria is o la te d fro m d ia m o n d b a c k m oth, Plutella x ylo stella L . p u p ae. Jp n . J. A ppl. E ntom ol. Z ool. 3 5 :2 4 7 -2 5 1 . K ieft, T . L. 1988. Ice n u c le a tio n activ ity in lichens. A ppl. E n viron. M ic ro b io l. 5 4 :1 6 7 8 -1 6 8 1 . K u n g , K-J. S ., M iln e r, M ., W y m an , J. A., F eld m an , J., and N o rd h eim , E . 1992. S u rv iv a l o f C o lo rad o p o ta to b e e tle (C o le o p te ra : C h ry so m e lid a e ) afte r e x p o su re to su b z e ro th e rm a l sh o c k s d u rin g d iap au se. J. E co n . E n to m o l. 8 5 :1 6 9 5 -1 7 0 0 . L ay n e, J. R ., L ee, R . E ., a n d H u an g , J. L. 1990. Inoculation trig g ers free zin g a t h ig h su b z ero te m p e ra ­ tu res in a fre e z e -to le ra n t fro g ( R ana sylvatica) an d in se ct ( E urosta solidaginis). C a n . J. 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R eview : In se c t co ld -h a rd in e ss an d ice n u ­ cle a tin g a c tiv e m ic ro o rg a n is m s in c lu d in g th eir p o ten tial use fo r b io lo g ic a l co n tro l. J. In sect P hysiol. 3 9 :1 -1 2 . L ee, R . E ., M u g n a n o , J. A ., an d T ay lo r, R. T. 1992a. E n d o g en eo u s c ry sta llo id sp h e res reg u late th e su ­ p e rc o o lin g p o in t o f th e gall fly, E urosta solidaginis. C ry o b io lo g y 2 9 :7 5 0 -7 5 1 . L ee, R. E ., S tro n g -G u n d e rso n , J. M ., L ee, M . R „ an d D av id so n , E . C . 1992b. Ice-n u c le a tin g activ e b a c ­ te ria d e c re a se th e c o ld -h a rd in e s s o f sto red g rain in sects. J. E con. E n to m o l. 8 5 :3 7 1 -3 7 4 . L ee, R . E ., S tro n g -G u n d e rso n , J. M ., L ee, M . R , G rove, K. S., an d R ig a , T. J. 1991. Iso latio n o f ice n u c le a tin g a c tiv e b a c te ria fro m in se c ts. J. E xp. Z ool. 2 5 7 :1 2 4 -1 2 7 . M iller, K. 1982. C o ld -h a rd in e ss strateg ies o f so m e a d u lt an d im m a tu re in se cts o v e rw in te rin g in in terio r A lask a. C o m p . B io c h e m . P h y sio l. 7 3 A :5 9 5 -6 0 4 . M iln er, M ., K u n g , K .-J. S ., W y m an , J. A ., F eld m an , J., an d N o rd h e im , E . 1992. E n h a n c in g o v erw in ter­ in g m o rta lity o f C o lo ra d o p o ta to b e etle (C o leo p tera: C h ry so m e lid a e ) b y m a n ip u la tin g th e te m p e ra ­ tu re o f its h a b ita t. J. E co n . E n to m o l. 8 5 :1 7 0 1 -1 7 0 8 . P o u leu r, S., R ic h a rd , C ., M a rtin , J.-G ., an d A n to n n , H . 1991. Ice n u c le a tio n activ ity in tw o Fusarium sp e cies. (A b str.) Int. C o n f. B io lo g ic a l Ice N u cleatio n , 5th. M a d iso n , W I. R o g e rs, J. S., S tall, R . E ., a n d B u rk e, M . J. 1987. L o w -tem p eratu re c o n d itio n in g o f th e ice n u cleatio n a c tiv e b a c te riu m , E rw in ia herbicola. C ry o b io lo g y 2 4 :2 7 0 -2 7 9 . S h ield s, E . J., a n d W y m a n , J. A. 1984. E ffect o f d efo liatio n a t sp e c ific g ro w th sta g es o f p o ta to yields. J. E co n . E n to m o l. 7 :1 1 9 4 -1 9 9 . S h im a d a , K. 1989. Ic e -n u c le a tin g a c tiv ity in th e alim en tary can al o f th e free zin g -to leran t p re p u p a e o f T richiocam pus p o p u li (H y m e n o p te ra :T e n th re d in id a e ). J. Insect P h y sio l. 3 5 :1 1 3 -1 2 0 .

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S h im ad a, K ., a n d R iih im a a , A . 1988. C o ld acc lim a tio n , in o c u la tiv e fre e z in g a n d slo w co o lin g : E sse n tia l facto rs c o n trib u tin g to th e fre e z e -to le ra n c e in d ia p a u sin g la rv a e o f C hym om yza costata (D ip tera: D ro so p h ilid ae). C ry o L ett. 9 :5 -1 0 . S o m m e, L. 1982. S u p e rc o o lin g a n d w in te r su rv iv al in terrestrial a rth ro p o d s. C o m p . B io ch em . P h y sio l. 7 3 A :5 1 9 -5 4 3 . S teig erw ald , K. A ., L ee, M . R ., L ee, R . E ., a n d M a rsh all, J. C . 1993. E ffe c t o f ic e n u c le a tin g ac tiv e b a c ­ te ria on in se c t su p e rc o o lin g c a p a c ity v aries w ith th e a n a to m ic site o f a p p licatio n . (A b str.) Int. C o n f. on B io lo g ical Ice N u c le a tio n , 6 th . L aram ie, W Y . S torey, K . B ., an d S to re y , J. M . 1988. F re e z e to le ra n c e in an im als. P h y sio l. R ev. 6 8 :2 7 -8 4 . S tro n g -G u n d erso n , J. M ., L ee, R . E ., an d L ee, M . R. 1989. Ice re g u la tin g b a c te ria p ro m o te tra n sc u tic u la r n u c le a tio n in in se cts. C ry o b io lo g y 2 6 :5 5 1 . S tro n g -G u n d erso n , J. M ., L ee, R . E ., a n d L ee, M . R. 1990a. N ew sp e c ie s o f ic e n u c le a tin g a c tiv e b a c ­ te ria iso la te d fro m in se cts. C ry o b io lo g y 27:6 9 1 . S tro n g -G u n d erso n , J. M ., L ee, R. E ., L ee, M . R ., an d R iga, T. J. 1990b. In g estio n o f ice n u c leatin g a c ­ tiv e b a c te ria in c re a se s th e s u p e rc o o lin g p o in t o f th e lad y b e e tle H ippodam ia convergens. J. In sect P h y sio l. 3 6 :1 5 3 -1 5 7 . S tro n g -G u n d erso n , J. M ., L ee, R. E ., a n d Lee, M . R . 1992. T o p ic a l ap p lic a tio n o f ice n u c leatin g b a c te ­ ria d ec re a se s in se c t c o ld to le ra n c e . A p p l. E n v iro n . M icro b io l. 5 8 :2 7 1 1 -2 7 1 6 . T su m u k i, H ., K o n n o , H ., M a e d a , T ., a n d O k a m o to , Y . 1992. A n ic e -n u c le a tin g activ e fu n g u s iso la te d fro m th e g u t o f th e ric e ste m b o re r, Child suppressalis W a lk e r (L ep id o p tera: P yralidae). J. In sect P h y sio l. 3 8 :1 1 9 -1 2 5 . Z a c h aria ssen , K. E ., an d H a m m e l, H . T. 1976. N u c leatin g a g en ts in th e h aem o ly m p h o f in se cts to leran t to freezin g . N a tu re 2 6 2 :2 8 5 -2 8 7 .

CH APTER 15

I c e N u c lé a t io n G e n e s a s R e p o r t e r s Nickolas J. Panopoulos

U s e s o f R e p o r t e r G e n e s in B io lo g ic a l R e s e a r c h O n e av e n u e fo r th e in v e s tig a tio n o f b io lo g ic a l p ro c e s s e s is to d e te rm in e h o w an d w h en re le v a n t g e n e s a re tu rn e d o n o r off, an d h o w th e ir ac tiv ity is m o d u la te d by e n v iro n m e n ta l a n d c e llu la r sig n a ls. S in c e m o st g e n e s in liv in g o rg an ism s d o n o t h a v e e a sily a s s a y a b le fu n c tio n s , in v e stig a tio n o f th e ir te m p o ra l a n d sp a tia l p a tte rn s o f e x p re s s io n p re s e n ts d iffic u ltie s . T h e c o n c e p t o f u sin g a g e n e w ith an e a s ily a s ­ sa y a b le p ro d u c t to “r e p o r t” th e e x p re s s io n o f a n o th e r g e n e w as a b re a k th ro u g h a n d h as se rv e d as a b a s is fo r th e d e v e lo p m e n t o f a n a ly tic a l to o ls in m o le c u la r b io lo g y fo r n ea rly 25 y ears. A t p re s e n t, a w id e v arie ty o f g e n e s an d p ro te in s are u sed as r e ­ p o rte rs o f tra n s c rip tio n , as p r o b e s fo r th e d is s e c tio n o f p ro te in to p o lo g y , lo c a liz a ­ tio n , an d se c re tio n , a n d a s in d ic a to rs o f o th e r c e llu la r, g e n e tic , an d en v iro n m e n ta l p ro c e s s e s (B ro o m e -S m ith e t al., 1990; G a lla g h e r, 1992; K o n c z e t al., 1990; M a n o il an d B e c k w ith , 1986; S ilh a v y an d B e c k w ith , 1985; S h a w e t al., 1987; S te w a rt an d W illia m s , 1992, 19 9 3 ). Ic e n u c le a tio n g e n e s are a re c e n t a d d itio n to th e ra n k s o f g e n e tic re p o rte rs . T h is c h a p te r w ill d isc u ss the fe a tu re s o f ice n u c le a tio n g e n e s th a t are g e rm a n e to th e ir u se a s “ ic e n u c le a tio n ac tiv ity (IN A ) r e p o rte rs .”

C o n v e n tio n a l V e r s u s I N A R e p o r te r s T h e m o s t fre q u e n tly u s e d r e p o rte rs in b io lo g ic a l r e s e a rc h e n c o d e e n z y m e s w h o se a c tiv ity can b e e a sily d e te c te d a n d q u a n tifie d , u su a lly by e m p lo y in g a c h ro m o g e n ic , flu o ro g e n ic , ra d io a c tiv e , o r im m u n o d e te c ta b le s u b stra te . S u ch g en e s, h ere re fe rre d to as c o n v e n tio n a l re p o r te r s , in c lu d e lacZ, p h o A , cat, uidA ( g u sA ), xylE , an d lux, w h ich re s p e c tiv e ly e n c o d e (5 -g alactosidase, a lk a lin e p h o s p h a ta s e , c h lo ra m p h e n ic o l ac e y ltra n s fe ra s e , P -g lu c u ro n id a s e , c a te c h o l 2 ,3 -o x y g e n a s e , a n d lu c iferase . T h e luc ife ra s e re p o rte rs d iffe r fro m th e o th e rs o n ly in th a t th e m e a su re d re a c tio n p ro d u c ts are th e p h o to n s e m itte d d u rin g th e c o n v e rsio n o f s u b s tra te , ra th e r th a n a c h e m ic a l su b sta n c e . IN A re p o rte rs d if fe r fro m c o n v e n tio n a l re p o rte rs in a fu n d a m e n ta l sen se; the sig n al d e te c te d is n o t d u e to e n z y m a tic c a ta ly sis b u t is, in stead , a p h y sic a l p h e ­ n o m e n o n (th e liq u id -to -s o lid p h a s e tra n sitio n o f w ate r). A n o th e r im p o rta n t d iffe r­ e n c e b e tw e e n IN A a n d c o n v e n tio n a l re p o rte rs is th e n a tu re o f th e d o se -re sp o n s e c u rv e o b ta in e d w h en th e a m o u n t o f th e r e p o rte r p ro te in is p lo tte d ag a in st the sig n a l 271

272

P a n o p o u lo s

it g e n e ra te s; c o n v e n tio n a l re p o rte rs ty p ic a lly p ro d u c e lin e a r re s p o n s e s , w h erea s IN A re p o rte rs g iv e a n o n lin e a r re s p o n s e (L in d g re n e t a l., 1989; S o u th w o rth e t al., 1988; se e a lso C h a p te r 5). In c o m p a r is o n to c o n v e n tio n a l re p o rte rs, IN A r e p o r te r s h av e b e e n u se d in r e la ­ tiv e ly few in s ta n c e s , a n d all p u b lis h e d e x a m p le s in v o lv e g ra m -n e g a tiv e p ro k a ry o te s. H o w e v e r, IN A r e p o r te r s c a n b e e m p lo y e d in a b ro a d s p e c tru m o f o rg an ism s: all g ra m -n e g a tiv e b a c te r ia stu d ie d to d ate, som e g ra m -p o s itiv e b a c te ria , y e a sts, and p la n ts c a n e x p r e s s fu n c tio n a l ice n u clei up o n in tro d u c tio n o f b a c te ria l ina gen es (B a rtle in et al., 1 9 9 2 ; D . A .B a rtle in , S. E . L in d o w , N . J. P a n o p o u lo s , S. P. L ee , and T . H . H . C h e n , u n p u b lis h e d ; S .E . L in d o w , u n p u b lish ed ; a n d D . P rid m o re , p erso n a l com m u n ica tio n ). T h e u s e o f a n ic e n u c le a tio n g en e as a tr a n s c rip tio n a l r e p o rte r w as first e x p lo re d in 1 9 8 9 (L in d g re n e t al., 1989). T h e o b je c tiv e o f th is a n d su b se q u e n t stu d ie s in th is la b o ra to r y w as to e s ta b lish the tra n s c rip tio n a l o rg a n iz a tio n o f gen es in th e 2 2 -k b h rp c lu s te r o f th e p la n t p ath o g en P seu d o m o n a s syrin g a e pv. phaseolicola an d to a n a ly z e th e ir te m p o ra l p a tte rn o f e x p re s s io n d u rin g p a th o g e n e s is and h y p e rs e n s itiv e n e c ro s is . F u sio n s b e tw e e n m o st o f th e se g e n e s an d th e lac o p ero n p ro d u c e d b y in s e rtio n a l m u ta g e n e s is w ith p H o H o l ( T n 3-lacZ Y A ; S ta c h e l e t al., 1985) s h o w e d n o d e te c ta b le e x p re s s io n in v itro (L in d g re n a n d P a n o p o u lo s , u n p u b ­ lished). T h e h rp g e n e s o f P. syrin g a e pv. p h a seo lico la h a v e p a rtic u la r p H , m ed iu m , a n d p la n t s ig n a l r e q u ire m e n ts fo r e x p re ssio n (R ah m e e t al., 1992), w h ic h w ere u n ­ k n o w n at th a t tim e . F u rth e rm o re , la cZ w o u ld n o t b e a u se fu l re p o r te r in g ree n le av e s, e s p e c ia lly a t e a rly sta g e s o f in fec tio n w hen th e b a c te ria l p o p u la tio n is sm all (u su a lly le ss th a n 106 C F U /c m 2). T h e in a Z re p o rte r e m p lo y e d in th is ea rly stu d y has b ee n u se d in s e v e r a l o th e r la b o ra to rie s in re se a rc h o n p la n t-b a c te riu m in te ra c tio n s (T a b le 1; A n d e rs e n , 1993; F e lla y et al., 1991; F re d e ric k , 1989; G e o rg a k o p o u lo s,1 9 9 3 ; G rim m a n d P a n o p o u lo s , 1989; H u y h n e t al., 1 9 8 9 ; L ee, 1993; L o p e r and L in d o w , 1994; M a e t al., 1991; R a h m e et al., 1991, 1 9 92).

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V a rio u s r e p o r te r s sa tisfy th e n e e d s o f p a rtic u la r a p p lic a tio n s to d iffe re n t d eg re es (M e ig h e n , 1 9 9 1 ; S h a w e t al., 1987; S ilh av y an d B e c k w ith , 1985; S te w a rt and W illia m s , 1 9 9 3 ). M o s t h a v e lim ita tio n s o r d is a d v a n ta g e s , an d n o n e sa tisfie s all th e o re tic a l an d p ra c tic a l re q u ire m e n ts o f d iffe re n t e x p e rim e n ta l n e e d s to th e sam e d e g re e . A c c o rd in g ly , th e re is a co n tin u in g need for n e w re p o rte rs th a t m ay b e a p ­ p ro p ria te in p a r tic u la r situ a tio n s. O n e esse n tia l p r e re q u is ite o f an y r e p o rte r a p p lic a ­ tio n is th e a b s e n c e o f e n d o g e n o u s ac tiv itie s th a t w o u ld in te rfe re w ith th e assay . IN A re p o rte rs sa tisfy th is r e q u ire m e n t in a la rg e n u m b e r o f c a s e s , sin c e e n d o g e n o u s ice n u c le a to rs a c tiv e a t w a rm a s sa y te m p e ra tu re s are c o n fin e d to a few o rg a n ism s th a t carry ina genes (se e C h ap ter 3). A lso, w arm -tem perature ic e nuclei (active ab o v e - 5 ° C ) a re ra re o r a b s e n t fro m e n v iro n m e n ta l sa m p le s, su ch a s so il, as w ell as fro m p lan ts, w h e n th e se a re n o t c o lo n iz e d b y Ic e + b a c te ria (as is tr u e fo r p la n ts g ro w n in the g re e n h o u se ). T h is is e s p e c ia lly im p o rta n t fo r the u se o f IN A r e p o rte rs in th e study o f g e n e a c tiv ity in p la n t p a th o g e n s o r e p ip h y tes, in so il, o r in o th e r e n v iro n m e n ta l s a m p le s. A s e c o n d im p o rta n t re q u ire m e n t fo r r e p o rte rs is th e a b ility to e x p re ss a p h e n o ty p e o r s ig n a l th a t c a n b e se n sitiv e ly d e te c te d a n d e a sily q u a n tifie d w ith m in im al s a m p le p ro c e s s in g in in d iv id u a l cells, in c e ll p o p u la tio n s , o r in v ario u s n a tu ra l se ttin g s u n d e r a w id e v arie ty o f co n d itio n s. B e s id e s th e se re q u ire m e n ts, re p o rte rs id e a lly s h o u ld n o t g iv e false p o sitiv es, re q u ire e x p e n s iv e e q u ip m e n t, em -

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p lo y h a z a rd o u s c h e m ic a ls , o r in v o lv e tim e -c o n su m in g p ro c e d u re s fo r the assay s o r fo r s a m p le p re p a ra tio n . IN A re p o rte rs cle a rly su rp ass m o s t c o n v e n tio n a l re p o rte rs in re g a rd to th e s p e e d o f th e assay co m m o n ly u sed (th e a s s a y its e lf ta k e s a b o u t 1 -5 m in). F u rth e rm o re , th e y are m u c h m o re se n sitiv e th a n c o n v e n tio n a l re p o rte rs (< 1 0 5fo ld m o re s e n s itiv e th a n lacZ), an d at le a st in so m e a p p lic a tio n s th e y h av e c o m p a ­ ra b le se n sitiv ity to lu c ife ra s e re p o rte rs (L in d g re n e t al., 1989).

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a n d I n a P r o te in F u s io n s G e n e r a l- P u r p o s e T r a n s p o s o n s a n d V e c to r s T h e ra n g e o f to o ls c u rre n tly a v a ila b le for the c o n s tru c tio n o f ina g e n e fu sio n s is so m e w h a t lim ite d . M o s t re s e a rc h a p p lic a tio n s o f IN A re p o rte rs u tiliz e th e T n i S p ic e tra n s p o s o n o rig in a lly c o n s tru c te d by L in d g ren e t al. (1 9 8 9 ). A fe w o th e r g en e fu sio n v e h ic le s a re a v a ila b le o r are cu rre n tly u n d e r c o n s tru c tio n . T h e ir sa lie n t fe a ­ tu res are b rie fly re v ie w e d h ere . W h e n m o re fle x ib ility o r a p a rtic u la r stru c tu ra l d e ­ sig n is n e e d e d f o r d iffe re n t a p p lic a tio n s , n ew v eh icle s a n d g en e fu sio n to o ls ca n be e a sily m a d e , ta k in g a d v a n ta g e o f th e ab se n c e o f c le a v a g e sites fo r m a n y re stric tio n e n d o n u c le a s e s in in a Z a n d /o r o th e r ina gen es. T h e tra n s p o s o n s d e s c rib e d b e lo w are use d fo r th e c o n s tru c tio n o f m a Z ::ta rg e t g en e fu s io n s by in v iv o in se rtio n al m u ta g e n e sis o f c lo n e d D N A fra g m e n ts. T h ese fu sio n s c a n se rv e th e n e e d s o f bo th m u ta tio n a l a n d e x p re s s io n an a ly sis o f th e ta rg e t gene. A few p la s m id v e c to rs e x ist th a t c a rry th e in a Z g e n e in v a rio u s re p lic o n s an d allo w fo r the c o n s tru c tio n o f such fu sio n s b y c lo n in g a D N A fra g m e n t u p strea m o f an ic e n u c le a tio n g en e. In m o st c a se s, th e ic e n u c le a tio n g e n e la ck s its n ativ e p ro m o te r. T n 3 -S p ic e l, 2 , a n d 3

T h e th re e T n i - S p i c e tra n s p o s o n elem e n ts are d e riv e d fro m p H o H o l, a T n ilacZYA tra n s p o s o n (S ta c h e l e t al., 1985), by r e p la c e m e n t o f the lacZYA se g m en t w ith a p r o m o te rle s s in a Z g e n e c lo n e d fro m p lasm id p M W S lO ( W o lb e r et al., 1986). T h e y a lso c o n ta in th e 2 .0 -k b S p 7 S m r-e n c o d in g “O m e g a ” fra g m e n t (P re n tk i and K rish , 19 8 4 ) to p r o v id e b e tte r se le c ta b le m a rk e rs f o r b a c te ria , su c h as P seudom onas, th a t a re re la tiv e ly re s is ta n t to fM actam a n tib io tic s . T h e T n J tra n sp o s a se g e n e ( tn p A ) is in a c tiv e in all th re e T n i- S p ic e e le m e n ts d u e to th e tru n c a tio n o f its C -te rm in u s in th e p H o H o l p ro g e n ito r an d the in se rtio n o f th e O m e g a fra g m e n t in th e C la l site o f p T n i- I c e . T n 3 -S p ic e ele m e n ts are u se d as p a rt o f a tw o -p la sm id sy stem , a lo n g w ith th e p S S h e p la sm id (C m r), w h ich p r o v id e s th e T n i tra n sp o s a se fu n c tio n in tra n s, a s in p H o H o l m u ta g e n e sis (S ta ch e l e t a l., 1985). T h e th re e T n J - S p ic e tra n s p o s o n s d iffe r fro m ea ch o th e r in re g a rd s to w h e th e r or n o t th e in a Z g e n e c a n b e fu sed to u p stre a m se q u e n c e s in fra m e. T n J - S p ic e 1 is the tra n s p o s o n o rig in a lly d e s c rib e d by L in d g re n et al. (1 9 8 9 ) as T n J - S p ic e . In th is e le ­ m en t, th e s e g m e n t p re c e d in g th e in a Z g en e has tra n s la tio n a l sto p c o d o n s in all th ree re a d in g fra m es; a s a re su lt, o n ly transcription al fu sio n s c a n b e m a d e. T h is is a d e ­ sira b le fe a tu re in r o u tin e g e n e tic an a ly sis, sin c e tra n s la tio n a l fu sio n s c o u ld p ro d u c e In a Z p ro te in s w ith d iv e rs e N -te rm in a l e x te n sio n s an d , th e re fo re , u n p re d ic ta b le a c ­ tiv ity . T n J - S p ic e 2 a n d T n J - S p ic e 3 (M .N . M in d rin o s , L .G . R a h m e a n d N .J. P a n o p o u lo s , u n p u b lish ed ) d iffe r fro m T n J - S p ic e 1 in th a t o n e o f th e th re e re a d in g fra m e s in th e s e g m e n t u p s tre a m o f in aZ d o e s not c a rry s to p c o d o n s ; th e re fo re , the fu sio n s g e n e ra te d b y th e se e le m e n ts ca n b e e ith e r tra n s c rip tio n a l (th e m a jo rity ) o r

Ic e N u c lé a tio n G e n e s a s R e p o rte rs

275

tra n sla tio n a l. T h e s p e c ia liz e d u s e s o f T n 3 -S p ic e 2 an d T n J - S p ic e 3 rem a in to b e e x ­ p lo red . T n 3 -N ic e

T n 3 -N ic e is e q u iv a le n t to T n i - S p i c e l , w ith a n e o m y c in -re sis ta n c e g en e fro m T n 903 re p la c in g th e S p r/S m r fra g m e n t (J. K ra u s a n d J. L o p e r, unpublished). T n 5-ina D e r i v a t i v e s

T n 5 -in a d e riv a tiv e s d e r iv e fro m th e T n 5 -/a c tra n s p o s o n s d e s c rib e d by S im o n et al. (1 9 8 9 ) by in se rtio n o f th e in a Z g e n e n e a r the te rm in u s o f th e left in v e rted re p e a t (IR l ). T h e y c a rry th e c o g n a te tra n s p o s a s e g en e in th e sa m e e le m e n t as in aZ a n d w ill p e rm it th e g e n e ra liz e d in s e rtio n o f th e r e p o rte r in th e b a c te ria l c h ro m o so m e at ra n d o m by s u ic id e m u ta g e n e s is , w h ich is n o t c u rre n tly p o s s ib le w ith the T n 3 -S p ic e tra n sp o s o n s. p L A F R 6 -in a Z

T h e p L A F R 6 -m a Z p la s m id is d e riv e d fro m th e b ro a d h o s t ra n g e v e c to r p R K 2 9 0 (D itta e t al., 19 8 0 ) a n d th u s c a n r e p lic a te in a b ro a d ra n g e o f g ra m -n e g a tiv e b a c te ­ rial h o sts. It c o n ta in s a p ro m o te rle s s in a Z g en e p lu s a m u ltilin k e r se q u e n c e an d sy n th e tic tra n s c rip tio n a l te rm in a to rs o n b o th sid e s (D . D a h lb e c k and B .J. S task aw ic z, un p ub lish ed ). T h e te rm in a to rs in su la te th e in a Z g e n e fro m th e tra n s c rip ­ tio n a l a c tiv ity o f v e c to r s e q u e n c e s ; as a re su lt, th e b a s a l le v el o f inaZ e x p re s s io n is v ery lo w (a lth o u g h th is m a y v a ry w ith th e h o st in w h ich th e p la sm id is p ro p a g a te d ). W h e n a p r o m o te r-c o n ta in in g D N A fra g m e n t is c lo n e d in fro n t o f in a Z in the p ro p e r o rie n ta tio n , th e le v el o f in a Z e x p re s s io n in c re a se s a c c o rd in g ly . PV S P 6 1 -i« a Z

p V S P 6 1 is a h y b rid p la s m id c o n s is tin g o f th e p V S l a n d p A C Y C 1 8 4 re p lic o n s (W . T u c k e r, p e r so n a l co m m u n ic a tio n ) an d is v e ry sta b le in E sch erichia coli an d P seud om on as sp e c ie s g ro w n in c u ltu re , the rh iz o s p h e re , o r th e p h y llo p la n e . T h e p ro m o te rle ss in a Z w as in s e rte d in to th e p U C 8 -d e riv e d m u ltilin k e r o f p V S P 6 1 in a d ire c tio n o p p o s ite to la c Z a n d a fra g m e n t c a rry in g th e p v d (p y o v e rd in e ) p ro m o te r fro m P. syrin g a e w as p la c e d u p stre a m . T h is re p o r te r e x p re s s e s iro n -re g u la te d IN A an d e x e m p lifie s th e p o s s ib ility o f b io se n sin g u sin g IN A re p o rte rs (L o p e r an d L in d o w , 1994). P r o c e d u r e s U s e d in T n 3 - S p ic e M u ta g e n e s is T h e T n i - S p i c e l tr a n s p o s o n is th e m o s t fre q u e n tly u se d ina fu sio n v eh icle. T h e p ro c e d u re s fo r its u se a re d e s c r ib e d in L in d g re n et al. (1 9 8 9 ) an d are b a s e d on th o se u se d fo r T n 3 -la c m u ta g e n e s is u sin g p H o H o l (S ta c h e l e t al., 1985). T h e p M B 8 d e riv e d re p lic o n o f th e tr a n s p o s o n d o n o r p la sm id c a n b e m a in ta in e d in m o st E. coli strain s b u t d isp la y s th e rm o s e n s itiv e re p lic a tio n in a p o lA 's m u ta n t C 2 1 1 0 (also d e s ­ ig n a te d S F 8 0 0 ) an d is u n a b le to r e p lic a te in n o n e n te ric b a c te ria l ho sts. A c c o rd in g ly , if th e ta rg e t D N A fra g m e n ts are c a rrie d on /;o /A -in d e p e n d e n t v ec to rs, th e tr a n s ­ p o so n in se rtio n d e riv a tiv e s o f th e ta rg e t p la sm id are s p e c ific a lly s e le c te d in th e fin al step (see b e lo w ). A g e n e ra l o u tlin e o f th e strain c o n s tru c tio n s an d ste p s fo llo w e d in th e is o la tio n o f T n i - S p i c e l in s e rtio n s in ta rg e t D N A is g iv e n b elo w :

1) T h e ta rg e t D N A is c lo n e d in a v e c to r w h o se r e p lic a tio n in E. coli is p o lA - in d e ­ p e n d e n t. C o s m id c lo n e s in th e co m m o n ly u se d b ro a d h o st ra n g e v e c to rs m ay be u se d d ire c tly fo r p T n i - S p i c e l m u ta g e n e sis.

276

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2) T h e ta rg e t D N A - c o n ta in in g p la sm id is in tro d u c e d in to an E. co li stra in th a t c a r­ rie s p T n i - S p i c e l ( tra n s p o s o n d o n o r p la sm id ) a n d p S S h e (e n c o d in g tran sp o sa se). T h is tr a n s f e r m a y b e a c c o m p lish e d by trip a re n ta l m a tin g , fo r ex a m p le u sin g th e h e lp e r p la s m id p R K 2 0 1 3 (D itta et al., 1980). 3) T h e s tra in c a r ry in g all th re e p la s m id s (th e ta rg e t, p T n 3 - S p ic e l, a n d p S S h e ) is allo w e d to c o n ju g a te w ith the p o lA 's re c ip ie n t, a g a in m o b iliz in g the targ et p la s m id w ith p R K 2 0 1 3 , in a trip a re n ta l m ating. 4 ) T h e c e lls in th e c o n ju g a tio n m ix tu re are p la te d o n a m e d iu m th a t c o n ta in s n a lid ix ic a c id , to w h ich o n ly th e p o lA

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Figure 5. Size distributions of bubbles in foams, comparing samples derived from freeze-concentrated and intact egg white, both with and without sucrose. A, Freeze-concentrated, with sucrose; B, not con­ centrated, with sucrose; C, freeze-concentrated, without sucrose; and D, not concentrated, without su­ crose. The histograms indicate a narrowing of the bubble size distribution in the foams made from concentrated egg white. The foams were observed at 40x magnification under a polarizing microscope, and an image analyzer (Carl Zeiss IBAS) was used to determine the size distribution of the bubble di­ ameters. Reprinted, with permission, from Kumeno et al. (1993a).

of some food materials. It can denature proteins to obtain gels in the cases of egg (Bridgman, 1914; Hayashi et al., 1989) soy protein (M atsumoto and Hayashi, 1990), meat (Suzuki et al. 1990), and milk. Pressure-processing also has the merit that fresh flavors and colors are usually retained. Our freeze-concentration process with bacterial ice nuclei also retains latent functions o f fresh materials (W atanabe et al., 1991b). This section illustrates the synergy of the two methods in the production of milk gels produced by a com bina­ tion of the freeze-concentration and pressurization techniques. Freeze-concentrated milk (both raw and market milk) is able to gel upon pres­ sure treatment, whereas neither unconcentrated nor freeze-dried milk will gel unless sucrose is also added. The gels produced from freeze-concentrated raw milk were

308

W a ta n a b e a n d A ra i

stronger and m ore viscoelastic than those from the freeze-concentrated market milk. These results suggest that very labile components in milk are involved in gelation. In this sense, freeze-concentration is favorable for concentration because it avoids protein denaturation (W atanabe et al., 1989). The textural properties o f pressure-induced gels from freeze-concentrated raw milk depended on pressure. Pressurization at more than 300 M Pa induced gelation, and increasing pressure made a gel stronger and m ore viscoelastic. W hen gels were heated, storage modulus decreased with temperature. The tem­ perature at which storage modulus approaches zero corresponds to the melting temperature of the gel (Shimizu et al., 1991). A remarkable decrease in the modulus was observed at about 40°C, and the modulus reached zero at 63-75 °C regardless of pressure and sugar concentration. As is well known (Bridgman, 1914; Hayashi et al., 1989; M atsum oto and Hayashi, 1990; Okamoto et al., 1990; Suzuki et al., 1990; Yamamoto et al., 1990; Ohmori et al., 1991; Suzuki and Suzuki, 1992), no protein denatures at 300 M Pa. W e speculate that native proteins contribute to pressureinduced gelation. The gel prepared from milk only by the combined method o f freeze-concentra­ tion and pressurization is novel as a food product. It is distinguished from milk jelly (produced with the aid o f gelling agents) by its whiteness, brightness, and fresh milk flavor. An optim ized procedure to make a pressure-induced milk gel is to use freeze-concentrated raw milk, to add sugar at 10% to the concentrated milk, and to compress it at m ore than 300 M Pa at a low temperature. F r e e z e - C o n c e n tr a tio n o f L e m o n J u ic e to R e d u c e V o lu m e

Lemon juice was also freeze-concentrated with the aid of bacterial ice nuclei. The presence of polyols reduces the amount of water that freezes during cooling (Franks, 1985). This means that ice formation is more difficult in a sugar solution. Lemon juice, however, could be efficiently concentrated with high solids recoveries (Table 1). On freeze-concentration, constituents with low boiling points were well retained (Fig. 6), whereas they decreased on vacuum-concentration, causing a change from the original flavor. W e regarded limonen (peak L) and citral (peak C) Fr e sh

Fr e e ze - co n ce n t r a t e d

R e te n tio n

tim e

V a cu u m - co n ce n t r a t e d

( m in )

Gas chromatograms of fresh, freeze-concentrated, and vacuum-concentrated lemon juice samples demonstrating the retention of volatile limonen (peak L) in comparison to the less volatile citral (peak C) in freeze-concentrated juice. (IS denotes the peak produced by the internal standard, npentadecane). Freeze-concentrated lemon juice had a solid content of 36% (see Table 1); lemon juice vacuum-concentrated at 10 torr also had a solid content of 36%. Reprinted, with permission, from Watanabe et al. (1989).

F ig u r e 6 .

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as easily volatile and not easily volatile components, respectively, and calculated the ratios o f limonen to citral (L/C) to be 8.63, 8.60, and 1.46 for the fresh, freeze­ concentrated, and vacuum-concentrated materials, respectively. Panelists in a sen­ sory test answered that the freeze-concentrated product was not significantly different in flavor from fresh material. F r e e z e - C o n c e n tr a tio n o f S t r a w b e r r y J u ic e

In the conventional production of strawberry jam , a strawberry paste is mixed with granulated sugar and then evaporated by heating at atmospheric pressure. The product loses its fresh flavor owing to evaporation of certain flavor components. Pressurization of the fruits (Horie et al., 1991) obviates the need for heat treatment, but the ja m ’s texture, particularly its thickness, must then be provided by adding large amounts of thickening agents such as pectin, because the method lacks a con­ centration step. Freeze-concentration can be used to remove excess water before pressurizing to sterilize the product and still retain a fresh flavor. W e have compared jam made in the conventional way with jam formed by freeze-concentrating strawberry juice at 5°C overnight (using bacterial ice nuclei) followed by pressurization to 400 MPa. W e measured the viscoelastic properties of the jam samples. None o f the meas­ ures differed significantly between samples (Table 3). Thus, a jam sample with normal viscoelastic and textural properties was produced without the use o f any heating process. The nonheated jam is superior in brightness (L value) and red color

T a b le

3. Viscoelastic, textural, and color properties of a nonheated straw berry jam sample' H ea te d “ N o n h e a ted

P r o p e r ty

Viscoelastic param eter' A pparent viscosity (Pa-s) at 14.6/s shear rate at 58.5/s shear rate Yield stress (Pa) Consistency index (P a-sn) Flow behavior index T extured H ardness (RU) Cohesiveness (RU) Adhesiveness (RU) Springiness (RU) C o lo r' Brightness (L value) Red (a value) Yellow (b value)

(c o n v e n tio n a l)

(M e a n

± SE)

(M ea n

± SE)

1.34 0.65 3.65 0.73 0.52

± 0 .3 0 ± 0.08 ± 1.23 ± 0 .1 9 ± 0.05

1.03 0.67 2.52 0.51 0.61

± 0 .2 1 ± 0.05 ± 0 .3 5 ± 0 .1 1 ± 0.03

0.117 0.635 0.115 2.781

± 0 .0 3 0 ± 0.025 ± 0 .0 2 5 ± 0 .6 9 5

0.143 0.629 0.165 2.367 7.8 15.2 5.8

± 0.012 ± 0.008 ± 0 .0 1 3 ± 0.058

7.2 13.4 5.4

“R eprinted, with permission, from W atanabe et al. (1991b). bJam sam ple produced by the conventional heating method. 'M easu red with a viscometer (H aake, R hotovisco RV-3) at 25°C. N o significant difference was observed in any of the param eters between the nonheated and the conventional samples. d M easured with a R heolom eter (Iio Denki). The conditions for m easurem ent were as follows: sam ple tem perature, 25° C; cycle speed, 12 cycles/m in; clearance, 2.0 mm; sample height, 1.2 cm; plunger diam eter, 4.0 cm; m otion num ber, 2; and load range, 0.5 kg. N o significant difference was observed in any of the param eters between the nonheated and the conventional samples. 'M easu red with a color difference m eter (N ihon D enshoku, ND -1001-DP).

310

W a ta n a b e a n d A ra i

R e t e n t io n

t im e

( m in )

F i g u r e 7. Gas chromatograms o f nonheated ( A ) and conventional (B) strawberry jam samples. Com­ parison indicates that more volatiles were retained during production of the nonheated jam. Headspace volatiles were trapped in a Tenax TA trapping tube; the headspace gas was injected into a gas chroma­ tograph using a fused silica capillary column coated with PEG 20M, with a flame ionization detector. Reprinted, with permission, from Watanabe et al. (1991b).

(a value) to the conventional jam , probably because the M aillard reaction does not proceed during production of the nonheated jam. Flavor was evaluated using the Tenax TA trapping technique (Tsugita et al., 1980) and gas chromatography (Tsugita et al., 1979). Figure 7 shows chromatograms obtained with the nonheated and the conventional ja m samples, suggesting that the former retained the original flavor components. Evidently, freeze-concentration with the aid of bacterial ice nuclei can perm it the manufacture of a nonheated jam with superior color and flavor. Similar procedures could be generally applied for the purpose of producing processed food items retaining original color and flavor qualities. F r e e z e - D r y in g w ith B a c t e r ia l I c e N u c le i

Demand is growing for a means of minimizing supercooling in the freeze-drying of foods. The higher the temperature at which freezing can be achieved, the more efficient the process. M oreover, the formation o f larger ice crystals imparts

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properties to the product that facilitate the powdering process subsequent to drying. W atanabe and Arai (1987) experimented with the use of bacterial ice nuclei to re­ duce supercooling in food freeze-drying. This application is best illustrated in the freeze-drying of two food items that are difficult to freeze: soy sauce and soybean paste. In the case o f soy sauce, a super­ cooling of only 2°C was observed in the presence o f bacterial ice nucleators; in soybean paste, a greater degree of supercooling was seen. For each of the food items, a porous structure with large cavities was observed when the sample with the Ina+ cells was freeze-dried. The porous products from both the soy sauce and the soybean paste could be easily pulverized. Both types of sample supercooled ap­ proximately 10 degrees m ore in the absence of the Ina+ cells. Applying bacterial ice nuclei to the freeze-drying o f food items that are difficult to freeze under proper conditions made it possible to shorten their freezing times and to obtain powdered products with greater efficiency. Nagashima and Suzuki (1985) stated that the dilution o f a sample with water was necessary for effective freezing of high-salt food items. The addition o f the Ina+ bacterial cells to such samples is expected to allow similarly effective freezing without dilution. It may result in lowered processing costs. A n is o t r o p ic T e x t u r in g w ith B a c t e r ia l I c e N u c le i

Texturing of proteins has been studied actively by many researchers for the pur­ pose o f producing meat analogs from plant materials (Feeney et al., 1982). A method o f freeze-texturing has long been practiced in Japan in making frozen soy­ bean curd. Generally, the water in an aqueous dispersion or hydrogel of proteins tends to supercool to a great degree. To break down such a supercooled state for ice crystallization, the tem perature has to be maintained much below the melting point o f the ice in the same system. This is economically disadvantageous. Another dis­ advantage is the disordered formation o f ice crystals and their omnidirectional growth, which produces an isotropic texture perceived as spongelike. Although the first disadvantage may be inevitable when conventional freezing is used, the second one can be removed by two devices for making ice crystals propagate in a particular direction. One is to use fibrous materials such as fish muscles (Lillford, 1985) and the other is to force the texture o f a frozen slurry to turn to one direction by melting part of the ice under added pressure (W ilding et al., 1984). Arai and W atanabe (1986) undertook experiments based on the idea that bacte­ rial ice nuclei would prevent supercooling of the bulk water in aqueous dispersions and hydrogels of food proteins and polysaccharides, and that this might modify ice crystallization so as to im prove their textures. As seen above (Fig. 3), egg white freezes at a warmer tem perature in the presence of bacterial ice nuclei. When tex­ ture was examined microscopically, bacterially nucleated frozen egg white was seen to contain large, long, m utually parallel ice crystals. The parallelism o f ice crystals suggests that an isotropic liquid such as egg white can be textured into a film or flake state. We attempted to impart textures to iso­ tropic liquids or gels of five proteinaceous and four polysaccharide com positions.1 A hot setting step was sometimes necessary to prevent the breakdown of frozen textures when water was added. Control samples without the ice nucleation-active The use o f degassed water to disperse some materials is important because dissolved gases are excluded from ice, forming bubbles that perturb the resulting texture.

'T e c h n ic a l n o te :

312

W a ta n a b e a n d A ra i

bacterial cells were similarly prepared with addition of water instead o f the bacterial cell suspension. The products frozen with the bacterial cells generally possessed directionally ar­ ranged textures, whereas the textures of the products without the cells were spongelike. Directional textures were obtained from raw egg white, bovine blood, 5 -15% soybean protein isolate, soybean curd, milk curd, 0.5-2% agar, 5-20% corn starch paste, and 0 .5 -2 % glucomannan. However, such a texture was not obtained from a calcium -bridged glucomannan gel at the concentrations tested (0.25-2% ); this may be due to its strong gel structure, which interfered with the directional growth of ice crystals. C o n c lu s io n s

Among various techniques for removal of water from foods, freeze-concentration is unique in em ploying the principle that solutes in aqueous media are excluded during the growth o f ice crystals. In the presence of added ice nucleation-active bacterial cells as ice nuclei, the bulk water in foods freezes at a subzero temperature near the melting point o f ice. For applications, we thus concentrated raw egg white and found that the product formed a hard gel when heated and also gave a fine foam when whipped. The freeze-concentrated product from fresh milk was characterized by forming a gel when pressurized. A similar technique was applied to fresh lemon juice to obtain a concentrated product still retaining its original flavor. As another application, strawberry paste was separated into juice and pulp fractions, and the juice fraction alone was freeze-concentrated. The pulp fraction was then put back together with sugar, pectin, and citric acid. W e thus succeeded in making a jam without heating, which, compared with conventional jam , was almost equal in texture and superior in fresh flavor and color. L ite r a t u r e C ite d Arai, S., and Watanabe, M. 1986. Freeze texturing of food materials by ice-nucleation with the bacte­ rium Erwinia ananas. Agric. Biol. Chem., 50:9-175. Bridgman, P. W. 1914. The coagulation of albumin by pressure. J. Biol. Chem., 19:511-512. Donovan, J. W., Mapes, C. J., Davis, J. G., and Garibaldi, J. A. 1975. A differential scanning calorimetric study of the stability o f egg white to heat denaturation. J. Sci. Food Agric. 26:73-83. Erikson, S. E., and Borden, R. E. 1955. Frozen eggs in relation to their use in cooked products. Ken­ tucky Agric. Exp. Stn. Bull. 635:3-23. Feeney, R. E„ Yamasaki, R. B„ and Geoghegan, K. F. 1982. Chemical modification of proteins: An overview. Adv. Chem. 198:3-55. Franks, F. 1985. Complex aqueous system at subzero temperatures. Properties of Water in Foods. D. Simatos and J. L. Multon, eds. NATO ASI (Adv. Sci. Inst.) Ser. Ser. E Appl. Sci. 90:497-509. Hayashi. R. 1987. Possibility of high pressure technology for cooking, sterilization, processing and storage of foods. Shokuhin to Kaihatsu, 22(7):55-62. Hayashi, R. 1989. Use o f high pressure in food processing and preservation. Pages 1-30 in: Use of High Pressure in Foods. R. Hayashi, ed. San-Ei Shuppan, Kyoto. Hayashi, R., Kawamura, Y., Nakasa, T., and Okinaka, O. 1989. Application of high pressure to food processing: Pressurization of egg white and yolk, and properties of gels formed. Agric. Biol. Chem. 53:2935-2939. Honma, K., Makino, T., Kumeno, K., and Watanabe, M. 1993. High-pressure sterilization of ice nucleation-active Xanthomonas campeslris and its application to egg processing. Biosci. Biotechnol. Biochem. 57:1091-1094. Horie, U., Kimura, K., and Hori, K. 1991, Development of processed foods from fruits by pressuriza­ tion. Nippon Nogeikagaku Kaishi 65:706-707 . Inukai, S., and Matsuda, N. 1980. Factors affecting heat resistance of bacterial spores. Part 1. Effect of pH on heat resistance o f genus Bacillus spores. Canners J. 59:219-224.

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Jeanes, A. 1974. Extracellular microbial polysaccharides: New hydrocolloids of interest to food indus­ try. Food Technol. 28:34-40. Kobayashi, M., and Nakahama, N. 1986. Rheological properties of mixed gels. J. Texture Stud. 17:161-174. Kumeno, K., Kurimoto, K., Nakahama, N„ and Watanabe, M. 1994. Functional properties of freeze­ concentrated egg white foam and its applicatios to food processing. Biosci. Biotechnol. Biochem. 58:447-450. Kumeno, K., Nakahama, N., Honma, K., Makino, T., and Watanabe, M. 1993. Pressure-induced ge­ lation of freeze-concentrated milk. Biosci. Biotechnol. Biochem. 57:750-752. Lindow, S. E. 1983. The role o f bacterial ice nucleation in frost injury to plants. Annu. Rev. Phytopathol. 21:363-384. Matsumoto, T„ and Hayashi, R. 1990. Properties of pressure-induced gels of various soy protein prod­ ucts. Nippon Nogeikagaku Kaishi 64:1455-1459. Nagashima, N„ and Suzuki, E. 1985. Freezing curve by broadline pulsed NMR and freeze-drying. Re­ frigeration Sci. Technol. 1985-1:65-70. Ohmori, T., Shigehisa, T., Taji, S., and Hayashi, R. 1991. Effect of high pressure on the protease ac­ tivities in meat. Agric. Biol. Chem. 55:357-361. Okamoto, M., Kawamura, Y., and Hayashi, R. 1990. Application of high pressure to food processing: Textural comparison of pressure- and heat-induced gels of food proteins. Agric. Biol. Chem. 54:183-189. Omran, A. M., and King. C. J. 1974. Kinetics of ice crystallization in sugar solutions and fruit juices. AIChE J. 20:795-803. Shimada, S., and Takada, Y. 1989. Effect of high pressure on the microbial behavior and death. Pages 31-52 in: Use of High Pressure in Foods. R. Hayashi, ed. San-Ei Shuppan, Kyoto. Shimizu, A., Kitabatake, N., Higasa, T., and Doi, E. 1991. Melting of the ovalbumin gels by heating: Reversibility between gel and sol. Nippon Shokullin Kogyo Gakkaishi 38:1050-1056. Shirai, Y., Nakanishi, K., Matsuno, R., and Kamikubo, T. 1985. On the kinetics o f ice crystallization in batch crystallizers. AIChE J. 31:676-682. Shirai, Y. Sasaki, K. Nakanishi. K., and Matsuno, R. 1986. Analysis of ice crystallization in continuous crystallizers based on a particle size dependent growth rate model. Chem. Eng. Sci. 41:2241-2246. Suzuki, C., and Suzuki, K. 1992. The protein denaturation by high pressure. Changes of optical rotation and susceptability to enzymic proteolysis with ovalbumin denatured by pressure. J. Biochem. 52:6771. Suzuki, A., Watanabe, M., Iwamura, K. Ikeuchi, Y„ and Saito, M. 1990. Effect of high pressure treat­ ment on the ultrsstructure and myofibrillar protein of beef skeletal muscle. Agric. Biol. Chem 54:3085-3091. Tsugita, T., Imai, T., Doi, Y., Kurata, T., and Kato, H. 1979. CC and GC-MS analysis of headspace volatiles by Tenax GC trapping techniques. Agric. Biol. Chem. 43:1351-1354. Tsugita, T„ Kurata, T., and Kato, H. 1980. Volatile components after cooking rice milled to different degrees. Agric. Biol. Chem. 44:835-840. Vali, G. 1971. Quantitative evaluation of experimental results on the heterogeneous freezing nucleation of supercooled liquid. J. Atmos. Sci. 28:402-409. Watanabe, M„ and Arai, S. 1987. Freezing of water in the presence of the ice nucleation active bacte­ rium Erwinia ananas, and its application for efficient freeze-drying of foods. Agric. Biol. Chem. 51: 557-563. Watanabe, M., Watanabe, J., Kumeno, K., Nakahama, N., and Arai, S. 1989. Freeze concentration of some foodstuffs using ice nucleation-active bacterial cells entrapped in calcium alginate gel. Agric. Biol. Chem. 53:2731-2735. Watanabe, M., Kumeno, K„ Nakailama, N., and Arai, S. 1990. Heat-induced gel properties of freezeconcentrated egg white produced using bacterial ice nuclei. Agric. Biol. Chem. 54:2055-2059. Watanabe, M., Makino, T., Kumeno, K., and Arai, S. 1991a. High-pressure sterilization of ice nuclea­ tion-active bacterial cells. Agric. Biol. Chem. 55:291-292. Watanabe, M„ Arai, E., Kumeno, K., and Honma, K. 1991b. A new method for producing a non-heated jam sample: The use of freeze concentration and high-pressure sterilization. Agric. Biol. Chem. 55:2175-2176. Watanabe, M., Watanabe, J., Makino, T., Honma, K., Kumeno, K„ and Arai, S. 1993. Isolation and cultivation of a novel ice nucleation-active strain of Xanthomonas campestris. Biosci. Biotechnol. Biochem. 57:994-995. Wilding, P., Lillford, P. J., and Regenstein, J. M. 1984. Functional properties of proteins in foods. J. Chem. Technol. Biotechnol. B 34:182-189. Yamamoto, K., Miura, T., and Yasui, T. 1990. Gelation of myosin filament under high hydrostatic pressure. Food Microstruct. 9:269-277.

C H A P T E R 18

T h e R o le o f N u c lé a t io n in C r y o p r e s e r v a t io n G re g o ry M . F a h y

In 1985, I organized a symposium for the Society for Cryobiology entitled “Physical Events at Low Tem peratures.” My first speaker, Don Rasmussen, spoke on the subject o f “N ucléation.” In discussing the concept of the symposium before it took place, a certain senior cryobiologist offered the opinion that nucléation is really beside the point since, first o f all, there is nothing to be done about it and, secondly, it is not really significant because it is what happens after nucléation that kills cells, not nucléation itself. As we now more generally appreciate, nucléation can in fact be modified and even largely avoided, and the site o f nucléation, which can determine whether cells die after nucléation takes place, is also under our con­ trol. Therefore, nucléation is indeed highly pertinent to cryopreservation, and it is the purpose of this chapter to provide a brief introduction to this subject. N u c l é a t i o n S i t e D e t e r m i n e s S u r v i v a l o f F r o z e n C e ll s a n d D e f in e s S lo w a n d R a p id C o o lin g

Figure 1 depicts the three possible fates that can befall a cell during cooling to cryogenic temperatures. Each fate is defined by the nature of the nucléation process involved. If nucléation is exclusively extracellular, the cell shrinks and tends to survive. If nucléation is intracellular, ice crystals form inside the cell and damage its internal structure, usually leading to cell death. Either situation may arise during continuous freezing or during discontinuous freezing in which abrupt temperature steps are imposed (see below). The third fate is that nucléation fails to occur either intracellularly or extracellularly— instead of freezing, the cell and its surrounding medium pass into the glassy state, i.e., they vitrify (Fahy et al., 1984). Very often, vitrified cells survive on warm ing (Fahy, 1988). As initially explained and quantitatively analyzed by M azur (Mazur, 1963), and as subsequently validated in detail mostly by M azur, Cravalho, and their colleagues, the factor that determines whether cells freeze externally or internally is the cooling rate or, more precisely, the thermal history. Cooling rate affects nucléation site pri­ marily for two reasons. 315

316

F ahy

First, the extracellular space is uniformly found to contain heterogeneous nucleators of thus far undefined nature that are more effective than any putative intracellu­ lar nucleators may be (Franks et al., 1983; Rail et al., 1983; Rasmussen et al., 1975). Consequently, in almost any biologically relevant situation short of ul­ trarapid cooling, crystallization will always begin extracellularly. Second, the ability o f the cell to respond osmotically to extracellular freezing is not instantaneous. The ice that forms in aqueous solutions is a pure phase that re­ jects any solutes that may be present. Extracellular nucleation therefore results in a reduction in extracellular solvent water but not in a loss of dissolved solutes. Thus, freezing produces an increase in extracellular solute concentration by a decrease in extracellular water concentration. This reduction o f extracellular water concentra­ tion creates a water concentration (more properly, water activity) gradient across the cell membrane that results in net diffusion of water from the intracellular to the extracellular space. This loss o f cell water in turn concentrates the intracellular sol­ utes and therefore lowers the freezing point of the cytoplasm until it is equal to the freezing point o f the extracellular space, thus eliminating the driving force for any intracellular freezing. B ut when cooling proceeds too quickly, the cell cannot lose water as rapidly as it is being lost from the extracellular space, and therefore the freezing point o f the cytoplasm fails to fall as rapidly as the tem perature of the cy­ toplasm, leading to the potential for intracellular nucleation. It should be realized that cells that can live in distilled water because they are surrounded by a cell wall dehydrate normally when frozen in distilled water. This is the case, for example, with yeast cells (Mazur, 1965b). This forces us to recognize that cellular dehydration is driven by the lower vapor pressure of ice in comparison to the vapor pressure o f intracellular water: the intermediary o f “osmotic pressure” E x t r a c e llu l a r Flu id

37°C

I n t r a c e llu la r Flu i d

Slow Cooling

-196°C ALIVE

DEAD

ALIVE?

Figure 1. Physiochemical processes during cryoperservation of cells— possible fates of cells cooled to low temperatures. Freezing can occur exclusively in the extracellular space (right), it can occur in both the extracellular and the intracellular space (middle), or it may occur not at all (left). Freezing may be accomplished either by continuous cooling protocols or by interrupted (two-step or three-step) cooling protocols (see text). The avoidance of ice altogether requires either ultrarapid cooling or, more practi­ cally, the use of very high concentrations of cryoprotective agents (see text). Adapted from Coger and Toner (1995).

N u c lé a tio n in C ry o p re s e rv a tio n

317

LU

et:

o cr

0.1

10

100

1000

COOLING RATE (°C/ min) Figure 2. Relationship between freeze-thaw survival (closed symbols) and the incidence of intracellular nucléation (open symbols). Circles: data for ova. Triangles: data for HeLa cells. Squares: data for human red blood cells (RBC). Reprinted, with permission, from Mazur (1988).

is not necessary, though it is usually present for most cells. Furthermore, the extra­ cellular solution, too, dehydrates entirely because its vapor pressure is greater than that of ice; once ice forms, both the extracellular and the intracellular compartments adjust themselves to it by the same mechanism. Given sufficient dilution of the wa­ ter in solution by solutes as they concentrate, the vapor pressure of water in solution can be lowered to equal that o f ice at any given temperature, and this is when the liquid solution comes into equilibrium with ice, and the formation of ice stops. The biological consequences of intracellular nucleation are shown in Figure 2, which plots the survival rate o f a variety of cell types against the rate at which they are cooled and relates cell death to the incidence o f intracellular ice formation. Al­ though different cooling rates are required to kill different cell types, cell death is closely associated in all cases with a rising likelihood o f intracellular freezing. B e­ cause cell death is associated with intracellular nucleation rather than with any spe­ cific absolute cooling rate, M azur has defined “fast” cooling as a cooling rate that induces intracellular nucleation and “slow” cooling as any lesser cooling rate, and it is understood that the specific cooling rate that is “fast” for one cell type may not be “fast” for another. Based on these phenomena, it is fair to say that the field of applied cryobiology as well as the possibility o f surviving freezing temperatures in nature exist largely because of the special nucleation properties of biological systems. C o n t r o llin g N u c le a t io n S ite D u r in g C r y o p r e s e r v a tio n b y F r e e z in g S u p e r c o o lin g

As noted above, intracellular freezing is only possible when the cytoplasm is su­ percooled.1 Although it is evidently possible for supercooled intracellular water to 'Although Franks has suggested the term “undercooling” in preference to “supercooling,” the more common usage is adopted here.

318

Fahy

f r e e z e d u e to t h e a c t i o n o f w e a k , t r u l y i n t r a c e l l u l a r n u c l e a t i n g a g e n t s ( F r a n k s e t a l., 1 9 8 3 ) o r v i a h o m o g e n e o u s n u c l e a t i o n ( R a i l e t a l., 1 9 8 3 ; R a s m u s s e n e t a l ., 1 9 7 5 ) , it is p r o b a b l y m o r e t y p i c a l f o r c e l l s c o n t a i n i n g s u p e r c o o l e d w a t e r to b e n u c l e a t e d b y g r o w t h o f ic e t h r o u g h th e c e l l m e m b r a n e ( M a z u r , 1 9 6 5 a ,b ; R a il e t a l ., 1 9 8 3 ) . T h i s h a s b e e n s u g g e s te d to o c c u r th ro u g h o rd in a ry m e m b ra n e p o r e s (M a z u r, 1 9 6 5 b ), th ro u g h m e m b ra n e d e fe c ts r e s u ltin g fro m a c ritic a l tra n s m e m b ra n e h y d ro s ta tic p r e s ­ s u re g r a d ie n t ( M u ld re w a n d M c G a n n ,

1 9 9 0 ) , b y d i r e c t i n j u r y to th e m e m b r a n e

c a u s e d b y c o n t a c t w ith ic e ( F u j i k a w a , 1 9 8 1 ) , b y r e o r g a n i z a t i o n o f m e m b r a n e c o m ­ p o n e n t s b y e x t r a c e l l u l a r ic e s o a s to r e n d e r t h e m e f f e c t i v e a t n u c l e a t i n g i n t r a c e l l u l a r w a te r ( T o n e r a n d

T o m p k in s ,

1 9 9 2 ), o r th ro u g h

o th e r m e c h a n is m s

( S te p o n k u s ,

1 9 8 4 ; D o w g e r t a n d S t e p o n k u s , 1 9 8 3 ; S t e p o n k u s e t a l., 1 9 8 5 ) . R e g a r d l e s s o f th e p r e c i s e c a u s e , t h e k e y to p r e v e n t i n g i n t r a c e l l u l a r n u c l e a t i o n is to m i n i m i z e i n t r a c e l ­ lu la r s u p e rc o o lin g . A l t h o u g h e m p h a s i s is u s u a l l y p l a c e d o n c o o l i n g r a t e a s t h e f a c t o r t h a t le a d s to i n t r a c e l l u l a r n u c l e a t i o n , t h e i m p o r t a n c e o f t h e i n it ia l c o n d i t i o n s o f e x t r a c e l l u l a r n u ­ c le a tio n s h o u ld n o t b e o v e r lo o k e d ( D ille r, 1 9 7 5 ). E x te n s iv e e x tr a c e llu la r s u p e r c o o l­ in g i m p l i e s e x t e n s i v e i n t r a c e l l u l a r s u p e r c o o l i n g . O n e e a s y m e t h o d t o m i n i m i z e s u p e r c o o l i n g d u r i n g f r e e z i n g is to u s e c o m m e r ­

Pseudom onas syringae f r o m S n o m a x T e c h n o l o g i e s , R o c h e s t e r , N e w Y o r k ) in th e f r e e z i n g

c ia lly a v a ila b le ic e -n u c le a tin g s u b s ta n c e s s u c h as S n o m a x ( fre e z e -d rie d

m e d i u m . I f o n e is f r e e z i n g t i s s u e s a m p l e s t h a t r e q u i r e s t e r i l i t y o r t h a t o t h e r w i s e s h o u l d n o t b e e x p o s e d to n u c l e a t i n g s u b s t a n c e s , m a n u a l s e e d i n g b y t o u c h i n g th e w a ll o f t h e c o n t a i n e r , a t a s i t e d i s t a n t f r o m t h e t i s s u e its e l f , w ith a p i e c e o f d r y ic e o r a w a n d p r e c o o l e d in li q u i d n i t r o g e n is a n e f f e c t i v e p r o c e d u r e a n d s h o u l d b e d o n e a s s o o n a s t h e t e m p e r a t u r e f a l l s s l i g h t l y b e l o w th e f r e e z i n g p o i n t o f t h e m e d i u m ( f o r e x a m p l e , F a n y , 1 9 S 0 b ) . S i n c e ic e p r o p a g a t i o n t h r o u g h t h e s a m p l e a f t e r m a n u a l s e e d i n g m a y r e q u i r e m i n u t e s t o c o m e to c o m p l e t i o n , it is i m p o r t a n t t o a l l o w f u ll ic e p r o p a g a t i o n b e f o r e p r o c e e d i n g w ith c o n t r o l l e d - r a t e f r e e z i n g s o t h a t c e l l s a r e a b l e to r e s p o n d f u l l y t o t h e l o w e r v a p o r p r e s s u r e o f lo c a l ic e c r y s t a l s a n d t h u s m i n i m i z e s u p e rc o o lin g . C e r ta in c o n tr o lle d - r a te f r e e z in g d e v ic e s c o n ta in a u to m a te d m e th o d s fo r in d u c in g n u c l e a t i o n in s a m p l e s a s th e y a r e c o o l e d to n e a r t h e i r f r e e z i n g p o i n t s . T h i s is a h i g h l y d e s i r a b l e f e a t u r e , b u t d i f f e r e n t m e t h o d s m a y n o t y i e l d th e s a m e r e s u l t s . M e r e l y c o o l i n g t h e s u r r o u n d i n g a t m o s p h e r e a b r u p t l y to a t e m p e r a t u r e w e ll b e l o w th e f r e e z i n g p o i n t a n d th e n r a i s i n g it a f t e r th e n u c l e a t i o n o f a d u m m y s a m p l e o c c u r s o r a f t e r a s e t p e r i o d o f t im e h a s e l a p s e d s h o u l d b e v i e w e d a s a n u n r e l i a b l e p r o c e ­ d u r e . T h e r e is n o g u a r a n t e e t h a t a d u m m y s a m p l e w ill n u c l e a t e a t th e s a m e t e m p e r a ­ t u r e a s th e a d j a c e n t c e l l s a m p l e o r th a t, in f a c t, a n y tw o c e l l s a m p l e s w ill n u c l e a t e a t th e s a m e t im e . T h e r e is t h e f u r t h e r p o t e n t i a l p r o b l e m o f l o s s o f c e l l s b y i n t r a c e l l u l a r f re e z in g a t th e w a lls o f th e ir c o n ta in e rs , s in c e th e n u c le a tio n p r in c ip le e m p lo y e d r e q u i r e s e x t e n s i v e s u p e r c o o l i n g a t th e c o n t a i n e r w a ll f o r its e f f e c t i v e n e s s . S o m e d e v i c e s a r e a b l e t o n u c l e a t e s t r a w s b y t o u c h i n g a ll s t r a w s a t t h e s a m e m o m e n t w ith a w a n d t h a t h a s b e e n s u f f i c i e n t l y p r e c o o l e d . I n p r i n c i p l e th i s a c c o m p l i s h e s g u a r a n ­ t e e d n u c l e a t i o n o f a l l s a m p l e s a t th e t i m e d e s i r e d w ith m i n i m a l c o n t a c t a r e a b e t w e e n t h e n u c l e a t i n g w a n d a n d t h e c o n t a i n e r a n d t h u s w ith m i n i m a l l o c a l c e l l lo s s . T h i s a p p r o a c h is t h e o r e t i c a l l y s u p e r i o r a n d w o u l d b e s e c o n d in e f f e c t i v e n e s s o n l y t o d i ­ r e c t i n t r o d u c t i o n o f n u c l e a t o r s t h r o u g h o u t t h e s a m p le . I t is i m p o r t a n t t o p o i n t o u t t h a t s u p e r c o o l i n g c a n in i t s e l f p r o v i d e a m e a n s o f p r e ­ s e rv in g s a m p le s fo r s ig n ific a n t tim e s , p a r tic u la rly n o n liv in g s a m p le s s u c h a s p u ri-

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fied proteins in solution (Franks, 1988; M athias, 1991). Since nucléation is difficult to prevent for lengthy periods in bulk samples, supercooling must generally be ac­ complished by subdividing the aqueous phase into many small droplets dispersed in a noncrystallizing organic phase (émulsification). Since each droplet has a low probability of containing a nucleator, and since homogeneous nucléation is highly improbable at temperatures above about -20°C , it is possible to maintain consider­ able quantities o f sample in a nonfrozen state for weeks or even for years by this approach (Franks, 1988; M athias, 1991). C o o lin g R a te

Not evident in Figure 2 is the general observation that, at lower cooling rates than indicated in Figure 2, cell survival increases as cooling rate increases, reflect­ ing reduced damage from time-dependent, low-temperature conditions other than intracellular freezing. (Injury caused by slow freezing is usually thought to result

—T e m p e ra tu re

(C )

Figure 3. Calculated loss of freezable water from yeast cells under different freezing conditions. Curve 1 = cell water content at equilibrium; 2 = cell water content based on the typical assumption that nu­ cléation takes place at the equilibrium freezing point (equal to TM) and that cooling rate (100°C/min) then remains constant despite release of the latent heat of fusion; 3 = cell water content based on the more realistic assumption that temperature rebounds to TM shortly after nucléation (or that nucléation occurs at 7'M) and that subsequent cooling is slow (0.5°C/min) until a constant cooling rate (again, 100°C/min) is imposed at -5 °C , causing a departure from the equilibrium curve; 4 = maximal hazard assumption, i.e., that the cell supercools 5°C prior to nucléation, and cools at a constant rate (100°C/min) thereafter, without thermal rebound. Inset: Hazard of intracellular nucléation expressed as the percent of cell water that is freezable as a function of temperature and thermal history (curve labels are the same as previously described). All curves calculated as described in the legend of Figure 1 of Fahy (1981a) according to methods detailed in Fahy (1980a, 1981a,b), using 1 M DMSO as the cryoprotectant.

from alterations in the composition and properties of the extracellular solution and is therefore termed “solution effects” injury.) Therefore, the purpose o f controlling cooling rate is to minimize both the total time required for freezing (thus minimiz­ ing “solution effects”) and the likelihood of intracellular nucleation. However, the attainment of these goals is more complex than this simple statement may imply. The likelihood o f intracellular nucleation depends on the kinetics o f cellular water loss during freezing, which are decidedly nonlinear. The basic issues are best discussed in terms of M azur’s model o f cell dehydra­ tion during freezing (M azur, 1963). As is evident from Figure 3, a cell cooling at an infinitely slow rate will generally dehydrate very rapidly within the first several de­ grees below the freezing point and then lose water much more slowly thereafter (curve 1). Cells cooled at constant rates, however (Fig. 3, curve 2), respond to subfreezing tem peratures rather ineffectively at first, experiencing the greatest super­ cooling ju st below their nominal freezing point. On the other hand, cooling at a predetermined constant rate despite rapid ice propagation through the sample just below the freezing point is a physically unrealistic situation in most cases due to liberation o f the latent heat o f fusion and consequent warming o f the environment back to ju st below the freezing point. The effect of slow dissipation of the heat of fusion by slow cooling followed by more rapid cooling is shown as curve 3 in Fig­ ure 3. Finally, the effect o f 5°C o f supercooling prior to freezing without thermal rebound during freezing is indicated as curve 4. The inset o f Figure 3 expresses these results explicitly in terms of the amount o f supercooled intracellular water, and thus the likelihood o f intracellular nucleation. This discussion makes it obvious that the occasionally expressed desire to force a preconceived constant cooling rate on samples even during the initial part o f the crystallization process in order to comply with the imperative of freezing at con­ trolled cooling rates is misplaced. The net effect o f such a m aneuver will be to con­ vert curve 3 into curve 2 or even into curve 4, i.e., to bias the system in favor of intracellular nucleation. Some have suggested that “solution effects” injury must be minimized by hastening the passage through the freezing point, but this is also gen­ erally incorrect. Solution effects injury is the result of severe dehydration for most cells and is not caused by the mild dehydration that occurs in the immediate vicinity of the freezing point. For example, Fahy (1980b) reported that the “freezing point isotherm” for tissue slices frozen in 15%, w/v, dimethyl sulfoxide was over at -10°C or above (corresponding to conversion of well under half of the volume o f the solution into ice), whereas injury became just detectable at -17°C (52% volume conversion), and severe only below -2 9 °C (>62% volume conversion). Finally, it should be understood that the commonly accepted imperative o f im­ posing a fixed cooling rate on samples during freezing, whether in the temperature region o f the freezing point or at lower temperatures, is not motivated by what is biologically optimal but by what is computationally and experimentally convenient. It was M azur who first urged cryobiologists to use constant cooling rates, but this suggestion was motivated in part by the fact that it is computationally easier to model and com pare the effects of constant cooling rates as described in Figure 3 than it is to model and com pare the effects of variable cooling rates that might in fact be more biologically meaningful. The basic situation can be visualized with the aid of the schem atic diagram s of Figure 4. Figure 4 represents three possible continuous cooling protocols. For simplicity, we make the usual assum ption that nucleation takes place extracellularly at the

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freezing point and that cooling proceeds without respect to the release of the latent heat o f fusion. The situation depicted on the left is the effect of cooling at a constant rate, and the resulting cell water response resembles curve 2 o f Figure 3. The situation depicted in the middle of the figure is a commonly encountered one, in which biologists without controlled-rate freezing equipment place their cells in a low-temperature freezer surrounded by some type of insulation in the hope that this will slow the cooling rate sufficiently. W hat theoretically happens in this situa­ tion, o f course, is that the cooling rate is initially high due to the initial large ther­ mal gradient between the sample and the environment and then falls progressively as this thermal gradient is dissipated. The cell water response is maximally adverse: the amount o f cell water at risk of nucleation is increased compared to the constant cooling rate protocol, while exposure time at lower temperatures, where “solution effects” injury proceeds at a maximum rate, is also maximized. The situation depicted on the right in Figure 4 shows the effects of a hypothetical cooling protocol tailored to the biological properties of the cell. To minimize super­ cooling, the cooling rate is first kept low to perm it near-equilibrium loss of cell water. As cell water content approaches the minimum permissible values, and the risk of “solution effects” injury therefore begins to rise, the cooling rate is progres-

—

T e m p e ra tu re

Figure 4. The effects of two commonly used freezing procedures (constant cooling rate, left; and expo­ nentially declining cooling rate, middle) vs. a proposed freezing procedure (continually rising cooling rate, right) that minimizes both “solution effects” injury (SEI) and the danger of intracellular nucleation (ICF). Dashed horizontal lines in middle and right upper panels represent the same cooling rate shown at the left, for comparison. Cell water content (lower panels) is compared with the equilibrium cell water content (eq), which is theoretically attained only at very low cooling rates. Horizontal solid line begins when cell liquid volume is reduced to 50% of initial volume, marking the onset of hazard for SEI. Note that in the SEI region, cooling rates higher than indicated by the horizontal dsashed line (upper panels) signify lower exposure times and therefore less injury. For further description, see text. All curves shown are schematic.

322

F ahy

sively increased. Slow cooling at lower temperatures can have only a marginal pro­ tective effect on the likelihood and quantity of intracellular ice formation, so there is little reason not to increase cooling rate progressively to minimize “solution ef­ fects” injury as cell dehydration permits. Figure 4 suggests that a cooling protocol similar to that proposed could give higher cell survivals than constant cooling rate protocols, and the biologically motivated protocols ought to be derivable from pre­ viously established data on the effect of constant cooling rates on cell survival. This novel idea has yet to be validated experimentally. However, it approximates what many investigators now do when they cool through the freezing point isotherm slowly, adopt a higher constant cooling rate to about -40°C , and then plunge their samples into liquid nitrogen. I n te r r u p t e d C o o lin g a n d W a r m in g P r o to c o ls

There are many laboratories that lack controlled-rate freezing equipm ent and that must preserve cells at cryogenic temperatures. Short of simple insertion into a freezer as discussed above, there are uncomplicated measures that can potentially achieve the purpose o f controlled-rate freezing, that is, avoidance o f intracellular nucleation with a minimum exposure time to potentially damaging conditions, with­ out the controlled-rate freezer. O f these, possibly the most important is two-step freezing. T w o -S te p F r e e z in g

In two-step freezing, the cryoprotected sample is first exposed directly to a sub­ zero bath held at some tem perature in the vicinity of - 2 0 to -30°C (W alter et al., 1975). It is thought that the temperature must be chosen to be high enough to pre­ vent intracellular nucleation upon exposure to the supercooling represented by this temperature, yet low enough so that sufficient dehydration will occur during a hold at this tem perature to prevent intracellular freezing upon subsequent transfer to li­ quid nitrogen. W alter et al. (1975) used freeze substitution to visualize intracellular ice crystals and reported that the surviving cells were those that were massively de­ hydrated and lacked clear-cut signs o f intracellular ice, whereas, the cells whose morphology indicated little dehydration contained intracellular ice and were killed. Questions about this standard approach to two-step freezing arise from consider­ ing the basic physics o f aqueous solutions. Cytoplasm in equilibrium with ice at a temperature o f —25°C will have a homogeneous nucleation tem perature of around -90°C (Fahy et al., 1984; Rail et al., 1983) (see also Figure 5), which is above the glass transition tem perature o f all common penetrating cryoprotectant solutions. Success o f two-step protocols involving temperatures near -25°C at the first step is probably due to the slow rate o f growth of nuclei formed after subsequent cooling to -90°C and below and the brief time available for growth and recrystallization during very rapid warming. Theoretically, two-step freezing should require an initial dehydration tempera­ ture in the vicinity o f about -40°C in order to avoid intracellular nucleation on sub­ sequent plunging into liquid nitrogen (Fahy et al., 1984), but this may not be possible because direct exposure to -40°C may itself induce intracellular nucleation before dehydration has time to occur (Diller, 1975). Kuwano et al. (1994), using a modified two-step protocol that avoided this nucleation problem by using slow rather than rapid cooling to the first holding temperature, reported that several strains o f Porphyra did show optimal survival when the initial holding temperature

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323

was -4 0 °C as predicted from phase diagram considerations (Fahy et al., 1984). Similarly, Alink et al. (1977) found that for adult rat heart cells, two-step cooling was ineffective because the temperature required in the first step to protect against damage upon plunging into liquid nitrogen was sufficiently low to kill most cells prior to plunging. Overall, survival tended to be higher when a protocol was employed in which the first holding temperature was gradually lowered before plunging the sample into liquid nitrogen. An alternative for cells that cannot tolerate long exposure times at high subzero temperatures would be to employ two steps prior to the -196°C step, e.g., -1 5 to -20°C , followed by transfer to a bath in the vicinity of -40°C. 0

^

- 4 0

* LU

cc =>

I
> -4-»

w /w

1 , 2 —p r o p a n e d i o l

47%

w /w

1 ,2 —p r o p a n e d i o l

600

in

R P S -2 ------- e ---------

400 50%

200

w /w

1 ,2 —p r o p a n e d io l ■ in R P S - 2

0 - 3 0

- 5 0

- 7 0

T e m p e ra tu re

- 9 0 (de g.

-1 1 0 C)

Figure 7. Effect of physiological solutes and small changes o f cryoprotectant concentration on cumula­ tive incidence of nucleation (CIN spectrum) of large volumes o f cryoprotectant during cooling. RPS-2 is a storage solution for mammalian kidneys; its composition and other experimental details are given in Fahy et al. (1990). Data were obtained by photographing 482-ml specimens, projecting each image onto a standard 18 x 24 in. page, then tracing and numbering each crystal, and finally correcting the results to 500 ml. For discussion, see text. Photographic views o f these data are given in Fahy et al. (1990).

326

F ahy

represent the cum ulative population size of visible ice crystals during vitrification of the m ajor portion o f the solution in the presence and absence of physiological vehicle solution solutes, each crystal representing the effect o f a single nucleation event. As can be seen, the number of nucleation events (critical nuclei) is large in the absence o f vehicle solution solutes (upper curve). Adding these physiological solutes dramatically reduces nucleation density (lowest curve), a factor that could be critical to the success o f organ vitrification. However, lowering the concentration of the 1,2-propanediol again produces large numbers of nuclei (middle curve). If each crystal is capable of damaging a blood vessel in an organ during cooling (Pollock et al., 1986), the result could be massive injury upon transplantation. If, on the other hand, each crystal can produce only local injury, then a consideration of the total volume fraction occupied by the crystals (small even for the upper curve) suggests that injury could be minimal. Until it becomes possible to vitrify organs without damaging regions that are not in contact with such growing crystallization centers, it will remain difficult to determine which of these two possible outcomes is the correct one. H o m o g e n e o u s N u c le a tio n o f C r y o p r o t e c t a n t S o lu t io n s

Classically, homogeneous nucleation is a rapid event that occurs at a sharply de­ fined temperature ( Th) that rises as the volume o f the sample increases (Angell, 1982). The distinctness o f homogeneous nucleation becomes blurred, however, un­ der conditions that impede the growth of ice, for two reasons. First, slow ice crystal growth delays and reduces the exothermic signal that serves as the means of detect­ ing homogenous nucleation, so that the temperature at which homogeneous nuclea­ tion is detected may be less than the temperature at which it actually occurs and will be dependent on cooling rate. M ore fundamentally, homogeneous nucleation itself becomes distributed over a significant temperature range because the probability of nucleation per unit time and per unit volume becomes low enough to preclude all possible nucleation from taking place at the first possible opportunity. This means that the actual extent of homogeneous nucleation may depend strongly on cooling rate when concentrations are high. Under such conditions, Th per se loses its mean­ ing, and what becom es meaningful is either the temperature at which homogeneous nucleation first begins to increase appreciably during cooling or the sum total of homogeneous nuclei that actually form under the specific circumstances at hand (the cumulative incidence of nucleation, or CIN spectrum). The former is the more conservative (and more easily determined) measure and may be extrapolated rea­ sonably well from the more dilute portion of the Th curve (Fahy et al., 1984). The CIN spectrum is increasingly inferable from accurate theoretical models of nuclea­ tion in concentrated cryoprotectant solutions (Bronsheteyn and Steponkus, 1995; Kresin and Korber, 1991; Karlsson et al., 1994). The complexities introduced by high concentrations (as well as by higher rates of temperature change) are extinguished at still higher concentrations, at which the probability of nucleation falls to negligible values, or growth o f the resulting ice embryos becomes vanishingly small, or both, rendering homogeneous nucleation moot (Fahy et al., 1984). Experimentally, it has been found that the incidence of visible ice vanishes (i.e., vitrification becomes evidently complete) at the same con­ centration that brings the onset temperature for homogeneous nucleation down to Tg, the glass transition temperature (when both Te and the extrapolated Th are based on a cooling rate o f about 10°C/min) (Fahy et al., 1984).

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327

Despite the com plexities of homogeneous nucléation, nuclei not detected on cooling can be detected by warming back to a sufficiently high temperature to permit them to grow and thus produce heat, provided concentration is low enough to permit crystal growth. An example of the application of this approach is given in Figure 8, wherein the data reflect the amount o f melting observed after holding at different tem peratures for 1.5 or 30 min. The 30-min peak clearly reflects nucléa­ tion more than ice crystal growth per se, since no signal is observed at the higher temperatures after 1.5 min, indicating no nucléation. Further, the upper limb o f the peak rises as temperature falls (and crystal growth rates diminish). Combining 18

16 14

E

12

03 V__

CO

°

CD N

6

2

0 -50

-60

-70

-80

-90

-100

H o l d i n g T e m p e r a t u r e / 0C F igure 8. Detection of “invisible” nucleation at low temperatures by its effects during warming to tem­ peratures permitting crystal growth. The vitrification solution used consisted of 6%, w/v, polyvinylpyr­ rolidone K30 (mean M„ 40,000 Da) plus 49%, w/v, D(l)FPio, which consists of 10%, w/v, 1,2propanediol and 39%, w/v, of a mixture of dimethyl sulfoxide and formamide in a 1:1 mole ratio. Samples were cooled to the temperatures indicated at 80°C/min and held for either 1.5 min (filled cir­ cles) or 30 min (open circles) before rewarming at 80°C/min. Data shown are the number of calories detected by differential scanning calorimetry (DSC) during melting of the resulting ice. DSC traces on cooling showed no evidence o f crystallization, and ice formation on warming appeared as devitrifica­ tion with an onset temperature of more than -70°C ; even at a warming rate of 10°C/min, devitrification became detectable only near -7 0 °C and peaked at -53°C . Therefore, ice crystal growth is minimal be­ low -70°C in this system, and the limiting factor for the signals shown is nucleation. Data were col­ lected by T. Takahashi (currently at the Japanese Red Cross, Sapporo, Japan) on 17 July 1984 at the American Red Cross in Bethesda, MD.

328

Fa hy

calorimetric analysis o f this kind with cryomicroscopy has allowed the contributions of nucleation and crystal growth to be separated from each other in accounting for the overall calorim etric record (Mehl, 1990, 1992, 1993). Ice formation is accom panied by an increase in the volume occupied by water molecules undergoing crystallization. For this reason, nucleation is inhibited by high pressures in much the way it is inhibited by solutes. In fact, Figure 9 reveals that the effects o f pressure and o f solutes on Th are essentially identical, and this has led to extensive experim entation to attempt to exchange elevated pressure for some of the potentially toxic cryoprotectant required for vitrification. Figure 9, however, obscures the fact that the mechanisms by which pressure and cryoprotective solutes depress Th are in opposition to each other (M acFarlane et al., 1992). Cryoprotectants form hydrogen bonds with water, in the process interfering with water-water hydrogen bonding but also increasing the effective volume occu­ pied by each water molecule, or imposing water “structuring.” The latter effect is in opposition to the structure-breaking effect of high pressure (M acFarlane et al.,

□

ETHYLENE GLYCOL •

GLYCEROL

A DIMETHYL SULFOXIDE O 1,2 PRO PA NE DIOL (P G ) »

0

-1 0

20% W /V ME2 S O +

-2 0

-3 0

-4 0

Tm ( ° C ) F igure 9. Comparison o f effectiveness of pressure and cryoprotective agents in depressing the homoge­ neous nucleation temperature of water. The depression of Th induced by solutes or by pressure is plotted against the equilibrium freezing point depression caused by pressure or solutes. Reprinted, with per­ mission, from Fahy et al. (1984).

N u c le a tio n in C ry o p re s e rv a tio n

-40

-50

-60

-70

-80

-90

-100

329

-1 1 0

T h a t 1 a t m o s p h e r e ( °C ) F igure 10. Lack of equivalence of salts and cryoprotectants in supporting depression of the homogene­ ous nucleation temperature o f water (7'h) by high pressures. NaCl and dimethyl sulfoxide are compared on an equal basis by determining the change in Th produced by application of 1,000 atm (1 kb, or 100 MPa) (vertical axis) at a previously existing 7h depression produced by either solute at ambient pressure (horizontal axis), without regard to the absolute concentration or specific chemical properties of either solute. The initial 1], represents a universal scale for which agents can be compared based only on their net effect on water. The points and dashed line referred to as 7^ + ATh = Tc define the ends of the curves, where Tj, is depressed until it coincides with the glass transition temperature. NaCl data derived from Kanno and Angell (1977); DMSO data are collected from Fahy et al. (1984) and MacFarlane et al. (1981).

1992). This is strikingly reflected in the antagonism that cryoprotectants produce to the ^-depressing effects o f high pressure (Fahy et al., 1984). As indicated in Figure 10, high pressures depress Th much more strongly in the presence of electrolytes (which, like pressure, are chaotropic, or structure-breaking) than in the presence of “kosmotropic,” or structure-making cryoprotectants such as dimethyl sulfoxide. This phenomenon limits the utility of pressure for facilitating vitrification and sug­ gests that more ionic cryoprotectants or vehicle solutions would be more advanta­ geous for high-pressure vitrification. H o m o g e n e o u s N u c le a tio n o f V it r if ie d S o lu t io n s

The storage time of vitrified systems is expected to be limited mostly by nuclea­ tion that may continue at a slow pace even below Tt . Nucleation below Te is possi­ ble because Ts, like Th at high concentrations, is not an absolute boundary between the liquid and solid states but is dependent upon cooling rate and upon the time scale of the observation in question. Given sufficiently long time scales (which lengthen rapidly as tem perature drops progressively below Tg), more and more nu­ cleation can occur. “Shelf life” in the vitrified state may therefore depend on the cooling rate, the storage tem perature, and the attainable warming rate.

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Fa hy

So far, hard data on nucleation rates below the measured Tg of solutions that are relevant for cryopreservation are meager (Angell and M acFarlane, 1982). Chang found devitrification temperature to fall by 5"C after only 180 min o f annealing of 55%, w/w, glycerol at 8°C below the measured Tg (Chang, 1992). However, this is a concentration that yields a doubly unstable glass (Fahy et al„ 1984), and it is not clear that similar changes would occur at higher concentrations. W illiams and col­ leagues (1990) have pointed out that the interfaces formed by spontaneous fractur­ ing in vitrified aqueous solutions can serve as extremely effective nucleating surfaces for ice, and it is important to avoid this artifact in studies of solution nu­ cleation rates below Tg. Mehl (1993) reported that the classically defined critical heating rate o f a vitrification solution stored for 6 months at about 10°C below the measured Tg was only moderately elevated in comparison to nonstored samples, due to the slow pace o f additional nucleation during the 6-month storage period. On the other hand, he pointed out that if damage is associated more with the size of each growing crystal than with the number of such crystals (M azur, 1965a, 1988), the critical warming rate for avoiding damage is essentially independent o f continuing nucleation during storage, since all nuclei will be the same size and will grow at the same rate on warming (Mehl, 1990; Mehl, 1993). Clearly, much additional research on sub-Tg nucleation and its biological consequences is needed. A v o id in g N u c le a tio n o r th e E ffe c ts o f N u c le a tio n D u r in g V it r if ic a t io n f o r C r y o p r e s e r v a tio n

During cooling to sub-Tg temperatures, nucleation may be a problem either in the temperature range of rapid ice crystal growth (about -3 0 to - 8 0 HC) or at tempera­ tures closer to Tg, which systems such as organs may experience for prolonged times due to the low cooling rates that attend the approach to storage temperatures close to Tg. The form er temperature range will be governed by heterogeneous nu­ cleation and the latter by hom ogeneous nucleation. C o n tr o llin g H e te r o g e n e o u s N u c le a tio n

Several approaches to avoiding heterogeneous nucleation or its consequences can be explored. For solutions containing an air-liquid interface, nucleation at this interface will inevitably occur unless special precautions are taken, perhaps in part because water vapor in the air is capable o f crystallizing and seeding the interface and in part because the surface energy of the interface favors nucleation (a factor that may explain in part why freezing-point osmometers successfully nucleate solu­ tions by generating turbulence and bubbles in them). W hatever the mechanism, it is possible to reduce or prevent this unique type of heterogeneous nucleation by layer­ ing an immiscible hydrocarbon phase over the solution to cover the interface. W illiams et al. (1990) found isopentane (2-methyl butane) to be an effective interface-protecting material; I have found methylcyclohexane and methylcyclopentane to be considerably more reliable. For noninterfacial heterogeneous nucleation, a direct approach would be to identify, characterize, and finally to inactivate or remove all or most heterogeneous nucleators. Despite many decades of research on water and supercooling, the nucleators present in ordinary beakers of laboratory water have evidently not been identified. Once such identifications are available, technologies such as monoclonal antibody production could be applied to the inactivation of the nucleators, or the nuclei could be rem oved by adsorption to appropriate immobilized ligands, and so

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forth. Less elaborate methods may also be helpful. For example, heating and/or subjecting the solution to high pressures (e.g., 2,000 atm or more) for a few hours prior to use to inactivate organic nucleators. A nother possibly feasible approach could be to emulsify a bulk aqueous solution in a reversible way, cool the emulsion to temperatures sufficient to nucleate droplets containing active heterogeneous nu­ cleating agents, separate the frozen droplets from the unfrozen droplets by centrifu­ gation or drop-sorting techniques, and then break the emulsion to give back the nucleator-depleted bulk solution. Even more feasibly, bulk solutions could be cen­ trifuged while being cooled to force the nucleators trapped in the developed crystals that they have nucleated to float to the surface (Fahy et al., 1990), where they can be removed along with the ice.

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Figure 11. Lack of effectiveness of antifreeze glycoproteins (AFGP, consisting of a physiological mix­ ture of AFGPs 1-8 of the Antarctic nototheniid fish Dissostichus mawsoni) on inhibiting ice crystalli­ zation rates in 40%, v/v, dimethyl sulfoxide in water. Filled circles, no AFGP; open circles, 2%, w/v, AFGP present. Data were obtained by timing the movement of an ice front down a standardized vertical channel in a plastic plate; the crystal orientation with respect to the axis of the channel was therefore random. AFGP was provided by A. L. DeVries.

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Parody-M orreale et al. (1988) presented evidence that antifreeze glycoproteins can directly inactivate nucleators by binding to their ice-nucleating sites. Another possibility is to use such proteins to block the growth of ice that is nucleated. How­ ever, as noted in Figure 11, there may be little effect in highly concentrated aqueous solutions, whose cryoprotectant content may already produce “antifreeze” effects superior to or com parable to those o f the natural proteins. Furthermore, it has been shown that using natural antifreeze proteins in combination with cryoprotectants may cause the ice that does form to become more lethal, actually increasing damage rather than decreasing it (Ishiguro and Rubinsky, 1994). This is due to crystal growth primarily in the usually unimportant c-axis direction, yielding thin needle-shaped crystals (for details, see Davies and Hew, 1990; Raymond and DeVries, 1977). Both of these problem s could be overcome by developing artificial molecules capable of inhibiting crystal growth in the direction of the c-axis. As effective as low molecular mass cryoprotectants are, their limited sizes prevent them from hav­ ing the type o f cooperative interactions with ice that several points o f contact would provide, so it is possible to imagine specifically engineered polymers that could be more effective. Such polymers should remain of relatively low molecular weight in comparison to the proteins, however, to permit them to adsorb onto the ice crystal lattice before it grows past them, a problem that may also contribute to the failure of natural antifreezes to inhibit crystal growth at temperatures below -3 0 HC (Fig. 11). Finally, the ability to inhibit simultaneously all crystallographic growth planes of ice, not just most such growth planes, should significantly outstrip the effectiveness o f and circum vent the hazards o f natural antifreeze proteins. Effectively, this would be tantamount to controlling the physics of ice and thereby changing the rules of the game of cryobiology. A v o i d in g t h e C o n s e q u e n c e s o f H o m o g e n e o u s N u c l e a t io n

Since hom ogeneous nucleation will generally be of importance only during warming from the vitrified state, when homogeneous nuclei can grow to damaging sizes (devitrification), the most direct solution to this problem is rapid warming. The ability to meet the challenge of rapid warming will depend on the stability of the cryoprotectant medium, exact details of the cooling regimen, the availability of appropriate warming technology, the biological effects of different ice crystal size distributions during rapid warming, the applicability of hydrostatic pressure to slow down crystal growth and/or to prevent additional nucleation on warming, and the ability to specifically modify the growing ice crystal/solution interface so as to re­ duce the growth rate o f the developing nuclei. With regard to the latter, Sutton and Pegg (1993) have reported that antifreeze peptides can be extraordinarily effective in blocking ice crystal growth during warming following previous homogeneous nucleation o f 2,3-butanediol solutions. The control of devitrification is a complex and fertile area for future investigation. D u r in g C o o lin g f o r C r y o u lt r a m ic r o t o m y

M icroscopists have devised several cryo techniques to reduce the artifacts asso­ ciated with frozen sections. Some of these involve the use o f high concentrations of agents such as sucrose, which do not penetrate living cells and therefore must either be used on dead cells or must be allowed to kill the cells before they can be exam­ ined. However, the same vitrification techniques being pursued by cryobiologists for maintaining the viability o f living systems could also allow biochemists and

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morphologists to obtain thin sections of living biological material without freezing artifacts and without chemical fixation or embedding. Using rabbit renal cortex and our standard vitrification solution VS41A, which vitrifies at ambient pressure (Mehl, 1993), we have been able to make sections over a very narrow temperature range (about —111.5 ± 1.0°C), tissue instability (crumbling or smearing) being ob­ served at higher or lower temperatures. This sectioning temperature is over 10°C above Tg, and therefore should permit considerable nucleation during sectioning, but it is well below the crystal growth range, so visible nuclei should be absent. D u r in g F r e e z in g o f C o m p le x S y s te m s

Although I have been pursuing the cryopreservation of mammalian organs by vitrification, there may be an alternative approach to the cryopreservation of com ­ plex systems. This approach would involve the deliberate induction of very large numbers of nucleation events (at least 1012/cm3, or about l/|im 3, or more) in the ex­ tracellular space, all at roughly the same temperature. In this way, paradoxically, the mechanical consequences o f nucleation might be avoidable by inducing nucleation on an unprecedented scale. M acFarlane and co-workers proposed one version of this approach, which in their scheme would be used to circumvent recrystallization and therefore devitrification-related injury in form erly vitrified organs (M acFarlane et al., 1981). The idea was to anneal formerly vitrified organs near Tg so as to allow nucleation densities to become so great that no growth o f the formed nuclei could occur during warming, since most freezable water would have already been converted into the nuclei them­ selves. Forsyth and M acFarlane (1986) later pointed out that very high nucleation densities can give the visual impression of vitrification, because the individual crystallites are too small to scatter light and too numerous and uniform to support substantial recrystallization (opacification) on warming. A more general strategy could be used to allow similar benefits to be obtained in nonvitrified systems being preserved by slow-freezing procedures. This would in­ volve introducing high quantities of nucleating agents in the simultaneous presence o f large quantities of antifreeze substances such as antifreeze proteins and cryoprotectant concentrations around 3 to 6 M (the latter to limit effects such as cell shrink­ age). This strategy is based on the common observation that ice per se need not be harmful to organized tissues or cells, provided its growth by normal mechanisms or through recrystallization is prevented (Mazur, 1965a, M azur, 1988). By inducing large numbers o f nuclei that cannot grow to damaging dimensions due to the pres­ ence of the antifreeze substances, extracellular ice could be rendered innocuous and inert, unable to reorganize during either cooling or warming into large grains that could damage the tissue. Even the water emerging from cells as they respond osmotically during freezing m ight be tied up in a series o f new nucleation sites, since nucleating substances not responsible for the formation of particular ice crystals may be excluded from those crystals and therefore concentrated in the remaining unfrozen liquid. As above in the discussion of designing specific ice growth inhibitors, this ap­ proach would essentially change the physics of ice, and thereby change many of the basic phenomena of cryobiology. Indeed, artificial, specific ice-binding substances should be more effective and more practical to use in this scheme than natural anti­ freeze proteins. Such a scheme would only work if the nucleators and antifreeze substances do not inactivate one another as cited above (Parody-Morreale et al.,

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1988). This, however, could be arranged by sterically designing one or both of the components o f this system (the nucleator or the ice-binding molecule) so as to pre­ vent contact at the active sites. Are there natural examples o f this strategy? Nucleating proteins in freeze-toler­ ant vertebrates are thought not only to induce but also to at least partially control ice crystallization (Storey and Storey, 1992), and antifreeze proteins in insects are similarly thought to be important for minimizing recrystallization (Storey and Sto­ rey, 1992). The observation o f both nucleating and antifreeze activity in the same organism, however, seems not to be generally recognized (Storey and Storey, 1992). W hether this is because there are no such organisms or because they have been overlooked is an open question. C o n c lu s io n

Nucleation plays a central role in applied cryopreservation, both for freeze pres­ ervation and for vitrification. Additional attention to the avoidance or minimization of nucleation or the control of its consequences could have major effects on the ef­ ficacy o f applied cryopreservation and could help to link applied cryopreservation studies to the cryobiology of organisms in nature. A c k n o w le d g m e n t s The au th o r thanks Joe W atson, Kris T hom pson, and M elissa C onsoli for photographic and graphics support, A. L. D eV ries for providing antifreeze glycoprotein, M. T oner for perm ission to publish a m odified form o f his figure as Figure 1, T. Takahashi for perm ission to publish Figure 8, J. M atthes for counting m ost o f the crystals o f Figure 7, and P. M. M ehl for interesting discussions. Supported by grants G M -17959, B S R G 2507-R R 05737, and a grant from the G. H arold and L eila Y. M athers Charitable F oundation.

L it e r a t u r e C ite d Alink, G. M ., V erheul, C. C ., an d O fferijns, F. G. J. 1977. T hree-step cooling: A preservation method for adult rat heart cells. C ryobiology 14:409-417. Angell, C. A. 1982. Supercooled water. Pages 1-81 in: W ater, A C om prehensive T reatise. Vol. 7, W ater and A queous Solutions at SubzeroTem peratures. F. Franks, ed. Plenum Press, N ew Y ork. Angell, C. A., and M acFarlane, D. R. 1982. C onductim etric and calorim etric m ethods for the study of hom ogeneous nucleation below both Th and T g. Adv. C eram ics 4:66-79. B ronshteyn, V. L., and S teponkus, P. L. 1995. N ucleation and growth o f ice crystals in concentrated solutions o f ethylene glycol. C ryobiology 32:1-22. Chang, Z. H. 1992. T he b iological and physical aspects o f cryopreservation by vitrification: A Droso­ phila melanogaster m odel. D issertation. State University o f New Y ork at Bingham ton, NY. Coger, R., and T oner, M . 1995. Preservation techniques for biom aterials. Pages 1557-1566 in: B iom edical E ngineering H andbook. C R C Press, B oca Raton. D avies, P. L., and H ew, C. L. 1990. B iochem istry o f fish antifreeze proteins. FA SEB (Fed. Am. Soc. Exp. B iol.) J. 4:2460-2468. D iller, K. R. 1975. Intracellular freezing: E ffect o f extracellular supercooling. C ryobiology 12:480-485. D ow gert, M . F., and S teponkus, P. L. 1983. E ffect o f cold acclim ation on intracellular ice form ation in isolated protoplasts. Plant Physiol. 72:978-988. Fahy, G. M. 1980a. A nalysis o f “solution effects” injury: Equations for calculating phase diagram in ­ form ation for the ternary system s N aC l-dim ethylsulfoxide-w ater and N aC l-glycerol-w ater. Biophys. J. 32:837-850. Fahy, G. M. 1980b. A nalysis o f “solution effects” injury: R abbit renal cortex frozen in the presence of dim ethyl sulfoxide. C ryobiology 17:371-388. Fahy, G. M . 1981a. S im plified calculation o f cell w ater content during freezing and thaw ing in nonideal solutions o f cryoprotective agents and its possible application to the study o f “solution effects” in ­ jury. C ryobiology 18:473-482.

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Fahy, G. M. 198 lb . Analysis o f “solution effects” injury: C ooling rate dependence o f the functional and m orphological sequellae o f freezing in rabbit renal cortex protected w ith dim ethyl sulfoxide. C ryo­ biology 18:550-570. Fahy, G. M. 1988. V itrification. Pages 113-146 in: Low T em perature Biotechnology: E m erging A ppli­ cations and E ngineering C ontributions. J. J. M cG rath and K. R. D iller, eds. Am erican Society o f M e­ chanical Engineers, N ew York. Fahy, G. M ., M acFarlane, D. R „ A ngell, C. A., and M erym an, H. T. 1984. V itrification as an approach to cryopreservation. C ryobiology 21:407-426. Fahy, G. M ., Saur, J., and W illiam s, R. J. 1990. Physical problem s w ith the vitrification o f large bio ­ logical system s. C ryobiology 27:492-510. Forsyth, M ., and M acFarlane, D. R. 1986. Recrystallization revisited. C ryo Lett. 7:367-378. Franks, F. 1988. Storage in the undercooled state. Pages 107-112 in: Low Tem perature Biotechnology: E m erging A pplications and E ngineering C ontributions. J. J. M cG rath and K. R. Diller, eds. A m eri­ can Society o f M echanical E ngineers, N ew York. Franks, F., M athias, S. F„ G alfre, P., W ebster, S. D., and Brow n, D. 1983. Ice nucleation and freezing in undercooled cells. C ryobiology 20:298-309. Fujikaw a, S. 1981. The effect o f different cooling rates on the m em brane o f frozen hum an erythrocytes. P ages 323-334 in: Effects o f Low Tem peratures on B iological M em branes. G. J. M orris and A. C larke, eds. A cadem ic Press, N ew York. Ishiguro, H., and R ubinsky, B. 1994. M echanical interactions betw een ice crystals and red blood cells during directional solidification. C ryobiology 31:483-500. K anno, H„ and A ngell, C. A. 1977. H om ogeneous nucleation and glass form ation in aqueous alkali halide solutions at high pressures. J. Phys. Chem . 81:2639-2643. Karlsson, J. O. M ., C ravalho, E. G ., and Toner, M. 1994. A m odel o f diffusion-lim ited ice growth inside biological cells during freezing. J. Appl. Phys. 75:4442-4455. Kresin, M ., and K orber, Ch. 1991. Influence o f additives on crystallization kinetics: Com parison b e­ tw een theory and m easurem ents o f aqueous solutions. J. Chem . Phys. 95:5249-5255. Kuwano, K., A ruga, Y., and Saga, N. 1994. Cryopreservation o f the conchocelis o f Porphyra (Rhodophyta) by applying the sim ple prefreezing system . J. Phycol. 30:566-570. M acFarlane, D. R., Angell, C. A, and Fahy, G. M. 1981. H om ogeneous nucleation and glass form ation in cryoprotective system s at h igh pressures. C ryo Lett. 2:353-358. M acFarlane, D. R., Forsyth, M „ and Barton, C. A. 1992. V itrification and devitrification in cryopreser­ vation. Adv. L ow -T em p. Biol. 1:221-278. M athias, S. F. 1991. U ndercooling— L ow tem perature w ithout freezing. T rends Biotechnol. 9:370-372. M azur, P. 1963. K inetics o f w ater loss from cells at subzero tem peratures and the likelihood o f intracel­ lular freezing. J. G en. Physiol. 47:347-369. M azur, P. 1965a. Physical and chem ical basis o f injury in single-celled m icro-organism s subjected to freezing and thaw ing. Pages 213-315 in: Cryobiology. H. T. M erym an, ed. A cadem ic Press, New York. M azur, P. 1965b. T he role o f cell m em branes in the freezing o f yeast and other single cells. Ann. New Y ork A cad. Sci. 125:658-676. M azur, P. 1988. S topping biological time: T he freezing o f living cells. Ann. New York Acad. Sci. 541:514-531. M ehl, P. M. 1990. E xperim ental dissection o f devitrification in aqueous solutions o f 1,3-butanediol. C ryobiology 27:378-400. M ehl, P. M. 1992. C ryom icroscopy as a support technique for calorim etric m easurem ents by DSC for the study o f the kinetic param eters o f crystallization in aqueous solutions. Part 1. Nucleation in the w a te r-1,2-propanediol system . T herm ochim . A cta 203:475-492. M ehl, P. M. 1993. N ucleation and crystal grow th in a vitrification solution tested for organ cryopreser­ vation by vitrification. C ryobiology 30:509-518. M uldrew, K., and M cG ann, L. E. 1990. M echanism s o f intracellular ice form ation. Biophys. J. 57:525532. Parody-M orreale, A., M urphy, K. P., C era, E. D ., Fall, R., D eV ries, A. L., and Gill, S. J. 1988. Inhibi­ tion o f bacterial ice nucleators by fish antifreeze glycoproteins. N ature 333:782-783. Pollock, G. A., Pegg, D. E., and H ardie, I. R. 1986. An isolated perfused rat m esentery m odel for direct observation o f the vasculature during cryopreservation. C ryobiology 23:500-511. Rail, W. F „ M azur, P., and M cG rath, J. J. 1983. Depression o f the ice-nucleation tem perature o f rapidly cooled m ouse em bryos by glycerol and dim ethyl sulfoxide. Biophys. J. 41:1-12. Rasm ussen, D. H., M acauley, M . N ., and M acK enzie, A. P. 1975. Supercooling and nucleation o f ice in single cells. C ryobiology 12:328-339.

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Raym ond, J. A., and D eV ries, A. L. 1977. A dsorption inhibition as a m echanism o f freezing resistance in polar fishes. Proc. N atl. A cad Sci. USA 74:2589-2593. Steponkus, P. L. 1984. R ole o f the plasm a m em brane in freezing injury and cold acclim ation. Annu. Rev. Plant Physiol. 35:543-584. Steponkus, P. L., Stout, D. G ., W olfe, J., and Lovelace, R. V. E. 1985. P ossible role o f transient electric fields in freezing-induced m em brane destabilization. J. M em brane Biol. 85:191-198. Storey, K. B., and Storey, J. M . 1992. N atural freeze tolerance in ectotherm ic vertebrates. Annu. Rev. Physiol. 54:619-637. Sutton, R. L., and Pegg, D. E. 1993. D evitrification in butane-2,3-diol solutions containing anti-freeze peptide. C ryo. Lett. 14:13-20. Toner, M ., and T om pkins, R. G. 1992. T ransport phenom ena during freezing o f isolated hepatocytes. A IC H E J. 38:1512-1522. W alter, C. A., K night, C. A ., and Farrant, J. 1975. U ltrastructural appearance o f freeze-substituted lym phocytes frozen by interrupting rapid cooling with a period at -2 6 ° C . C ryobiology 12:103-109. W illiam s, R. J., M ehl, P., and C arnahan, D. L. 1990. Ice nucleation and growth in vitrifiable solutions. Pages 71-86 in: C ryopreservation and Low Tem perature Biology in Blood T ransfusion. C. Th. Sm it Sibinga, P. C. D as, and H. T. M erym an, eds. K luw er A cadem ic, Boston.

C H A P T E R 19

A p p lic a tio n s o f B io lo g ic a l Ic e N u c le a to r s in S p r a y -Ic e T e c h n o lo g y R ic h a r d J . L a D u c a , A . F r a n k lin R ic e , a n d P a t r ic k J . W a r d

Ice nucleation-active (Ina+) bacteria have been successfully utilized in a number of commercial application areas that take advantage o f the ability of these m icro­ organisms to efficiently initiate freezing events at high subzero temperatures (for reviews see W arren, 1987; M argaritis and Bassi, 1991; Gurian-Sherman and Lindow, 1992, 1993). In large part, the commercial potential and value of Ina+ bacteria is directly proportional to the savings in energy costs that can be realized when freezing events are initiated at higher subzero temperatures with Ina+ bacteria. Earlier chapters of this book have discussed the application of Ina+ bacteria in the control o f insect pests (Chapter 14), the use of ice nucléation genes as reporters (Chapter 15), and the impact of biological ice nucleators in food processing (Chapter 17). Spray-ice applications of Ina+ bacteria will be the focus of this re­ view. The largest volume commercial use of Ina+ bacteria is today found in the sprayice application area of artificial snowmaking. In this application area, the ability of Ina+ bacteria to efficiently and cost effectively generate artificial snow has been shown to offer significant advantages over non-biologically nucleated snowmaking systems. Further commercial evaluation o f Ina+ bacteria in spray-ice applications is the focus of efforts in both academia and industry. Issues relating to the commercial manufacture o f Ina+ bacteria and use of these biological ice nucleators in spray-icetype product application areas such as artificial snowmaking, natural thermal stor­ age in ice ponds, freeze crystallization, weather modification through cloud seeding, and arctic ice island construction will be described. F e r m e n t a t io n a n d R e c o v e r y o f I n a + B a c te r ia In d u c ti o n o f t h e ln a + P h e n o t y p e in Ic e N u c le a t io n - A c t iv e B a c te r ia

Economically viable utilization of ice nucleation-active bacteria for commercial spray-ice-type product applications requires that the ice nucléation frequency of warm-temperature nuclei (defined as the ratio o f cells active in ice nucléation at -5 °C to the total number o f bacterial cells in a population of Ina+ bacteria) be as 337

338

L a D u c a , R ic e , a n d W a rd

close to one as possible. Historically, the frequency of expression of the Ina+ pheno­ type in pure cultures of ice nucleation-active bacteria has been shown to be highly variable (M aki and W illoughby, 1978; Lindow et al., 1982; Lindow, 1983; Yankofsky et al., 1983; Hirano et al., 1985). The source of this variability in the frequency and level o f Ina+ expression has been attributed to physiological responses of the Ina+ bacteria to nutritional and environmental signals (Hirano et al., 1985; Rogers et al., 1987; O bata et al., 1990; Pooley and Brown, 1991; Lawless and LaDuca, 1992; Nemecek-M arshall et al., 1993). Lindow et al. (1982) showed that the ratio between the number of ice nuclei and the number of bacterial cells in a culture can vary with incubation temperature, growth medium composition, culture age, and genotype. Nucleation frequencies appear to be most dramatically impacted by temperature. W hen ice nucleation-active bacteria are allowed to incubate at low temperatures (not conducive to cell growth), a shift in the frequency o f warm-temperature ice nuclei can be observed (Rogers et al., 1987). This process, perhaps the result of the maturation o f the nucleation site on the cell surface, affects the number and type (Type 1, 2, or 3) of ice nucleation sites observed in the microbial population. As Ina+ bacteria progress through a growth cycle in culture, key growth-promot­ ing nutrients are consumed at stoichiometric levels. Depletion of key nutrients sig­ nal the end o f the active growth phase and progression into the stationary growth phase. Elevated nucleation frequencies in Pseudom onas syringae have been tem­ porally linked to the transition of cells from the exponential to the stationary phase of growth (Lindow et al., 1982; Hirano et al., 1985; Deininger et al., 1988; Luquet et al., 1991; Pooley and Brown, 1991). Nemecek-M arshall et al. (1993) found that nutritional starvation for nitrogen, phosphorous, sulfur, or iron, but not carbon, at 32°C coupled with a shift to 14 to 18°C will lead to the rapid induction of warmtemperature ice nuclei. Expression of ice nuclei may also be regulated by other nu­ tritional factors. K ozloff and co-workers have shown that the addition to minimal media of sugars, such as inositol, mannose, and glucosamine in com bination with M n2+ will enhance ice nucleus formation (Turner et al., 1990; K ozloff et al., 1991a,b). They suggested that these nutrients are components of the stepwise as­ sembly o f Ina+ protein aggregates in the cell membrane, a process proposed to in­ volve covalent m odification of the protein by glycosylation and anchoring to phosphatidylinositol (K ozloff et al., 1991a,b). Numerous studies have focused on physiological and genetic responses of microorganisms to changes in the availabil­ ity of nutrients (Ingrahm et al., 1983; Matin et al., 1989; Blum et al., 1990). Nutri­ ent depletion resulting from the lack of carbon, phosphorus, or nitrogen and related physiological stresses including heat shock, osmotic shock, and oxidative stress in bacteria may prom ote the expression of unique sets of genes that function to miti­ gate the effects o f an environmental stress (Groat et al., 1986; Blum et al., 1990; Jenkins et al., 1991; M cCann et al., 1991). Expression o f these genes is usually ac­ complished through the use o f a non-housekeeping-type transcriptional promoter. This type o f regulation is similar to that observed in the expression o f low-temperature (Jones et al., 1987) or starvation (Matin et al., 1989) genes. Other unique nu­ cleotide sequences, termed gearbox-type promoters, are known in genes whose products are required at higher relative amounts when growth rates approach zero (Aldea et al., 1990; V incente et al., 1991). Nemecek-M arshall et al. (1993) sug­ gested that the regulatory region of the inaZ gene preceding the translational start codon has a high degree o f homology to the -3 5 and - 1 0 regions o f the consensus gearbox prom oter proposed by Aldea et al. (1990). Regions of homology were

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identified at 57 and 38 nucleotides, respectively, from the translational start codon of the inaZ gene. This inaZ putative prom oter sequence, AAGCAA(13 bp)CGGCTGCT, has a high degree o f homology to the consensus gearbox pro­ moter sequence identified in Escherichia coli, CTGCAA(14-16 bp)CGGCAAGT (Vincente et al., 1991). The homology o f these regulatory regions suggests that a gearbox-type or related prom oter structure may function to temporally control ex­ pression of the Ina+ protein following nutritional limitation in the culture medium (Nemecek-M arshall et al., 1993). L a r g e - S c a le M a n u f a c t u r e o f ln a + B a c t e r ia

Successful commercialization of spray-ice nucleation technology in all applica­ tion areas is based on the ability to manufacture the product (i.e., Ina+ bacteria) at minimal cost with the highest quality. It is essential that the development of fermen­ tation and recovery processes include optimization with respect to total activity produced in the fermentor, recovery process yield, and minimization of total pro­ cessing time. Generally, strain selection, choice of raw materials, fermentation pa­ rameter definition, recovery methodology, and ability to scale-up collectively prevail in implementing a process that is economically favorable. The commercialization of ice nucleation bacteria started in 1985 with the devel­ opment o f SNOMAX® Snow Inducer (Genencor International, Rochester, New York) for the artificial snowmaking industry. Fermentation and recovery processes have been described for the large-scale production of microorganisms having high levels of Type 1 and Type 2 ice nuclei (Lynn and Noto, 1988; Hendricks et al., 1992; Lawless and LaDuca, 1992). To date, these processes are considered state-ofthe-art for commercial manufacturing and will be described in some detail. F e r m e n t a t io n o f ln a + B a c te r ia

Optimal growth conditions for microorganisms expressing ice nucleation activity have been under investigation since their discovery in the early 1970s. Tradition­ ally, fermentation conditions have varied widely with regard to the nutritional and environmental conditions necessary to achieve cell growth and ice nucleation activ­ ity (Lindow et al., 1978; M aki and W illoughby, 1978; Kozloff et al., 1983). Ex­ trapolation of these conditions and methods to large scale often resulted in less than optimal expression of the Ina+ phenotype on a per cell basis as well as high cost for the growth medium raw materials. In the manufacture of Ina+ bacteria for spray-ice applications, optimal growth conditions require the generation of high cell densities with high nucleation frequencies (i.e., the ratio of cells active in Ina+ at -5 °C to the total cell number in the bacterial population must be as close to one as possible) in a low-cost production medium. Initial improvements in the large-scale manufacture o f Ina+ bacteria focused pri­ marily on the nutritional conditions necessary to develop high cell density fermen­ tation processes. H endricks et al. (1992) described a process using controlled fermentation pH, mannitol as a carbon source, and yeast extract as a nitrogen source for the generation of P. syringae Ina+ bacteria. This process produced greater than 1011 ice nuclei per gram o f dry cells in the reactor with fermentor productivities ex­ ceeding 10" ice nuclei per liter per hour. Further medium development led to the use o f a chemically defined medium for the manufacture of Ina+ bacteria (Lynn and Noto, 1988). Fermentation of Ina+ bacteria in a chemically defined medium, as compared to media utilizing more complex sources o f carbon and nitrogen (e.g.,

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yeast extract), has resulted in enhanced efficiencies in Ina+ yield and recovery in large-scale processes. Further enhancements to the fermentation process for manu­ facturing Ina+ bacteria have sought to leverage the environmental conditions that impact Ina+ expression in P. syringae. To achieve maximal cell densities and ice nucleation frequencies in a large-scale fermentation process, program m ed fermentation strategies have been designed to take into account the temperature dependence of nucleation frequency in P. syrin­ gae as well as to take advantage of the correlation between tem poral expression of warm-temperature ice nuclei in the growth cycle and depletion o f key nutrients (Lawless and LaDuca, 1992). In submerged cultures o f P. syringae that use these strategies, maximal nucleation frequency at -5 °C can approach one active nucleus out o f every one to 10 cells. These levels of expression have been accomplished by supplying stoichiom etrically limiting quantities of nitrogen in the fermentation me­ dium such that at the end of the active growth phase, there is insufficient nitrogen remaining to inhibit ice nucleus expression and formation during the subsequent stationary growth phase (Lawless and LaDuca, 1992). By programming a tempera­ ture downshift during fermentation to coincide with the nutritional depletion of ni­ trogen, maximal nucleation frequencies and cell densities can be obtained. Figure 1 shows a schematic for this high productivity process. To take full advantage o f the economy associated with large-scale fermentation, it is essential that fermentation development work take place in order to predict the behavior of the process on a large scale. This can be accomplished by either scaleup or scale-down methodology. It involves understanding the fermentation response to changes in environmental parameters to ensure optimum performance at the in­ creased scale. Although behavior of a population o f m icroorganisms should be in­ variant to scale, the behavior o f factors affecting the surrounding environment is more difficult to predict (e.g., oxygen transfer, broth mixing characteristics, and shear). Scale-up can have considerable costs in both time and equipm ent but incurs less risk than scale-up o f a process based solely on theoretical models. Understand­ ing o f the nutritional and environmental factors that affect the induction of ice nu­ cleation activity associated with P. syringae has enabled the commercialization of this particularly interesting application of industrial biotechnology.

FROZEN

SHAKE

V IA L

FLA S K

SEED FERM ENTOR

P R O D U C T IO N FERM ENTO R

F ig u re 1. G enencor International’s high-productivity process. T he final production ferm entor involves a two stage ferm entation. F irst biom ass is increased by ferm entation at optim um growth tem perature. Second, Ina+ ind u ctio n takes place by rapid ferm entor cooling w hen nutrient lim itation brings cell mass into stationary grow th phase. F erm entor harvest occurs at m axim um Ina+ (6 -1 0 h r post induction).

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Ice nucleation-active bacteria are currently being produced by the thousands of liters per fermentation, with productivities reaching one active ice nucleation site per cell at -5 °C . Associated with the large-scale manufacture of any industrial fer­ mentation process are larger risks. One o f the risks that can virtually shut down production without advanced warning is the susceptibility of bacteria to bacterio­ phage infection.

P. syringae a n d

S u s c e p t ib ilit y to B a c t e r io p h a g e In fe c t io n s

Naturally occurring strains o f P. syringae are susceptible to lytic infection by bacteriophage. It is clear that cell lysis due to phage infection during fermentation is detrimental to the production o f ice nucleation activity and should be guarded against in the production environment. Lytic events in the large-scale manufacture o f any microbial product including Ina+ bacteria have generally been dealt with through two parallel approaches. In the first approach, microbial isolates that are insensitive to lytic infection by isolated species of bacteriophage are isolated as spontaneous mutants. Bacterial isolates resistant to a broad range o f phage can be obtained by selecting spontaneous phage-resistant mutants following batch or se­ quential exposure o f the phage-sensitive Ina+ bacteria to numerous serotypes of bacteriophage. In this way, a family o f bacteriophage-resistant Ina+ producers can be generated to minimize susceptibility of the commercial process to lytic fermen­ tation failures. A second and essential approach to minimize lytic infections has been to control, through enhanced cleaning and m onitoring procedures, all avenues of bacteriophage introduction into the fermentation process. For this reason it is es­ sential to manufacture under sterile conditions, abide by all current good manufac­ turing practices, and regularly monitor the air both in the fermentation area and at the intake for process air quality. R e c o v e r y o f Ic e N u c le a t io n A c ti v it y

The design o f a process for the recovery of ice nucleation activity involves the integration o f many factors. Such factors can and should include recovery process yield, total processing time, and final desired product form. O f these considerations, the final product form is determined once the final customer has been identified, and the custom er’s needs and applications are understood. Final package size, product shelf life, environmental impact, and shipping and handling constraints must be defined before the entire downstream process can be developed. It is es­ sential that the recovery methodology be cost effective on a large scale and meet all the customer requirem ents and specifications (W heelwright, 1991). The large-scale purification of active ice nucleation bacterial fermentation broths has presented several m ajor purification challenges. These challenges stem from the susceptibility o f the ice nucleation protein to dénaturation by temperature, and sus­ ceptibility of the active site to incur damage over time in high osmotic strength, aqueous environments. An example of large-scale recovery o f ice nucleating bac­ teria is the production o f SNOM AX Snow Inducer. This product is recovered on the large scale through a series of unit operations that help to preserve the integrity of the ice nucleation protein all the way from fermentation harvest broth to delivery to the final customer. Figure 2 is a schematic of the process typical for the large-scale recovery o f SNOM AX. In the production o f SNOM AX, the fermentation broth at harvest (peak Ina+ ac­ tivity) is sent downstream for cell separation. Since the bacterial cells are the prod-

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F igure 2. Typical com m ercial-scale recovery process for bacterial ice nuclei.

uct of interest, cell concentration is essential to reduce the volume needing to be processed in further downstream refinements. Typically, cell concentration can take place by centrifugation or permeable membrane separation. In the case of SNOM AX, ultrafiltration is preferred. Ultrafiltration uses microporous polymeric membranes driven by liquid pressures to separate the cells from the spent fermenta­ tion broth. Ultrafiltration takes advantage of the sieving mechanism of the mem­ brane based on m olecular weight. Thus cells and high molecular weight components are concentrated on one side o f the membrane, while smaller mole­ cules, such as water, salts, and low molecular weight proteins, pass through the po­ rous skin o f the membrane. Concentration continues until the cell slurry reaches approximately 25% dry solids. It is this high osmotic strength aqueous environment that causes Ina+ dénaturation to occur rapidly, damaging the ice nucléation protein. To stop the dénaturation process, the cell slurry is rapidly fed under pressure in thin streams into liquid nitrogen. The interfacial surface interactions with liquid nitrogen cause the streams o f cell mass concentrate to freeze as pellets. The ice nucléation activity o f cells in the pellets is much more stable than in the slurry. The frozen pellets are subsequently freeze-dried. Freeze-drying at low temperature is advanta­ geous because it minimizes loss o f heat-labile products such as the Ina+ protein. The freeze-dried Ina+ product is relatively stable at room temperature, but shelf life can be extended through packaging and long-term storage at subzero temperatures. Packaging of the material is best conducted with an inert gas purge to prevent oxi­ dation o f the ice nucléation protein over time. It is also recommended that ultravio­ let light-insensitive materials be used to store the final product. For product applications o f Ina+ bacteria, some regulatory agencies require that the cell viability of P. syringae be reduced. This helps to ensure that the number of viable organisms in the environment does not increase beyond already existing natural Ina+ bacterial populations due to the use and release of Ina+ bacteria. Beta irradiation of SNOM AX powders is routinely performed to reduce cell viability and in some instances to produce a sterile product. Effective cell inactivation can be achieved through low-dose beta irradiation with minimal Ina+ loss. C o m m e r c ia liz a tio n o f S p r a y -Ic e T e c h n o lo g ie s S n o w m a k in g

The production of artificial snow at ski resorts worldwide has become an eco­ nomic necessity to ensure adequate snow coverage under variable weather condi­

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tions throughout the ski season. To this end, it is estimated that worldwide annual snow production requires approximately 10 billion U.S. gallons (37.8 billion liters) o f water to generate 60 million acre feet (70 billion cubic meters) of snow. Typical ski areas utilize an estim ated 500 to 750,000 U.S. gallons of water to generate 3 to 4 acre feet (3,500 to 4,000 m3) o f snow each day of operation. Marginal weather conditions both early and late in the ski season typically limit the amount, as well as quality, o f man-made snow that can be produced, resulting in shorter operational seasons and reduced revenues for ski operators. During the ski season, temperatures above -2 ° C dry bulb lim it effective operation o f snow-making equipm ent re­ sulting in poor quality and quantity o f artificial snow generated. O perators of these facilities must thus balance the costs associated with generation o f artificial snow against the benefits o f an extended season and the amount of skiable terrain that can be m aintained throughout operational periods and extremes of weather fluctuations. In the production of artificial snow, water is commonly sprayed through nozzles under air pressure in order to facilitate supercooling and nucleation of water drop­ lets. The atomizing nozzles typically employed in this application of spray-ice tech­ nology promote the quick formation o f fine water droplets, typically 200 to 700 urn in size, significantly enhancing air-water heat exchange rates and formation o f nu­ cleated droplets. Com pressed air primarily promotes formation of atomized water droplets, however pressure changes resulting from expansion of air across the ori­ fice o f the nozzle further enhance cooling of the droplets and the crystallization process. Thus, in the absence of heterogeneous sources of ice nucleators, artificial snowmaking is primarily affected by water flow rates and temperature, air pressure, ambient temperatures, and relative humidity. Biological sources o f ice nucleators are effective in enhancing the temperature range at which artificial snow can be generated (W oerpel, 1980). The presence of biological ice nucleators in water droplets generated in standard snowmaking equipment initiates ice formation without the need to supercool the water by the amount that is typically required in their absence. Commercialization of biological ice nucleators in this application area has been accomplished by SNOMAX Tech­ nologies, a division o f Genencor International, Inc., with the introduction of SNOMAX. SNOM AX is a freeze-dried powder containing Ina+ P. syringae that facilitates efficient production o f man-made snow at temperatures as high as 1.1 °C dry bulb. In typical snowmaking applications, 270 g o f SNOMAX is used to treat 100,000 gallons of water. Inclusion of this biological ice nucleator increases the volume and quality o f man-made snow produced and extends the temperature range at which snow can be made utilizing standard snowmaking equipment (Liao and Ng, 1990). Snow densities are reduced by 10 to 15% in man-made snow generated with biological ice nucleators. Further, snow volumes generated in the presence of SNOM AX have been observed to increase by 25 to 60% (compared with snow vol­ umes generated in the absence o f the nucleator) depending on the equipment used and atmospheric conditions. Since its introduction in 1985, SNOM AX has been used at the Calgary and Albertville Olympic Games and in 1994 was used in all machine-made snow gen­ erated for the Lillehammer Olympic games in Norway. Through the use of SNOMAX, biological ice nucleators are utilized routinely as snowmaking additives in ski areas throughout North America, South America, Australia, New Zealand, Japan, and Europe.

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The application o f P. syringae as an ice nucleator in the manufacture o f artificial snow has previously raised questions concerning the release of microorganisms into alpine environments. The P. syringae cell, which is encapsulated in the ice crystals of man-made snow, can contact soil and natural waters from snow melt runoff. Studies designed to evaluate the fate of Ina+ P. syringae in alpine soils, waters, and synthetic snow samples have shown that ambient environmental conditions (e.g., freeze-thaw cycles, sunlight, and soil contact) severely limit the survival of this mi­ croorganism (G oodnow et al., 1990) subsequent to its use as a snow inducer. N a tu r a l T h e r m a l S t o r a g e

Natural thermal storage systems, also known as “ice ponds,” are simple outdoor man-made ponds equipped with centrifugal pumping systems that atomize recircu­ lating pond water via overhead-mounted spray system assemblies (Fig. 3). These same pumps also deliver 1.7°C ice water to an “interfacing” heat exchanger located within the facility of choice requiring cooling, replacing a conventional chlorinated fluorocarbon (CFC) chiller and associated cooling tower system. Ice ponds may be either constructed open to the environment for seasonal operation or housed com­ pletely enclosed within insulated structures for year-round operation. Ice ponds are based upon the am bient freezing, thawing, and recirculation of water, requiring a seasonal winter climate with 4,000 (or more) heating-degree days (HDD) per year typically required for economic operation. Several attempts have been made in the past to economically capture wintertime coldness in the form o f frozen water as perhaps the ultimate in environmentally compatible refrigeration. M ost noteworthy is the Prudential Insurance Company’s 1979 installation o f a 4,000 m2 ice pond for year-round comfort cooling at one of their commercial buildings (12,000 m2) in Princeton, New Jersey.

F ig u re 3. Ice pond schem atic for enhanced natural therm al storage

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Since the introduction o f commercially available biological ice nucleation-active products, energy efficiency and economic viability o f these ambient systems have been substantially im proved. Unlike conventional, mechanical ice thermal storage systems, these “enhanced” ice ponds dramatically reduce both power and energy requirements while shifting electrical loads to off-peak hours of utility demand, all without the use o f refrigerant gases. An additional im portant feature of the ice pond is its ability to reject sensible heat directly to the atmosphere without consuming valuable stored ice. It is estimated that 40% of the total heat load can thus be re­ jected directly to the environm ent via evaporative cooling. Theoretical calculations of cost savings achieved via elimination of conventional refrigerant com pressors are substantiated by two industrial installations located in upstate New York: an 80 refrigeration-ton system at Kutters Cheese (Corfu, New York) and a 500 refrigeration-ton system at Genencor International, Inc. (Rochester, New York). At Kutters Cheese (a cheese-making company), the plant owner wanted to sup­ plement existing CFC-based refrigeration capacity with natural thermal storage to meet seasonal peak production demands without additional investments in compres­ sor capacity. Based on a seasonal mode of operation and a 6,900-HDD weather profile, the enhanced ice pond constructed in 1989 was operated continually for 5 months with a seasonal average energy efficiency o f 0.05 kW/ton compared with existing electrical, mechanical equipment operating at 1.0 kW/ton, a 20:1 reduction in energy consumption. Similar results have been obtained at Genencor Interna­ tional’s biochemicals production facility in Rochester, New York, where a 1,200-m2 ice pond has been in seasonal operation since 1991. In both cases, energy cost sav­ ings were sufficient to offer a 3-year simple pay-back period of invested capital. Ice ponds do have limitations. Given the 4,000-HDD economic restriction, the southern half of the United States is unsuitable for economic application of this technology. Ice ponds also have aesthetic limitations and require land— a combina­ tion not usually suited for high-density, high-rise central business district environ­ ments. A m b ie n t F r e e z e - C r y s t a liiz a t io n a n d W a te r P u r if ic a tio n

Natural thermal storage systems can also be effectively utilized as low-cost, freeze concentration machines for the effective separation of impurities from water. W ater desalination is an excellent example of such an application. Natural freeze concentration systems are based on the fact that many inorganic materials are preferentially excluded from ice crystals during the freezing process. In the case of sea water, for example, freezing of the brackish water will result in the formation o f a low-salt ice mass and a high-salt concentrate. By linking two to four ponds in series and feeding sequential ponds with concentrate from the prior stage, water of higher potable quality can be produced in the form of ice. Removing ice from the final-stage ice pond provides both cooling and “fresh” water. In Octo­ ber, 1984, engineers from NOVA Inc., an engineering firm in Gaithersburg, M ary­ land, in conjunction with the New Y ork State Energy Research and Development Authority (NYSERDA) constructed such a system in Greenport, New York, for the production of potable water from sea water. The study concluded that using snow­ making machines to spray and collect sea water in a series of ice ponds could pro­ duce drinking water for $1.50 to $3.00 per 1,000 gallons, or about the same unit cost as the then state-of-the-art reverse osmosis machines. The system produced 10

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million gallons per year o f desalinated water using a 1.5-acre reservoir and an 8,000-gallon per m inute pumping system. The addition o f Ina+ bacteria in these systems should provide even greater efficiencies in freeze-crystallization applica­ tions, given the wide diversity o f freezing-point depressions found in aqueous sys­ tems containing various inorganic impurities. W e a th e r M o d if ic a t io n

W eather modification (cloud seeding) is the intentional treatment o f individual cloud or storm systems for the purpose of altering the physical processes leading to the formation and growth of water droplets and ice crystals in clouds. M ost conti­ nental precipitation is produced by ice crystal formation in supercooled clouds, known as the cold rain process. However, many regions have only a few ice nuclei in the atmosphere, which reduces the efficiency of the cold rain process. For this reason, artificial ice nuclei are introduced through conventional weather modifica­ tion techniques in order to enhance the precipitation process. Once ice has formed, the ice crystals accumulate moisture and grow in size and mass until they fall as ei­ ther rain or snow depending on the temperature of the atmosphere near the ground. Cloud-seeding agents have proven to be useful in fog and stratus dispersal, winter and summer precipitation augmentation, and hail suppression (W eather M odifica­ tion Association, 1984). Very much has been learned in the past 20 years regarding the ability of certain strains of naturally occurring bacteria to efficiently nucleate the formation of ice at slight supercoolings (Gregor, 1967; Schnell and Vali, 1972, 1976; Maki et al„ 1974; Vali et al., 1976; M aki and W illoughby, 1978; Lindow et al., 1978, 1982; Yankofsky et al., 1981; Levin et al., 1987). M ost naturally occurring ice nuclei found in the atm osphere are not active at temperatures warmer than about -10°C . For this reason, there is continued interest in searching for novel ice nuclei that can convert liquid water to ice crystals at the lowest degree of supercooling. Although dry ice is a very good source o f warm-temperature ice nuclei, it has limitations in handling, storage, bulk density, and dispersal (Woodley and Henderson, 1990). Recently, Genencor International, Inc. commercialized P. syringae (SNOMAX W eather M anager) for use as an atmospheric ice nucleus alternative. To date, this is the only existing commercial bacterial ice nucleation product used to induce pre­ cipitation from supercooled clouds. Figure 4 shows a series of electron micrographs of increasing magnification of an ice crystal captured after SNOM AX aerosol was introduced into a -2 0 °C supercooled cloud. It is evident that the bacterial particle initiated ice crystal formation because the rod-shaped bacterial particle is symmetri­ cally centered in the hexagonal ice crystal formation. Atmospheric tests of SNOM AX W eather Manager have been performed during the past few years. Initial quantitative atmospheric tests indicate that P. syringae may prove to be a valuable tool for artificial nucleation in the atmosphere (Rogers et al., 1987; W ard and DeM ott, 1989; Jung, 1990; W oodley and Henderson, 1990). Continued research in capturing warm-temperature Ina+ bacteria in quantity at low cost could eventually result in a displacement of silver iodide and solid carbon dioxide technologies, and become widely used in weather modification. A r c tic S p r a y - ic e C o n s t r u c t io n

Low-tem perature sea water is an abundant resource in arctic regions that can be used in numerous spray-ice technology applications for the construction of load-

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I----------------------1

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(15.360X ) I-------------------- 1 2 p m

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I--------------------- 1

15 H m

(30,000X ) I---------------- 1

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F ig u re 4. E lectron m icrographs o f biologically seeded ice crystals. Panels A to D show increasing m agnifications o f a transm ission electron m icrograph o f an ice crystal captured after SNOM AX aerosol introduction into a - 2 0 ° C supercooled cloud. R eprinted, w ith perm ission, from W ard and DeM ott (1989).

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bearing structures including aircraft runways, roads, ice berms, and ice islands for offshore drilling. Construction o f ice structures in the absence of ice nucleants can be initiated only when ambient air temperatures are sufficiently low to cause rapid freezing o f sea water. The efficiency o f droplet formation and freezing of the sea water in these high-volume sprays depends upon the temperature o f the air, the sa­ linity of the water, and the efficiency of heat exchange between the air and sprayed droplet. In addition, other factors, including water volume and velocity at the spray nozzle, trajectory o f the stream spray, surface tension o f the sea water, and wind speed, affect the efficiency of spray-ice formation. In contrast to snowmaking appli­ cations, successful ice construction requires that the frozen droplets that form in the sprayed sea w ater that falls to the surface have a liquid content (Owen et al., 1987). Additional freezing that occurs after impact positively influences the strength of the sprayed-ice structure. Heterologous ice nucleants in the form of ice nucleation-ac­ tive P. syringae have been successfully applied in arctic ice construction to effec­ tively reduce supercooling in sprayed sea water streams (Owen et al., 1987). This work dem onstrated that more rapid freezing of single water droplets and of larger mass impounded waters can be obtained in the presence o f the Ina+ bacteria, result­ ing in enhanced efficiencies of ice formation as well as extended tem perature ranges for use of spray ice in ice construction applications.

Sum m ary

The ability o f biological ice nucleators to minimize supercooling o f aqueous so­ lutions and facilitate ice crystal formation at high subzero tem peratures has resulted in numerous opportunities for use of these microorganisms in spray-ice applica­ tions. Efforts to identify and characterize species of Ina+ bacteria in conjunction with efforts to biochem ically and physiologically characterize ice-nucleating mechanisms have been critical in the design and implementation o f commercial processes for the manufacture o f biological ice nucleators. Fermentation and recov­ ery processes have been established that result in high-level expression of Ina+ in P. syringae ice nucleators. Nutritional and environmental signals appear to be key physiological effectors required for the efficient expression of Ina+ in P. syringae and are expected to play key roles for expression of this activity in other species of Ina+ bacteria. The use o f biological ice nucleators in spray-ice applications including snow­ making, weather m odification, thermal storage, arctic ice construction, and freeze crystallization and effluent processing has been expanding since the commercial introduction o f Ina+ products in 1985. The primary role of ice nucleators has been to reduce the supercooling of aqueous solutions used in these applications, thereby elevating nucléation temperatures and reducing the amount of energy required to complete the freezing process. These energy savings have resulted in significant reductions in the costs associated with generation of spray-ice products. In addition to energy savings, the enhanced freezing efficiencies resulting from the use o f bio­ logical ice nucleators in snowmaking applications also improve the quality of the final product. This combination of economic as well as quality enhancements in spray-ice applications should provide the basis for expanded use o f biological ice nucleators in this application area as well as form a platform for generation of new uses of these unique microorganisms.

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L it e r a t u r e C ite d Aldea, M ., G arrido, T., Pla, J., and V incente, M. 1990. D ivision genes in Escherichia coli are expressed incoordinately to septum requirem ents by gearbox prom oters. E M B O J. 9:3787-3794. Blum , P. H., Jovanovich, S. B., M cC ann, M. P., Schultz, J. E ., Lesley, S. A., Burgess, R. R., and M atin, A. 1990. C loning and in vivo and in vitro regulation o f cyclic A M P-dependent carbon starvation genes from Escherichia coli. J. B acteriol. 172:3813-3820. Deininger, C. A., M ueller, G. M ., and W olber, P. K. 1988. Im m unological characterization o f ice nu­ cleation proteins from Pseudomonas syringae, Pseudomonas fluorescens, and Erwinia herbicola. J. B acteriol. 170:669-675. Goodnow, R. A., H arrison, M . D., M orris, J. D., Sweeting, K. B „ and LaDuca, R. J.1990. Fate o f ice nucleation-active Pseudomonas syringae strains in alpine soils and w aters and in synthetic snow sam ples. Appl. E nviron. M icro. 56:2223-2227. G regor, P. H. 1967. A tm ospheric m icrobial cloud system s. Sci. Progr. O xford 55:613-628. Groat, R. G., Schultz, J. E ., Z ychlinsky, E., B uckm an, A., and M atin, A. 1986. Starvation proteins in Escherichia coli: K inetics o f synthesis and role in starvation survival. J. Bacteriol. 168:486-493. Gurian-Sherm an, D., and L indow , S. E. 1992. Ice nucleation and its application. Curr. Op. Biotechnol. 3:303-306. Gurian-Sherm an, D., and L indow , S. E. 1993. B acterial ice nucleation: Significance and m olecular b a­ sis. FA SEB (Fed. A m Soc. Exp. Biol.) J. 7:1338-1343. Hendricks, D., W ard, P. J., and O rrego, S. A. Production o f m icroorganism s having ice nucleation ac ­ tivity. 1992. U.S. p aten t 5,137,815. H irano, S. S., B aker, L. S., and U pper, C. D. 1985. Ice nucleation tem perature o f individual leaves in relation to population sizes o f ice nucleation active bacteria and frost injury. Plant Physiol. 77:259265. Ingrahm , J. L., M aaloe, O ., and N eidhardt, F. C. 1983. R egulation at the whole cell level, pages 349370. in: G row th o f the B acterial Cell. Sinauer A ssociates, Inc., Sunderland, MA. Jenkins, D. E., Auger, E. A., and M atin, A. 1991. Role o f RpoH, a heat shock regulatory protein, in Escherichia coli carbon starvation protein synthesis and survival. J. Bacteriol. 173:1992-1996. Jones, P. G ., V anB ogelen, R. A., and N iedhardt, F. C. 1987. Induction o f proteins in response to low tem perature in Escherichia coli. J. B acteriol. 169:2092-2095. Jung, J. A. 1990. Prelim inary field experim ents o f SNOM AX® on cum ulus m ediocris clouds to artifi­ cially induce the production o f ice particles. J. W eather M odification 22:153-157. Kozloff, L. M ., Schofield, M . A., and Lute, M. 1983. Ice nucleation activity o f Pseudomonas syringae and Erwinia herbicola. J. B acteriol. 153:222-231. Kozloff, L. M., Turner, M. A., and Arellano, F. 1991a. Form ation o f bacterial m em brane ice-nucleating lipoglycoprotein com plexes. J. Bacteriol. 173:6528-6536. Kozloff, L. M „ Turner, M . A., Arellano, F „ and Lute, M. 1991b. Phosphatidylinositol, a phospholipid o f ice-nucleating bacteria. J. B acteriol. 173:2053-2060. Lawless, R. J., and L aD uca, R. J. 1992. Ferm entation o f m icroorganism s having ice nucleation activity using a tem perature shift. 1992. U.S. patent 5,153,134. Levin, Z., Y ankofsky, S. A ., Pardes, D., and M agal, N. 1987. Possible application o f bacterial conden­ sation freezing to artificial rainfall enhancem ent. J. Clim . A ppl. M eteor. 26:1188-1197. Liao, J. C., and Ng, K. C. 1990. E ffect o f ice nucleators on snow m aking and sprayfreezing. Ind. Eng. Chem . R es. 29:361-369. Lindow, S. E ., A m y, D. C ., and Upper, C. D. 1978. Erwinia herbicola: A bacterial ice nucleus active in increasing frost injury to com . Phytopathology 68:523-527. Lindow , S. E., H irano, S. S., B archet, W. R., A m y, D. C., and U pper, C. D. 1982. R elationship betw een ice nucleation frequency o f bacteria and frost injury. Plant Physiol. 70:1090-1093. Lindow, S. E. 1983. T he role o f bacterial ice nucleation in frost injury to plants. Annu. Rev. Phytopathol. 21:363-384. Luquet, M. P., C ochet, N ., B ouabdillah, S., Pulvin, S., and Clausse, D. 1991. Som e characteristics o f a biological ice nucleating agent: Pseudomonas syringae. C ryo Lett. 12:191-196. Lynn, S. Y ., and N oto, G. D. 1988. Ferm entation o f m icroorganism s having ice nucleation activity u s­ ing a defined m edium . E uropean patent 272669. M aki, L. R., Galyan, E. L., C hein, M. C., and Caldw ell, D. R. 1974. Ice nucleation induced by Pseudo­ monas syringae. A ppl. M icrobiol. 28:456-459. M aki, L. R „ and W illoughby, K. J. 1978. B acteria as biogenic sources o f freezing nuclei. J. Appl. Meteriol. 17:1049-1053. M argaritis, A., and B assi, A. S. 1991. Principles and biotechnological applications o f bacterial ice

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nuclei. C rit. Rev. B iotechnol. 11:277-295. M atin, A. E ., A uger, E. A ., B lum , P. H., and Schultz, J. E. 1989. G enetic basis o f starvation survival in nondifferentiating bacteria. A nnu. Rev. M icrobiol. 43:293-316. M cCann, M. P., Kidw ell, J. P., and M atin, A. 1991. The putative sigm a factor K atF has a central role in developm ent o f starvation-m ediated general resistance in Escherichia coli. J. Bacteriol. 173:41884194. N em ecek-M arshall, M ., L aD uca, R. J., and Fall, R. 1993. High-level expression o f ice nuclei in a Pseudomonas syringae strain is induced by nutrient lim itation and low tem perature. J. Bacteriol. 175:4062-4070.' O bata, H., T okuyam a, T ., K aw ate, S., Hori, H., and Higashi, Y. 1990. Culture conditions o f Erwinia uredovora in reference to its high ice-nucleating activity o f the culture supernatant. Agric. Biol. C hem . 54:2171-2174. Owen, L. B., M asterson, D. M ., and Green, S. J. 1987. Rapid construction o f ice structures with chem ically treated sea w ater. U.S. patent 4,637,217. Pooley, L., and Brow n, T. A. 1991. Effects o f culture conditions on expression o f the ice nucleation phenotype o f Pseudomonas syringae. FEM S (Fed. Eur. M icrobiol. Soc.) M icrobiol. Lett. 77:229232. Rogers, J. S., Stall, R. E „ and Burke M . J. 1987. Low -tem perature conditioning o f the ice nucleation active bacterium , Erwinia herbicola. Cryobiology 24:270-279. Schnell, R. C ., and V ali, G. 1972. A tm ospheric ice nuclei from decom posing vegetation. Nature 236:163-165. Schnell, R. C., and V ali, G. 1976. B iogenic ice nuclei. Part I: T errestrial and m arine sources. J. Atmos. Sci. 33:1554-1564. Turner, M . A., A rellano, F., and Kozloff, L. M. 1990. Three separate classes o f bacterial ice nucleation structures. J. B acteriol. 172:2521-2526. Vali, G., C hristensen, M ., Fresh, R. W ., G ayan, E. L., M aki, L. R., and Schnell, R. C. 1976. B iogenic ice nuclei. Part II: B acterial sources. J. Atm os. Sci. 33:1565-1570. Vincente, M „ K ushner, S. R., G arrido, T., and Aldea, M. 1991. T he Role o f the “G earbox” in the transcription o f essential genes. M ol. M icrobiol. 5:2085-2091. W ard, P. J., and D eM ott, P. J. 1989. Prelim inary experim ental valuation o f SNOMAX® snow inducer, Pseudomonas syringae , as an artificial ice nucleus for w eather m odification. J. W eather M odification 21:9-13. W arren, G. J. 1987. B acterial ice nucleation: M olecular biology and applications. Biotechnol. Genet. Eng. Rev. 5:107-135. W eather M odification A ssociation. 1984. Som e facts about seeding clouds. The A ssociation, P.O. Box 8116, Fresno, CA 93747. W heelw right, S. M . 1991. Protein Purification— Design and Scale-U p o f D ow nstream Processing. H anser Publishers, N ew York. W oerpel, M. D. 1980. Snow M aking. U.S. patent 4,200,228. W oodley, W . L., and H enderson, T. J. 1990. A tm ospheric tests o f an organic nucleant in supercooled fog. J. W eather M odification 22:127-132. Y ankofsky, S. A ., L evin, Z., Barthold, T., and Sanderm an, N. 1981. Som e basic characteristics of bacterial freezing nuclei. J. A ppl. M eterol. 20:1013-1019. Y ankofsky, S. A., N adler, T. N., and Levin, Z. 1983. Induction o f latent freezing nucleus capability in an ice n ucleation-active bacterium . Curr. M icrobiol. 9:263-268.

G lo ssa r y

a c c li m a t i o n — The

ability to increase tolerance to a given environmental stress, usually triggered by an environm ental cue. In reference to cold, it means an in­ crease in tolerance to subzero temperatures, a l p h a - h e l i x — A term used to describe the secondary structure of a protein. The polypeptide chain turns regularly on itself every 3 -6 amino acids due to hydro­ gen bonding betw een the amino acids. This results in a rigid structure and is of­ ten found in globular protein, a m o r p h o u s l a y e r — An especially thickened layer of the cell wall in the vicinity of the pit m em brane of a xylem ray cell. It is also referred to as the protective layer, although its function is poorly understood, a n a e r o b i c s t r e s s — A stress imposed on an organism as a result of the absence of free oxygen or air. (See also ice encasement.) a n n e a l — The process of holding a substance at a temperature below the melting transition in order to change the glass transition characteristics. Annealing may either allow for com plete crystallization (see devitrification and recrystalliza­ tion), so that glasses do not form with subsequent cooling, or it may enhance the glass transition signal if the glass is particularly stable, a n t e m e l t i n g — A m inor endotherm ic event that occurs prior to the true melting transition. This phenom enon can be detected upon warming previously frozen aqueous solutions of sugars with specific concentrations. Although antemelting events remain unexplained, it is believed that they reflect a partial melting of a highly concentrated domain. Similar pretransitions are also observed in lipids and are believed to reflect changes in crystalline structure. In this respect, ante­ melting events may be classified as cooperative transitions, a n t i f r e e z e p r o t e in — A protein that lowers the freezing point o f an aqueous solu­ tion w ithout low ering the vapor pressure (and therefore the melting temperature) of aqueous solutions. The depression of the freezing point by an antifreeze pro­ tein may exceed that of the m elting point by a large factor. (See thermal hys­ teresis, thermal hysteresis protein.) a p o p l a s t — The nonliving part o f the plant external to the plasma membrane that comprises the extracellular spaces, the cell walls, and dead xylem elements, b e t a - s h e e t s — A folding pattern resulting from hydrogen-bonded lateral associa351

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tions o f extended polypeptide chains into sheets, formed by parts of globular proteins and prim arily due to the presence o f large numbers o f glycine residues, b o u n d w a t e r — W ater that has thermodynamic and motional properties that differ from pure water because o f the association o f bound water with a macromolecular surface. Also known as vicinal water. c h i l li n g i n j u r y — Injury incurred by biological material due to low nonfreezing tem peratures (e.g., 15 to 0°C or lower if supercooling occurs). In this phenom e­ non, ice does not occur in the tissue (contrast with Cold injury), c h il lin g r e q u i r e m e n t — Low-tem perature requirement to overcom e rest endodormancy (in seeds and buds), c o l d i n j u r y — Injury incurred by biological material due to the crystallization of water at tem peratures below 0°C. The term is often used interchangeably with w inter injury, freezin g injury, and fro st injury. c o l d p r o t e c t i o n — M ethods of guarding against injury from temperatures below 0°C. The term is often used interchangeably with frost protection and commonly refers to protection of blossom s in the spring, c o l d s h o c k — Im position of a brief, nonfreezing temperature resulting in a strain to the organism s; may or may not induce further acclimation, c o o p e r a t i v e t r a n s i t i o n — A phase transition in which the reorganization of mole­ cules occurs before the actual transition temperature. This type of transition is described as an order-disorder transition in which domains of the new phase form in different parts o f the old phase, and the presence o f the new phase fa­ vors the form ation o f more o f that phase. In a differential scanning calorimetry (DSC) trace o f a cooperative transition, the heat capacity changes continuously before the actual phase transition temperature, rather than abruptly as is ob­ served in true first-order transitions. Often DSC traces o f water m elting in bio­ logical tissues resem ble cooperative transitions. In a multicomponent, m etastable system, cooperative transitions can lead to phase separations. For ex­ ample, in a mixed lipid system, the ordering o f one lipid com ponent can lead to the separation and ordering o f a different lipid component, c r it i c a l t e m p e r a t u r e — The lowest ambient temperature at which the whole plant or parts o f a plant can endure for 30 minutes without injury. This is not to be confused with the chem ical definition of critical temperature, which is the tem­ perature of vaporization o f a liquid, c r y o p r e s e r v a t i o n — A process in which biological m aterials are stored at subfreez­ ing tem peratures in order to m aintain the chemical and physical integrity of the tissues. C ryopreservation implies that viable tissues are m aintained in suspended animation. It is believed that the formation of aqueous glasses (vitrification) prevents the lethal form ation o f ice crystals and this in turn allows tissues to be cryopreserved. c r y o p r o t e c t a n t — Any com pound or agent that protects cells from freezing and chilling injury. Consists of two classes: penetrating (e.g., dimethyl sulfoxide and sugars) and nonpenetrating (e.g., polyvinylpyrrolidone), c y t o r r h y s i s — Collapse o f the cell wall with the protoplasm attached due to a loss o f water— in contrast to plasmolysis, where the protoplasm shrinks and sepa­ rates from the cell wall, d e e p s u p e r c o o l i n g — M aintenance of the liquid state at tem peratures well below the freezing point o f the liquid. For example, water freezes at 0°C if nucleated with ice; however, in the absence o f nucleators, water can remain in a meta­

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stable liquid state to -3 8 °C . Xylem ray parenchym a and dorm ant hardy flower buds deep supercool from - 2 0 to -5 0 °C and - 1 0 to -4 0 °C , respectively. d e h a r d e n in g — Synonym ous with deacclimation, primarily to warm temperatures. The loss of freezing resistance in plant tissues, organs, and cells. d e v i t r i f i c a t i o n — A crystallization process that occurs upon slow warming of a glass to a tem perature above the glass transition temperature. Because the pro­ cess liberates heat, it is identified by an exothermic event after the glass melting transition and before the m elting transition. If the substance crystallizes (i.e., is annealed) at the devitrification temperature, a glass will not form upon subse­ quent cooling. Devitrification events occur because the liquid phase formed af­ ter the glass melts is unstable at the low tem perature, and the solid phase is the equilibrium phase. d if f e r e n t i a l s c a n n i n g c a l o r i m e t r y ( D S C ) — A technique that measures the change in heat capacity o f a sam ple with temperature. In DSC, the sample temperature is kept identical to that o f a reference, while the external temperature is raised or lowered. The energy that is applied to the sample or released from the sample to keep the sample isotherm al with the reference is measured. Because heat capac­ ity changes during a phase transition, DSC can be used to quantify the energy of the transition as well as the temperature of the transition. d if f e r e n t i a l t h e r m a l a n a l y s i s ( D T A ) — A technique that measures a change in tem perature in a sam ple undergoing a phase transition. In DTA, the sample temperature is com pared with that of a reference while the system is cooled or warmed. During a phase transition, the temperature of the sample rises (exothermic) or falls (endotherm ic) compared with that o f the reference. DTA can be used to determ ine the tem perature of a phase transition. d y n a m ic m e c h a n i c a l a n a l y s i s ( D M A ) — A technique that measures the viscoelas­ tic properties o f a substance. A substance is given an oscillating (dynamic) de­ forming stress (stretching, bending, or pressure), and the work done by deform ing the object is m easured (mechanical analysis) as an elastic or storage modulus. Elastic properties are dependent on the m olecular properties o f the material. For exam ple, crystalline structures have larger moduli (they do not deform easily), and am orphous polymers have smaller moduli. When the sub­ stance is restored to its original shape (relaxed), energy is usually dissipated as heat. The loss m odulus (also m easured by DM A) describes the energy loss, and Tan 8 is a function that compares the storage modulus with the loss modulus. Tan 8 peaks at the glass transition temperature, at which the storage modulus is great relative to the loss m odulus. A related property, strength, is the stress needed to break a material. In most cases, increased strength is due to increased crystallization. e l e c t r o l y t e c o n d u c t i v i t y t e s t — Loss of electrolytes from cells following an injuri­ ous stress due to a loss of the semipermeability properties o f the membranes. e l e c t r o l y t e l e a k a g e — A technique used to determ ine cell or tissue viability by es­ timating membrane integrity. Following a stress, the tissue is shaken in a given quantity of w ater for a predeterm ined period, and the electrical conductivity of the effusion is determ ined. The tissue is then killed, either in liquid nitrogen or by heat, shaken for a given time, and the final conductivity determined. Viabil­ ity is assessed either at the stress level that results in a 50% loss in electrolytes (relative to that o f killed tissue) or at the m idpoint between the maximum and minimum loss of electrolytes.

354

G lo s s a r y

e v a p o r a t i v e c o o l i n g — Cooling

due to the vaporization o f a liquid. In the context of cold hardiness, it is the reduction in temperature of biological m atter due to the loss o f water to the atmosphere, e x t r a c e l l u l a r f r e e z i n g — The crystallization o f apoplastic water, i.e., water not con­ tained within the cytoplasm o f living cells, e x t r a o r g a n o r e x t r a t i s s u e f r e e z i n g — M echanism o f freezing tolerance in which water is translocated from supercooled tissues or organs to nucleation centers in adjacent tissues (extratissue freezing) or outside the organs (extraorgan freez­ ing). f i r s t - o r d e r t r a n s i t i o n — a phase transition in which the interm olecular interactions change abruptly within a narrow temperature range (87), where T is the transi­ tion tem perature. Ostensibly, this results in a discontinuity in reaction rates, m olecular mobilities, and the energy content of the system. First-order transi­ tions are defined because the first derivative o f chemical potential with respect to tem perature is discontinuous. An explanation follows: at the transition tem­ perature o f a first-order transition, the chemical potentials of both phases are equal (by definition). However, the change in the chemical potential with re­ spect to tem perature (holding pressure constant) (8^/87% , is different on each side o f the transition tem perature. This difference (8A|i/87')p = -A.S’m (the sub­ script m denotes “per m ole”). Thus, first-order transitions are associated with a discrete change in entropy. Equilibrium exists at the transition tem perature; thus, ASm = AHim/T * 0 (the subscript t denotes “transition”). Thus, first-order transi­ tions are also associated with a change in enthalpy (phase transition enthalpy). Since H is discontinuous at the transition temperature (AH * 0), its derivative (8 ///8 7 )p or the heat capacity (Cp) is infinite at that temperature. Differential scanning calorim etry measures heat capacity and first-order transitions are de­ picted by a peak at the transition temperature. Freezing and m elting transitions are first-order transitions, f l o o d i n g — The partial or com plete covering of an organism by water, resulting in anaerobic stress. f r e e w a t e r — W ater that can be rem oved from polym eric surfaces gravimetrically. Its properties resem ble w ater in dilute solutions. Also known as bulk water. f r e e z e a v o i d a n c e — Lack of water crystallization in- tissues or cells at subzero tem­ peratures due to the absence o f either intrinsic or extrinsic nucleators. The tissue supercools and thereby escapes injury due to the effects of ice. f r e e z e d e h y d r a t i o n — Loss of symplastic water due to the vapor pressure deficit created by ice in the extracellular spaces. The degree o f dehydration is a func­ tion of tem perature; as the tem perature decreases, freeze-dehydration increases, resulting in a concentration of the cell contents, f r e e z e d e s i c c a t i o n — See Freeze dehydration. f r e e z e t o l e r a n c e — The ability o f a cell or tissue or whole organism to tolerate the presence of ice in the apoplastic spaces, f r e e z i n g i n j u r y — Injury incurred by biological material due to the presence o f ice. f r e e z i n g n u c l e a t i o n a s s a y — Detection and quantitative assessment o f ice nuclea­ tion active substances by dispersing them in distilled water and determ ining the freezing tem peratures o f drops or other units of the sample, f r e e z i n g p o i n t — The tem perature at which freezing nucleation is initiated, f r o s t — A deposit o f one o f several forms of ice crystals as a result of sublimation o f water vapor on objects colder than 0°C.

G lo s s a ry

a d v e c t i o n f r o s t — Occurs

355

from the m ovement of large cold air masses into an area for several days resulting in severe low temperatures and often accom ­ panied by strong winds, b la c k f r o s t — A dry freeze that occurs when the dew point is low, preventing air vapor crystallization on objects and resulting in the internal freezing o f vege­ tation. h o a r f r o s t / w h i t e f r o s t — A deposit o f interlocking ice crystals formed by direct sublimation of air vapor on objects, r a d i a t i o n f r o s t — O ccurs on calm, clear nights when there is unimpeded radia­ tion from the earth resulting in strong temperature inversions. Usually occurs in the early m orning hours and is characterized by relatively mild subfreezing temperatures. f r o s t h a r d e n i n g — An increase in the freeze tolerance of a plant. Depending upon the plant species and environm ental stimulus, frost hardening can be divided into one, two, or three stages. In many species, frost hardening is restricted to the second stage. f i r s t s t a g e — Triggered by short day environment, which stimulates the produc­ tion o f a translocatable hardiness-prom oting factor. Predominantly an active metabolic process. Found in woody plants, e.g., red-osier dogwood, s e c o n d s t a g e — T riggered by low temperatures o f less than 10°C. Both meta­ bolic and physical changes are involved. Found in winter annuals and woody plants not sensitive to photoperiod, t h ir d s t a g e — Triggered by subzero temperatures. Found in hardy and some her­ baceous plants, e.g., winter cereals, that have been exposed to prolonged freezing tem peratures, resulting in physical alterations. In some cases, cryoprotective agents are excreted from the sym plast to the apoplast to protect the plasma mem brane or modify ice growth, f r o s t h e a v i n g — Partial or com plete uplifting of a surface due to expansion of ice in underlying m aterial or tissue, f r o s t p l a s m o ly s i s — Separation o f the dead protoplast from the cell wall following a lethal stress. Prim arily due to the inability of the cell to reabsorb and maintain turgor following a lethal freezing stress, f r o s t p r o t e c t i o n — M ethods o f guarding against freezing injury. Commonly refers to protection of blossom s in the spring. The term is often used interchangeably with cold protection. g e l p h a s e ( p o l a r l i p i d ) — In the context of a polar lipid, the gel phase is a m olecu­ lar crystal with up to three two-dimensional (2-D) lattices (rectangular, oblique and hexagonal), each with specific chain-packing subcells. (The 2-D hexagonal lattice should not be confused with the hexagonal phase, which is a 1-D lamellar liquid crystal.) In the rectangular and oblique lattice, the hydrocarbon chains are tightly packed in layered arrays that are held together by van der W aals forces. In the hexagonal lattice, the aliphatic chains lose some of the specific chainchain interactions, are m ore loosely packed, and have greater rotational m obil­ ity. The crystalline structure with nonspecific chain packing may be stable at higher tem peratures than the more tightly packed crystals with specific chainchain interactions. Because the van der W aals interactions that make the crystal are relatively weak, m elting of the crystal is associated with a relatively small enthalpy change. The melting tem perature is also rather low compared with other crystals. Polar lipids with shorter chain lengths and a greater proportion of

356

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unsaturated fatty acids have fewer intermolecular interactions, less stable crys­ talline structure, and low er m elting temperatures than do polar lipids with longer chain lengths and a sm aller proportion of unsaturated fatty acids. The presence of water as a dispersion medium separates the molecular species, further weak­ ening the van der W aals attraction and resulting in lower melting temperatures. The polym orphic (solid-solid) transitions between crystalline states and the solid-liquid m elt o f the crystal are first-order phase transitions. Liquid crystal­ line to gel phase transitions may occur in cells when they are dried or cooled and may be deleterious in themselves or may result in phase separations that could be deleterious. Gel can also be used to describe a soap with a lamellar hexagonal liquid crystalline structure, g la s s — A m etastable state that exists below the equilibrium crystallization tempera­ ture. Glasses, which are often called amorphous solids, are distinguished from stable solid phases in that they lack crystalline structure. Glasses are distin­ guished from supercooled liquids in that they are highly viscous (viscosities of 1015 poise or more, com pared with 10“2 poise for liquid water at 0°C). Clearly, these distinctions are som ewhat arbitrary, since the number o f defects (breaks in uniformity present in most crystals) and viscosity are relative terms. The highly viscous nature o f glasses inhibits the molecular mobility required to form the molecular bonds of the equilibrium crystalline phase. Thus, glasses are kinetically, but not therm odynam ically, stable. It is a matter of some debate whether water that is associated with m acromolecular surfaces (bound water or unfrozen water) is m etastable (and therefore, by our definition, a glass). W hile the mo­ tional characteristics o f bound water suggest extreme viscosity, thermodynamic considerations (i.e., the strength of water and molecular surface interactions) suggest that a system in which water is associated with m acrom olecular surfaces may be at a lower energy state than water associated with other water molecules (i.e., the form er situation may be the equilibrium phase), g l a s s f o r m e r s — Substances that have the ability to avoid the crystallization process when cooled below the freezing point and eventually vitrify at the glass transi­ tion tem perature. Glass formers can be defined as pure substances that form glasses (i.e., silica), or solutes that enhance the glass-forming characteristics of the solution. Crystallization is avoided by minimizing the difference between the freezing tem perature and the glass transition temperature, and by reducing the rate at which crystals grow. Solutes can modify the phase transition behavior of solvents by lowering the temperature of freezing and by increasing the strength and num ber o f solvent-solute intermolecular interactions (thereby in­ creasing the viscosity o f the solution). Glass formers in aqueous systems are hy­ drophilic. Effective aqueous glass formers are important to cryopreservation efforts because they obviate the need for cooling at extremely high rates and storage at extrem ely low temperatures. Glass formers also may be im portant for deep supercooling, since they may inhibit the lethal crystallization of water during extrem e cold. D im ethyl sulfoxide, ethylene or propylene glycol, acetamide, glycerol, and sucrose-sugar mixtures are commonly used in cryo­ preservation protocols because o f their glass-forming tendencies, g l a s s s t a b i l i t y — The rate at which a metastable glass converts to the equilibrium crystalline condition (the process of devitrification). Since this transition re­ quires m olecular redistribution, it can be likened to a transport process; thus, the tendency to m ove is described by the difference in chemical potential between

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the glass and the equilibrium phase and the ability to move is described by the conductivity (a function o f viscosity). Since viscosity is a function of the interm olecular interactions and the temperature, one can conclude that glass stability is a function of the chem ical potential difference, the nature of intermolecular bonding and the tem perature. The first two factors are functions of the concen­ tration and properties o f the glass former. Glasses become increasingly stable as the temperature is lowered below the glass transition temperature. A stable glass can remain for m illennia and an unstable glass may last for a few hours. Pres­ ently, there is little docum entation of the stability of aqueous glasses and this is a problem when trying to predict the longevity o f cryopreserved tissues, g l a s s t r a n s i t i o n — A transition involving an abrupt change in the viscosity of a sub­ stance near the glass transition tem perature ( Te), even though there is no abrupt change in the chem ical potential or enthalpy o f the substance at this tem pera­ ture. There is some debate as to whether formation of an aqueous glass repre­ sents a discontinuity in the properties of water at all, and whether glasses formed by different methods can be considered equivalent. The abrupt increase in vis­ cosity at the transition tem perature has not been explained but is a function of the chemical com position o f the solution and probably a function o f the inter­ m olecular bonding strength (£ a in the Boltzmann distribution), h a r d in e s s p r o m o t e r ( s ) — Naturally occurring substance(s) synthesized in plants that induce(s) freezing resistance. (See also translocatable hardiness promoter), h e t e r o g e n e o u s ic e n u c l e a t i o n — The formation of an ice embryo of critical size on a solid or liquid substrate, followed by free growth o f the embryo from the su­ percooled liquid or supersaturated vapor phase. The substrate serves to increase the probability of critical em bryo formation by reducing the number of mole­ cules needed for critical size, and by the interfacial energy between the substrate and ice. The tem perature or supersaturation needed for heterogeneous ice nu­ cleation is largely determ ined by the properties of the substrate, h o m e o h y d r i c p la n t — (also spelled homoiohydric). A plant that maintains a con­ stant water status independent of its environment, h o m o g e n o u s i c e n u c l e a t i o n — The formation o f a critical-size ice embryo by the random clustering o f molecules in supercooled water or in supersaturated vapor. A fter achieving the critical size, the embryo becom es thermodynamically stable and grows freely. Tem perature at which this occurs is -38°C . h y s t e r e s i s p r o t e in — See antifreeze protein. ic e e n c a s e m e n t — The partial or com plete covering o f an organism by ice, resulting in anaerobic stress. ic e n u c l e a t i o n — An event triggering the conversion of supercooled water (a meta­ stable phase) into ice (a stable phase). (See hom ogenous ice nucleation, hetero­ geneous ice nucleation, ice nucleus, ice-nucleating site.) i c e n u c l e a t i o n - a c t i v e ( I n a +, I c e +, o r I N A ) — Organisms, e.g., bacteria, fungi, and lichens, that have ice nucleation activity at temperatures slightly below 0°C (i.e., - 2 to -5°C ). i c e n u c le a t i o n a c t i v i t y ( I N A ) — A measure of the ability of a substance — either biogenic (e.g., bacteria or lichens) or nonbiogenic (e.g., silver iodide)— to cata­ lyze ice formation, thereby limiting the supercooling of water, ic e n u c le a t i o n t h r e s h o l d — See threshold temperature. i c e n u c le u s ( i c e e m b r y o ) — Technically, the icelike cluster of water molecules that serves as the origin o f crystallization. In practice, biologists also use this term

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synonym ously with ice nucleation site, extracellular freezing. in t r i n s i c i c e n u c l e a t o r — Substances within plant tissues and organs capable o f ini­ tiating heterogeneous ice nucleation. k il l i n g t e m p e r a t u r e — The temperature at which an organism cannot recover from freezing injury as m easured by a viability test. L T 50— The m inim um tem perature at which 50% of the population survives, l a m e l l a r p h a s e — Orientation o f phospholipids of m embranes with the polar head groups exposed to an aqueous environment and the acyl side chain oriented toward the center o f the phospholipid bilayer, li q u id c r y s t a l l i n e p h a s e ( p o l a r li p i d ) — A molecular crystal that has some longrange order (van der W aals), but these interactions are so weak that the crystal possesses virtually no rigidity and thus has properties o f both liquids (ability to flow) and crystals (aligned in rodlike or lamellar configuration). In the smectic phase, the m olecules align themselves in layers (the familiar bilayer construc­ tion o f m em branes, also known as La or lamellar phase). In the nematic phase, liquid crystals are not layered, but retain a parallel alignment. The type o f liquid crystal is dependent on the m olecule and the temperature. Liquid crystal forma­ tion can be induced by heating or adding water. In biology, the liquid crystalline phase usually refers to the La-lamellar phase (the native bilayer configuration of membranes); however, hexagonal phases are also considered liquid crystalline, l o w - t e m p e r a t u r e e x o t h e r m s ( L T E ) — Exotherms that appear at a lower tempera­ ture than the large exotherm that represents freezing of intracellular water. The low-tem perature exotherm s represent a small percentage o f the total water, l o w - t e m p e r a t u r e i n j u r y — Ambiguous term used for injury occurring from tem­ peratures above freezing (chilling injury) to tem peratures as low as -196°C . m e lt in g — A phase change in which a solid turns to a liquid. W hen the solid is a crystal, the reaction is endotherm ic and is a first-order transition. When the solid is a glass, the reaction is a second-order phase transition. Because there are no kinetic lim itations with the formation of nuclei and growth of crystals, the tem­ perature o f fusion represents the equilibrium condition. That is, it is independent o f the rate of cooling or warm ing or the presence of nucleators. m e lt in g p o in t d e p r e s s i o n — The decrease in melting point temperature of a solid due to the addition of solutes, m e m b r a n e f l u i d i t y — The physical state of membranes determ ined by fatty acid chain length, lipid saturation level, sterol components, and possibly proteins. Physiologically active m em branes are in a relatively fluid, flexible liquid state that changes to a solid gel-like structure at lower temperatures, m e m b r a n e p e r m e a b i l i t y — The degree to which a membrane will allow a solvent or solute to penetrate, m e ta s ta b le p h a s e — Liquid at a temperature below the melting point (supercooling), or vapor at higher vapor pressure than the saturation vapor pres­ sure (supersaturated vapor), m i n i m u m s u r v i v a l t e m p e r a t u r e — Lowest temperature above which cells either tolerate or avoid freezing and below which they are killed, n u c l e a t i n g s i t e — Specific locations on the surfaces o f substrates where heteroge­ neous nucleation is promoted, n u c le a t i o n r a t e — The rate at which subcritical embryos of the condensed phase reach critical size in hom ogeneous nucleation. The same definition can be api n t e r c e l l u l a r f r e e z i n g — See

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plied to heterogeneous nucleation if it is restricted to identical nuclei, tem perature at which hom ogeneous or heterogene­ ous nucleation takes place in a supercooled liquid, o r t h o d o x s e e d — A seed that survives almost com plete dehydration and can there­ fore be stored under low relative humidity and low temperatures for extended times. M ost agronom ically im portant grain crops produce orthodox seeds (e.g., wheat, corn, soybean, bean). It is believed that the extreme viscosity of water in desiccated seeds prevents deteriorative reactions and allows seeds to be easily cryopreserved. p h a s e — The physical nature of matter, usually referring to substances in a solid, liquid, or gaseous form . Each phase is marked by distinct thermodynamic and motional properties that are a function o f the interm olecular interactions of the substance. p h a s e d i a g r a m — A way to depict the combinations of tem perature-pressure or tem perature-concentration at which a phase is most stable. Generally, phase dia­ grams depict the equilibrium (i.e., most stable) condition, and the lines of the phase diagram represent the pressure-tem perature-concentration conditions in which the chem ical potential of the two phases is equal. However, more re­ cently, phase diagram s have been used to give tem perature and concentration relationships in which m etastable states can be found, p h a s e s e p a r a t i o n — T he process o f dispersion or demixing a solution. Phase sepa­ rations occur when im m iscible substances are dispersed without an emulsifying agent (e.g., the difference between oil and vinegar salad dressing and m ayon­ naise is the proteinaceous em ulsifier in the latter). Phase separations also occur when an aqueous solution of low molecular weight solutes freezes at tem pera­ tures above the eutectic tem perature (pure water freezes leaving behind a more concentrated liquid solution). Phase separations are believed to occur in biologi­ cal membranes, w here lipids of the same species may interact and crystallize at the freezing tem perature. This may result in the efflux o f proteins, the crystalli­ zation o f other lipid m oieties (a cooperative process), and the general disruption of the sem iperm eability of the membrane. Phase separations eventually occur in glasses (this is observed in a stale lollipop where the sugar forms crystals.). The latter two exam ples o f phase separations are believed to be important m echa­ nisms in desiccation and freezing injury. Addition of emulsifying agents and vitrification are good ways to avoid phase separations, p h a s e t r a n s i t i o n — An abrupt change in the intermolecular association o f a sub­ stance that gives rise to a change in structure or mobility. (See also freezing, melting, glass, and first- and second-order transitions.) For membranes, the temperature at which lipids adopt a hexagonally packed structure and enter a gel phase, thus losing their diffusional freedom, p it m e m b r a n e — Area o f a cell wall that traverses a pit, which in turn is an area where there is an absence of secondary cell wall. In ray cells of xylem, the pit mem brane is com posed of three layers: 1) an outer layer of electron-dense m a­ terial; 2) a m iddle layer o f primary cell wall derived from both the ray cell and adjoining vessel elem ent; and 3) an inner, am orphous layer, p la s m o ly s i s — The withdraw al of water from a cell due to a lower water potential external to the cell, resulting in contraction of the protoplasm and separation from the cell wall. p r i m a r y p r o t e in s t r u c t u r e — The number of polypeptide chains in a protein, their n u c le a t i o n t e m p e r a t u r e — The

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sequences of amino acids, and the location of inter- and intrachain disulfide bonds. p r o t e c t a n t s — M olecules added to a biological material that help to prevent the deleterious effects o f a stress. For freezing or desiccation stresses, protectants may prevent phase transitions or phase separations o f the aqueous or lipid do­ mains. Thus, protectants may be important in lowering the freezing temperature, forming glasses, acting as emulsifiers, and/or acting as plasticizers. Studies have also suggested that desiccation and freezing result in peroxidation. Protectants, in this case, would scavenge free radicals or protect molecules from free radical attack. The m ode o f action and nature of protectants may vary with different stresses; however, it is generally accepted that these m olecules prevent injury rather than repair dam ages after the stress has been removed. q u a t e r n a r y p r o t e i n s t r u c t u r e — The manner in which two or more folded polypeptide chains o f proteins are held together and oriented in space with re­ spect to one another. q u e n c h c o o l i n g — Rapid cooling of a sample by immersion into a m edium (e.g., Freon 22 or isopropane) cooled by liquid nitrogen (-196°C ) or liquid helium (-268°C ). s e c o n d - o r d e r t r a n s i t i o n — A phase transition in which there is no abrupt change in the first derivative of the chemical potential (See first-order transitions) before and after the transition tem perature. Consequently, there are no abrupt changes in entropy or enthalpy and therefore no measurable transition enthalpy. How­ ever, the second derivative o f the chemical potential [related to the heat capacity (A///A7)p] is discontinuous. This is observed as a shift in the baseline in differ­ ential scanning calorim etry thermograms. The change in heat capacity at the transition suggests an abrupt change in the internal energy and/or a change in the ability to expand and contract. Glass transition and transitions from conduc­ tivity to superconductivity are considered second order. s e c o n d a r y p r o t e i n s t r u c t u r e — The location and extent o f the two regular protein conform ations (See alpha-helix and beta-sheets), as well as irregular conform a­ tions along the polypeptide chain. s t r u c t u r a l w a t e r — A type o f bound water that is believed to be important to the structural stability of the macromolecule. This water may be an integral part of the m acrom olecule and is therefore not considered in the argum ent of whether bound w ater is a glass. s u p e r c o o l i n g — M aintenance o f the liquid state at temperatures below the freezing point of the liquid. For exam ple, water freezes at just below 0°C if nucleated with ice; how ever in the absence of active nucleators, water can remain in a m e­ tastable liquid state to -3 8 °C . Many herbaceous plants supercool in the range of - 2 to —10°C, flow er buds at - 1 0 to -40°C , and xylem ray parenchym a at - 2 0 to -5 0 °C . s u p e r c o o l i n g p o i n t ( S C P ) — The temperature at which an organism, specifically an insect or other ectotherm , begins to freeze after various amounts of supercooling. s y m p l a s t — The living part of the plant that is contained within the plasm a mem­ branes of the cells. t e m p e r a t u r e o f c r y s t a l l i z a t i o n — See supercooling point. t e r t i a r y p r o t e i n s t r u c t u r e — The three-dimensional arrangem ent of all protein at­ oms in space, w ithout regard to the relationship with neighboring molecules or

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subunits. As a rule, a protein has the unique three-dimensional arrangement de­ termined by its am ino acid sequence, t h e r m a l h y s t e r e s i s — The difference between freezing and melting temperatures, t h e r m a l h y s t e r e s i s p r o t e i n — An alternative term for antifreeze protein, t h e r m a l ly s t i m u l a t e d d e p o l a r i z a t i o n c u r r e n t ( T S D C ) — A technique that meas­ ures the electrical current generated during the thermal relaxation of a polarized substance. The technique involves polarizing a substance with an electric field at a given tem perature (usually at room temperature) and then quench cooling. The field is then turned off. During controlled heating, a current is generated when ions migrate, dipoles disorient, or trapped m olecules are released. Because the tem perature of relaxation is specific for various water-solute interactions, the technique is useful in determ ining the mobility of different pools of water in a complex system. t h e r m o g r a m — The data recorded by differential thermal analysis (DTA) and dif­ ferential scanning calorim etry (DSC). In DTA, thermograms give tem pera­ ture/change in tem perature data, and in DSC, thermograms give temperature (or time) vs. pow er data. W hen substances undergo first-order transitions, DTA and DSC therm ogram s exhibit peaks in the exothermic or endothermic directions. W hen substances undergo a second-order transition, DSC thermograms som e­ times show a change in the baseline (referring to a shift in power) but more of­ ten show endotherm ic (perhaps antemelting or cooperative transitions) and/or exothermic (devitrification) events before the first-order melting transition, t h r e s h o l d t e m p e r a t u r e — The tem perature at which the rate of heterogeneous ice nucleation at a particular ice nucleation site becomes active and results in the crystallization of water, t r a n s i t i o n e n t h a l p y — The energy required to drive a transition (endothermic) or the energy released during a transition (exothermic). Commonly denoted as A//„ the transition enthalpy is measured by integrating the area of a peak in a differ­ ential scanning calorim etry thermogram. In first-order transitions, AH, * 0, and in second-order transitions, AH, = 0. t r a n s i t i o n t e m p e r a t u r e — The tem perature at which a substance undergoes a phase change. Pure substances are identifiable by their equilibrium transition tempera­ tures. Transition tem perature is dependent on the composition of the system and the pressure. Generally, high concentrations of solutes decrease or increase the fusion or glass transition temperature. Transition temperatures for freezing and vaporization are dependent on the presence o f nuclei or surfaces to form vapor; thus, they may not represent the equilibrium temperature. Rate of cooling is im­ portant to the freezing tem perature, but it is not a determinant (except for in­ strument thermal conductivity limitations) in the melting transition, u n d e r c o o l i n g — See supercooling. u n f r o z e n w a t e r — The portion of water that remains unfrozen at subzero tem pera­ tures. U nfrozen w ater can be detected in aqueous mixtures of most polymers, as well as some low m olecular w eight solutes, although the properties of the unfro­ zen phase may differ in the two situations. The presence o f unfrozen water in polym eric m ixtures suggests that the bound (or vicinal) water-surface interac­ tions perturb the interactions o f vicinal water with the water that is removed from the polym er surface (free water). Unfrozen water is often referred to as bound water; it may also be classified as a glass, v i t r e o u s — In glassy state; a substance that is extremely viscous but has amorphous

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characteristics. (It is not crystalline.) process by which a glass is formed. In biology, this term is usually used in the context of cryopreserving tissue when an aqueous glass is desired. To vitrify a substance, it must be cooled rapidly enough to temperatures well below the glass transition so that crystallization is inhibited. The required rate of cooling depends on the chemical composition o f the substance (concentration o f glass formers, water content for biological materials) and the size o f the substance. For a hydrated unprotected substance, required cooling rates may be in excess of 20,000°C/second. With some dehydration and with high concentration of protectants, the cooling rate required to vitrify tissue may be dram atically reduced.

v i t r i f i c a t i o n — The

In d e x

Abies firm a, 183 abscisic acid (A B A ), 121, 122, 124 -1 2 5 acclim ation, 116, 121, 165, 176, 178, 194, 201, 227 acclim ation cycle, 193-194 acid fuchsin dye, 190 adipokinetic horm one, 206 Adorybiotus coronifer, 212 aerial dispersal, 52 Alaskozetes antarcticus, 204 aliphatic alcohol, 25 Alnus tenuifolia, 31 alpha-helices, 103 alpine soils, 344 am ino acids, 25 anaerobiosis, 257 antagonistic organism s, 145 A ntarctic, 206 Antheraea polyphemus, 211 antibiosis, 2 4 4 -2 4 5 , 253 antibodies, 75, 9 1 -9 2 antifreeze proteins, 75, 122, 129, 191, 202, 2 0 4 205, 332, 333 aphids, 208 apoplast, 143, 144, 148, 163, 169 apoproteins, 213 apple, 165, 168, 178, 195 apricot, 195, 196 Arabidopsis thaliana, 124, 126 arabinosyl, 121 artificial snow , 339 assay for ice nuclei, 11, 65, 153, 277 atm ospheric aerosol, 19 atm ospheric ice nuclei, 22, 29, 36, 44 azalea flow er buds, 115 azosulfam ide, 190 b acteria ( See also individual species) antagonism , 125, 145, 245, 246, 247, 249 antibiosis, 2 4 4 -2 4 5

as biological insecticide, 206 cell viability, 342 com m unities, 45 ecology, 4 1 -5 7 epiphytic, 137 g u t flora, 233 phyllosphere, 4 4 -4 5 raindrop m om entum , 51 B acterial Ice N ucléation D iagnostic (BIN D ), 284 bacterial ice nuclei. (See also ice nucléation proteins) assay for ice nuclei, 65 assem bly and turnover, 7 5 -7 8 biochem ical characterization, 63 contribution o f dead bacteria to, 146 discovery, 29-38 ecology, 4 1 -5 7 grow th conditions, 64 grow th cycle, 338 inhibitors, 125-126 tem perature classes, 6 5 -6 8 , 76, 80, 96, 284, 338 tem perature-labile, 67 bacterial outer m em branes, 101, 247 anchoring to m em branes, 112 cell localization, 69 influence on protein structure, 112 phospholipids, 71 protein aggregation w ithin, 69, 110-113 vesicles, 70 bacterial population, 145, 241, 244, 272 com petitive exclusion, 245, 249 dynam ics, 45 m icroclim ate m odification studies, 51 size, 2 3 9 -2 5 2 bactericide, 125, 145 application frequency, 242 control o f plant frost injury, 2 4 1 -2 4 4 preventive and eradicative, 242 -2 4 3 resistance, 2 4 3 -2 4 4

363

364

In d e x

bacteriophage infection, 341 b acteriophage-resistant Ina, 341 barley, 125 bean, 47, 49, 140, 246 bean le a f beetle, 206, 258 beta irradiation, 342 beta-sheets, 103 beta-sitosterol, 122 Betula lenta, 171 Betula papyrifera, 171 bim olecular interaction, 113 biological control, 206, 253 advantages and disadvantages, 2 6 6 -2 6 7 C olorado potato beetle, 2 6 3 -2 6 6 field efficacy, 249 o f frost dam age, 244 o f insect pests, 2 5 7 -2 6 7 legal challenges, 251 o f stored.product pests, 2 6 1 -2 6 3 biological insecticides, 206, 257 black body radiation, 116 black locust, 124 black raspberry, 188 blood osm olality, 227 blueberry, 188, 192 body size, 2 2 8 -2 2 9 Bolitophagus reticulatus, 206, 210 box turtles, 222, 233 Bracon cephi, 203, 204, 211 Brassica napus , 126 brom egrass, 121, 125 Bromus inermis, 126 bulk w ater, 10 bullfrog, 228 Buxus microphylla, 147 cabbage, 123 calcium phosphate, 207 calorim eter, 14 cam pestrol, 122 canola, 122, 124 Carya ovata, 166, 169 cavitation, 119, 168 cD N A clones, 124

Celastrus arbriculatus, 192 cell death, 317 cell w all, 121 m icrocapillaries, 190 porosity, 171 structure and d eep supercooling, 169 cellular dehydration, 257, 316, 320 cellulase, 175 centipede (Lithobius forficatus), 204, 210 Ceratoma trifurcata, 206, 258 Ceruchus piceus, 206, 213, 215 chaperonins, 129 ch aracteristic tem perature, definition, 11 characteristics o f effective freezing nuclei, 23 characteristics o f fungal ice nucleation sites, 154-155

chem ical factors, 22 chem ical potential, 117 cherry, 125, 188, 195 Chilo suppressalis, 154, 259 Chlorella, 123 chloroplasts, 123 chorus frog, 222, 231 Chymomyza costata, 257 Chrysemyspicta, 221, 223 Cisseps fulvicollis, 210 Citrobacter, 291 cloud cham ber experim ents, 8 cloud seeding, 25, 37, 3 4 4 -3 4 5 cold acclim ation, 116, 124, 165, 176, 17 8 ,2 2 7 cold shock, 257 cold-labile enzym e, 129 cold-regulated genes and proteins, 124, 126 C ollem bola, 206 colligative, antifreeze, 2 0 2 -2 0 4 , 205 properties, 163 C olorado potato b eetle (Leptinotarsa decemlineata), 2 6 3 -2 6 6 com m ercial application, 239, 257, 271, 283, 299, 3 1 5 ,3 3 7 detection o f food pathogens, 2 8 3 -2 9 6 freeze-crystallization applications, 346 spray-ice technology, 337 use o f com petitive bacteria, 252 com petitive exclusion, 245, 249 condensation freezing, 6, 19 contact freezing nucleation, 6, 19 convergent lady beetle ( Hippodamia convergens), 206, 258 cooling rate, 196, 225, 315, 319 copper com pounds, 241 com , 32, 47, 129, 247 Com us florida, 171 costs o f registration o f a b iological pesticide, 252 cotton, 140 covellite, 24 crane fly ( Tipula trivittata), 210 critical em bryo size, 5 ■ critical size, 2 cryobiology, 317 cryom icroscopy, 258 cryopreservation, 211, 3 1 5 -3 3 4 by vitrification, 3 2 4 -3 3 0 m am m alian organs by vitrification, 326, 333 plant germ plasm , 149 tw o-step freezing, 322 cryoprotectants, 203, 232, 328, 333 cryoprotective effect, 123 cryoprotective proteins, 129 cryoultram icrotom y, 332 Cryptolestes ferrugineus, 261 Cryptopygus antarcticus, 206

Cucurbita maxima, A1 cum ulative freezing distributions, 264 cum ulative incidence o f nucleation, 326

In d e x

cupric iodide, 24 currant, 188 cyanobacteria, 151, 152 cyclohexim ide, 124, 176 cytorrhysis, 164 D 20 , 65, 75 deacclim ation, 191 deep supercooling. ( See also p lan t cold­ hardiness, supercooling) acclim ation cycle o f buds, 1 9 3 -1 9 4 buds, 183, 188-189 distribution o f plants, 168, 194 -1 9 5 m echanism in dorm ant buds, 189-193 tissue injury, 166 xylem parenchym a cells, 163 dehydration, 1 6 8-169, 178, 194 dehydrin, 124 Dendroides canadensis , 205, 206, 207 deposition nucleation, 6, 19 depression o f freezing point, 163 desiccation, 169, 170, 230 detection o f freezing, 11 devitrification, 332 diam ondback m oth (Plutella xylostella ), 258 diazines, 25 differential scanning calorim etry, 14, 164 differential therm al analysis (D T A ), 14, 164, 195. {See also therm al analysis) dim ethyl sulfoxide, 203, 320 distribution o f ice, 144 dose-response curve, 278 drop-freezing assay, 11, 13, 65, 153, 277 evaluation of, 15 D TA . See differential therm al analysis earthw orm , 202 ectotherm s, 221 electron m icrographs, 346 electroporation, 129 Eleodes blanchardi, 204, 205, 211 em bryos, ice, 2, 23, 111, 213, 214 em ulsions, 14 endogenous ice nucleators, 208 endotherm s, 235 Enterobacter agglomerans, 206, 233, 258, 259 Enterobacter taylorae, 44, 206, 258 E nvironm ental Protection A gency, 251 equilibrium freezing point, 118, 202, 226 Erwinia, 278, 299 Erwinia amylovora, 241, 245, 252 Erwinia ananas, 43, 85, 302 Erwinia herbicola, 43, 52, 69, 71, 74, 80, 85, 145, 206, 240, 245 Erwinia uredovora, 43 Escherichia coli, 85, 93, 283, 291 ethylene glycol, 203 Eurosta solidaginis, 207, 209, 210, 258 eutectic point, 119 evolution, 89, 156, 208, 223 o f freeze tolerance, 212

365

extensin, 122 extracellular freezing, 117, 118, 143, 167, 189, 316 extracellular ice nucleators, 203 extraorgan freezing, 149, 192 Fahrenheit, 8 fat body, 209 fatty acid unsaturation, 122 ferm entation o f Ina+ bacteria, 339 fir, 188, 190, 192 fish, 204 flow ering dogw ood, 174, 188, 192 food processing and technology anisotropic texturing w ith bacterial ice nuclei, 3 1 1 -3 1 2 applications, 299 freeze-concentration, 299 freeze-concentration o f egg w hite, 302 -3 0 6 freeze-concentration o f ju ic e , 309 -3 1 0 freeze-concentration o f m ilk, 3 06-308 freeze-drying w ith bacterial ice nuclei, 310— 311 freeze-texturing, 299 m onitoring food for pathogens, 283 forsythia, 183, 189, 191. 192 frass, 210 Fraxinus pennsylvanica, 168 freeze avoidance, 201, 2 0 7 -2 0 8 , 2 2 5 -2 2 6 freeze-concentration, 3 0 2 -3 1 0 freezing inhibitors, 121 freezing injury cells, 315 discovery, plant bacterial, 3 2 -3 8 freeze-induced dehydration injury plant, 1 17118 m em brane, 118 freezing nucleation. (See also ice nucleation) characteristics, 23 definition, 6 historical survey o f experim ents, 8 m acroscopic factors, 19 freezing point, 124 hysteretic, 202 isotherm , 320 m olal, 202 nonequilibrium , 204 o f cytoplasm , 316 freezing tolerance extraorgan freezing, 149, 192 freezing at a high tem perature, 209 insects, 257 invertebrates, 201 plants, 115-117. (See also plant cold­ hardiness) role o f bacteria, 55 vertebrates, 221, 225, 232 freezing-point depression, 346 freezing-point osm om eter, 330 frost control, 2 3 9 -2 4 1 , 242, 244, 250

36$

In d e x

field efficacy o f biological control, 249 frost injury, 3 2 -3 5 , 115, 2 3 9 -2 4 3 , 249, 250 frost-sensitive plants, 239, 240, 242 fructose, 123, 204 fruit trees, 47, 165. ( See also individual species) fungal ice nucléation, 151 potential applications, 157 proteinaceous ice n u cléatio n sites, 155 selective advantages, 156-157 fungi. ( See Fusarium ) Fusarium, 137, 151, 154, 157, 258 Fusarium acuminatum, 154, 259, 261 Fusarium avenaceum, 154, 261 gam m a irradiation inactivation analysis, 155 g arter snake ( Thamnophis sirtalis), 223 gastropod, 214 g earbox-type prom oters, 338 glass, 123 glass form ation, 119, 120 glass transition tem perature (7g), 119, 322, 324 glucose, 123, 203 glycerol, 203, 208, 257 Glycine max, 47, 140, 177 g lycoprotein antifreeze. See antifreeze protein g lycosylation, 338 g oldenrod gall fly (Eurosta solidaginis), 207, 209, 210, 258 g rape, 183, 195, 196 gut evacuation, 206 Gynaephora groenlandica, 210 hardiness. See p lan t cold-hardiness hardw oods, 168 heat o f fusion, 138, 140, 224, 320 heat-shock protein, 129 heat-stable proteins, 124 hem icellulase, 175 hem olym ph, 203 heptane, 14 heterogeneous ice nucléation, 1, 66, 118, 137, 145, 163, 169, 201, 203, 316 b iological system s, 5 cryoprotectant solutions, 325 definition, 3 electric effects, 23 m odes of, 5 volum e dependence, 19 heterogeneous n ucléation tem perature ( 7 h e t ), 325 hibem aculum , 210, 221 high-tem perature ex o th erm (H TE ), 164, 183, 188 Hippodamia convergens, 206, 258, 261, 266 hom ogeneous ice nucléation, 1, 4 - 5 , 118, 125, 137, 163, 190, 201, 203, 205, 318, 319, 327 cryoprotectant solutions, 326 definition, 3 vitrified solutions, 3 2 9 -3 3 0 volum e dependence, 19 hom ogeneous ice n ucléation tem perature (Th),

118, 324

Hordeum vulgare, 126 hydration state, 2 2 9 -2 3 0 hydrostatic pressure, 318 hydrostatic tension, 169 hydroxyl groups, 213 hydroxyproline, 121 Hyla versicolor, 222 hysteretic freezing point, 202 ice c-axis, 332 em bryos, 2, 23, 1 1 1 ,2 1 3 ,2 1 4 Ic (cubic), 105 Ih (hexagonal), 105 ice (deletion m utant), 54, 56, 2 4 7 -2 5 2 ice crystallization tem perature (Tc), 224. (See also supercooling point) ice m inus strains survival, 248 ice nucleation activity, 149, 203 associated w ith plants, 145 disruption by chem ical agents and enzym es, 72 d roplet assay for, 11, 65, 153, 277 ice nucleation frequency, 63 ice nucleation gene. (See also ina) advantages and disadvantages o f reporters, 2 7 8 -2 7 9 construction o f gene and protein fusions, 274 evolutionary relationships, 8 9 -9 0 expression, 9 3 -9 4 horizontal dissem ination, 86 physiological regulation, 81 repeated m otifs, 86 sequences, 8 5-91 ice nucleation plant, 115 citrus, 148 cooling rate, 142 ice nucleation sites, 144, 149 ice propagation, 191 intrinsic ice nucleators, 125, 130, 145, 146, 249 intrinsic nucleation sites, 137, 147-149, 189 leaf-derived nuclei, 31 leaves, 147-148 m oisture, 142 nucleation sites, epiphytic bacteria, 137 ray parenchym a and pitch cells, 120 specim en size, 139, 140 w oody stem tissues, 146 ice nucleation protein, 258. ( See also bacterial ice nuclei) anchoring to m em branes, 112 expression, 93 identification, 9 1 -9 2 im m unology, 69 kinetics o f assem bly, 96 lattice-m atch, 110 m odels of, 79, 104 oxidation, 342 plants, 130

In d e x proteolysis, 77 purification and characterization, 78, 92 repetitive dom ain, 102, 107 requirem ent o f m em brane phospholipids, 71 sequence, 85, 9 1 -9 2 ice nucleation spectrum , 205, 264. (See also droplet-freezing assay) ice nucleation tem perature, 139 ice n ucleation-active com ponents, 148 ice n ucleation-active sites, 9 3 -9 4 , 141, 143 ice nucleator, 66, 115 in blood, 233 insect gut, 206 ice ponds, 3 4 4 -3 4 5 Ice+ phenotype, 41, 5 3 -5 7 , 72, 85, 86, 233, 2 3 9 253 im m ersion freezing, 6, 10 im m unology, 69, 213 ina. (See also ice nucleation gene) gene fusions, 274 ina-transduction assays, 284, 287, 289, 292, 294 detection lim its, 292 inaZ transform ants, 69, 130 inhibition o f ice nuclei, 75 inoculative freezing, 142, 143, 201, 202, 210, 230, 233, 257 inositol, 212, 338 insect, 10, 204, 257 insect gut, 258 insertional m utagenesis, 274 intracellular freezing, 116, 118, 209 intracellular ice, 143, 189 intracellular nucleation, 320 intracellular nucleators, 316 intracellular supercooling, 318 intrinsic ice nucleation. See ice n ucleation plant Iphthimus laevissimus, 205 Ips acuminatus , 202 irreversible chem isorption, 22 isogenic ice m utant strain, 252 Jerusalem artichoke, 122 ju niper, 188 ju v en ile horm one, 206 kaolinite, 24

tem perature.stability, 155 threshold tem peratures for freezing, 153 lipoprotein ice nucleator (LPIN ), 205, 207, 212 258 chain-form ing behavior, 212, 214 liposom es, 122, 213 Listeria monocytogenes, 283, 285, 294 Lithobius forficatus, 204, 210 Lobelia telekii, 56, 125, 147 lognorm al frequency distribution, 45 low nucleation tem peratures, 208 low -tem perature conditioning, 6 8 -6 9 low -tem perature exotherm (LT E), 164, 166-167, 175-176, 183, 188, 189, 191, 193 LT50, 120 Lycopersicon esculentum, 47, 129, 140, 141, 249 m acerase, 174 m aize, 140 M alpighian tubules, 207, 212, 258 Malus pumila, 165 Manduca sexta, 215 m annitol, 123, 204, 339 m annose, 338 m arker exchange, 276 m arker-exchange m utagenesis, 248 m ealw orm larvae (Tenebrio molitor), 258 Megachile rotundata, 205 Melampus bidentatus, 214 m elting point, 1, 190, 194 depression, 203, 204 m elting point, therm al hysteresis, 204 m em brane injury during freezing, 118 m em ory effect, preconditioning, 22 m ercuric iodide, 24 m etaldehyde, 25 m etastable states, 1, 118, 120 m inerals, 24 m ite, 204 m odels o f Ina proteins, 7 9 -8 0 , 104 m olal freezing point, 202 m onitoring food sam ples for pathogens, 283 m ountain spiny lizard (Sceloporus jarrovi), 225 m ussel (Mytilus edulis), 204, 212 m utualistic relationship betw een fungus and host, 259 Mytilus edulis, 204, 212

Lacerta vivipara, 232 lactate, 204 lam ellar-to-hexagonal II phase transition, 118 L anthanum nitrate, 171 larch, 188, 190, 195 lead iodide, 24 Lecanora dispersa, 153 Leptinotarsa decemlineata, 263—266 lethal low tem perature, 257 lettuce, 192 lichen, 56, 140, 153 lichen ice nucleation sites pH, 156

367

natural therm al storage, 344-345

Nicotiana tabacum, 124, 130 n onequilibrium freezing, 117, 204

Nostoc, 151 n uclear m agnetic resonance, 164 n ucleating site, definition, 5 nucleation, definition, I n ucleation frequency, 146 nucleation rate, 3 orange, 249 organ v itrification, 326, 333

368

In d e x

Oryzaephilus surinamensis, 261 123, 209, 227 o s m o t i c p r e s s u r e , 316 o s m o t i n , 125 o x a l i c a c i d , 176 O x y t e t r a c y c l in e , 241 o s m o tic e f fe c t,

painted turtles (Chrysemys picta), 221, 233 pea (Pisum sativum), 47, 147 peach, 125, 140, 174, 178, 1 83-196 pear, 195, 249 pectinase, 175, 177 PEG, 203 Peltigera, 152 pesticide, 252 phase change, 1 Phaseolus vulgaris, 140 phloroglucinol, 25 phosphatidylethanolam ine, 71 phosphatidylinositol, 72, 76, 212, 338 phospholipase, 118, 213 phospholipids, 71, 122 phyllosphere, 44-^15 phytopathogenic host range, 53 Pisum sativum, 4 7 , 147 pit m em brane, 1 7 3 -177. ( See also deep supercooling) plant cold-hardiness. ( See also deep supercooling, ice n u cleation plant) abscisic acid (A B A ), 1 2 1-122, 124-125 freeze-induced d ehydration injury, 117 freezing injury, 3 2 -3 8 , 116 freezing pattern in do rm an t buds, 189 frost-sensitive plants, 239, 240, 242 hardiness categories o f plants, 120 ice distribution, 144 ice form ation, 140, 143 intracellular freezing, 116-117 m olecular biology o f cold acclim ation, 126 polysaccharides, 121, 125 rate o f ice pro p ag atio n , 117 tissue supercooling, 145, 253 plasm a m em brane, 122, 209 Plutella xylostella, 258 Podarcis muralis, 231 polyols, 202, 257 polypeptides, bo ilin g -stab le, 129 p olysaccharides, 121, 125 poplar, 195 Populus, 119 pore size, 169-171 pore structure, 190

Porphyra, 'i'll potato, 249, 250, 121 proline, 204 protein, 63, 85. ( See also antifreeze protein, ice n ucleation protein) algorithm s for predicting secondary structure, 103 antifreeze p ro tein s, 75, 122, 129, 191, 202,

2 0 4 -2 0 5 , 332 apoproteins, 213 cold-regulated, 126 heat-shock, 129 heat-stable, 124 hydrophilic, 120, 124 repetitive dom ain, protein, 102, 107 requirem ent o f m em brane phospholipids, 71 sequence, 85, 9 1 -9 2 tertiary structures of, 101 proteoliposom es, 213 proteolysis, 77 Prunus, 117, 125, 192 Prunus besseyi, 188 Prunus maaki, 195 Prunus padus, 191, 195 Prunus pennsylvanica, 188, 195 Prunus persica, 171 Prunus serotina, 188, 191 Prunus virginiana, 191 Pseudomonas chlororaphis, 55 Pseudomonas fluorescens, 43, 47, 85, 92, 206, 233, 245, 248, 259, 266, 284 Pseudomonas putida, 44, 233, 259, 266 Pseudomonas syringae, 32, 35, 4 3 -5 6 , 6 3 -7 0 , 74, 80, 8 5 ,9 2 , 94, 130, 145 Pseudomonas viridiflava, 43, 70, 85 Pseudopleuronectes americanus, 211, 235 pum pkin ( Cucurbita maxima), 47 PVP, 203 Pytho deplanatus, 211 radiation frost, 116 raffm ose, 119, 123 raindrop m om entum , 51 rate, o f ice propagation plant, 117 o f solidification, 11 recom binant bacteria, 69, 250-251 R ecom binant D N A A dvisofy C om m ittee, N ational Institutes o f H ealth, 250-251 recom binant ice nucleation strains, 251 recrystallization, 333 red ash (Fraxinus pennsylvanica), 168 red o sier dogw ood, 195 red raspberry, 188 repeated m otifs, 86 repetitive dom ain o f protein, 102, 107 reporter genes, 277, 283 advantages and disadvantages, 2 7 8 -2 7 9 dose.response curve, 271 properties o f ina genes, 272, 274 use in biological research, 271 reverse osm osis m achines, 345 Rhabdophaga strobiloides, 208 Rhagium inquisitor, 205 Rhizoplaca chrysoleuca, 151, 153-157 Rhododendron japonicum, 183, 189, 190, 192, 193, 194 Rhododendron kosterianum, 190

In d e x

Rhododendron mollis, 183, 192 Rhododendron roseum, 192 Rhyzopertha dominica, 262 rice stem borer (Chilo suppressalis), 154, 259 ring porous xylem , 168 rusty grain b eetle (Cryptolestes ferugineus), 261 rye (Secale cereale), 120, 122, 147-148

Salix babylonica, 165, 171 Salmonella, detection of, 2 8 3 -2 9 6 salt, 258 scanning differential calorim eters, 195-196 scanning electron m icroscopy, 164, 189 scanning tunneling m icroscopy, 215 sea w ater, 345

Sceloporus jarrovi, 225 Secale cereale, 120, 122, 147 -1 4 8 seed crystal, 205 shagbark hickory (Carya ovata), 166, 169 silicone oils, 14 silver iodide, 9, 22, 2 3 -2 4 Sitophilus granarius, 261 SN O M A X snow inducer, 318, 339 snow m aking, 3 4 2 -3 4 4 sodium chloride, 201 Solanum, 120, 124 Solanum acaule, 117, 121, 122 Solanum cardiophyllum, 120 Solanum commersonii, 120, 124, 126, 130 Solanum tuberosum, 121, 263 solute, 203 solution effects, 320, 321 sorbitan tristearate, 14 sorbitol, 123, 257 soybean (Glycine max), 47, 140, 177 Sphenomorphus tympanum, 232 spiders, 204spinach, 123, 124 spray-ice construction, 346, 348 technology, 337 stachyose, 119, 123 stag beetle (Ceruchus piceus), 206, 213, 215 steroids, 25 stigm asterol, 122 stom ata, 143, 144 straw berry, 56 streptom ycin, 241, 243 sucrose, 119, 123, 203 supercooling, 201, 3 1 7 -3 1 9 . (See also deep supercooling) capacity, 2 2 6 -2 3 2 plant, 116, 118, 240 size, 257 stochastic and tem poral factors, 2 3 1 -2 3 2 vertebrates, 223 w ater, 1 ,8 , 163 supercooling point, 206, 2 23, 257 supercooling point depression, 203, 204 supersaturated cell solutions, 119

369

supersaturated sugar, 119 supersaturated vapor, 1 surface disinfectants, 145 surfactants, 266 sw eet cherry, 192, 195 sym plast, 169 sym plastic w ater, 163 tardigrade (Adorybiotus coronifer), 212 target theory, 67 tem perature classes bacterial ice nuclei, 6 5 -6 8 , 76, 80, 284 fungi, 151 ice nucleation sites, 338 tem perature o f crystallization, 224, 257. (See also supercooling point) tem perature w indow , 240 Tenebrio molitor, 258 tension stressed, 119 tertiary structures o f proteins, 101 Thamnophis sirtalis, 223 therm al acclim ation, 116, 227 therm al analysis, 153, 183, 195-196. (See also differential therm al analysis, plant cold­ hardiness) m easurem ent o f bud hardiness, 195-196 therm al hysteresis, 204 therm al im aging, 139 therm ocouple, 195 therm ogram , 164 therm opile, 195 threshold tem perature, 75, 81 definition, 5 for freezing in lichens, 153 size-dependence of, 93 thylakoids, 124 tim e dependence, 20 tim e lag, 22 Tipula trivittata, 205, 212 tobacco (Nicotiana tabacum), 124, 130 tom ato (Lycopersicon esculentum ), 47, 129, 140, 1 4 1 ,2 4 9 transgenic plants, 130 transposons, 97, 274 trap crop, 266 Trebouxia, 151 trehalose, 119, 204, 257 Trichiocampus populi, 258 Triticum aestivum, 117, 123, 124, 126, 191 turtle hatchlings, 229 T w een 80, 266 tw o-step freezing, 322

Uloma impressa, 207 ultraviolet lig h t-in sen sitiv ity , 342 undercooling, 163, 317. (See also supercooling) urea, 25, 203 Usnea, 153 vapor pressure deficit, 169

370

In d e x

equilibrium , 209 gradient, 169 ice, 316 vaterite, 24 vectors, 274 Veronica persica, 147 Vespula maculata, 212 Vibrio fischeri, 283 Vicia faba, 147 Vicia saliva, 147 v itrification, 324, 326 cells, 315 cryopreservation, 315 volum e dependence, 19 w all lizard ( Podarcis muralis), 231 water, bulk, 10 conservation, 209 desalination, 345

m etastable, 120 m igration, 192, 194 purification, 345 w ater activity, 316 w ater potential, 231 w ater vapor, supersaturated, 1 w ater-binding tem plates, 79 w eather m odification, 346 w heat (Triticum aestivum), 117, 123, 124, 126, 191 w heat stem saw fly (Braetin cephi), 203, 204, 211 w illow , 195 w inter flounder (Pseudopleuronectes americanus), 211, 235 w inter rye, 120, 122

Xanthomonas campestris, 44, 55, 85, 299, 302 xylem , 115, 120, 144, 163, 165, 168, 171, 188, 191, 193