Synthetic and Degradative Processes in Marine Macrophytes: Proceedings of a Conference held at Bamfield Marine Station Bamfield, Vancouver Island, British Columbia May 16–18, 1980 9783110837988, 9783110084900

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Synthetic and Degradative Processes in Marine Macrophytes

Synthetic and Degradative Processes in Marine Macrophytes Proceedings of a Conference held at Bamfield Marine Station Bamfield, Vancouver Island British Columbia May 16-18,1980 Editor L. M. Srivastava

W DE G_ Walter de Gruyter • Berlin • New York 1982

Editor Lalit M. Srivastava Department of Biological Sciences Simon Fräser University, Burnaby, B. C„ V5A 1S6 Canada

CIP-Kurztitelaufnahme

der Deutschen

Bibliothek

Synthetic and degradative processes in marine macrophytes : proceedings of a conference, held at Bamfield Marine Station, Bamfield, Vancouver Island, British Columbia, May 16-18,1980/ ed. L. M. Srivastava. - Berlin ; New York : de Gruyter, 1982 ISBN 3-11-008490-2 NE: Srivastava, Lalit M. [Hrsg.]; Bamfield Marine Station

Library of Congress Cataloging in Publication

Data

Main entry under title: Synthetic and degradative processes in marine macrophytes. Includes bibliographical references and index. 1. Marine algae-Congresses. 2. Marine algae culture-Congresses. 3. Kelp-Congresses. 4. Marine algae-British Columbia-Congresses. I. Srivastava, L. M. (Lalit Mohan), 1932QK570.2.S96 639 81-19513 ISBN 3-11-008490-2 AACR2

Copyright © 1982 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Kupijai & Prochnow, Berlin. - Binding: Dieter Mikolai, Berlin. Printed in Germany.

CONTENTS List of c o n t r i b u t o r s

VII

Preface

XI

Opening Remarks R.F. Scagel Part I .

1 S t r a t e g i e s of growth and reproduction

Physiology of carbon f i x a t i o n in benthic marine a l g a e . J . Willenbrink

7

Seasonal p a t t e r n s of carbon a s s i m i l a t i o n and u t i l i z a t i o n by kelps. K.H. Mann, A.R.O. Chapman and J . Gagne Ehgelmann's theory:

23

The compelling l o g i c .

J . Ramus

29

Seasonality in l a r g e r brown algae and i t s possible regulation by the environment. K. Luning

47 Part I I .

Nutrition and c u l t u r e

Inorganic n u t r i t i o n of marine macro-algae in c u l t u r e . J . Mslachlan Nutrient uptake and growth in the Laminariales and other macrophytes: A consideration of methods. P . J . Harrison and L.D. Druehl

71

99

Nitrogen n u t r i t i o n of Macrocystis. W.N. Wheeler

121

Uptake of inorganic ions and t h e i r long distance t r a n s p o r t in Fucales and l a m i n a r i a l e s . J.Y. Floc'h

139

Translocation of organic compounds in l a m i n a r i a l e s . K. Schnitz

167

VI Part III. Marine macrophytes in coastal ecosystems The regulation of macroalgal associations in kelp forests. M.S. Foster

185

Holes for detritus in complementing productivity of coastal systems. J.R. Sibert

207

Degradation of the kelps Macrocystis integrifolia and Nereocystis luetkeana in British Golunbia coastal waters. L.J. Albright, J. Chocair, K. Masuda and M. Valdes

215

Part IV. Polysaccharides, kelp farming and harvest Toward improved understanding of polysaccharide synthesis and storage in marine algae. R.G.S. Bidwell

237

Fanning Macrocystis at coastal and oceanic sites. W.J. North, V. Gerard and J. Kuwabara

247

Macrocystis harvest strategy in British Golunbia. L.M. Goon Subject index

265 283

LIST OF CONTRIBUTORS Asterisk indicates author of a paper in this volume. *L. ALBRIGHT, Department of Biological Sciences, Simon Fraser University, Burnaby, B.C. V5A 1S6 CANADA *R.G.S. BIDWELL, Wallace, R.R. #1, N.S. BOK 1Y0 CANADA E. CABOT, Bamfield Marine Station, Bamfield, B.C. VOR 1B0 *L.M. COON, Marine Resources Branch, Parliament Building, Victoria, B.C. V8V 1X5 CANADA *L. DRUEHL, Deptartment of Biological Sciences, Simon Fraser University, Burnaby, B.C. V5A 1S6 CANADA M.J. DUNCAN, Botany Department, University of British Columbia, Vancouver, B.C. V6T 1W5 CANADA *J.Y. FDX'H, laboratoire de Physiologie ysgétalé, Faculté des Sciences, Université'de Bretagne Occidentale, F-29283-BREST. Cedex, FRANCE R. FOREMAN, Bamfield Marine Station, Bamfield, B.C. VOR 1B0 CANADA *M.S. FOSTER,

MDSS

Landing Marine Laboratory, P.O. Box 223, ffoss Landing,

CA 95039 USA *V.A. GERARD, Caltech Kerchkoff Marine Laboratory, 101 Dahlia St., Corona Del Mar, CA 92625 USA *P.J. HARRISON, Department of Oceanography, University of British Columbia, Vancouver, B.C. V6T 1W5 CANADA C.S. LOBBAN, Biology Department, University of New Brunswick, P.O. Box 5050, St. John, N.B. E2L 4L5 CANADA *K. LUNING, Biologische Anstalt Helgoland, Zentrale, Palmaille 9, 2 Hamburg 50 WEST GERMANY

VIII *K.H. MANN, Bedford I n s t i t u t e of Oceanography, Box 1006, Dartmouth, N.S. B2Y 4A2 CANADA T. IVfcCCNNAUGHEY, Department of Oceanography, U n i v e r s i t y of Washington, S e a t t l e , WA 98195 USA *J. McLACHLAN, NRC A t l a n t i c Regional Laboratory, 1411 Oxford S t . , Halifax, N.S. B3H 3Z1 CANADA T.F. MUMFQRD, J r . , D i v i s i o n of Marine Land Management, Department of Natural Resources, Olympia, WA 98504 USA *W.J. NORTH, Cal tech Kerchkoff Marine Laboratory, 101 Dahlia S t . ,

Corona

Del Mar, CA 92625 USA *J. RAMUS, Duke u n i v e r s i t y Marine L a b o r a t o r y , P i v e r s I s l a n d ,

Beaufort,

N.C. 28516 USA R. SCAGEL, Botany Department, University of B r i t i s h Golumbia, Vancouver, B.C. V6T 1W5 CANADA *K. SCHMITZ, Botanisches I n s t i t u t der U n i v e r s i t ä t Köln, G y r h o f s t r a s s e 15, 5 Köln 41 (Lindenthal) WEST GERMANY * J . SIBERT, Department of F i s h e r i e s and Oceans, B i o l o g i c a l

Research

S t a t i o n , Nanaimo, B.C. V9R 5K6 CANADA L.M. SRIVASTAVA, Department of B i o l o g i c a l

Sciences,

Simon

Fraser

University, Burnaby, B.C. V5A 1S6 CANADA R. WAALAND, Botany Department, U n i v e r s i t y of Washington, S e a t t l e , WA 98195 USA *W.N. WHEELER, Department of Biological Sciences, Simon Fraser University, Burnaby, B.C. V5A 1S6 CANADA J.N.C. WHYTE, F i s h e r i e s and Marine S e r v i c e , 6640 N.W. Marine D r i v e , Vancouver, B.C. V6T 1S6 CANADA

IX *J. WILLENBRINK, Botanisches Institut der Uliver si tat Köln, Gyrhofstrasse 15, 5 Köln 41 (Lindenthai) WEST GERMANY OTHER PARTICIPANTS

C. Bell, R. Booking, R. Boal, M. Bonettemaker, R. Bunting, B. Carowsell, M. ESbijan, I.M. German, J. Lindsay, K. Lloyd, G. Magensen, B. Pakula, L. Quain, A. Quek, K. Resell, P. Ryan, B. St. Clair, R. Saunders, M.L. Shih, M. Shivji, R. anith, S. 3nith-Pakula, G. Stalder, A. Stewart, D. Trotter, T. T\jcminen, I. \felji, S. Villeneuve

PREFACE Seaweeds a r e important s o u r c e s o f f o o d , s e v e r a l

phycocolloids,

pharmaceuticals and, more recently, biomass for energy.

Their continued

and successful commercial u t i l i z a t i o n depends on our understanding of their growth and reproduction, t h e i r s t r a t e g i e s and adaptations that enable them to exploit their environment, and their place in the coastal ecosystem of which they are such an important part. the brown

Among the seaweeds,

(Phaeophyta) and red (Rhodophyta) algae are s p e c i a l l y

important, not because they are highly v i s i b l e , but because they form the b a s i s f o r much of the seaweed industry.

Despite t h e i r importance,

however, these macrophytes, which include the "redwoods of the s e a , " are poorly studied and very few volumes have been exclusively devoted to them. The present volume is a record of the proceedings of a small conference held at the Bamfield Marine Station, Vancouver Island, British Columbia on May 1 6 - 1 8 ,

1980 on the s y n t h e t i c and degradative aspects of marine

macrophytes. Over the years, a snail but active group of researchers studying various a s p e c t s o f m a c r o p h y t e s , e s p e c i a l l y k e l p growth and p h y s i o l o g y , reproduction, chemical constituents, mariculture and degradation, has come together in B r i t i s h Columbia and i t seemed appropriate that the Bamfield Marine Station, where much of this work i s being done, be the s i t e of this f i r s t conference.

Since the board and residence f a c i l i t i e s at Bamfield

were very limited, only a small number of participants could be i n v i t e d . While this resulted in a narrowed scope for the conference, i t permitted a greater exchange of ideas on the topics that were covered than i s usually possible in l a r g e r conferences.

Also, the unhurried contact of people

from different disciplines under the idyllic setting of a fishing v i l l a g e on the P a c i f i c ocean, f a r from the madding crowd, proved to be a most valuable experience. The aim of the conference, s e t by the Organizing Committee, was to emphasize marine macrophytes as a natural resource and t h e i r prudent management.

Since kelp are the predominant macrophytes along the P a c i f i c

coast o f North America, they received the most attention.

A mixture of

topics on growth and reproduction, n u t r i e n t a v a i l a b i l i t y and supply,

XII macroalgal associations and degradation, and on kelp farming and harvest, was proposed and within that framework, individual speakers were left free to develop their themes and highlight what they thought was important. During the conference it was agreed by the participants that the papers and relevant discussions, which ware being taped, be published as a single volume.

On returning to their respective homes, the authors prepared

their manuscripts according to the publisher's instructions and sent them back to me, in most cases promptly and without any nudging.

In two cases,

however, I am still awaiting the manuscripts. It has been my very pleasant duty to compile these proceedings.

The

papers, all of them by experts in their fields, are of consistently high quality and, having spent the better part of my research career with higher plants, I am impressed by the range of unique and in sane cases untouched problems that marine macrophytes offer for inquisitive, bright, and resourceful young minds.

At times I have changed the order and

grouping of papers from that followed in the conference for better continuity and readibility.

I would like to take this opportunity to

thank all contributors, authors and discussants, who made this volume possible, and who so patiently and ungrudgingly bore the brunt of my editorial scissors. Many individuals and institutions cooperated and helped in the production of this book.

Financial support for the conference was provided in part

by Simon Fraser University, in part by the Marine Resources Branch, Department of Environment, Government of British Golunbia, and in part by the participants who not only generously donated their time, but also bore a substantial part of their travel fare to Vancouver and back.

Our

special thanks are due to the Director and staff of Bamfield Marine Station who provided the facilities for holding the conference and to numerous students, staff and faculty who volunteered their time and effort to running the various errands associated with the conference.

I vrould

specially like to thank Larry Albright, Mia Bonettemaker, Bea Donald, Louis Druehl, Walter Griba, Lin Kemp, Carolyn T&te, and Carole Thompson, all from Simon Fraser University, and Ron Foreman from University of British Golunbia and Bamfield Marine Station.

XIII Funds for preparing this volume for publication were provided by Simon Fraser University.

It is not often that the editor of a book is blessed

with a typist who is a specialist in that field and who also owns a word processor.

I am extremely grateful to Bill Wheeler for being here at the

right time to do the typing for this book and to decipher many mumblings on the recording tape.

I hasten to add, that while the typographical

errors may be Bill's or the word processor's, the editorial mistakes are all mine. Finally, I would like to thank Walter de Gruyter for publishing

this

volume and for being very patient with an editor who failed to meet several deadlines.

July 1981

Laiit M. Srivastava

Organizing Committee: L. Albright, L. Oruehl, R. Fbreman C.L. Kemp, L.M. Srivastava

OPENING REMARKS

R.F. Scagel Department of Botany, University of B r i t i s h Columbia, Vancouver, B.C. Canada

Qi behalf of the Management Council of the Western Canadian U n i v e r s i t i e s Marine B i o l o g i c a l S o c i e t y , t h i s Cbnference.

I am pleased to welcome the participants to

This s o c i e t y i s a consortium o f f i v e

universities,

comprising the University of Alberta, the University of B r i t i s h Colunbia, the University of Calgary, Simon Fraser University and the U n i v e r s i t y o f Victoria.

Although the S o c i e t y has not given any s p e c i a l mandate to

marine plant b i o l o g i s t s a t the Bamfield Marine S t a t i o n , i t has been highly supportive of their e f f o r t s , and i s very appreciative of the high p r o f i l e given to t h i s station by marine plant researchers, e s p e c i a l l y through the contributions of Drs. Druehl, Foreman and Srivastava. Although Dr. Srivastava was most circunspect when he i n v i t e d me t o g i v e some opening remarks a t t h i s Conference, I detected that the reason was that I might be regarded as a senior marine plant c i t i z e n - hopefully not yet a f o s s i l

specimen.

And when I examined the r o s t e r of

invited

p a r t i c i p a n t s , which included former graduate s t u d e n t s , post d o c t o r a l f e l l o w s , i n d i v i d u a l s whom I had met as graduate students elsewhere, and colleagues whom I had introduced to l o c a l k e l p beds o r known f o r many y e a r s , a l l o f whom are now i n t e r n a t i o n a l l y known for t h e i r research on seaweeds, i t seemed that I had l i t t l e choice but to a c c e p t the l a b e l the f a c e o f t h i s e v i d e n c e .

in

Furthermore, i t i s 34 years ago t h i s month

t h a t , as a graduate student, I s e t forth in a 9 - f o o t rowboat to spend the summer studying a bed of Macrocystis near Port Hardy, B r i t i s h Columbia ( 1 ) , a t the commission o f the B r i t i s h Columbia P r o v i n c i a l Department, and at a stipend of $150.00 per month.

Fisheries

At t h a t time i t was

t h i s Department t h a t was responsible for licensing seaweed harvesting in B r i t i s h Colunbia.

Hence, perhaps I can give an h i s t o r i c a l perspective t o

t h i s Conference, with some emphasis on the l o c a l s c e n e .

What i s the

2 present state of fundamental knowledge concerning seaweeds? What are the prospects for application of the great wealth of expertise that is outlined in the programme of this conference?

Wisdom doesn't necessarily

come with age, but perhaps one can learn something from past mistakes! Early in the 20th Century the United States Government became concerned about the extent to which it relied on imported sources of potash for agricultural fertilizer.

This led to a massive report (2) published by

the United States Department of Agriculture in 1912 on fertilizer resources, including kelps as a source of potash, and extensive surveys of these marine plant resources of the Pacific Cbast of the United States, including Alaska, were undertaken.

Shortly thereafter, in 1916, the

Fisheries Research Board of Canada (3) carried out a survey of the kelp beds along the coast of British Columbia, again with a view to possible utilization of kelp resources as a source of potash.

During Wbrld War I

years (1914-1918), when the source of potash from the German mines at Stassfurt was cut off, the United States produced sane potash from kelp (e.g., in 1917, 3,572 tons of potash were produced).

However, shortly

thereafter extensive deposits in Chile became available to world commerce and it was no longer economically feasible to use seaweed resources as a source of fertilizer potash. Although interest in kelp as a commercial source of potash disappeared, an even greater interest in kelps as source of phycocolloids soon emerged and the foundation was laid for the modern algin industry in California based on Macrocystis.

An increasing demand for algin, and the shortage of

supplies of agar from Japan, resulting from World War II (1939-1944), again gave an impetus to the seaweed industry on the Pacific Coast of North America.

However, despite the availability of exploitable natural

kelp resources in British Cblimbia, attempts to develop a seaweed industry of any magnitude have still not met with success. In the mid-forties, there was renewed interest on the British Columbia coast in seaweeds, and same funds were provided for surveys and research by the British Golimbia Provincial Government (through the Provincial Department of Fisheries and the British Cblunbia Research Council), the Canadian Federal Government (through the Fisheries Research Board), and a

3 private industry in Maine that was interested in agar.

A kelp company was

f i n a l l y established, tried to get into operation in a plant constructed on Deer Island in the v i c i n i t y of Hardy Bay, and almost

succeeded by 1947.

From the beginning, i t was planned that t h i s company would produce a l g i n , although t h e r e were a l s o plans t o produce seaweed m e a l , p i l l s , p o s s i b l y even f e r t i l i z e r .

and

Apart from producing some p i l l s and a small

amount of meal, t h i s company never achieved f u l l production and soon went i n t o r e c e i v e r s h i p a t a loss of several hundred thousand d o l l a r s . Despite the a v a i l a b i l i t y o f s u f f i c i e n t k e l p ( m a i n l y M a c r o c y s t i s )

t o have

maintained a modest o p e r a t i o n and s u f f i c i e n t p r i v a t e c a p i t a l to have a n t i c i p a t e d s u c c e s s , the company f a i l e d apparently b e c a u s e o f

poor

management. In the m i d - s i x t i e s t h e r e was another attempt t o e s t a b l i s h an a l g i n industry in B r i t i s h Columbia - t h i s time at the north end of the Queen Charlotte Islands.

Et>r reasons of apparent poor management and f i n a n c i a l

problems t h i s e f f o r t also soon f a i l e d , at a considerably greater financial loss than the e a r l i e r attempt in the mid-forties. Ihere has been a renewed i n t e r e s t and support o f the B r i t i s h Columbia P r o v i n c i a l Government in both fundamental and applied research on marine plants in the past decade.

This renewed i n t e r e s t on both kelps as well as

on o t h e r phycocolloid-producing marine algae, i f matched with financial b a c k i n g and e n t r e p r e n e u r i a l

skill,

may l e a d t o t h e

successful

e s t a b l i s h m e n t o f a healthy seaweed industry in B r i t i s h Columbia.

Marine

plant s c i e n t i s t s in B r i t i s h Columia f a c e a s p e c i a l c h a l l e n g e in t h i s developnent. The r e s e a r c h a c t i v i t i e s to be reported on and the p r e s e n c e o f many d i s t i n g u i s h e d s c i e n t i s t s as participants in t h i s Conference are bound to have a s i g n i f i c a n t impact on local i n t e r e s t s and w i l l undoubtedly have a p o s i t i v e i n f l u e n c e on the development o f a healthy seaweed industry in B r i t i s h Columbia.

Whatever small r o l e I have played as a pioneer in

encouraging and developing marine phycology in B r i t i s h Cblunbia i s more than amply rewarded in finding an assemblage o f e x p e r t i s e such as i s represented here today meeting in B r i t i s h Golunbia and a t the Bamfield Marine S t a t i o n .

4 References 1.

Scagel, R.F.: British Columbia Dept. Fish. No. 1, pp. 1-70 (1948).

2.

Cameron, F.K.: U.S. Senate, Doc. No. 190, 62nd Congr., 2nd Sess., pp. 1-290 (1912).

3.

Cameron, A.T.: The commercial value of the kelp-beds of the Canadian Pacific coast. A. Preliminary report and survey of the beds. Gontr. Canadian Biol, for 1914-15, pp. 25-39 (1916).

Part I .

S t r a t e g i e s of growth and reproduction

PHYSIOLOGY OF CARBON FIXATION IN BENTHIC MARINE ALGAE

J . Willenbrink Botanisches I n s t i t u t , Universität Köln D-5000 Köln-Lindenthal, F.R. Germany

Life would not be p o s s i b l e without the l i g h t energy o f the sun.

The

process o f capturing and u t i l i z i n g t h i s energy has been perfected over millions of years of plant evolution.

In photosynthesis, l i g h t energy i s

converted in a s e r i e s of steps into chemical energy which can then be used by t h e o r g a n i s m f o r v a r i o u s m e t a b o l i c

processes.

The

typical

photoautotrophic growth of algae includes both t h i s energy conversion and carbon f i x a t i o n , also called "dark reaction," in which the chemical energy in the form of ATP and NADPH2 i s used to bring C0 2 i n t o reduced o r g a n i c compounds. I would l i k e to concentrate here on some aspects of the physiology o f CO^ - f i x a t i o n r a t h e r than on i t s b i o c h e m i s t r y .

The f i r s t section will be

devoted to a u t o t r o p h i c C0 2 - f i x a t i o n , the second to l o c a t i o n o f t h e photosynthetic carton reduction cycle in the algal c e l l , the third to CO^ - f i x a t i o n by systems o t h e r than RuBP-carboxylation, and the fourth and l a s t bo regulation of C02 - f i x a t i o n .

Autotrophic CO2 -Fixation Marine a l g a e a r e exposed t o c a r b o n i c a c i d (H„CO,) and i t s i o n s , ?— ' bicarbonate (HCO^ ) and carbonate (CO^ ) as well as to C0 2 , the l a s t one a t a very l i m i t e d concentration.

In addition, an increase in pH r e s u l t s

in an i n c r e a s e in the amount o f b i c a r b o n a t e and carbonate in the sea water. These algae are able to take up C0 2 which r e a d i l y d i f f u s e s a c r o s s membranes

(25)

RuBP-carboxylase.

and

can be d i r e c t l y

used f o r c a r b o x y l a t i o n

cell by

Many s p e c i e s a l s o use b i c a r b o n a t e but t h i s must be

© 1982 Walter de Gruyter & Co., Berlin • New York Synthetic and Degradative Processes in Marine Macrophytes

8 converted to CC^ before i t i s assimilated. e i t h e r of

Such a conversion may occur in

two ways: d i r e c t uptake o f b i c a r b o n a t e by the c e l l s and

subsequent hydrolysis to CC>2 by carbonic anhydrase (25) a process which m i g h t n o t be p r e s e n t in a l l a l g a e ( 1 1 ) ; second, b i c a r b o n a t e may be converted to CC^ before i t has been taken up by the plant.

In sea w a t e r ,

bicarbonate concentration i s more than 100-fold t h a t o f CC^f and hence, the l a t t e r process i s quite l i k e l y .

Both carbon sources may play a role

in the same species, for as Kremer and Schmitz (15) have shown r e c e n t l y the l i t t o r a l species of Fucus platycarpus may have the same r a t e o f CC>2 - f i x a t i o n in submerged as well as in emerged s t a t e . The important steps in incorporation o f CC^ i n t o organic compounds and t h e i r subsequent reduction, the "dark r e a c t i o n , " have been elucidated by C a l v i n and h i s g r o u p .

By 1969 t h e e x i s t e n c e

of the

reductive

pentosephosphate c y c l e , the Calvin c y c l e , was established for numerous chlorophytes, but t h e r e was very l i t t l e published information on CC^ - f i x a t i o n in other algae, e s p e c i a l l y marine algae.

The pioneering vork by

Yamaguchi e t a l . (35) on CC^ - f i x a t i o n by brown algae was a s t e p forward in t h i s d i r e c t i o n . CO^ - f i x a t i o n in brown algae Yamaguchi e t a l . (35) incubated fronds of the P a c i f i c brown a l g a , E i s e n i a 14 b i c y c l i s , with C-bicarbonate sea water in the l i g h t and followed the intramolecular distribution of label in mannitol, which i s known to be the main assimilate of these a l g a e , in 1 t o 60 min p e r i o d s . The r a t i o o f 14 14 total C to C in C^ and Cg positions in the mannitol molecule decreased from 8 . 0 a f t e r 1 min to about 3 a f t e r 60 min, which suggested a trend toward a uniform d i s t r i b u t i o n

of the l a b e l .

These r e s u l t s

subsequently confirmed in our l a b o r a t o r y u s i n g Fucus s e r r a t u s

were and

Dictyota dichotoma. In both species, the r a t i o of t o t a l "^C to "^C in C, 14 and C, p o s i t i o n s d e c r e a s e d in e x p e r i m e n t s from 6 s t o 600 s C b assimilation ( 3 2 ) . These r e s u l t s imply that mannitol may be synthesized via the r e d u c t i v e pentosephosphate c y c l e through 3-carbon compounds. Yamaguchi already had demonstrated enzyme a c t i v i t i e s in the crude e x t r a c t o f Dictyota dichotoma for hexose-diphosphatase, and g l u c o s e - p h o s p h a t e - i s o m e r a s e ,

mannitol-l-phosphatase,

which can be involved in mannitol

9 biosynthesis.

Because of the high mucilage content and the presence of

large amounts of phenolic compounds, progress in enzyme work on brown algae was not really possible until technical improvements in the isolation of enzymes could be made, for instance, by addition of polyvinyl pyrrolidone (PVP), bovine serum albumin (BSA) and cysteine.

Nordhorn et

al. (23) demonstrated the presence of both glyceraldehydephosphatedehydrogenase and mannitol-l-phosphate-dehydrogenase in Fucus serratus, both of which are involved in mannitol biosynthesis.

In 1973, evidence

was presented for RuBP-carboxylase in Laminaria, which catalyzes CC^ binding in the light (29).

Fig. 1: Labelling pattern of assimilation products of the blade of Laminaria saccharina. Data are percentage of total ethanol-water-soluble radiocarbon, present after 10 - 600 s of photosynthesis (after 17). In other work on CC^ -fixation by brown algae done in our laboratory, it has been established that the intermediates of CO„ -fixation and 14 assimilates show clear kinetics of the distribution of C with increasing 14 time of exposure of the frond to CC>2 (12,17). Figure 1 presents data on short-term experiments with Laminaria saccharina. The rapid decrease of relative label in PGA corresponds to the well established fact of PGA as being the first product of carboxylation in the light.

The label in

sugar-phosphates also decreases with time, as expected, whereas mannitol,

10 serine/glycine and alanine become h e a v i l y l a b e l l e d with time, both in relative and absolute terms.

It should be noted that the total percentage

of label in the four amino acids listed here exceeds that in mannitol and accounts for more than 50% of the total radioactivity present in the frond a f t e r 10 min.

Similar experiments were carried out with some other brown

algae which a l l showed a s i m i l a r pattern of l a b e l l i n g

(e.g. Giffordia

mitchellae and Laminaria hyperborea), except that in Fucus the increase in 14 C-labelled amino acids was s l i g h t l y less ( 1 4 ) .

These data correspond

w e l l with the r e s u l t s o f Hellebust and Haug (9) who found amino acids, especially alanine and aspartate, more strongly labelled than mannitol in 3 hour incubation experiments with Laminaria d i g i t a t a . % 80

\

3-PGA

**£

Sugar-m-P v

40-

*••..

.o^"" a

1 2

.a-''

"v.

Sugar-b-P..*"

1

1 6

_Mannitol ..rv«*'.o*""* 1 1—i 10 30

1 60

1 180

S

Fig. 2: Time-course of ^ C in various phosphate e s t e r s and in mannitol a f t e r 2 to 180 s C - p h o t o s y n t h e s i s by b l a d e a r e a s o f Laminaria hyperborea. Blades were pre-illuminated f o r at l e a s t 10 min p r i o r t o exposure to radiocarbon. Data a r e p e r c e n t a g e o f t o t a l ethanol-water-soluble radiocarbon ( a f t e r 33). Btir Laminaria hyperborea, short-term incubations down to 2 s show an even 14 more pronounced decrease of r e l a t i v e C l a b e l in PGA ( F i g . 2 ) . By separating sugar-biphosphates (SBP) from sugar-monophosphates (SMP), two

11 subsequent peaks could be shown:

the peak for SBP might represent mainly

RuBP but f r u c t o s e biphosphate i s not excluded, the peak for SMP might 14 contain various sugar-monophosphates. The subsequent r i s e of C-mannitol 14 with a corresponding decline in C - l a b e l l e d PGA and sugar phosphates, i n d i c a t e s t h a t mannitol i s one o f the main assimilates in brown algae (33). CX>2 - f i x a t i o n in marine red and green algae Fbr Rhodophyceae, Bean and Hassid (3) presented evidence that f l o r i d o s i d e became labelled in these algae.

- f i x a t i o n , and that Calvin c y c l e was operating

A study on Porphyra and o t h e r g e n e r a r e v e a l e d

in

that

floridoside and digeneaside were the most strongly labelled carbohydrates; mannitol could not be detected ( 1 6 ) .

The pattern of

14

C - l a b e l l i n g in the

p h o t o s y n t h e t i c i n t e r m e d i a t e s r e f l e c t s the role of glycerophosphate as a precursor of floridoside.

Glycine and s e r i n e , compared with brown a l g a e , 14 carry a l e s s e r but s t i l l a remarkably high percentage of t o t a l C-label. The prevalence of amino acids in rhodophycean photosynthesis becomes even more obvious in long-term experiments.

In Pterosiphonia dendroidea, 75%

of the t o t a l assimilated carbon was distributed in the amino acid fraction ( 1 3 ) , a f a c t which emphasizes the extraordinarily high rate of amino acid synthesis in photosynthetic carbon fixation in many marine algae. Information on CC>2 - f i x a t i o n in marine chlorophytes i s r e l a t i v e l y meagre. Percival (24) described that in Ulva lactuca sucrose was the most h i g h l y 14 labelled sugar, with some C - l a b e l in g l u c o s e , f r u c t o s e , x y l o s e , and myoinositol,

in experiments longer than 20 min.

Et>r the siphonaceous

green alga, Caulerpa simpliuscula, Howard and coworkers (10) have shown soluble

, 3 - p o l y g l u c a n s as important labelled carbohydrates, whereas

s u c r o s e , as might have been e x p e c t e d , p l a y s only a minor r o l e .

The

authors presented data which show an exceptionally high label in sugar phosphates, e s p e c i a l l y phosphoglyceric acid and fructosebiphosphate, with incubation time o f up to 40 min, and suggest a role for these substances as a storage pool.

That speculation may be q u e s t i o n a b l e , but the o t h e r

d a t a presented by these authors show a r e l a t i v e l y slow s y n t h e s i s o f carbohydrates, and, hence, the high pool o f phosphate e s t e r s appears reasonable.

Again, the radioactivity of amino acids increased markedly,

12 alanine became labelled most rapidly, followed by glycine and o t h e r s , but unfortunately the data given by the authors do not allow a calculation of percentage of t o t a l "^C in the amino acid f r a c t i o n .

S i t e of Photosynthetic Carbon Reduction in Algae Fran the points of view of energetics as well as our understanding o f the compartmentation inside a photosynthesizing c e l l , i t i s important to know whether the chloroplasts are the s i t e s of the Calvin c y c l e , and f u r t h e r , where in the c e l l the s y n t h e s i s o f the f i n a l a s s i m i l a t e takes place. Ihere i s good evidence in the l i t e r a t u r e from higher p l a n t s t h a t the enzymes o f the Calvin cycle are exclusively located in the chloroplasts, whereas the enzymes involved in sucrose biosynthesis may be active outside these organelles ( 5 ) . chloroplasts

Bidwell e t a l .

( 4 ) reported on p r e p a r a t i o n s o f

from A c e t a b u l a r i a m e d i t e r r a n e a which c a r r i e d

out

photosynthetic a c t i v i t y in v i t r o that was i n d i s t i n g u i s h a b l e in r a t e and products from p h o t o s y n t h e t i c a c t i v i t y in i n t a c t c e l l s .

Since t h e s e

preparations were able to synthesize s e q u e n t i a l l y o l i g o s a c c h a r i d e s from sucrose and were shown to be r e l a t i v e l y c l e a n ,

the c h l o r o p l a s t

Acetabularia has been suggested as the s i t e o f s y n t h e s i s o f

of

sucrose.

However, these preparations s t i l l contained cytoplasm, as was shown l a t e r (34). From the above review, i t might be concluded that sucrose i s adjacent to but outside the chloroplasts.

synthesized

In order to t e s t whether or not

c h l o r o p l a s t s in brown a l g a e ( p h a e o p l a s t s ) a r e the s i t e o f

mannitol

synthesis, we t r i e d to i s o l a t e p h a e o p l a s t s a f t e r _in vivo exposure o f fronds of Laminar ia hyperborea and Fucus s e r r a t u s to "^CC^ f o r v a r i o u s times.

I t was impossible to i s o l a t e p h a e o p l a s t s from

hyperborea

fronds, but with F. serratus there was p a r t i a l success using a nonaqueous procedure ( 3 0 ) .

After extraction and chromatography, distribution of the label in the individual photoassimilates and t o t a l 14 C between phaeoplasts 14 and the remainder of the c e l l was analyzed. After 10 s C -assimilation, more than 80% of a l l "^C-mannitol was present within the plastid f r a c t i o n , but a f t e r 180 s the major p a r t was found in the remainder of the c e l l

13 (Fig. 3).

These data support the idea that mannitol is synthesized inside

the phaeoplast.

14 F i g . 3: Time-course of C -incorporation into mannitol present in phaeoplasts isolated nonaqueously from fronds of Fucus serratus which were exposed to H C0,~- seawater for 10 - 180 s in the lightT Note the high percentage of labelled mannitol present in the phaeoplast fraction after 10 s exposure of the frond to radioactive bicarbonate, and i t s subsequent and rapid decline (modified after 30). In a second approach, we used aqueously isolated phaeoplasts from F. serratus (23).

With these preparations, i t has not been possible so f a r

to obtain rates and pho tosyn the tic activities comparable to those reached for higher plant c e l l s , but the phaeoplast f r a c t i o n seems to be able to synthesize mannitol.

In addition, 0„ evolution by the phaeoplasts shows -2 -1 l i g h t saturation at 58 c a l m * h ^; O^ e v o l u t i o n i s temperature insensitive between 4 C and 24 C, and above 24 C the rate of apparent photosynthesis decreases rapidly.

This sensitivity to even a s l i g h t r i s e

in temperature above 24 C i s t y p i c a l f o r benthic brown algae.

DCMU, a

competent inhibitor of photosystem I I , has a half-maximal inhibition at 5 _7 x 10

M.

Finally, the

vivo absorption spectrum exhibits a peak f o r

fucoxanthin which coincides with the action spectrum indicating that

14 fucoxanthin i s involved in l i g h t r e a c t i o n s ( 2 3 ) .

The d i s t r i b u t i o n o f

enzymes involved c a s t s some doubt on mannitol being synthesized in the phaeoplasts.

So f a r , mannitol-P-dehydrogenase has been found mainly

o u t s i d e the phaeoplasts, a contradiction which cannot be resolved as y e t . I t i s noteworthy in t h i s connection that phaeoplasts in brown a l g a e a r e surrounded by p e r i p l a s t i d a l c i s t e r n a e o f the ER system ( 6 ) , which may contain enzymes of hexitol biosynthesis.

Presumably, the p e r i p l a s t i d i c

network has not been separated completely from the phaeoplasts during isolation.

From an e n e r g e t i c

point

of

v i e w and

considering

compartmentation, mannitol may well be synthesized outside the phaeoplasts i f phosphate exchange a c r o s s

t h e p l a s t i d membrane i s l i n k e d

to

triosephosphate transport, as stated by Heber ( 8 ) .

Other C0 2 -Fixation Reactions

Whereas CC>2 - f i x a t i o n o u t l i n e d so f a r i s i n t i m a t e l y r e l a t e d t o energy conversion, further CC>2 - f i x a t i o n reactions are important f o r a l l growth in autotrophic as well as in heterotrophic organisms.

These reactions are

well known for animal and higher p l a n t systems, but they have r e c e i v e d l i t t l e attention for algal c e l l s , e s p e c i a l l y marine algae.

Fbr instance,

for marine algae there are no data in the l i t e r a t u r e on the s y n t h e s i s o f a r g i n i n e and prymidines, dependent on the a c t i v i t y of an enzyme which catalyzes carboxylation of ammonia yielding carbamoyl-phosphate. Other carboxylating enzymes (excluding the biotin-dependent enzyme which needs CO^ only as a c a t a l y s t to s t a r t f a t t y acid s y n t h e s i s ) , a r e mainly involved in a n a p l e r o t i c p r o c e s s e s which supply carbon s k e l e t o n s

for

biosynthesis of various products, and in the absence of which intermediary pathways l i k e g l y c o l y s i s or the Krebs-cycle would be depleted of t h e i r intermediates.

An example i s phosphoenolpyruvate-carboxylase whose

r e a c t i o n y i e l d s o x a l o a c e t a t e , the acceptor for acetyl-OoA and substrate for aspartate biosynthesis. organisms s t u d i e d .

This enzyme i s p r e s e n t i n most o f

In t h i s c o n n e c t i o n ,

it

the

should be noted t h a t

PEP-carboxykinase i s a highly active enzyme in some marine algae ( 2 9 ) .

15

Table I : Carbon f i x a t i o n in the l i g h t and under dark conditions in young and old parts of blades of Laminaria hyperborea. Light f i x a t i o n >omol C/dm / h Young frond February, 4 March, 4 C April, 5 C May, 8 C Old frond February, 4 C March, 4 C April, 5 C May, 8 C

15

Dark f i x a t i o n pmol C/dm / h

15 23

4.1 3.6 4.2 2.9

(27.3) (13.8) (28.0) (12.6)

23 35 36 37

1.4 1.5 1.5 0.8

( ( ( (

26

6.1) 4.3) 4.1) 2.2)

Figures in parentheses represent % of carbon fixed in l i g h t . Gompiled a f t e r ( 3 3 ) .

a

T h i s enzyme used t o be c o n s i d e r e d a d e c a r b o x y l a t i n g enzyme, b u t an i n c r e a s i n g number o f s t u d i e s in h i g h e r p l a n t s i n d i c a t e t h a t i t has a carboxylating

function.

Akagawa e t a l .

(1,2)

p r e s e n t e d d a t a on

c a r b o x y l a t i o n r a t e s in seme species of P a c i f i c brown algae and i d e n t i f i e d PEP-carboxykinase as the enzyme responsible. processes

as a f f e c t e d

by age o f

fronds

Our studies on carboxylation in Laminariales

revealed

e x c e p t i o n a l l y high r a t e s o f dark f i x a t i o n o f C0 2 in the meristematic zone or young parts of the f r o n d s .

In Laminaria hyperborea p h o t o s y n t h e t i c

r a t e s in young and old p a r t s were in the same r a n g e , 12 t o 40 jamol CO. - 2

dm

- 1

h

, a t 4 C which i s the normal ambient temperature in the North Sea

during e a r l y spring (T&ble I ) .

However, dark f i x a t i o n was much higher in

the young than in the old p a r t s o f the f r o n d , namely 13 t o 28% o f

the

corresponding l i g h t f i x a t i o n i n t h e young frond and 2 t o 6% in the old frond ( 3 3 ) .

P r i n c i p a l l y the same c o r r e l a t i o n can be deduced from d a t a on

Macrocystis i n t e g r i f o l i a , Nereocystis luetkeana and s e v e r a l o t h e r brown algae ( 3 1 ) .

The pathway of

14

C - i n c o r p o r a t i o n in the dark i s

completely

d i f f e r e n t from t h a t in the l i g h t , and l a b e l from dark f i x a t i o n can e n t e r phosphate e s t e r s or mannitol only by gluconeogenesis (see Fig. 5 ) .

16

V

• •

O •f jui oa\

/

D

8''° °°° 2

4 6810 2 0 3 0 60 YOUNG

BASAL

180

600S

2

PGA SMP SOP

* N\

\

\

O

4 6810203060

180

b

600S

OLD PHYLLOID

Fig. 4: Kinetics of 14, ^C-incorporation intoraalate(MAL), aspartate (ASP), mannitol, phosphoglycerate (PGA), and sugar mono- or di-phosphates (SMP or SDP) in young and old parts of blades of Laminaria hyperborea. Samples were exposed to H CCL - seawater in the light for 2 - 600 s; ambient t e m p e r a t u r e 4 C, February. Data presented as % of total ethanol-water-soluble C (modified after 33). Short term

14

C02~incubation experiments, 2 to 600 s duration, show that in

the young frond two types of carboxylation reactions occur in the light (33). A coupling of these carboxylating reactions, as in C-4 plants, is a tempting speculation, but tvo types of evidence indicate that both systems wark independently.

The ratio of labelling of the first carboxylation

products, phosphoglycerate to malate/aspartate, is 70% to 30% after 2 to 4 14 s C02~incubation of frond segments under continuous light (Fig. 4, left

17 s e t of drawings).

Secondly,

the s h i f t of short

term

labelled

i n t e r m e d i a t e s , a f t e r a subsequent transfer of the labelled frond samples to a C0 2 -free sea water shows a marked i n c r e a s e in

14

C-RuBP, but only a

marginal increase in "^C-PEP, which suggests that both systems are a c t i v e in the l i g h t but only one of them (RuBP carboxylase) i s l i g h t dependent. There i s no doubt that both systems are involved in metabolism o f carbon. Figure 5 summarizes a p o s s i b l e scheme l i n k i n g the s i t e o f a c t i o n o f PEP-carboxykinase to intermediary metabolism and v a r i o u s

synthetic

pathways in marine algae.

MANNITOL

jPEP-Carboxykinase (Carboxylase) OA A-—»-ASPARTATE

KREBS-CYCLE

OXOGLUTAR / GLUTAMATE

PORPHYRINS

Fig. 5: Flow o f carbon between C a l v i n - c y c l e and K r e b s - c y c l e in brown a l g a e , with s p e c i a l r e f e r e n c e to r i b u l o s e b i p h o s p h a t e - c a r b o x y l a s e and phosphoenolpyruvate-carboxykinase as primary s i t e s o f CC^- f i x a t i o n . Anino acids are underlined.

18 Regulation of CC^ -Fixation in Marine Algae It is obvious that among external factors light and temperature play the dominant role because for marine organisms inorganic carbon supply and oxygen level seem to be maintained more or less constant throughout the whole year. However, there is same evidence that nutrient supply, mainly of nitrogen, governs growth rate in sane species (7). As far as light is concerned, irradiation leads to species-specific enhancement of CC>2 -fixation.

Fbr species of Laminaria, Macrocystis, Nereocystis, and Fucus,

light saturation is reached at about 200 foot candles (Fig. 6? see reference 31). Consequently, these species behave, in a classical scheme, as shade plants which presumably is of ecological significance.

It was

pointed out at the beginning, that light influences CC^ -fixation by providing ATP and NADPH2, whose availability in light provides the striking difference between photosynthesis and dark fixation; for it is not until reduction of carbon has occurred that a real gain is made by the plant.

As shown by short-term light/dark experiments, ATP-synthesis and

corresponding decrease in ADP occurs with the same velocity in both higher plant and brown algal cells (23).

Fig. 6: Dependence of net photosynthesis on incident light (in ft. candles along x axis) in fronds of Macrocystis integrifolia. Results frcm 2 experiments, open and closed circles, are shown. Assimilation rate measured by C- fixation (modified after 31).

19 I t i s well known from higher plants that they have evolved a long distance transport system for assimilates, which i s of s i g n i f i c a n t advantage in the carbon economy of the organism.

Mobilization of food m a t e r i a l stored in

o l d e r p a r t s of the t h a l l u s and t r a n s l o c a t e d t o young growing t i s s u e s , o c c u r s in higher organized brown a l g a e ,

also.

Evidence f o r

translocation was presented by Luning for Laminaria hyperborea.

such

Growth of

t h i s alga s t a r t s during a season when l i g h t intensity in the s u b l i t t o r a l zone exceeds the compensation point only f o r a few hours d a i l y

(21).

Growth measurements c l e a r l y demonstrated a dependence of the young frond on materials translocated from the old one ( 2 0 ) . By feeding a d i s t i n c t 14 part of the old frond with C-sea water, i t was shown t h a t some hours l a t e r r a d i o a c t i v e m a t e r i a l moved mainly towards the new frond where the l a b e l was concentrated in the b a s a l zone ( 2 7 ) .

D e t e c t a b l e movement

occurred only in the growth period of the new frond.

In a f i r s t , rather

rough c a l c u l a t i o n , i t was shown that about 50% of the growth of the young blade was due to translocation of newly synthesized material and the other 50% to t r a n s l o c a t i o n o f mobilized m a t e r i a l from the old frond

(22).

Translocation i s by no means s p e c i f i c f o r Laminaria hyperborea but has been shown for a l l Laminariales studied so f a r (see Scbmitz, t h i s volume). Leaving aside the interesting question of the transport process i t s e l f ,

it

may be mentioned t h a t these plants seem to translocate what they produce as photoassimilates; in the conducting system, there are both high amounts of amino acids and mannitol (26-28). The question a r i s e s whether or not the high r a t e o f dark f i x a t i o n in a young frond contributes to i t s growth.

In order to answer that question,

i t must be remembered that a n a p l e r o t i c r e a c t i o n s become e f f i c i e n t only when they are provided with reduced materials. main t r a n s l o c a t e

is mannitol,

As reviewed e a r l i e r , the

a h i g h l y reduced compound.

The

e s t a b l i s h m e n t o f a system which combines light-dependent carboxylating reactions with a supply of reduced material could have been a necessity in the course of evolution considering the biotope of t h i s perennial alga.

20

iiumIM

BASAL

i i i i i—i i i i i i

APICAL

emoles

JF M A M J J A S O N D J F M A

JFMAMJJASONDJFMA

aj moles

jumóles

i iii ii ii ii iiii i

i i i i i i i i i i i I M—r JFMAMJJASONDJFMA

JFMAMJJASONDJFMA

Fig. 7: Seasonal changes of enzyme a c t i v i t i e s (measured a t 25 C) in basal (young) and apical (old) parts of fronds of Laminaria hyperborea. April v a l u e s a r e s e t as 100%, bars i n d i c a t e standard d e v i a t i o n . RuBP-C: r i b u l o s e - 1 , 5 - b i s p h o s p h a t e - c a r b o x y l a s e ; PEP-CK: phosphoeno1pyruvatec a r b o x y k i n a s e ; GA-3-P-DH(NADP): NADP-dependent gly c e r a l d e h y d e 3-phosphate- dehydrogenase. A b s c i s s a : a b b r e v i a t i o n s mark the months, beginning with J = January (modified a f t e r 1 9 ) . Therefore, t r a n s l o c a t i o n of a s s i m i l a t e s

in L a m i n a r i a has b o t h a

q u a n t i t a t i v e and q u a l i t a t i v e s i g n i f i c a n c e f o r the formation of a new thallus. all

But, one has to take into account the f a c t that young tissues of

Laminariales

tested

so

far

exhibit

high

activities

of

21 PEP-carboxykinase, n o t o n l y t h o s e which a r e c h a r a c t e r i z e d by s e a s o n a l growth p e r i o d s .

Measurements of s e v e r a l enzymes involved in c a r b o x y l a t i o n

and mannitol b i o s y n t h e s i s have been c a r r i e d o u t f o r two y e a r s in o r d e r

to

c l a r i f y w h e t h e r o r n o t a s e a s o n a l a d a p t a t i o n e x i s t s a t the enzyme l e v e l (18,19).

As shown in F i g . 7, the enzymes i n v o l v e d i n c a r b o x y l a t i o n and

m a i n t e n a n c e of a r e d u c t i o n p o t e n t i a l e x h i b i t peaks in March through May. But one h a s t o c o n s i d e r t h a t enzyme s t u d i e s a r e n o r m a l l y made u n d e r standard c o n d i t i o n s , f o r i n s t a n c e , a t 25 C.

Temperature in v i v o , however,

c h a n g e s d r a s t i c a l l y in the course of the y e a r .

Consequently, t h e s e d a t a

were r e c a l c u l a t e d on t h e b a s i s of t h e _in v i v o t e m p e r a t u r e s and a c t i v a t i o n e n e r g i e s of t h e enzymes.

the

T h i s c o r r e c t i o n l e d , a s could be

e x p e c t e d , t o a maximum a c t i v i t y in summer.

I t s h o u l d be m e n t i o n e d

that

t h e maximum a c t i v i t y f o r mannitol-P-dehydrogenase was not reached u n t i l September, which s u p p o r t s the idea t h a t mannitol s y n t h e s i s i s h i g h e s t t h a t time of t h e y e a r (19).

at

Figure 7 a l s o shows t h a t f o r both young and

old p a r t s of the frond a compensatory i n c r e a s e i n t h e amount of enzymes t a k e s p l a c e i n e a r l y s p r i n g when e n z y m a t i c a c t i v i t y s h o u l d be lowest because of the low temperature in the s e a .

References 1.

Akagawa, H., Ikawa, T . , Nisizawa, K.:

Bot. Mar. 15, 126-133 (1972).

2.

Akagawa, H . , Ikawa, T . , N i s i z a w a , K . : 999-1016 (1972).

3.

Bean, R.C., Hassid, W.Z.:

4.

Bidwell, R.G.S., Levin, W.B., S h e p h a r d , D . C . : 70-75 (1970).

5.

Bird, I . F . , Cornelius, M . J . , Keys, A . J . , Whittingham, Phytochemistry 13, 59-64 (1974).

6.

B i s a l p u t r a , T . : ^ n A l g a l P h y s i o l o g y and B i o c h e m i s t r y Stewart, e d . ) , Blackwell, Oxford, pp. 124-160 1974

7.

Chapman, A.R.O., Markham, J.W., Luning, K.: (1978).

8.

Heber, U.:

9.

H e l l e b u s t , J . A . , Haug, A.:

10.

toward, R . J . , G a y l e r , K . R . , G r a n t , B . R . : (1975).

Plant Cell Physiol.

13,

J . B i o l . Chem. 212, 411-425 (1955). Plant Physiol.

J . P h y c o l . 14,

45,

C.P.: (v«.u.x-. 195-198

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22 11.

J o l i f f e , E.A., Tregunna, E.B.:

12.

Kremer, B.P.: Untersuchungen zur Biochemie und Physiologie von Mannit in benthischen marinen A l g e n . D i s s e r t a t i o n s s c h r i f t , Universität Bonn 1973

Phycolcgia 9, 292-303 (1970).

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14.

Kremer, B.P.: In, Handbook o f P h y c o l o g i c a l Methods. (J.A. Hellebust, J.S. Craigie, eds.) Cambridge Univ. Press, Cambridge, pp. 269-283 1978

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Kremer, B.P., Schmitz, K.:

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Kremer, B.P., Vögl, R.:

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Kremer, B.P., Willenbrink, J . :

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Küppers, U.: Enzymologie der C02-Fixierung bei Laminaria hyperborea: Jahresperiodische, umweltbezogene Veränderungen von EnzymaktivItäten. Dissertationsschrift Universität Köln 1978

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Mar. Biol. 2, 218-223 (1969).

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Luning, K., Schmitz, K., Willenbrink, J. : (1973).

Mar. B i o l . 23, 275-281

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Nordhorn, G., Weidner, M., Willenbrink, J . : 153-165 (1976).

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Schmitz, K.: Zum Assimilattransport in marinen Algen: Struktur und Funktion der Siebelemente bei den Laminarlales. Habilitationsschrift Universität Köln 1976

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Schmitz, K., Lüning, K., Willenbrink, J . : 418-429 (1972).

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Weidner, M., Küppers, U.:

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Willenbrink, J . , Kremer, B.P.:

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Plant Cell Physiol.

SEASONAL PATTERNS OF CARBON ASSIMILATION AND UTILIZATION BY KELPS1

K.H. Mann, A.R.O. Chapman and J. Gagné Department of Biology, Dalhousie University Halifax, N.S., Canada B3H 4J1

Earlier wark had shown that in St. Margaret's Bay, Nova Scotia, where nutrients limit growth in summer, Laminaria longicruris shows two major adaptations to maximizing annual production.

During winter, when

nutrients are abundant, it stores nitrate to a concentration 28,000 times ambient, and uses the stored nitrate to prolong growth into late spring and early summer.

In late summer it stores carbon in the form of mannitol

and laminaran, then uses it for growth in fall, when nutrients are available but light is limiting. It is now shown that this pattern is not invariate.

Studies at a site

where nutrients were never limiting showed that under these conditions L. longicruris had negligible storage of either nitrate or carbohydrate. Instead variation in growth rate paralleled variation in light flux. At a site where nutrients were limiting for a short period in summer, there was winter storage of nitrate but greatly reduced storage of carbohydrate in summer. A study of Laminaria solidungula in the Arctic showed that its growth was strongly controlled by availability of nitrogen and was out of phase with variation in light flux. Cn the other hand, Laminaria pallida in South Africa experiences no nutrient limitation so that variation in growth rate follows the seasonal light pattern.

It is concluded that for all these

species photosynthesis tends to be maximal in summer and minimal in winter, and that growth follows the same pattern provided nutrients are 1

The full text of Dr. Mann's paper delivered at Bamfield Meetings appears

in:

Primary Productivity in the Sea. (P.G. Falkowski, ed.). Brookhaven

Symposia in Biology 31 Plenum, N.Y., pp. 363-380

1980

© 1982 Walter de Gruyter & Co., Berlin • New York Synthetic and Degradative Processes in Marine Macrophytes

(ed. note).

24 not l i m i t i n g .

When they are l i m i t i n g , nutrient storage and carbon storage

are used as devices for maximizing growth.

I t i s not known to what extent

the presence or absence of storage phases in L. longicruris i s g e n e t i c a l l y controlled.

DISCUSSION LUNING:

J u s t a comment, Dr. Mann.

Nitrogen i s c e r t a i n l y a v i t a l factor

f o r growth, but I wonder whether n i t r a t e i s d i r e c t l y r e g u l a t i n g and inducing these seasonal p a t t e r n s o f the kelp plants?

Ihere are indeed

d i f f e r e n t s t r a t e g i e s , but one can also say that there i s a general pattern o f seasonal periodicity in kelp plants.

Sane of these patterns a r e : that

they form the blade, or the majority of the blade at a very e a r l y time in the y e a r .

In the A r c t i c Laminaria s o l i d u n g u l a , the blade i s formed

already in darkness so t h a t , during the short period of l i g h t that i t has, the blade i s ready for capturing l i g h t quanta.

In Laminaria l o n g i c r u r i s ,

where t h e r e i s not so much n u t r i e n t l i m i t a t i o n , t h e r e i s a s e a s o n a l pattern of a minimum growth of 0 . 5 cm per day in November and a maximum in A p r i l , May and June.

So there i s a seasonal pattern even there.

Another

thing which you did not mention, most of these plants make sori in Autumn, when t h e growth r a t e goes down.

I t h i n k you a r e r i g h t , n i t r a t e

determining the seasonal patterns, but maybe only as one of v i t a l — l i k e L i e b i g ' s conditions, a minimun f a c t o r . i n c r e a s e the growth r a t e limitation, experiment.

Ibny Chapman was able to

i n a k e l p b e d , where t h e r e was

by p o u r i n g n i t r a t e

is

factors

i n t o the water.

nitrate

That was a good

But what we need a r e tank experiments where one can show

whether n i t r a t e i s inducing these t h i n g s or not.

Somehow I doubt that

these complicated p l a n t s have survived f o r m i l l i o n s o f y e a r s j u s t by reacting to n i t r a t e concentrations in the water. SRIVASTAVA:

In your growth measurements, did you also measure the weight 2

of the plants or was i t only the increase in surface area, cm /day? MANN:

The f i r s t year of sampling — the data that I have shown — did not

take i n t o account the biamass production.

But there i s a second year o f

25 d a t a , which i s n e a r i n g i t s end o f c o l l e c t i o n , which i n c l u d e s biomass production.

I t l o o k s t o me v e r y much a s i f

t h e p l a n t s t h a t cope with

nitrogen l i m i t a t i o n do so so e f f e c t i v e l y that they produce almost a s much as those that have no nitrogen l i m i t a t i o n s .

C e r t a i n l y t h e i r suimed growth

increments are almost as g r e a t in S t . M a r g a r e t ' s Bay a s t h e y a r e a t s i t e a t the south end of the province.

the

This i s of i n t e r e s t because, there

i s a reference made to Ttiny Chapman's e x p e r i m e n t i n which he

increased

growth r a t e in simmer by adding n u t r i e n t s in S t . Margaret's Bay.

There i s

a snag t o t h i s and t h a t i s t h a t , w h i l e t h o s e p l a n t s were growing

in

summer, they were not building up carbon r e s e r v e s and so they did not grow in f a l l when t h e i r c o l l e a g u e s — the ones growing on the carbon —

did.

I t looks as i f

reserves

the combined s t r a t e g i e s of s t o r i n g nitrogen and

s t o r i n g carbon can lead to as much production in the c o u r s e of a y e a r a s by p l a n t s which are not s u b j e c t to n u t r i e n t l i m i t a t i o n in summer. BIEWELL:

There i s a p a r a l l e l here from our work with Chondrus in c u l t u r e .

If you add a s u b s t a n t i a l amount of nitrogen you do indeed g r e a t l y increase the growth of the p l a n t s b u t , a s you would g u e s s , you do not g e t v e r y l a r g e carbohydrate formation a t the same time, and since we are i n t e r e s t e d in carbohydrate t h i s i s r e a l l y counter-productive.

However, you can t a k e

the p l a n t s , make them grow f a s t by adding n i t r o g e n , and by a subsequent treatment in which you put the p l a n t s under r a t h e r s t r e s s f u l

conditions

f o r a short period of time, you can g e t than to convert a l a r g e proportion o f t h e c a r b o n t h a t t h e y l a i d down, much o f carbohydrates.

i t a s p r o t e i n , back

into

In other words, i t seens t h a t they do have the c a p a c i t y to

backtrack a t t h a t point and prepare f o r an expected bad s i t u a t i o n .

Well,

then you simply harvest them when they have a maximim carbohydrate l e v e l . You can indeed b e n e f i t from both s i d e s of t h a t q u e s t i o n , f i r s t , by adding the n i t r o g e n t o make them grow b i g ,

then w i t h h o l d i n g i t t o make them

convert p r o t e i n s into carbohydrates. I was a l s o i n t e r e s t e d

in your o b s e r v a t i o n a b o u t l e a k a g e .

We d i d some

experiments with a u n i c e l l u l a r f r e s h w a t e r a l g a , O o c y s t i s , growing in a rather eutrophic s i t u a t i o n in Hamilton Harbour. photosynthesis and so c a l l e d p h o t o r e s p i r a t i o n . c u l t u r e s , made them e s s e n t i a l l y

We were interested in i t s We c a r e f u l l y p u r i f i e d

the

u n i - a l g a l and a x e n i c , as nearly as we

c o u l d , and found that there was no l e a k a g e o f carbon and no d e t e c t a b l e

26 photorespiration.

This was of concern because Harris had done many

experiments on algae from Hamilton Harbour and had found that there was a l o t of photorespiration and a l o t of leakage.

So we thought i f we added

Hamilton Harbour back t o these algae they might be happier.

And sure

enough, as soon as we added back some o f the b a c t e r i a , and other much smaller algae or a l g a l - l i k e organisms that were in t h e r e , the Oocystis then began not only t o leak carbon in large amounts but this carbon was then oxidized by these symbiotic organisms, and we g o t the appearance of photorespiration.

I t was not true photorespiration, in f a c t , but i t vould

seem that, in the absence o f associated h e t e r o t r o p h i c organisms, the Oocystis did not leak any carbon, i t j u s t kept i t that there may be inter-organism

inside.

It

suggests

mechanisms which could control leakage.

This may be one of the reasons why so many widely d i f f e r e n t r e s u l t s have been obtained, sometimes frcm the same organism, regarding the leakage of glycolate and other similar organic compounds into the sea. MANN:

Well, that is an interesting angle.

Fran the papers I have seen,

no one seems to have stated under what nitrogen conditions they carried out experiments to determine carbon leaks; i t does seem t o me that there i s a l o t of c i r c u m s t a n t i a l evidence pointing to release of carbon when growth is limited by nitrogen shortage and not a t other times.

If

the

experiments are done with an abundant n i t r o g e n then there may be no release. BIDWELL:

The same situation applies to lichens where the export of carbon

from algae increases dramatically at the time the lichens are undergoing their most active growth or sporulation and again there would seem t o be some inter-organism communication system which suggests the control of leakage — w e l l , leakage may be the wrong word — l o s s of excess carbon perhaps. DOBBAN:

Dr. Mann, how does reproduction t i e i n t o your s t o r y ?

When do

these d i f f e r e n t plants reproduce and how much carbon goes into i t ?

I

wonder i f that is where your 30% carbon went. MANN:

Tbny Chapman claims to have calculated that no more than about 1%

o f the carbon budget of these algae goes into reproduction.

That i s the

27 reason ws l e f t i t out. LOBBAN:

I wonder i f they keep growing a t the same r a t e , while t h e y

reproduce? MANN:

Klaus Luning made the point that f a l l i s the time when they produce

t h e i r s o r i and f a l l

i s the time they a l l show a dip in growth.

But we

have taken our cue from Tbny Chapman whose e s t i m a t e s o f the biomass o f products r e l e a s e d do not allow f o r much more than 1%.

Ihese actively

growing plants replace t h e i r blade 5 times or more per y e a r .

Thus, the

biomass of a blade i s no more than 20% of t o t a l production, and since the biomass of the spores released i s only 5% of the biomass of the blade then you are down to 1% of the annual production. LOBBAN:

They also tend to lose quite a l o t o f t i s s u e along with spores

and the whole sorus goes as w e l l .

Also, some o f them reproduce

all

through the winter as well, do they not? MANN:

I have very l i t t l e doubt t h a t the d i p in growth in f a l l

is

a s s o c i a t e d with the reproduction phase, but what i s so striking to me i s that sane cease growth completely in the f a l l and sane grow l i k e seeds — drawing down t h e i r carbon reserves. SCHMITZ:

Gould you t e l l us where in the c e l l or the p l a n t mannitol

is

being stored? MANN: No, sorry. DUNCAN:

In the measurements o f nitrogen in the ambient water was i t

always j u s t n i t r a t e , or were t h e r e any measurements made o f o r g a n i c nitrogen as well? MANN: ND. and n i t r i t e . DUNCAN:

I t i s a l l inorganic nitrogen, but i t included ammonia, n i t r a t e Nobody has looked a t organic nitrogen in the water.

I ask t h i s because, there was an 11 year study from Plymouth in

the English Channel showing that when the inorganic nitrogen was down the organic nitrogen was up and, i f i t was averaged out over a long period o f t i m e , t o t a l nitrogen was e s s e n t i a l l y the same a l l the time,

they were

28 looking primarily at planktonic organisms and found that those that could utilize organic nitrogen did so and increased their population while the other plankton, which used the inorganic nitrogen, were going down. There is a possibility of the same thing happening with the larger algae. MANN:

I have heard this to explain dinoflagellates replacing diatoms.

Dinoflagellates seem to be able to use organic nitrogen.

Ebr macroalgae,

Ibny Chapman wanted to test the idea that the only way these algae could survive in the Arctic was by taking up organic materials, that is, by being heterotrophic, and he felt that he had demonstrated adequately that there is enough photosynthesis to account for the observed production. All you need to invoke for winter growth is the utilization of carbon reserves. DUNCAN: And no uptake! Maybe both! MANN:

There are people in oceanography at Dalhousie, who work with

organic nitrogen in the water who say that most of it is refractory, in the sense that bacteria can not get at it.

So they wonder what else

does? DUNCAN: Naval Antia showed that a number of planktonic organisms other than dinoflagellates take up and use amino acids. MANN: Yes, we had a colleague from Southhampton with us for a term, and he maintained that as soon as any amino acid was released into the water there were bacteria ready to take it.

And that they were much more

efficient at taking it up than any plant that you could name. DUNCAN: Then they can release ammonium which is available to the plants. MANN: Yes, sure. But then you are back to inorganic cycles.

ENGELMANN'S THEORY: THE COMPELLING LOGIC

J . Ramus Botany Department and Marine Laboratory, Duke University Beaufort, North Carolina, USA 28516

The coincidence of wavelengths of maximum transmission through the marine water column (green l i g h t ) and maximum photosynthesis by red seaweeds (green light) crystallized a compelling logic, the theory of complementary chromatic a d a p t a t i o n .

The theory was o r i g i n a l l y proposed by T.W.

Engelmann (1,2) after he produced crude photosynthetic action spectra f o r nunerous algae by an ingenious 0 2 bioassy and a projected microspectrum. The action spectra of the green algae and diatoms were relatively inactive at wavelengths between the two major absorption peaks of chlorophyll, the s p e c t r a l region here designated the "green window."

However, the action

spectra of red algae were remarkably d i f f e r e n t : p h o t o s y n t h e s i s was sensitized in the green window, the s p e c t r a l region of phycoerythrin absorption.

The p r i n c i p l e presumably limiting the lower extreme o f

seaweed v e r t i c a l d i s t r i b u t i o n was the a b i l i t y to capture light in the green window.

Thus the color of red seaweeds carrying large quantities of

phycoerythrin relative to other antenna pigments complements the color of the submarine l i g h t f i e l d .

Engelmann's t h e o r y was sensu

strictu

genotypic, making no allowance for phenotypic color p l a s t i c i t y , and applied solely to seaweeds.

In addition, the theory was a priori: the red

seaweeds had evolved for effective light absorption in the deep sea. Engelmann's theory here s h a l l be distinguished from i t s

extension,

formulated by his student Gaidukov (3), to include phenotypic adjustment in the pigment systems of prokaryotic algae to colored l i g h t .

He observed

that blue-green algae became green in red l i g h t , blue in green l i g h t , yellow in blue-green l i g h t and blue in yellow l i g h t .

Oddly enough,

Engelmann's f i r s t theory was formulated for seaweeds, yet his observations were based on only two filamentous red (Callithamnion and Ce rami urn) and possibly one green (Cladophora) seaweed; the bulk of the species observed

© 1982 Walter de Gruyter & Co., Berlin • New York Synthetic and Degradative Processes in Marine Macrophytes

30 were diatoms and freshwater green algae.

Nevertheless, subsequent work by

others fleshed out the theory, although i t was subject to criticism.

The

controversy is exhaustively reviewed in the treatise on photosynthesis by Rabinowitch (4,5) and w i l l not be recapitulated here. of

A f u l l exposition

the theory p r o v i d e s that the green seaweeds ( c a r r y i n g

only

chlorophylls) occupy the most restricted vertical distribution (a narrow zone near the surface), the browns (carrying fucoxanthin) an intermediate d i s t r i b u t i o n and the reds (carrying phycoerythrin) the greatest vertical distribution.

The implications were clear: the vertical d i s t r i b u t i o n of

seaweeds i s imposed solely by the color of the submarine light f i e l d and there e x i s t s no other mechanism f o r f i l l i n g insertion of b i l i p r o t e i n s .

the green window save

The logic is so manifest that the theory has

persisted in the face of considerable contrary evidence.

For example,

believing Engelmann fundamentally correct, the esteemed Rabinowitch (5) summarized:

"WDuld i t not be strange i f the appearance of orange or red

pigments in deep-water algae would only be a coincidence, and these pigments were helpless in performing the task so obviously set to the plants by the character of the light in which they l i v e — to catch and u t i l i z e for their maintainance and propagation radiations in the middle of the v i s i b l e spectrum, which are the only ones to reach them in some intensity." Tt> detract from Ehgelmann is a mistaken enterprise.

His experiments in

photobiology were bold f o r the time arri elegant in design.

Indeed, the

experiments have withstood the scrutiny of r e p e t i t i o n in the subsequent century.

Yet Engelmann's theory as i t flowed from the primary data was

just that, a theory, an axiom he never tested.

The inference was so

strong that in the last century the theory has been elevated to principle without appropriate verification.

Now the burden of testing Engelmann's

hypothesis, or of devising alternative hypotheses, is l e f t to us. In the broadest sense, we are seeking mechanisms which structure seaweed communities.

Seaweeds are a l i f e form, and their assignment to three

divisions of the plant kingdom acknowledges their separate phylogenies. I t follows that unique genetic repertoires have in the course of evolution converged in niches broadly similar in terms of physical properties and b i o l o g i c a l interactions.

The distinct genetic repertoires are manifest

31 biochemically in the structure of the photosynthetic unit (PSU), primarily in the kinds of pigments in the antenna since we know the rest of the PSU to be evolutionarily conservative (6).

Engelmann of course knew nothing

of the modern concept of the PSU, but he knew of the major differences in antenna pigments giving red, green and brown seaweeds.

The theory of

complementary chromatic adaptation then serves as the basis f o r the formulation of a s p e c i f i c (and t e s t a b l e ) question: does the spectral d i s t r i b u t i o n of the submarine light f i e l d structure seawsed communities? I t is this question which we shall now systematically address.

However,

the question is immediately exacerbated by the simple f a c t that the absorption of light by a water column is wavelength dependent, v i z . change in l i g h t quantity with depth is always accompanied by change in spectral distribution.

Further, water columns vary in the wavelength of maximum

transmission, hence the optical codification of coastal and oceanic waters by Jerlov (7).

Any attempt to assess the structuring r o l e of

spectral

d i s t r i b u t i o n must separate color from quantity and examine their e f f e c t s independently. There are at l e a s t two broad approaches to the analysis of community structure.

In the f i r s t , we assume that Nature has already tested the

hypothesis - we need merely to analyse e x i s t i n g v e r i f i c a t i o n or negation. descriptive ( 8 ) .

communities

for

The analysis may be either functional or

In the functional analysis, component species are

experimentally manipulated, usually removed, then the impact on total corariunity structure is appropriately assessed over time.

The works of

Paine and Vadas ( 9 ) , Dayton ( 8 ) , Lubchenco (10) and Sousa (11) leave us with a sobering thought - seaweed community structure is a consequence of many mechanisms acting in concert, v i z . physical factors, plant-plant interactions and plant-animal interactions.

The notion in question here,

that l i g h t , and s p e c i f i c a l l y the color of l i g h t , is the sole mechanism structuring seaweed conmunities is summarily naive. that l i g h t plays a role.

There is no question

This is easily demonstrated by removing canopy

species, upon which the obligate understory species die out and the newly opened free space is occupied by fugitive species (8).

Whether i t is the

quantity of l i g h t (photon flux density, PFD) or q u a l i t y distribution) remains moot.

(spectral

32 D e s c r i p t i v e a n a l y s i s of community s t r u c t u r e i s h e r e the b a t h y m e t r i c q u a n t i f i c a t i o n of r e l a t i v e

s p e c i e s a b u n d a n c e s , o r p e r h a p s more

appropriate, c l a s s abundances, i n s o f a r as we a d d r e s s c l a s s s p e c i f i c PSU structure.

Tt> our p u r p o s e , we are immediately confronted by a profound

dilemma - in what u n i t s a r e abundances t o be measured: t h e number of i n d i v i d u a l s p e r s p e c i e s , % cover p e r s p e c i e s , biomass per s p e c i e s , productivity per species?

Said o t h e r w i s e the q u e s t i o n i s : on which of

t h e s e parameters does natural s e l e c t i o n operate?

Although c r u c i a l t o the

d i s c u s s i o n , t h i s i s not to be resolved here. Nevertheless, there are several bathymetric analyses of seaweed v e r t i c a l d i s t r i b u t i o n which a r e of use t o t h i s t r e a t i s e .

Larkum e t a l .

(12),

i n v e s t i g a t i n g a c l i f f in c l e a r oceanic water of the Mediterranean, found t h a t brown seaweeds dominated the upper 15 m, greens dominated from 15 t o 75 m ( t h e bottom) and the reds made l i t t l e contribution to bicmass a t any depth,

l b extrapolate the occurence of seaweeds below 75 m, the z o n a t i o n

in a submarine cave was q u a n t i f i e d .

Here, siphonaceous green seaweeds

p e n e t r a t e d some d i s t a n c e i n s i d e on the n e a r - v e r t i c a l w a l l s and were e v e n t u a l l y replaced by encrusting red species.

The t r a n s i t i o n from green

to red species occurred a t the PFD equivalent to a depth a t 80 m o u t s i d e t h e c a v e , with r e d s becoming sparse a t the equivalent of 120 m. i n s t a n c e , red seaweeds penetrated "deeper" than o t h e r t y p e s .

In t h i s However,

Larkum e t a l . (12) calculated t h a t the proportion of l i g h t a v a i l a b l e f o r p h o t o s y n t h e s i s by red and g r e e n seaweeds a t v a r i o u s d e p t h s i n t h e s e oceanic Jerlov type I I (7) waters i s v i r t u a l l y t h e same down t o 150 m, w e l l below t h e compensation depth f o r a l l species.

They a l s o noted t h a t

in the submarine cave and on t h e n o r t h - f a c i n g c l i f f , l i g h t i s a l r e a d y c o n f i n e d to the blue wavelengths well before the t r a n s i t i o n from green t o red seaweeds o c c u r s .

Thus, Larkum e t a l . (12) concluded t h a t

light

quantity r a t h e r than s p e c t r a l d i s t r i b u t i o n was the major regulating f a c t o r f o r the p a t t e r n of v e r t i c a l d i s t r i b u t i o n . Doty e t a l . (13) investigated v e r t i c a l d i s t r i b u t i o n of Hawaiian seaweeds seaward of the algal r i d g e . The r a t i o of brown to green species below both 10 m and 50 m was 0.5 and below 90 m was 0.0.

The r a t i o of red t o g r e e n

s p e c i e s below 10 m was 3.0 , below 50 m was 2.2 and below 90 m was 0 . 7 .

33 Thus, there was a progressive enrichment f o r green s p e c i e s with depth. Titlyanov (14), working in the turbid waters of the Gulf of S t . Peter the Great, reported the r a t i o of brown to green species at depths l e s s than 2 ra to be 2.1 and at depths exceeding 12 m to be 1.7, while the r a t i o of red to green at depths l e s s than 2 m to be 3.4 and g r e a t e r than 12 m to be 3.7. Hs concluded by stating that the species composition a t the g r e a t e s t depths of habitation i s not correlated with taxonomic groups of s p e c i f i c pigmentation i . e . , c l a s s e s . Schneider ( 1 5 ) , studying the s p a t i a l d i s t r i b u t i o n s of seaweeds on the c o n t i n e n t a l s h e l f of the C a r o l i n a s , reported t h a t 25% of the green species, 16% of the brown and 28% of the reds occurred at depths greater than 50 m. Early on, Oltmanns (16) proposed t h a t the v e r t i c a l d i s t r i b u t i o n s e a w e e d s was c o n t r o l l e d by l i g h t q u a n t i t y a l o n e .

of

Titlyanov (14)

i n v e s t i g a t e d a contiguous shallow g r o t t o and submarine c l i f f .

The

s i t u a t i o n appeared an i d e a l t e s t : the grotto represented a gradient in l i g h t quantity alone while the adjoining submarine c l i f f represented both a g r a d i e n t in l i g h t quantity and a change in spectral distribution.

The

species common to neighboring w e l l - l i t areas are absent inside the grotto. The g r o t t o supports s h a d e - t o l e r a n t s p e c i e s which grow nearby in deep crevasses or underwater c l i f f s .

A c r i t i c a l o b s e r v a t i o n i s t h a t the red

seaweeds P t i l o t a f i l i c i n a and Symphocladia l a t i u s c u l a living in the grotto and in c l i f f c r e v a s s e s near the s u r f a c e appear again in the open a t a depth of 10-15 m.

Thus, Titlyanov (14) concludes that their occurrence i s

a function of light quantity alone and the change in spectral distribution i s a minor contributor to the v e r t i c a l s t r a t i f i c a t i o n of seaweeds.

It

should be noted here t h a t the s p e c t r a l d i s t r i b u t i o n of l i g h t along a horizontal line of sight i s skewed in favor of shorter wavelengths, v i z . sidewelling l i g h t entering a submarine grotto i s predominantly blue (17)! Always irksome in t h i s context a r e the pavement r e d s , which, when occurring in a f l o r a , appear to p e r s i s t to depths where no other seaweeds exist.

Goreau (18) has speculated that the success of encrusting r e d s a t

low PFD i s due to a slow r a t e of a t t r i t i o n r a t h e r than to exceptional u t i l i z a t i o n of available l i g h t .

Their c a l c a r e o u s nature and p r o s t r a t e

habit i s a deterrent to both grazing and physical erosion.

In conclusion,

i t would appear that analyses of community s t r u c t u r e , whether using the

34 functional or descriptive approaches, f a i l to v e r i f y Engelmann's t h e o r y . However, i t i s not l i k e l y ( f o r the reasons noted) t h a t the e x i s t i n g analyses of seawsed community structure could e i t h e r verify or negate the paradigm. The second approach to testing Engelmann's theory i s experiment designed to e l i m i n a t e a l l s t r u c t u r i n g mechanisms save l i g h t , then i s o l a t e the e f f e c t s o f s p e c t r a l d i s t r i b u t i o n from PFD.

These are t e s t s

focused

s p e c i f i c a l l y on the PSU, c o r r e l a t i n g i t s s t r u c t u r e and function with depth.

For example, Levring (19) noted t h a t p h o t o s y n t h e s i s and PFD

decrease

i n a l o g a r i t h m i c manner with depth.

He reasoned t h a t

if

photosynthesis at tvro d i f f e r e n t d e p t h s , m and n , a r e P^ and P n and the corresponding PFD values are Em and E^, then the quotient of the slopes of the two curves,

can be calculated by the relationship: log (P m /P n ) ^m-n log < V * h >

I f photosynthesis were independent of spectral composition but dependent on PFD, then P and E vrould reduce proportionally with depth and q = 1.

If

photosynthesis wsre dependent on spectral composition and indpendent o f PFD, then P and E would not reduce proportionally with depth and q would be g r e a t e r o r l e s s impoverishment

than u n i t y ,

q > 1 representing

and q < 1 r e p r e s e n t i n g

a spectral

a

spectral

enrichment.

Photosynthesis (P) and i r r a d i a n c e (E) were measured a t depths m and n where E was l e s s than s a t u r a t i n g . coastal

Levring compared q values in turbid

(green) and c l e a r o c e a n i c waters ( b l u e )

continental s h e l f .

over the

Carolina

For Ulva lactuca in n e r i t i c waters q increased with

depth from 0 . 9 2 t o 1 . 3 5 ( p r o g r e s s i v e s p e c t r a l impoverishment) and in oceanic water q decreased from 1.18 to 0.79 ( s p e c t r a l e n r i c h m e n t ) .

For

G r a c i l a r i a f o l i i f e r a in n e r i t i c waters q decreased from 0 . 9 9 to 0 . 7 2 (progressive

spectral

enrichment)

and i n o c e a n i c water q v a r i e d

e r r a t i c a l l y and the data are best interpreted as s p e c t r a l

indifference.

For Fucus vesiculosus in both n e r i t i c and oceanic waters the q v a l u e s a r e b e s t i n t e r p r e t e d as spectral indifference. the expectations of the theory.

These data do not conform to

Dring ( 2 0 ) , using a computer model which

35 calculated photosynthesis from a c t i o n s p e c t r a and underwater s p e c t r a l d i s t r i b u t i o n d a t a , simulated photosynthesis as a function of depth and water type for many species of seaweeds.

Ihe species of red seaweeds were

b e s t adapted chromatically to photosynthesize at a l l depths (including 0 m) in a l l except the c l e a r e s t o c e a n i c w a t e r s .

The r e s u l t s show l i t t l e

c o r r e l a t i o n with p a t t e r n s o f v e r t i c a l distributions of green, brown and red seaweeds, and Dring (20) concludes t h a t complementary chromatic adaptation may be of l i t t l e importance in determining v e r t i c a l zonation of seaweeds.

As i f anticipating t h i s c o n c l u s i o n , Drew ( 2 1 ) c a l c u l a t e d an

increase in photosynthetic e f f i c i e n c y f o r Udotea p e t i o l a t a (a g r e e n ) , Peysonnelia squamaria (a red) and Padina pavonia (a brown) with depth, in that order, in the clear oceanic waters o f f Malta. I f in f a c t p h o t o s y n t h e s i s and p a t t e r n s o f v e r t i c a l d i s t r i b u t i o n

are

dependent p r i m a r i l y on l i g h t quantity and independent of l i g h t quality, then green and brown seaweeds must also have e f f e c t i v e mechanisms f o r the absorption o f l i g h t in the green window (500-600 nm).

Brown seaweeds

carry both fucoxanthin, the in vivo absorption spectrum o f which extends out to 580 nm, and chl c , and e f f e c t i v e l y absorb the e n t i r e PAR s p e c t r a l domain.

Yokohama and Kageyama ( 2 2 ) , comparing the in vivo absorption

s p e c t r a o f f i v e shallow-water and six deep-water green seaweed species, discovered an unexpected absorption peak a t 540 nm in deep-water s p e c i e s which was not present in shallow-water species.

Ihe pigment absorbing

l i g h t in t h i s region was identified chromatographically as siphonoxanthin. Siphonoxanthin i s c h a r a c t e r i s t i c of many siphonaceous greens (23), so the occurrence of the pigment in s p e c i e s o f Cladophora, Codium and Valonia came as no surprise. unexpected.

Ihe f a c t that Ulva japonica c a r r i e d the pigment i s

Kageyama e t a l . (24) showed an e f f i c i e n t e x c i t a t i o n energy

transfer from siphonoxanthin to chl a by fluorescence emmission s t u d i e s . In U. japonica, the e f f i c i e n c y of transfer a t 540 nm i s 50% g r e a t e r than for the shallow water species U. pertusa containing no siphonoxanthin. Photosynthetic e f f i c i e n c y for U. japonica collected from a depth o f 20 m was equal in "green l i g h t "

(simulated deep c o a s t a l water) t o "white

l i g h t , " whereas U. pertusa from shallow water was 30% l e s s e f f i c i e n t "green l i g h t " than in "white l i g h t " ( 2 2 ) .

in

There seems l i t t l e doubt t h a t

U. japonica has evolved an e f f i c i e n t mechanism f o r the capture o f green

36 l i g h t , allowing the species to e x i s t in spectrally limited l i g h t f i e l d s . The siphonaceous green Ostreobium growing endolithically in the coral head Favia and beneath the outer layer of zooxanthellae is subject to a unique spectral

distribution.

PAR w a v e l e n g t h s r e l a t i v e t o f a r - r e d are

transmitted through zooxanthellae in very low r a t i o s ( 2 5 ) .

Action spectra

for Ostreobium reveal that the "red drop" does not occur near 700 nm as expected, but r a t h e r beyond 740 nm.

I t would appear t h a t Ostreobium

p o s s e s s e s c h l o r o p h y l l , both antenna and trap, with absorption maxima in the far-red. I t has been demonstrated that the green window in seaweeds can be f i l l e d by c h l o r o p h y l l absorption alone.

The argument i s a complex one and has

been put forth in d e t a i l elsewhere ( 2 6 ) .

In summary, as the concentration

o f chlorophyll increases, the spectral region of low s p e c i f i c absorptance ( t h e green window) i n c r e a s e s more r a p i d l y than the r e g i o n s o f s p e c i f i c absorptance (the blue and red absorbing r e g i o n s ) .

high

Thus, Ulva

lactuca with high c o n c e n t r a t i o n s o f c h l o r o p h y l l absorbs l i g h t almost uniformly a c r o s s PAR wavelengths (26).

IVbst seaweeds, however, are very

heterogeneous systems with r e s p e c t t o the absorption o f l i g h t .

The

absorptance of the siphonaceous green seaweed Codium f r a g i l e i s always near 1, independent o f chlorophyll concentration within natural ranges, due to the e f f e c t s of heterogeneous absorption and multiple s c a t t e r

(26).

The u t r i c l e s forming the c o r t i c a l tissue enhance the capture of incident l i g h t due, among

other t h i n g s , to the waveguide f u n c t i o n o f the t h i n

peripheral layer of cytoplaan in which the chloroplasts are arrayed. the length o f the o p t i c a l

path i s made l a r g e r and i n c r e a s e s

Thus the

p r o b a b i l i t y of photon - chloroplast encounter, independent of wavelength. Many siphonaceous greens have a c o r t e x o f s i m i l a r s t r u c t u r e ,

perhaps

c o n t r i b u t i n g to t h e i r presence in even the weakest of submarine l i g h t fields. I t i s well known that antenna pigment concentration varies inversely with PFD, e . g . the pigment concentration of a species in a strong l i g h t f i e l d compared t o a weak l i g h t f i e l d i s lower, a s i s u s u a l l y the r a t i o o f a c c e s s o r y t o primary antenna pigments.

These quantitative adjustments

a r e , within genotype, a phenotypic expression of PSU p l a s t i c i t y .

37 The PSU of red seaweeds has undergone the most intensive investigation, prompted by the phenotypic corollary of Engelmann's axiom.

In this

context, his theory is extended to include phenotypic adjustments to light quality and/or quantity.

Aside from chl a, the red seaweed antenna

contains phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (APC). Here, the specifics for the model of the PSU are controversial.

Initially

it was thought that the role of the APC was solely to transfer excitation energy from phycobilisomes to the traps, but Larkum and Weyrauch (27) have shown that APC harvests a substantial amount of light.

Further, larkum

and Weyrauch (27) maintain that the phycobiliproteins are in a sense independent of both photosystems and transfer absorbed light energy preferentially to PS II with excess energy spilling over to PS I.

On the

other hand, Ley and Butler (28) suggest that the phycobiliproteins are associated entirely with PS II. Waaland et al. (29) demonstrated that adjustment of the PSU in Griffithsia pacifica is effected by PFD and not by spectral distribution. PE/chl a ratios (mg/mg) varied from 0.67 at 1000 f.c. to 1.8 at 8 f.c., the greatest variation occurring below 300 f.c. The correlation between phycobiliprotein concentration and phycobilisome density was tight, indicating that perhaps the PSU number varied here.

Ramus et al. (30)

reported for Chondrus crispus and Porphyra umbilicalis in situ an increase in total antenna pigment concentration and a PE/ chl a and PC/ chl a enrichment with decreasing PFD.

Plants suspended at varying depths in the

water column (where both spectral distribution and PFD change) were compared with plants frcrn intertidal habitats where only the PFD changed. Antenna pigment concentration varied in a similar manner, leading to the conclusion that these red seaweeds were responding to PFD alone. The adjustment of the PSU was reversible and did not require cell division. Rhee and Briggs (31) reported that the chlorophyll variation in Chondrus crispus was rot correlated with depth, but PE did increase with depth. The evidence indicated that the phycobiliprotein/ chl a variations are correlated with PFD, yet it is not known whether different spectral distributions of equal quantum flux density will produce the same adjustments.

38 For green seaweeds, an i n c r e a s e in t o t a l c h l o r o p h y l l and c h l b/ c h l a r a t i o i s correlated with a decrease in PFD ( 2 2 , 2 6 , 3 0 , 3 2 ) . r a t i o s v a r y from 0 . 4 4

Chl b/ c h l a

i n high PFD environments to 1 . 1 2 in low PFD

environments, while t o t a l chlorophyll can vary up to 1 5 - f o l d in ulva and 8-fold in Codium (26).

Apel e t a l ^ (33) r e l e a s e d PS I I from the PSU o f

Acetabularia mediterranea by d e t e r g e n t treatment and showed i t to be enriched in chl b.

In that the chlorophyll-protein complex photoreduced

dichlorophenolindophenol, but not NADP+, they concluded i t was PS I I . Siphonoxanthin transfers absorbed energy to chl a in Ulva j a p o n i c a ( 24) , yet

its

photosystem

association

i s unkown.

Molar r a t i o s

siphonoxanthin/ c h l a range from 0 . 2 5 in Codium f r a g i l e Valonia macrophysa.

to 0 . 3 5

of in

Carotenes, of course, are present in abundance in the

c h l o r o p l a s t s o f green seaweeds ( 2 3 ) , but as evidenced from enhancement spectra (34,35) and fluorescence e x c i t a t i o n measurements ( 2 4 ) , they may participate l e s s e f f i c i e n t l y in energy t r a n s f e r . The t h r e e major antenna pigments in the brown seaweeds Ascophyllum nodosum and Fucus vesiculosus increase with decreasing PFD.

However, c h l

c / chl a r a t i o s remained c o n s t a n t , while f u c o x a n t h i n / c h l a a c t u a l l y declined; an increase in fucoxanthin was not as g r e a t as in c h l a f o r these seaweeds (36). Mishkind and Mauzerall (6) have s p e c i f i c a l l y addressed the notion o f l i g h t - m e d i a t e d s t r u c t u r a l modification of the PSU.

Their d e f i n i t i o n of

PSU size i s a f u n c t i o n a l one and c o n s i s t e n t with t h a t o f Emerson and Arnold ( 3 7 ) : the molar r a t i o o f c h l o r o p h y l l t o 0^ formed in a s i n g l e s a t u r a t i n g turnover flash (in t h i s case the flashes were pulsed a t 100 ms intervals).

The average PSU s i z e f o r the green seaweeds i s s i m i l a r to

that of Chlorella and higher p l a n t s , v i z . 1500-2500 c h l / O2 and about 1 / 3 t h a t s i z e f o r brown and red seaweeds.

In the browns and reds, a large

complement of non-chlorophyll accessory pigment i s compensated f o r by a reduction in c h l o r o p h y l l c o n t e n t , but by no means do accessory pigments replace a l l the c h l o r o p h y l l .

The data o f Mishkind and Mauzerall

(6)

suggest that Ulva lactuca adjusts i t s PSU to low PFD in the same manner as higher p l a n t s , i . e . by increasing the number of PSUs rather than antenna size.

For Ulva, PSU s i z e

chlorophyll concentration.

is

independent over a 4 - f o l d range

Ft>r porphyra umbilicalis, PSU s i z e

of

increases

39 with chlorophyll content as PFD decreases as does apparently the number of "traps." There are in some cases s t r u c t u r a l m o d i f i c a t i o n s o f the c h l o r o p l a s t or c e l l which accompany a d j u s t m e n t in the PSU.

Unfortunately, the e f f e c t s

of structural modifications on quantum capture capacity or photosynthesis have not been q u a n t i f i e d , leaving t h e i r significance to be demonstrated. Ulva fenestrata taken from the dim r e c e s s e s o f a submarine g r o t t o had l a r g e r c h l o r o p l a s t s with denser thylakoid packing than c o u n t e r p a r t s growing in the i n t e r t i d a l ( 3 8 ) .

An increase in phycobilisome d e n s i t y was

correlated with a decrease in PFD in G r i f f i t h s i a p a c i f i c a ( 2 9 ) , while the s i z e o f p h y c o b i l i s o m e s remained c o n s t a n t .

C h l o r o p l a s t s o f the

siphonaceous green seawsed Halimeda tuna growing a t 16 m were l a r g e r than those of the same species growing near the surface; however, no difference was noted in the lamellar system ( 3 9 ) .

Deep-water p l a n t s have t h i n n e r

segments, l a r g e r u t r i c l e s and t h i n n e r c e l l w a l l s than shallow-water p l a n t s , and the c h l o r o p l a s t s are d i s t r i b u t e d over a wider area but in fewer layers (40). Variations in the PSU have been c o r r e l a t e d with _in s i t u p h o t o s y n t h e s i s (26,36,41).

Gomparing photosynthesis normalized t o c h l a the apparent

e f f i c i e n c y of low PFD acclimated seaweeds d i f f e r s frcm high PFD acclimated counterparts.

At the surface, the high PFD acclimated seaweeds a r e most

e f f i c i e n t , while at depth the low PFD seaweeds are most e f f i c i e n t .

When

in s i t u photosynthesis i s normalized to biomass, the compensation depth for low PFD seaweeds i s always g r e a t e r .

Thus, an adjustment in the PSU to

low PFD enhances photosynthetic performance a t depth, and t h i s i s true for a l l seaweed c l a s s e s . Given the richness of mechanisms f o r the capture o f l i g h t in the green window d e s c r i b e d h e r e , i t appears that green and brown seaweeds, as well as reds, have in the course of evolution undergone complementary chromatic adaptation.

Where then was Engelmann's logic erroneous?

Action spectra

for photosynthesis are t r a d i t i o n a l l y produced with monochromatic The s p e c t r a have been v e r y u s e f u l

light.

f o r deducing the mechanisms o f

photosynthesis, e . g . the celebrated "red drop" and "Ehierson enhancement."

40 .8

.1

O

.

Cryptopleura crispa

phallus

0

500

400

600 wavelength (nm)

700

F i g . Is Absorptance and action s p e c t r a ( w i t h and without 546 nm supplementary background light) for the red seaweed Cryptopleura crispa. Redrawn from Fork (35). But for e c o l o g i c a l purposes, the c l a s s i c a l method may be misleading. Submarine l i g h t f i e l d s , at least at or above the compensation depth for seaweeds (here set at tvro to three orders of magnitude below surface PFD) are always broadband (42). function of water type.

Further, spectral distribution varies as a

In Jerlov I (clear oceanic) waters, the spectral

d i s t r i b u t i o n at compensation depth is 400-520 nm with maximum PFD at 480 nm (blue^green l i g h t ) .

At the other extreme, in Jerlov 9 (turbid neritic)

waters, spectral distribution at compensation is 500-680 nm with maximum PFD at 580 nm (yellow-orange l i g h t ) .

Thus submarine light f i e l d s are not

as spectrally pure as conceived in Engelmann's theory. In that the s p e c t r a l d i s t r i b u t i o n o f

submarine l i g h t above the

photosynthetic compensation depth i s always broadband and of variable c o l o r , action spectra might more a p p r o p r i a t e l y be produced with supplementary weak background l i g h t . revealing.

When done so, the results are

For example, in the red seaweed Cryptopleura crispa the action

spectrum produced with 546 nm supplementary light more closely " f i t s " the

41 absorptance spectrum than one produced in the absence of supplementary light (Fig. 1, reference 35).

It can be inferred frcm action spectra with

background light near 680 nm of the green Ulva and the brown Endarachne binghamiae that the decline in photosynthesis in the spectral range of the green window is not so severe as in monochromatic light (35).

Had

Engelmann produced his photosynthetic action spectra for red seaweeds in supplementary background light, he may never have logically inferred the theory of complementary chromatic adaptation.

Acknowledgements I wish to thank Matt Dring and Klaus liming for stimulating discussion, and Ken Mann and Anne Walter for critically reading this manuscript.

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31.

Rhee, C., Briggs, W.R.:

Vision Res. 15, 1063-1070 (1975).

Ann. N.Y. Acad. Sei. 1091, 127-167 (1963). Bot. Mar. 11, 72-80 (1968). Limnol. Oceanogr. 26, 271-284 (1981). Proc. Intl. Seaweed Symp. 6, 151-159 (1969). Bot. Mar. 20, 433-436 (1977).

J. Phycol. 5, 281-284 (1969).

Biol. Bull. 134, 411-424 (1968).

J. Phycol. 14, 352-362 (1978). Photochem. Photobiol. 25, 65-72

Biochim. Biophys. Acta 462 , 290-294 J. Phycol. 10, 193-198 Mar. Biol. 37,

Bot. Gaz. 138, 123-128 (1977).

32.

Keast, J.F., Grant, B.R.:

33.

Apel, K., Bagorad, L., Wbodcock, C.L.F.: 568-574 (1975).

J. Phycol. 12, 328-331 (1976).

34.

Haxo, F.T., Blinks, L.R.:

35.

Fork, D.C.: In Photosynthetic Mechanisms in Green Plants, pp. 352-361. Publ. No. 1145 NAS-NRC, Washington, D.C. 1963

36.

Ramus, J., Lemons, F., Zimmerman, C. : (1977).

37.

Bnerson, R., Arnold, W.:

38.

Titlyanov, E.A., Mashansky, V.F., Glebova, N.T.: 59, 1553-1558 (1974).

39.

Cblombo, P.M., Orsenigo, M.:

40.

Golombo, P.M., Orsenigo, M., Solazzi, A., 1tilomio,C.: Mar. Ocean. 6, 197-208 (1976).

41.

Ramus, J., Beale, S.I., Mauzerall, D.: (1976).

42.

Dartnall, H.J.A.: In Vision in Fishes (M.A. Ali, ed.), pp. 543-563, Plenum Press, New York 1975

Biochim. Biophys. Acta 387,

J. Gen. Physiol. 33, 389-422 (1950).

Mar. Biol. 42^, 293-30 3

J. Gen. Physiol. 15, 391-420 (1932). Bot. Zh. S.S.S.R.

Phycologia 16, 9-17 (1977). Mem. Biol.

Mar. Biol. 2Z' 231-238

43 DISCUSSION BIDWELL: What strikes me in this total analysis is that, figuratively speaking, if Engelmann had stood on his head, he would have seen the whole problem from a different perspective. What I am getting at is, instead of being concerned about the addition of pigments, as the seaweed goes deeper and deeper, for capturing the light in the green window, what he really shoud have considered is, as the seaweed comes closer to the surface it has to get rid of pigments to avoid being cooked.

The only pigments that

can successfully be gotten rid of are the supplementary pigments, it must not get rid of chlorophyll, because without at least some chlorophyll it cannot do any photosynthesis.

So perhaps, if Bigelmann had looked at the

problem that way, he would never have come up with the concept of complimentary pigmentation.

There were sane very early experiments by

Wilstatter, who found that bean plants and some other species had far more chlorophyll in them than was necessary for the amount of photosynthesis that they did; they could in fact photosynthesize very effectively if bleached, with a very small amount of pigment left, provided there was lots of light. RAMUS:

These organisms always optimize photosynthesis, they never

maximize it. BIDWELL: Right. FOSTER:

How about other ways of coping with light intensity like changing

respiration? RAMUS: Vie have not done any wark like that personally, and I have not seen any evidence for that in the literature. FOSTER:

There are some deep growing seaweeds that seemingly grow very

slowly.

It seems to me that vrould be an entirely different strategy — do

not change anything, just grow slowly. BIDWELL: With respect to your remark about respiration, I presume it was photorespiration you were thinking of, I do not think most algae exhibit photorespiration.

They may have the metabolism for photorespiration but

44 we have never been able to measure i t in any s p e c i e s we have analyzed, e s p e c i a l l y under conditions that are physiologically similar to those the p l a n t would e x p e r i e n c e

in the s e a .

Some p e o p l e have m e a s u r e d

p h o t o r e s p i r a t i o n in algae, but usually there has been something peculiar about their measuring system, e i t h e r they have had the p l a n t in a l i t t l e c o n t a i n e r or sealed

i n a s m a l l amount o f s e a w a t e r and g e n e r a l l y

illuminated with a very bright l i g h t and so on — the plant was under very unnatural c i r c u m s t a n c e s . there

i s no way t h e y

Most o f t h e s e plants do not photorespire, so could

compensate

for

reduced

light

by

photorespiration. LUNING:

We have done some work t h a t shows t h e c a r o t e n o i d

content

increases with l i g h t i n t e n s i t y . RAMUS: Vfe have some data that says that i t does, and there i s some o t h e r data t h a t says i t does as w e l l .

I do not think there i s any question

about t h a t . WILLENBRINK:

But I think i t depends on the location of these carotenoids,

not a l l are located in the photosynthetic unit (PSU). RAMUS:

Right.

WILLENBRINK:

Most o f them are o u t s i d e and a r e well known to have a

protective function, at l e a s t well known from higher plants.

I doubt that

t h i s would be d i f f e r e n t in algae. RAMUS:

I think that i s to protect them from superoxides.

WILLENBRINK: algae.

Also, I would l i k e to add a b i t about the colour o f the red

I t depends f i r s t on the g e n e t i c a l b a s e s and s e c o n d l y , on the

pigment content.

There are some Chondrus in Helgoland t h a t a r e dark red

even in the uppermost i n t e r t i d a l . RAMUS:

Vfe are now finding good c o r r e l a t i o n s between the composition o f

the p h o t o s y n t h e t i c u n i t and the n i t r o g e n "loading r a t e " in the water column.

I t would seem that the composition of the photosynthetic u n i t

is

as f i n e l y tuned to ambient nitrogen as i t i s to l i g h t and maybe even more f i n e l y tuned to ambient nitrogen than to l i g h t .

This i s not unexpected

45 because the bulk of the combined nitrogen in a seaweed cell is in fact either in a photosynthetic unit or in RuBP carboxylase.

That is the bulk

of the protein in the cell. BIEWELL:

Yes, we noticed this in Chondrus. The rate of maximum, possible

attainable photosynthetic capacity under optimal conditions, which is never reached in the sea, is highest in March, it rises rapidly through February to late March. summer.

Then it drops precipitously throughout the

But the actual photosynthetic rate in the sea increases

continuously until July; during that period there is a tremendous build up of photosynthetic pigments and photosynthetic proteins and presumably rapid assimilation of nitrogen. I would also like to respond to Dr. Willenbrink's comment on colour of Chondrus.

I do not know what it is like in Helgoland, but in Nova Scotia,

you generally see, particularly later in the season, it is green on the surface, and it is a beautiful dark red deep down. And the same way when we grow it in culture, if we give it too much light, it just turns green. It does not die, it just turns green. DUNCAN:

In the work that you have done, particulary with the red algae,

have you seen any changes in membrane structure that may be related to quantities of accessory vs. other pigments. RAMUS: DUNCAN:

No, we have not looked at the structure of the chloroplasts. Ihere might be some relationship here because the structure of

the chloroplasts in these different groups is quite different and so are the accessory pigments. WAALAND: We did some experiments with the red alga, Griffithsia, and found that the variation in intensity changed the number of phycobi1isanes per unit area of thylakoid membranes.

But we did not do any experiments

with different colours of light. RAMUS:

The other person that I know who has looked at red algal

chloroplast structure as a function of light intensity is Elizabeth Gantt. Several Italians have looked at the siphonaceous green, Halimeda tuna, and

46 again that was a function of depth, where both quantity and q u a l i t y \ changing.

But they did see changes in structure of the chloroplasts.

did Titlyanov in Vladivostok in Ulva f e n e s t r a t a .

SEASONALITY IN LARGER BROWN ALGAE AND ITS POSSIBLE REGULATION BY THE ENVIRONMENT

K. Luning Biologische Anstalt Helgoland, D-2192 Helgoland, Federal Republic of Germany

Introduction S e a s o n a l i t y o f kelp performance in the f i e l d i s manifested in various ways, such as the seasonal course of vegetative growth and reproduction of sporophytes, or the development and f e r t i l i z a t i o n of gametophytes and the subsequent appearance of young sporophytes, which in many s p e c i e s and a t many locations occur most cottmonly in winter and spring.

As will be shown

in the f i r s t part of the present paper, photoperiod can be ruled out as a t r i g g e r i n g f a c t o r in the development o f the gametophyte in the various s p e c i e s o f Laminariales i n v e s t i g a t e d so f a r .

Gametophyte

fertility

depends on certain conditions of l i g h t q u a l i t y , irradiance and temperature which, in combination with the reproductive p a t t e r n o f the sporophyte, lead to the production of young sporophytes mainly in winter and spring. As to the growth and reproductive a c t i v i t y of the sporophytes, the factors inducing and synchronizing these p r o c e s s e s with the seasons are s t i l l l a r g e l y unknown due to l a c k o f experimental d a t a .

Some r e c e n t work

discussed in the second p a r t o f t h i s paper shows that the nutrient and temperature cycles influence the growth performance of the sporophytes.

Gametophyte Development and I t s Regulation by Environmental Factors The f a c t that a t elevated temperatures (15-20 C) the gametophytes o f the L a m i n a r i a l e s grow v e g e t a t i v e l y and form filamentous t h a l l i which ultimately become macroscopic was discovered by Schreiber ( 4 6 ) .

Schreiber

( 4 6 ) , and subsequently o t h e r s ( e . g . , Yabu, 50), also reported that once the gametophytes are in the filamentous s t a g e , f e r t i l i t y can be induced

© 1982 Walter de Gruyter & Co., Berlin • New York Synthetic and Degradative Processes in Marine Macrophytes

48 simply by transfer of the cultures to lower temperatures, e . g . , 5 C. Mare recent work has shown that, in a d d i t i o n to lower temperature,

the

gametophyte c u l t u r e must also be supplied with a certain quantum dose of blue l i g h t to induce f e r t i l i t y ( 3 4 , 3 5 ) . spectrum,

taken

at

15 C, p a r t s of

As can be seen from an action the UV and the blue range,

with

wavelengths up to 512 nm, are e f f e c t i v e in inducing egg production female gametophytes, whereas the green and red range are ineffective ( F i g . 1 ) .

in

completely

In contrast to an e a r l i e r report (34), the green or

red range is ineffective not only at 15 C, but also at 5 and 10 C (31).

Wavelength

(nm)

Fig. 1: Laminaria s a c c h a r i n a . Action spectrum f o r induction of egg production in female gametophytes a t 15 C. Plants were grown in red fluorescent l i g h t at 15 pE m s f o r 14 days and then irradiated with 15 jiE m s a t each wavelength f o r 48 h. P e r c e n t a g e o f 500 f e m a l e gametophytes with eggs was determined 8 days l a t e r . UV irradiation was for 72 h at 5 >JE M s , and results were adjusted by comparison with a control at 434 nm. Values plotted are based on results of 10 experiments. \fertical bars: 95% confidence limits. (Data from r e f . 35) Ihe blue l i g h t requirement for the induction of f e r t i l i t y increases in an exponential way with increasing

temperature (31).

As can be seen frcm

49 f i g u r e 2, which shows t h e blue l i g h t r e q u i r e m e n t f o r i n d u c t i o n

of

f e r t i l i t y in 50% of the female gametophytes of t h r e e Laminaria s p p . , a -2 • -2 quantum dose of 90 pE cm i s required a t 5 C, 110 >jE cm a t 10 C, and —2 —2 230 cm f o r L^ hyperborea and L^ s a c c h a r i n a , and 560 p'E cm f o r L. d i g i t a t a a t 15 C. Even higher quantum doses are required a t 18 C. Ihe

F i g . 2: Quantum dose of b l u e l i g h t (400-512 nm) r e q u i r e d t o i n d u c e f e r t i l i t y in 50% of the female gametophytes of Laminaria h y p e r b o r e a ( c i r c l e s ) , L. d i g i t a t a ( s q u a r e s ) , and L. s a c c h a r i n a ( t r i a n g l e s ) a t d i f f e r e n t t e m p e r a t u r e s . V e r t i c a l bars Tndicate confidence l i m i t s a t p= 0.05. (Data from r e f . 31)

50 blue light requirement for the induction of gametophyte fertility in the Californian species of Macrocystis pyrifera and Pterygophora cal ifornica, as determined at 15 C, is of a similar order to that for

hyperborea and

L. saccharina (38). The blocking effect of elevated temperatures in regard to induction of fertility, apparent in figure 2 for the temperature range of 10 to 18 C, is complete at 20 - 22 C, which is also the upper range for survival of the various European species of Laminaria (19,31). Only a few distorted sporophytes, or none at all, are formed at these high temperatures (19). Although the basic mechanism of the blue-light mediated morphogenesis is not yet understood, the relationship between the quantum dose of blue light and temperature explains why the common practice of lowering the temperature, without increasing irradiance, serves to induce fertility in laminarian gametophytes. Figure 3 summarizes the physiological requirements of the gametophytes of three European Laminaria spp. The embryospore, formed by the zoospore, germinates and empties within 24 h to form the primary cell of the gametophyte even in darkness. The primary cell can survive in the dark for at least 5 months.

In white fluorescent light the vegetative growth

of the primary cell is light-saturated at 20-30 pE m - 2 s _ 1 (1000-1500 lux).

The gametophytes, in the form of the primary cell, are killed at 10

times higher photon flux density and in direct sunlight, at about 350 jjE -2

m

-1

s

, they are killed within minutes.

Vegetative growth of the

gametophytes increases with temperature and exhibits a broad optimum between 10 and 18 C.

The effect of temperature on vegetative growth,

therefore, is opposite to that on fertility, since the percentage of fertile gametophytes at a given quantum dose of blue light increases with decreasing temperature.

As soon as the primary cell of the gametophyte

has increased to a certain critical diameter, due to photosynthesis and growth, the availability of blue light determines whether the pathway to fertility is opened or whether further vegetative growth occurs, such that filamentous thalli are produced.

It is also indicated in figure 3 that a

spermatozoid-releasing and -attracting substance is released by the female gametophyte at the moment the egg leaves the oogonium, which is a matter of seconds (37).

51

Fig. 3: Scheme sunmarizing morphological development and p h y s i o l o g i c a l requirements of gametophytes of three European s p e c i e s o f Laminaria ( L . hyperborea, L. d i g i t a t a , L. saccharina). SR: spermatozoid r e l e a s i n g and a t t r a c t i n g substance i s r e l e a s e d i n t o the water. See t e x t for further explanation. (Data taken from r e f . 31) Ihere is every reason to expect that the a v a i l a b i l i t y of blue l i g h t in the sea determines the maturation of gametophytes in s i t u , as i t does in the laboratory.

The blue l i g h t requirement of about 200 JJE c m - 2 a t 15 C f o r

the f e r t i l i t y of laminarian gametophytes i s similar to that of same other blue light-mediated morphogenetic responses (for examples and r e f e r e n c e s see 35) .

However, i t should be stressed that the magnitude of t h i s blue

light requirement

i s r a t h e r h i g h , compared t o t h a t o f t h e

light-mediated photoperiodic response in Scytosiphon lomentaria ( 3 5 ) .

blue In

S. lomentaria a 50% inhibition of the formation of e r e c t t h a l l i i s brought about by 0 . 0 0 2 juE c m - 2 o f blue l i g h t ,

i . e . , a t a quantum dose 1 0 5 x

smaller than that required for the gametophyte response o f the Laminaria spp. a t 15 C.

It) i l l u s t r a t e t h i s d i f f e r e n c e ,

i t may be said that the

photoperiodic response of S. lomentaria i s 50% i n h i b i t e d by g i v i n g the a l g a a n i g h t - b r e a k o f 10 s duration f o r 10 successive long nights with

52 -2

-1

very low photon flux density ( 2 p E u s ) blue l i g h t (quantum dose per n i g h t : 0.002 >iE cm_ 2 ) . In c o n t r a s t , a 50% response f o r l a m i n a r í a n gametophytes is achieved by irradiating them with blue l i g h t f o r 28 h at a —2 —1 flux density of 20 jjE m

s

.

I f cool white fluorescent l i g h t , with 1/5

of the total v i s i b l e quanta in the blue range, i s used, one vrould obtain a 50% f e r t i l i t y only a f t e r 28 x 5 h, o r 5.8 days in continuous l i g h t , or a f t e r 11.6 days i f a 12:12 LD cycle i s used. The e c o l o g i c a l cultivating

s i g n i f i c a n c e of these magnitudes has been t e s t e d

by

laminarian gametophytes in the l a b o r a t o r y in simulated

underwater l i g h t f i e l d s which d i f f e r in their blue l i g h t content, and also by comparing percentage f e r t i l i t y of gametophytes cultivated in the sea at d i f f e r e n t water depths where they receive d i f f e r e n t quantum doses of blue light

(31).

In a f i e l d experiment i t was found that the gametophytes

which where formed by zoospores of Laminaria saccharina and in e a r l y

November s u r v i v e d

hyperborea

the dark winter months as u n i c e l l u l a r

gametophytes due to lack of l i g h t even at moderate water depths (36) . the b e g i n n i n g of

By

February, the water suddenly became c l e a r and the

experimental gametophytes at 2 m water depth matured within 24 days, the f e m a l e s were a l l few-celled stage.

in the u n i c e l l u l a r s t a g e , and the males were in a During these 24 days a quantum dose o f blue l i g h t of

_2

165 JJE cm

was recorded by an automatic underwater l i g h t measuring

station at 2 m water depth (31, 36).

Water temperature at this time was 5

C. Thus, the quantum dose obtained was s u f f i c i e n t f o r even a 100% f e r t i l i t y , since in the l a b o r a t o r y experiments a quantum dose o f 90 JJE _2

cm

o f blue l i g h t at 5 C was s u f f i c i e n t f o r a 50% f e r t i l i t y of

gametophytes ( F i g . 2).

the

In another experiment, which also was started from

zoospores, but near the end of the sporing season (February, March), gametophytes a t 2 m water depth matured within 20 days, the fanales were again in the unicellular stage. During this period the gametophytes a t 2 m water depth had received more than 200 juE cm- 2 week - 1 . In contrast, the gametophytes a t 5 m water depth, which on the average received less than -2

50 ¿iE cm

-1

week

of blue l i g h t , formed 2 or 3 v e g e t a t i v e c e l l s in the

female or up t o 10 c e l l s in the male gametophytes (31). e x p e r i m e n t was d i s c o n t i n u e d

at

this

stage,

it

Although the

is unlikely

that

multicellular, ultimately macroscopic, gametophytes vrould have been formed

53 even a t 5 m w a t e r d e p t h , s i n c e t h e b l u e l i g h t s u p p l y a t increased

this

depth

c o n s i d e r a b l y d u r i n g t h e f o l l o w i n g weeks and a c o n s e q u e n t

f e r t i l i z a t i o n of the gametophytes i s more l i k e l y .

In f a c t ,

filamentous,

macroscopic gametophytes have never been found in n a t u r e . The f i n d i n g t h a t under s u f f i c i e n t l i g h t s u p p l y t h e f e r t i l i z a t i o n gametophytes

in the

field occurs

i n a few weeks c o r r o b o r a t e s

of the

o b s e r v a t i o n s made f o r p a c i f i c L a m i n a r i a s a c c h a r i n a by H s i a o and Druehl (16).

They found t h a t the gametophytes of t h i s s p e c i e s frcm March t o May

formed oogonia in 12-16 days and a n t h e r i d i a in 10-14 d a y s , and t h a t from November t o J a n u a r y the oogonia were formed in 20-26 days and a n t h e r i d i a in 15-18 d a y s . At Helgoland, where underwater l i g h t c o n d i t i o n s d e t e r i o r a t e

considerably

d u r i n g t h e w i n t e r months - a s i t u a t i o n comparable t o A r c t i c c o n d i t i o n s in regard to seasonal l i g h t supply - t h e r e o c c u r s a r e s t i n g p e r i o d of

the

g a m e t o p h y t e s in t h e u n i c e l l u l a r s t a g e f o r 2-3 months, due t o a l a c k of l i g h t for photosynthesis.

This i s f o l l o w e d by a sudden v e g e t a t i v e and

r e p r o d u c t i v e development w i t h i n 2-3 weeks when l i g h t i s again a v a i l a b l e , a s i t u a t i o n obtained in e a r l y spring near H e l g o l a n d and i n l a t e summer i n Arctic l o c a l i t i e s

(3).

Since s u f f i c i e n t l y high t e m p e r a t u r e s prevail

d u r i n g these p e r i o d s , a v a i l a b i l i t y

of s u f f i c i e n t i r r a d i a n c e f o r g r o w t h

and f o r i n d u c t i o n of f e r t i l i t y seems t o be the main f a c t o r r e g u l a t i n g t h e seasonal gametophytic development a t t h e s e l o c a t i o n s . Among o t h e r e n v i r o n m e n t a l f a c t o r s t h a t m i g h t r e g u l a t e

the

seasonal

d e v e l o p m e n t of the gametophytes, photoperiod has been found not t o a f f e c t the i n d u c t i o n of f e r t i l i t y in the gametophytes of L a m i n a r i a d i g i t a t a ,

L.

hyperborea, L. s a c c h a r i n a , L. j a p ó n i c a , Macrocystis p y r i f e r a , and Chorda filum (32).

Although more s p e c i e s should s t i l l be i n v e s t i g a t e d t o confirm

this observation,

i t may be t r u e t h a t p h o t o p e r i o d i s m , which i s such a

c h a r a c t e r i s t i c phenomenon among S c y t o s i p h o n a l e s ( 3 2 ) , d o e s n o t gametophytic development in the L a m i n a r i a l e s .

regulate

The y e a r l y c o u r s e o f

n u t r i e n t l e v e l s in the sea a l s o does n o t seem t o a f f e c t the s e a s o n a l i t y of g a m e t o p h y t e d e v e l o p m e n t ; t h e s e g a m e t o p h y t e s m a t u r e even a t v e r y low c o n c e n t r a t i o n s of n i t r a t e , p r o v i d e d t h e y a r e g i v e n s u f f i c i e n t l y h i g h i r r a d i a n c e s in white f l u o r e s c e n t l i g h t (Lüning, u n p u b l i s h e d ) .

In P a c i f i c

54 Laminaria saccharina gametophytes, maintained in axenic c u l t u r e a t 10 C and i l l u m i n a n c e o f 850 l u x , f e r t i l i t y was not observed a t n u t r i e n t concentrations lower than 5 pM NO^- and 10

PO^ ( 1 5 ) .

However, these

experiments need to be repeated with higher irradiances. Temperature may r e g u l a t e gametophytes.

the s e a s o n a l development o f

I f spores are shed in a season in which water temperatures

are s u f f i c i e n t l y high t o block the f e r t i l i z a t i o n gametophytes,

laminarian

of the

resulting

p a r t i a l l y o r c o m p l e t e l y , a seasonal p a t t e r n in the

occurrence of young sporophytes in the f i e l d may be c r e a t e d . i s provided by Chorda filum.

An example

The gametophytes of t h i s species mature at 5

and 10 C, but not a t 15 C, even at high irradiances in white fluorescent light (32).

Near Helgoland the sporophytes appear in April and dissappear

by mid September (25).

Since the water temperature in May already exceeds

10 C, the gametophytes o f t h i s s p e c i e s should be t o t a l l y blocked from becoming f e r t i l e during simmer and autumn by elevated temperatures. may explain why only one generation of macroscopic t h a l l i produced per y e a r . fertilization

Another example o f

of gametophytes,

This

in C^ filum

is

temperature c o n t r o l

of

a l t h o u g h n o t from t h e o r d e r

of

Laminariales, i s provided by Desmarestia v i r i d i s .

This i s also an annual

alga in which the gametophytes can be f e r t i l i z e d only at temperatures of 5 and 10 C, but not at 15 C ( 3 2 ) . In conclusion, the seasonal development of the gametophytes o f Laminaria s p p . , and perhaps a l s o o f o t h e r genera of the Laminariales seems to be related to the a v a i l a b i l i t y of s u f f i c i e n t l y high i r r a d i a n c e , and t o the reproductive patterns of the sporophytes (see below). be synchronized with a s p e c i a l

timing

signal,

I t does not seem to

such a s a

suitable

photoperiod; and temperature and n u t r i e n t conditions do not seem to be limiting factors under most ecological s i t u a t i o n s .

A s i m i l a r conclusion

was drawn by Hsiao and Druehl (16) in regard to in s i t u gametogenesis o f L. saccharina and the seasonal appearance of young sporophytes o f A l a r i a t e n u i f o l i a , Costaria costata and L. saccharina fron B r i t i s h Columbia ( 9 ) .

55 Phenolog ical Patterns in Sporophytes of Laminaria spp. The seasonal e v e n t s in sporophyte performance of a few perennial representatives of the Laminariales, f o r which detailed information is available about vegetative growth as well as reproductive activity, will be exemplified in the following section by the phenological patterns of four Laminaria spp. (Table I ) . In Laminaria hyperborea vegetative growth of the blade is s t r i c t l y limited to the period from December to June, the f a s t e s t expansion in area occurring in April/early May (22,23,27,28).

Also, stipe elongation

(22,27) as well as an increase in stipe diameter (17) are fastest during this period although growth activity in these two parameters continues at a slower rate during the second half of the year. activity of

The reproductive

hyperborea sporophytes is also confined to a limited

Table I . Laminaria spp.: Seasonal patterns of v e g e t a t i v e growth and reproduction in sporophytes. V: vegetative growth. S: sori present on blades. Underlined letters: seasonal peak. Letters in brackets: v a l i d for only part of the month. Dash: no activity. Species

L. hyperborea

L. d i g i t a t a

locality

J

F

M A

I s l e of Man Helgoland

S V S

s

s (S)

V S

V V (V) s (S)

Calvados

V S S

V

V

-

-

Wales Helgoland L. saccharina

L. angustata

Devon and Argyll Wales Helgoland

V j>

Hokkaido

V

( * ) - This paper.

s s

M J

A

s

0

s -

V

s -

V

s

s

V

s

s

s V

V

V

V

V

-

-

-

-

s

s

V. V

s

V

V

V

V

D

Ref

s (V)

s

(21) (27,28) (*)

V

(5,40)

s. s. s. s. s s s s s s s s s s s (S)

V. V. V. V

V.

N

_ __ _ s s s

V

s. S. s s s _s S (S) s s (S) s

J

s

V

s

V

s

V

V

V

V

s. s. (S) s s (S) s s s s

V

s

s

V

V

s. s

V

s

(6) (42) (*) (39) (39) (42)

(*) (13) (13)

56 period — from September t o e a r l y April on the I s l e of Man and from November to early April a t Helgoland (Table I ) . winter sporing

species,

L. hyperborea i s thus a

although i t should be emphasized t h a t the

appearance of sorus precedes the appearance of new blade by up to 3 months and reproductive a c t i v i t y is largely finished when the maximum vegetative growth o c c u r s (Table I ) . present.

In summer only v e g e t a t i v e sporophytes a r e

The species seems to react in a similar way throughout i t s whole

geographical range ( 2 1 ) , from Portugal up to the North Cape of Norway (20,24). In contrast, blade growth in

digitata and

saccharina i s continuous

during the year, but maxim an elongation rates occur, as in L^ hyperborea, during the f i r s t part of the year (Table I ) .

At Helgoland, L. digitata is

the e a r l i e s t of a l l three Laminaria spp. to produce sori in the course of the year.

The f i r s t sporogenous plants from which one may obtain zoospore

release appear by May, whereas in L^ saccharina they do not appear u n t i l mid September.

The l a s t sporogenous plants of L. d i g i t a t a a t Helgoland

are seen up to December and of

saccharina up to March of the next year.

Thus, in these two species also, as in JJ^ hyperborea, the reproductive a c t i v i t y i s stopped p a r t l y or completely when rapid vegetative growth takes place in early spring. been recorded for

A somewhat similar reproductive p a t t e r n has

digitata and L. saccharina from Wales ( 4 2 ) ; f o r L.

saccharina, no sporogenous plants can usually be found on the Port Erin breakwater in summer ( 2 1 ) .

L. digitata has also been reported to behave

a s a summer and autumn sporing s p e c i e s along the c o a s t of Calvados (France), with a maximum number of sporogenous p l a n t s and maximum spore emission occurring from June to October (6; for discussion of conflicting reports, see 11).

However, a t the l o c a l i t y c i t e d , sporogenous plants also

occur during the r e s t of the y e a r , but a t a lower frequency.

The same

case, with sporogenous plants a l l year round, has been reported for L. saccharina from France ( 4 5 ) , and from Devon and Argyll (British I s l e s ) by Parke ( 3 9 ) .

These findings are somewhat a t variance with those of Pees

(42) and Kain ( 2 1 ) ; however, this variance may be due to the e x i s t e n c e of l o c a l , genetically different populations.

L. saccharina from Helgoland,

in which no sporogenous p l a n t s can be found in the field frcm April to August, definitely represents a genetically d i f f e r e n t form, with smooth

57 blades, as opposed to the form with rugose blades otherwise common in Europe (29, 33). In Laminaria angustata from Hokkaido (japan) the maximum vegetative growth occurs from May to July, as reported by Hasegawa (13). The plant first becomes sporogenous in October, at a minimum age of 8 months.

In the

second year the sporogenous activity begins in July, reaches a maximum in October and ceases in the following February.

In its third year the plant

is sporogenous from October to the next January, again with a peak in October (data in T&ble I refer to third year). The seasonal patterns of vegetative growth and reproduction are thus similar to the European L. saccharina. The general picture, then, is that the adult sporophytes of Laminaria spp. considered here have a seasonal peak of vegetative growth in spring and a reproductive maximim in autumn or winter, the exact time period depends on the species and perhaps also on location.

Vegetative growth is minimal

when maximum reproduction occurs. After the seasonal peak of vegetative growth the fronds require a period of several months to form mature sori. In L. hyperborea this period lasts for at least 3 months (Isle of Man, June to August, Table I) or 5 months (Helgoland, June to October, TSble I).

In addition, it is known that L. hyperborea sporophytes must have a

minimum age of 15 months to become sporogenous (21).

For L. saccharina

from the British Isles, Parke (39) reported that sori are not formed on blade portions younger than 6 months, and for Helgoland Lj_ saccharina Liining (30) reported that the corresponding age may be 4 months.

"Resting

periods" of similar magnitude may be valid for L^ digitata, and the minimum age of a sporophyte of this species to became sporogenous has been reported to be 18-20 months (41). New sporophytes of the three European species of Laminaria, of a size just visible to the naked eye, appear in the field in most localities mainly in the winter and early spring.

This is to be expected from the sporing

periodicity indicated in Table I. Using sterilized stones of about 10 kg, which were exposed for two-month periods in the upper sublittoral in the Isle of Man, Kain (21) found that juvenile sporophytes of Laminaria spp. were formed in great quantity on stones exposed during November/December

58 and January/February, a few were also found on stones exposed during March/April, but none en stones exposed during the rest of the year.

The

largest L^ hyperborea sporophyte which developed in the sea at moderate water depth in October/November after 33 days consisted of 167 cells (22), equivalent to a length of about 0.2 irm (18).

In another f i e l d experiment,

which was conducted in May, Kain (18) found that only 25 days frcm spore release wsre required for the formation of 0.6 mm long sporophytes (1000 c e l l s ) ; frcm this she calculated a total of 54 days for the formation of 1 cm long sporophytes.

Ihese data are consistent with those from laboratory

experiments in continuous l i g h t , in which 2-3 cm long sporophytes of L. hyperborea, L.

d i g i t a t a and L^ saccharina have been grown from

gametophytes within 2 months (30). These results indicate that young sporophytes of a size just visible to the naked eye would appear in the f i e l d with a lag phase of several months after the seasonal reproductive peak of the sporophytes.

P o s s i b l e Role of Environmental Factors in Regulation of Sporophyte Periodicity For those laminarian species which show a clear seasonal pattern of sorus formation and for those in which production of the new blade commences abruptly, as in Laminaria hyperborea in December, the question a r i s e s : Does the plant receive an environmental signal and, i f so, what is i t s nature?

Ftor sorus formation in

saccharina, Parke (39) assumed that

"the development of the reproductive tissue in plants of this species is controlled by some factor or factors within the frond tissue of the plant, not by factors external to the p l a n t . "

Lobban (26) suggested that in

Macrocystis spp. the apical meristem might "leave some inhibitor or repressor in the laminae that are to remain s t e r i l e . "

I t is possible,

therefore, that for frond tissue in Laminaria spp. the distance from the meristem, and thus the age of a particular frond portion, may be an important precondition for onset of sorus formation. However, a completely internal control does not seem very l i k e l y f o r Laminaria hyperborea , in which plants of a l l age groups (maximum possible

59 age: 15-18 years; 24) stop growth by early summer, become sporogenous from autumn onwards and commence new growth by December (Table I).

Internal,

annually cycling rhythms of activity would be badly put out of step due to the innate variability of living things with time, as pointed out by Villiers (48).

If some environmental factor induces and synchronizes kelp

performance with the seasons, then, at first glance temperature or nutrient concentration or photoperiod might all represent such a factor. However, so far our knowledge in this respect is almost nil, and, hence, in the following, I will attempt to pull together the few fragments of information which may suggest directions for future research. In a few cases, such as Alaria esculenta (47), plants have been reported to become sporogenous following their transfer from the sea in a vegetative stage.

However, in these cases, the plants may have been

induced to become sporogenous while still in the sea before their transfer to the laboratory.

Only rarely has it been possible to cultivate the

sporophytes of Laminariales completely to the stage of sorus production. Sanbonsuga and Hasegawa (43; see also 14) achieved sorus production in Undaria pinnatifida (length of sporogenous plants: 15-60 cm) and in Costaria costata (maximum length of sporophytes obtained: 50 cm; sorus formation after a certain decrease in length), both of which are annual plants.

Sori were formed in these two species at temperatures above 22 C,

at an illuminance of 2500 lux and at 18 h light per day.

In the field, at

ftokkaido, both species begin to form sori from early summer onwards and attain full maturity by mid summer.

In additional experiments, Sanbonsuga

and Hasegawa (44) found that temperature (10-12 C or 18-20 C), illuminance (2500 or 5000 lux), and photoperiod (6 or 18 h light per day) are unlikely to represent triggering factors for sorus formation in Costaria costata, since sorus formation occurred in laboratory-reared sporophytes of this species under all conditions stated. These authors made the interesting observation that a period of retarded frond growth always preceded the onset of sorus formation, and that "the retardation of frond growth and onset of zoosporangium formation could be induced in mature plants, but not in the immature, by decreasing nourishment in the culture medium." Low nitrate content is known for several algae to be a precondition for onset of reproduction (see review by Dring, 8), and has, in addition,

60 d e f i n i t e l y been shown t o be r e s p o n s i b l e

for

slow growth

rates

of

Laminaria l o n g i c r u r i s during summer o f f the coast of Nova Scotia (2). observation

of

Sanbonsuga and Hasegawa

The

( 4 4 ) on i n d u c t i o n o f

formation by low nutrient content should be taken as a p o s s i b l e

sorus

starting

point f o r f u r t h e r analysis of the problem. However, as shown b e l o w , under i d e n t i c a l e n v i r o n m e n t a l

conditions

d i f f e r e n t Laminaria spp. r e a c t in a s p e c i e s - s p e c i f i c way, and i t to

believe

requirement.

that

this

could

L. saccharina, and

laboratory-raised sporophytes o f

nutrient

L^ d i g i t a t a ,

hyperborea were transferred by mid February

(blade

5 - 2 5 cm) i n t o the sea near Helgoland and t h e i r growth was

followed u n t i l November.

The experimental plants o f

growth c o m p l e t e l y i n J u l y ,

saccharina

considerably during t h i s month, wheras first

to a species-specific

This i s evident from f i g u r e 4 which summarizes an experiment

performed by l i n i n g ( 3 0 ) . lengths:

be due

the

i s hard

in

September.

reduced

hyperborea stopped its

growth

rate

d i g i t a t a reduced i t s growth r a t e

The most p u z z l i n g

a s p e c t in r e g a r d t o

these

s p e c i e s - s p e c i f i c patterns o f seasonal growth reduction was the abrupt stop Water temperature i n

this

month surpassed 15 C, but the plants did not resume growth when i t

in growth o f the

hyperborea plants in July.

fell

below 15 C in October.

Irradiance was s t i l l saturating f o r photosynthesis

in August, and n i t r a t e concentration was s t i l l near enough t o the g r o w t h - s a t u r a t i n g

as h i g h as 8 pM NO^ - r

concentration of

reported f o r L. saccharina sporophytes by Chapman e t a l .

10 JJM NO^ (4).

Reduced

summer growth under c o l d , NO^ - - e n r i c h e d c o n d i t i o n s has a l s o been reported

f o r A l a r i a esculenta

from Newfoundland by Buggeln ( 1 ) ,

s p e c u l a t e d whether an endogenous,

who

t r u e c i r c a n n u a l rhythm may n o t be

involved in bringing about t h i s reduced growth in summer. In many higher plants o f the temperate r e g i o n , a suspension of growth and i n d u c t i o n o f dormancy i s common a f t e r the plants have recorded a s i g n a l from the environment, namely a decrease in d a y l e n g t h , which o c c u r s from l a t e June onwards in the Northern hemisphere ( 4 8 , 4 9 ) . signal

This photoperiodic

r e p r e s e n t s an " e a r l y warning system" and o c c u r s , when g r o w t h

c o n d i t i o n s , such as temperature, irradiance and nutrient supply are favourable.

still

In t h i s c o n t e x t , i t should lae c l a r i f i e d that plants which

61

20 - -40

JUNE

|

JULY

|

AUGUST

[SEPTEMBER I

OCTOBER

| NOVEMBER

20--300 „

tvmperolur«

L. digitata

1

F i g . 4 : Helgoland, 1975. Seasonal course o f t e m p e r a t u r e , n i t r a t e concentration in seawater (from r e f . 12), photon flux density a t 2 m water depth (from r e f . 36), daylength (according to Smithsonian Meteorological Tables), and of frond area increase in experimental sporophytes o f three Laminaria spp. (bar graphs), cultivated at 2 m water depth. F i l l e d area in outline drawings: new-formed t i s s u e . (Data taken fran r e f . 30)

62 reduce t h e i r growth r a t e due t o t h e l a c k o f some f a c t o r ,

such a s

n u t r i e n t s , and resume t h e i r growth when t h i s lack has been r e c t i f i e d , are by d e f i n i t i o n only in a " q u i e s c e n t s t a t e "

(48).

The Nova

Scotian

Laminaria l o n g i c r u r i s , which i s reported to resume growth in summer a f t e r artificial

i n c r e a s e of n i t r a t e c o n t e n t in the sea water ( 2 ) , may be an

example showing quiescence.

In c o n t r a s t , the ceassation o f growth by L.

hyperborea in summer may r e p r e s e n t t r u e "dormancy," which can only be broken by another environmental s i g n a l .

In higher plants, t h i s may tie

photoperiod, o r , more o f t e n , a minimum period o f temperatures below 5 C (48).

Some of the photoperiodic responses reported in marine algae may be

interpreted in these terms.

Fbr instance the swarmers of the e r e c t t h a l l i

of the brown alga Scytosiphon lcmentaria form minute p r o s t r a t e which do n o t form any new f r o n d s , in long days ( 3 2 ) .

systems,

The c r i t i c a l

daylength f o r t h i s r e a c t i o n i n c r e a s e s toward the North P o l e l a t i t u d i n a l ecotypes of t h i s species.

in

the

The ecological significance of the

response i s particulary evident in s u b a r c t i c S^ l o m e n t a r i a , in which a small percentage of the swarmers s t i l l form e r e c t fronds a t 16 h l i g h t per day, but not at 18 h l i g h t per day or in continuous l i g h t , so t h a t by midsummer only p r o s t r a t e crusts are formed, which probably have a b e t t e r chance of survival under ice than a new generation of e r e c t fronds. The photoperiodic induction in Scytosiphon lcmentaria occurs over a broad temperature r a n g e .

This i s not so f o r the photoperiodic induction of

tetrasporangium formation by short days in the T r a i l l i e l l a - p h a s e o f the red a l g a Bonnemaisonia hamifera

(32),

which o c c u r s o v e r a narrow

temperature range around 15 C and r e q u i r e s , in addition, low nutrient concentrations.

Thus, i t may be that the temperature and nutrient content

o f the water are a d d i t i o n a l f a c t o r s and l i m i t the range o f

possible

photoperiodic induction to a certain set of environmental c o n d i t i o n s .

For

obvious reasons, photoperiod ism in marine algae has been searched for and discovered so far mainly in smaller forms (32; see also reviews by Dring, 7,8).

N e v e r t h e l e s s , i t seems more important now to ascertain whether or

not photoperiodic reactions are also involved in the c o n t r o l o f s e a s o n a l patterns of kelp sporophytes.

63 References

1.

Büggeln, R.G.: J. Phycol. 14, 156-160 (1978).

2.

Chapman, A.R.O., Craigie J.S.: Mar. Biol. 40, 197-205 (1977).

3.

Chapman, A.R.O., Lindley, J.E.: Mar. Biol. 57, 1-5 (1980).

4.

Chapman, A.R.O., Markham, J.W., Luning, K. : J. Phycol. 14, 195-198 (1978).

5.

Cosson, J.: Bull. Soc. linn. Normandie ji, 246-281 (1967).

6.

Cosson, J.:

7.

Dring, M. J.: Bi Photobiology of Microorganisms (P. Halldal, ed.), pp. 345-368, Wiley & Sons, London 1970

8.

Dring, M.J.: Di Algal Physiology and Biochemistry (W.D.P. Stewart, ed.), pp. 814-837, Blackwell, Oxford 1974 Dring, M.J., Luning K.: Planta (Berl.) 125, 25-32 (1975).

9. 10.

Soc. Phycol. de France 21, 28-34 (1976).

Druehl, L.D., Hsiao, S.I.C.: 1207-1211 (1977).

11. Gayral, P., Gosson, J.:

J. Fish. Res. Board Canada 34,

Synop. FAO Peches 89, pp. var. (1973).

12. Harms, E., Hagmeier, E.: In B.A.H. Jahresberichte 1975, pp. 21-27, Westholsteinische Verlagsdruckerei Boysens & Go, Heide, F.R.G. 1976 13.

Hasegawa, Y. : (1972).

Bull. Hokkaido reg. Fish. Res. Lab. 2A, 116-138

14.

Hasegawa, Y., Sanbonsuga, Y.: In Contributions to the Systematics of Benthic Marine Algae of the Northern Pacific (I.A. Abbott, M. Kurogi, eds.), pp. 109-116, Japanese Society of Phycology, Kobe, Japan 1972

15.

Hsiao , S.I.C., Druehl, L.D.: Can. J. Bot. 51, 829-839 (1973).

16. Hsiao , S.I.C., Druehl, L.D.: J. Phycol. 9, 160-164 (1973). 17.

Kain, J.M.:

J. mar. biol. Ass. UK 43, 129-151 (1963).

18.

Kain, J.M.:

J. mar. biol. Ass. UK 45, 129-143 (1965).

19. Kain, J.M.:

J. mar. biol. Ass. UK 49, 455-473 (1969).

20.

Kain, J.M.:

21.

Kain, J.M.: J. mar. biol. Ass. UK 55, 567-582 (1975).

FAO Fish. Synop. 87

65 pp. (1971).

22. Kain, J.M.: J. mar. biol. Ass. UK 56, 267-290 (1976). 23. Kain, J.M.: J. mar. biol. Ass. UK 56, 603-628 (1976). 24. Kain, J.M.: Mar. Biol. Ann. Rev. 17, 101-161 (1979). 25.

Kornmann, P. , Sahling, P.-H.: 1-289 (1977).

Helgoländer wiss. Meeresunters

26.

lobban, C.S.: Phycologia 17, 196-212 (1978).

27.

Luning, K.: Mar. Biol. 2, 218-223 (1969).

28.

Luning, K.:

In Ft>urth European Marine Biology Symposium (D.J. Crisp,

64 ed.), PP. 347-361, University Press, Cambridge 1971 29. Lüning, K.: Helgoländer wiss. Mseresunters. 27, 108-114 (1975). 30. Lüning, K.: Mar. Ecol. Prog. Ser.

195-207 (1979).

31. Iiining, K.: J. Phycol. 16, 1-15 (1980). 32. tuning, K.: In The Shore Environment, Vol. 2. Ecosystems (J.H. Price, D.E. Glrrine, W.F. Farnham, eds.) , pp. 915-945, Academic Press, London 1980 33. Lüning, K., Chapman, A.R.O., Mann, K.H.: Phycologia 17, 293-298 (1978). 34. Liining, K., Dring, M.J.: Planta (Berl.) 104, 252-256 (1972). 35. Lüning, K., Dring, M.J.: Mar. Biol. 29, 195-200 (1975). 36. lüning, K., Dring, M.J.: Helgoländer wiss. Meeresunters. 32, 403-424 (1979). 37. Lüning, K., Müller, D.G.: Z. Pflanzenphysiol. 89, 333-341 (1978). 38. Lüning, K., Neushul, M.: Mar. Biol. 45, 297-309 (1978). 39. Parke, M.: J. mar. biol. Ass. UK 27, 651-709 (1948). 40. Perez, R.: Proc. Int. Seaweed Symp. 6, 329-344 (1969). 41. Perez, R.: Rev. Trav. Inst. Peches Marit. 35, 287-346 (1971). 42. Rees, T.K.: Proc. Swansea Sei. Fid. Nat. Soc. J., 33-37 (1928). 43. Sanbonsuga, Y., Hasegawa, Y.: Bull. Hokkaido, reg. Fish. Lab. 32, 41-48 (1967). 44. Sanbonsuga, Y., Hasegawa, Y.: Bull. Hokkaido, reg. Fish. Lab. 35, 198-202 (1969). 45. Sauvageau, C.: Man. Acad. Sei. Inst. Fr. 56, 1-240 (1918). 46. Schreiber, E.: Planta 12, 331-353 (1930). 47. South, G.R.: Helgoländer wiss. Meeresunters. 20, 216-228 (1970). 48. Villiers, T.A.: Dormancy and the Survival of Plants, Edward Arnold, London 1975 49. Vince-Prue, D.: Photoperiodism in Plants, McGraw Hill, London 1975 50. Yabu, H.: Mem. Eac. Fish. Hokkaido Univ. 12, 1-72 (1964).

DISCUSSION FOSTER: I was curious. Having kept these gametophytes for 5 months in the sea, was there not enough cumulative blue light to set those off over that period?

65 LÜNING:

In winter our storms begin in October and then the water i s

p r a c t i c a l l y brown u n t i l February.

That i s the normal s i t u a t i o n .

Fran

year to year, there may be a phase of two weeks when suddenly the storms c e a s e and a l l fertilizations.

this dirt

f a l l s down, then c e r t a i n l y t h e r e might be

As I s a i d we have an A r c t i c c o n d i t i o n , we have d i r t

instead o f i c e , therefore, I gave the example frcm the I s l e of Man, where we have sporophytes from November, December onwards as you would expect. DRUEHL:

Vfe have used the blue l i g h t , or Lüning e f f e c t , very successfully.

There is one species, however, t h a t t o t a l l y f a i l s to respond, t h a t Postelsia.

is,

I t i s s t r i c t l y an i n t e r t i d a l plant, and obviously experiences

very d i f f e r e n t l i g h t regimes.

I was wondering i f you could comment on

this? LUNING:

I have a whole c o l l e c t i o n o f laminarian gametophytes,

40-50

s p e c i e s , and there are c e r t a i n l y 15 or so that we can not f e r t i l i z e a t all.

So there i s more than j u s t blue l i g h t e f f e c t .

observations.

These are j u s t

first

I should a l s o say t h a t i t i s improbable t h a t such a

pho tomor phog ene t i c pigment e x i s t s in only one form.

A l l such pigments

which have been found — phytochrome, rhodopsin, e t c . — e x i s t in two forms and now, f o r the l a s t two y e a r s or s o , the p h o t o b i o l o g i s t s are discovering

that blue l i g h t e f f e c t

blue-yellow e f f e c t s .

i n h i g h e r p l a n t s may a l s o be

We are nowhere near the end of these investigations,

and I do not know what happens and what other substances may be there. DUNCAN:

You talked about the single female c e l l s .

Were you talking about

Laminaria hyperborea, and were these in p l a s t i c dishes? LUNING: glass

All three Laminaria species did the same. slides,

They were growing on

b u t i n P l e x i g l á s c o n t a i n e r s f i l l e d with P r o v a s o l i .

Provasoli has about 300 micromolar n i t r a t e , but we know t h a t even a t 1 micromolar n i t r a t e ,

i f you always renew the medium, you can f e r t i l i z e

them. DUNCAN:

You never got multicellular females?

LÜNING:

No.

Well, I g o t them a t depths o f s i x m e t r e s , but I did not

continue t h e r e .

We need more experiments.

When I was in Santa Barbara,

66 we put Macrocystis down and saw some multicellular gametophytes, but again sane of the c e l l s had formed eggs. WILLENBRINK:

How s p e c i f i c are the a t t r a c t a n t s in the c a s e o f the t h r e e

Laminaria species? LUNING:

That i s easy.

Everybody releases everybody, there.

Macrocystis,

Laminaria hyperborea — we mix them a l l . WILLENBRINK: LUNING:

But not the fucoserraten.

Our Laminaria do not r e a l l y extract Fucus spermatozoids.

As you

may know, t h e r e are now f i v e c l a s s e s o f sex a t t r a c t a n t s in the plant kingdom.

The f i r s t one, found in 1968 was found in a f u n g u s .

remaining four have a l l been from brown algae (EDITOR'S note:

The

Here Dr.

Lüning reviewed the i n t e r e s t i n g work o f Dr. D.G. Müller o f Konstanz, F.R.G. and the p r o p e r t i e s o f ectocarpen, multifiden, fucoserraten, e t c . Por an introductory reference to that work see Science 212, 1040 [1981] ) . In the laminariales i t seems that Macrocystis, Laminaria, A l a r i a , and so on, a l l produce the same substance. the same peak.

Qi the gas chromatogram i t i s always

But t h e r e are tremendous d i f f i c u l t i e s

concentrate the substance for further characterization.

in t r y i n g

to

Laminaria i s the

only case, so f a r , where t h i s gas also releases the spermatozoids. FDX'H:

Une question concernant l a croissance de Laminaria hyperborea.

Es-ce que l e carmencement e s t toujours en Decembre, jamais avant? LUNING:

Always Dacembre.

SRIVASTAVA:

A small question about the blue l i g h t e f f e c t .

I am t r y i n g

to f i g u r e out whether i t i s a morphogenetic e f f e c t in the sense that the sperms or eggs are actually formed a f t e r blue l i g h t i s shone or whether i t i s simply a membrane e f f e c t - t h a t they are formed e a r l i e r , and simply released by shining of blue l i g h t . LUNING:

No.

I t i s a complete change in the program.

t h e oogonium i s c o n t r o l l e d controlled by red l i g h t .

The formation o f

by t h e b l u e l i g h t , but the r e l e a s e

We do not know much more about i t .

is

67 SRIVASTAVA:

From what you are saying it seems to me more likely that it

probably is some kind of a hormone release which can trigger the morphogenetic process. LUNING: Yes. Ihat is possible. But the hormone, of course, is only a secondary cause. DRUEHL:

Considering the general nature of this sex attractant in

Laminaria, one would expect to see a great deal of hybridization in nature in Laminaria. LUNING:

I do not think that is a problem. Attracting a spermatozoid does

not mean much.

It is the intrusion of the spermatozoid and acceptance of

the nucleus, that is where all the blocks begin.

It seems to be a

principle of nature to have this rather unspecific release as we have seen already in Fucus serratus and F. vesiculosus, probably also in others. Now if there is a population of Fucus serratus, there is normally only Fucus serratus there, and these things certainly act only over a very short distance.

Therefore, there is not much danger that hybridization

would occur. Muller has put it the other way around.

He said, " maybe

the fact that F^ serratus produces the same attracting substance as F. vesiculosus is like a kind of chemical warfare, to attract the others." SCHMITZ:

Are there any analogs to the sex substance in the laminariaceae

which could be applied, perhaps to vegetative cells, to elicit the same response? LUNING: No, not yet. They are still trying to establish the structure.

Part I I .

Nutrition and culture

INORGANIC NUTRITION OF MARINE MACRO-AICAE IN CULTURE1

J . ffcLachlan Atlantic Research laboratory, National Research Gouncil of Canada, 1411 Oxford S t . , Halifax, N.S., Canada B3H 3Z1

Introduction U n t i l a b o u t 25 y e a r s a g o , r e l a t i v e l y few s p e c i e s o f marine a l g a e , including both u n i c e l l s and macrophytes, were being c u l t u r e d .

Culturing

o f s p e c i e s in the l a t t e r category was in many instances limited to spore germination and perhaps s e v e r a l subsequent c e l l d i v i s i o n s .

Improved

marine media became a v a i l a b l e in the l a t t e r p a r t o f the 1 9 5 0 ' s r e s u l t i n g in a dramatic i n c r e a s e not only in the v a r i e t y o f available

(64)

species

i n c u l t u r e but a l s o in the number o f workers using a l g a l

cultures as research t o o l s . advancements

Bold in 1974 (10) concluded t h a t a l l major

i n p h y c o l o g y d u r i n g t h e p a s t s e v e r a l d e c a d e s were

a t t r i b u t a b l e to the a v a i l a b i l i t y and use o f a l g a l c u l t u r e s .

Certainly,

even a c a s u a l perusal of current l i t e r a t u r e v e r i f i e s that algal cultures are used in a large variety of research p r o j e c t s , although the majority of investigations have utilized unicellular species. I t was not until the immediate past decade that a t t e n t i o n was focused on t h e c u l t u r i n g o f marine m a c r o - a l g a e .

Since t h e n , t h e r e has been a

c o n s i d e r a b l e i n c r e a s e in the v a r i e t y o f i n v e s t i g a t i o n s

undertaken,

although compared with unicellular species, vrork with marine macro-algae s t i l l remains limited and information on t h e i r nutritional requirements i s meagre.

I believe t h i s to be the r e s u l t of two f a c t o r s , probably the most

important being the absence of any e s t a b l i s h e d c u l t u r e c o l l e c t i o n s macrophytes.

This i s in s p i t e o f the obvious need f o r c u l t u r e s ,

for as

emphasized by Bold ( 1 0 ) , and repeated p l e a s f o r the e s t a b l i s h m e n t o f collections (62,63,68). 1

Consequently, studies that have been done

NRCC Na. 18248

© 1982 Walter de Gruyter & Co., Berlin • New York Synthetic and Degradative Processes in Marine Macrophytes

72 generally r e f l e c t the interests of those who have isolated the organisms. Maintenance of macro-algae in culture perhaps requires more time and space than that required for unicellular species, but even so the situation with micro-algae i s becoming desperate ("Phycological Newsletter", vol. 15, No. 3, 1979).

Secondly, only a few macro-algae have been isolated into axenic

culture.

Many of the s t u d i e s undertaken have not required axenic

cultures; moreover, investigators and reviewers have not demanded them, but a t one time i t was impossible to publish on 'bacterized' unicellular species.

As a r e s u l t of t h i s p a u c i t y of axenic c u l t u r e s o f m a r i n e

macrophytes, there i s only limited information on organic nutrition, and this i s fragmentary and sometimes c o n t r a d i c t o r y .

I w i l l not consider

o r g a n i c nutrition as this aspect has been reviewed by Fries (24). emphasise, though, t h a t t h i s f i e l d

I must

i s wide o p e n , and b e c a u s e o f

developmental p r o c e s s e s a s s o c i a t e d with macrophytes, many exciting and c h a l l e n g i n g problems await p h y c o l o g i s t s .

Information on

inorganic

n u t r i t i o n a l requirements i s e q u a l l y scanty, partly because macro-algae have generally been cultured in media previously designed f o r c u l t u r e of unicellular

species.

S e v e r a l o f t h e s e media have been g e n e r a l l y

s u c c e s s f u l , thus obviating the n e c e s s i t y f o r d e s i g n i n g new or s p e c i a l media.

Moreover, t h e r e h a s been a g e n e r a l l a c k of employment of

a r t i f i c i a l media, and most have been singularly disappointing.

Because of

the l i m i t e d variety of studies on macrophytes in culture, i t i s d i f f i c u l t t o e s t a b l i s h general p r i n c i p l e s .

Instead I can only point to a few

interesting phenomena, and suggest areas where d e f i c i e n c i e s in information urgently require attention.

Essential Elements Nutritional studies must be done with c u l t u r e s , and of course a b s o l u t e requirements can only be established through culture studies.

Criteria

for an absolute requirement were established by Arnon (4) , based on the postulate that deficiency of a nutrient renders i t impossible for the alga to grow, t o p h o t o s y n t h e s i s e , o r t o c o m p l e t e the v e g e t a t i v e reproductive stages in i t s l i f e history.

and

The deficiency must be s p e c i f i c ,

i . e . only that nutrient can prevent or correct the deficiency.

Moreover,

73 the nutrient must be involved directly in the nutrition of the plant and not related to a chemical or biological condition of the medium (4). The latter condition is especially difficult to resolve because of the balance and interaction of elements in the concentrated and complex marine type of media. Only in a very few instances have absolute requirements been established for marine algae. Tàble I: Essential elements in plant nutrition Macronutrients Metals

Non-Metals

Potassium Sodium Calcium Magnesium

Carbon Hydrogen Oxygen Phosphorus Nitrogen Sulphur

Micronutrients Metals

Non-Metals

Iron Gopper Zinc Molybdenum Cbbalt Vanad ium

Boron Silicon Chlorine Iodine Branine

ffc>re than 100 years ago, both Knop and Sachs formulated media for growth of higher plants in hydroponic culture (22), thereby identifying the essential major nutrients. Molish, nearly 80 years ago, noted that the mineral nutrition of algae was not unlike that of higher plants (64), a conclusion that has been repeatedly substantiated.

Indeed, Knop's medium

is still commonly used, not only for higher plants but also for various other plants including freshwater algae.

Sachs included NaCl in his

medium and these two elements are now considered essential for plants (Table I).

It is interesting that sodium and chlorine are the predominant

elements in seawater (Table II), and, hence, in marine media, whereas nitrogen and usually calcium dominate media for freshwater algae and terrestrial plants. Those elements considered essential for plants are listed in Table I, and all of these have been identified as being required by at least one algal species (22, 59). Bromine is now regarded as essential for several marine macro-algae (25,50) and must be added to the list of required elements for plants.

It is unnecessary to emphasise that if an absolute requirement

74 Table i l : Major elements in seawater of 35 °/oo s a l i n i t y ( a f t e r Collier [ 1 6 ] ) . Element

g/kg

mmol/kg

10.556 0.380 1.272 0.400 0.0085

459.02 9.72 52.30 9.98 0.15

18.980 0.065 0.001 2.649 0.140 0.026

535.30 0.81 0.05 27.57 2.29 0.42

Cations *Sodium *Potassium *Magnesiun *Calcium Strontiun Anions *Chlorine *Bromine Fluorine *Sulfate *Bicarbonate *Boric acid * Essential for plants.

i s demonstrable then that nutrient must tie present in the environment o f the plant.

But i t i s important to appreciate that the nutrient must be in

an assimilative form, an important consideration with marine media.

As i s

to be e x p e c t e d , a l l elements known to be e s s e n t i a l for plants occur in seawater ( T a b l e s I I ,

III).

Moreover, the major c o n s t i t u e n t s o f

this

medium, except f o r strontium and f l u o r i n e , are required by plants, and other than c h l o r i n e are m a c r o n u t r i e n t s (Table I ) . numerous o t h e r e l e m e n t s ;

except

Seawater c o n t a i n s

f o r those p r e s e n t in very minute

concentrations, these are l i s t e d in TSble I I I .

Unlike the major elements,

only a few of these minor elements are known to be e s s e n t i a l for plants. The e s s e n t i a l elements are accumulated in the tissues of algae over t h e i r c o n c e n t r a t i o n in seawater (Table IV, c f . with Tables I I and I I I ) .

The

concentration factor of seme of the trace elements may be many thousand fold.

The elemental composition of the ash of macro-algae i s similar to

that of phytoplankton, and i n t e r e s t i n g l y , the c o n c e n t r a t i o n o f elements

75 considered adequate for higher plants (22) is similar to that found in marine algae.

T&ble hi. Minor (trace) elements3 in seawater of 35°/oo salinity (after Goldberg [29]). Element *Si A *N Li Rb *P *I Ba In *Pe Al *Zn *MD As *Cu U *Mn Ni *V Ti Sn Sb a

mg/1 3.0 0.6 0.5 0.17 0.12 0.07 0.06 0.03 re, L.F., Traquair, J.A. : Planta 128, 179-182 (1976). 55. Morel, F.M.M., Rueter, J.G., Anderson, D.M., Guillard, R.R.L. : J. Phycol. 15, 135-141 (1979). 56. Myklestad, S., Eide, I., Melsom, S. : Proc. Int. Seaweed Symp. 9, 143-151 (1979). 57. Neilson, A.H., Larsson, T.: Physiol. Plant. 48, 542-543 (1980). 58.

North, W.J., Wheeler, P.A.: Proc. Int. Seaweed Symp. 9y 67-78 (1979). 59. O'Kelley, J.C.: Di Algal Physiology and Biochemistry (W.D.P. Stewart, ed.), Cambridge University Press, Cambridge, pp. 610-635, 1974

97 60. Pedersen, M.:

Physiol. Plant. 22, 680-685 (1969).

61. Percival, E., McDowell, R. H. : Chemistry and Enzymology of Marine Algal Polysaccharides, Academic Press, London 1967 62.

Provasoli, L.: Proc. Int. Seaweed Symp. _4, 9-17 (1964).

63. Provasoli, L.: In Culture and Collections of Algae (A. Watanabe, A. Hattori, eds.), Japanese Soc. Plant Physiol., pp. 63-75 1968 64.

Provasoli, L., Mclaughlin, J.J.A., Droop, M.R.: Arch. Mikrobiol. 25, 392-428 (1957).

65. Schreiber, E.: Wiss. Meeresuntersuch. N.F. 16, 1-34 (1927). 66.

Schonbeck, M.W., Norton, T.A.: Bot. Mar. 22, 233-236 (1979).

67.

Shephard, D.C.: In Methods in Cell Physiology, Vol. 4 (D.M. Prescott, ed.), Academic Press, New York, pp. 49-69 1970

68. Starr, R.C.: Proc. Int. Seaweed Symp. ]_, 3-6 (1972). 69. Stoffyn, M.: 70.

Science 203, 651-653 (1979).

Tbpinka, J.A.: J. Phycol. 14, 241-247 (1978).

71. Tbpinka, J.A., Rabbins, J.V.:

Limnol. Oceanogr. 21, 659-664 (1976).

72. Ukeles, R.: In Marine Ecology Wiley and Sons, London, pp.367-466 73.

III, pt. 1 (O. Kinne, ed.), John 1976

Underwood, E.J.: Prog. Water Itech. _LL, 33-45 (1979).

74. von Stosch, H.A.: Naturwissenschaften 49, 42-43 (1962). 75. von Stosch, H.A.:

Proc. Int. Seaweed Symp. 4, 142-150 (1964).

76. von Stosch, H.A.: Proc. Int. Seaweed Symp. 6, 389-399 (1969). 77.

Wheeler, W.N.: Hi Synthetic and Degradative Aspects of Marine Macrophytes (L.M. Srivastava, ed.), Walter De Gruyter, Berlin 1981

78. Whyte, J.N.C., Ehglar, R.J.: Tfech. Itept. Fish. Mar. Ser. Ottawa 509, (1974). 79. Whyte, J.N.C., Ehglar, J.R.:

Bot. Mar. 23, 13-17 (1980).

80. Whyte, J.N.C., Ehglar, J.R.:

Bot. Mar. 23, 19-24 (1980).

81. Wbolery, M.L., Lewin, R.A.: Phycologia 12, 131-138 (1973). 82. Young, D.N., Howard, B.M., Fenical, W.: (1980).

J. Phycol. 16, 182-185

83. Young, E.G., Langille, W.M.: Can. J. Bot. 36, 301-310 (1958).

98 DISCUSSION BI DWELL:

I have a comment related to Dr. Willenbrink's observations this

morning about C^ fixation in photosynthesis of marine plants.

The

observation of sodium as a required nutrient is interesting because in higher plants sodium is a required nutrient only for those plants which have a C^ photosynthetic system, such as conventional C^ plants and crassulacean plants.

These plants always have PePC and usually the PePCk

and the inference is that the sodium requirement is related to the C^ carboxylation.

The interesting thing is that sodiuri is a required element

for the blue-green algae also, but I do not think that they are C^ plants; they do have an active C^ metabolism, however. NORTH:

I would like to emphasize the importance of contamination in trace

metal studies.

One of our graduate students made a calculation that the

amount of zinc in submicron particles contained in 1 liter of air in an ordinary urban environment, if allowed to settle in a petri dish with normal sea water, could double the amount of zinc in that water. McLACHLAN:

It is not necessarily the concentration of the elements , but

the form in which they exist relative to each other that is important.

It

is this interaction that we have probably not appreciated sufficiently in the past.

Now there are computer programs available and these

calculations can be made.

This will help a lot.

FOSTER: Another problem seems to be in the heavy metal contamination of the reagents that are used.

In some wark done in our laboratory we find

routinely that any culture we get frcm the culture collection, the heavy metal content arri everything else is on the order of one magnitude higher than the heavy metal content in the ocean. McLACHLAN:

I think it would now be possible (with the use of computers,

E3.) to correct for these errors.

Also there have been available for seme

time chelated resins and I think they can be used to clean up some of these media with very high salt concentrations, something we have not been able to do before.

NUTRIENT UPTAKE AND GROWTH IN TOE IAMINARIALES AND OTHER MACROPHYTES: A CONSIDERATION OF METHODS

P . J . Harrison Departments of Botany and Oceanography, U n i v e r s i t y of B r i t i s h Columbia, Vancouver, B.C. V6T 2B1, Canada L.D. Druehl Bamfield Marine S t a t i o n , Bamfield, B.C. VOR 1B0, Canada

Introduction The growth r a t e of seaweeds may be l i m i t e d by s e v e r a l e n v i r o n m e n t a l f a c t o r s such as l i g h t , temperature and n u t r i e n t s . Ihe l a t t e r f a c t o r has j u s t begun t o r e c e i v e a t t e n t i o n and t h i s review w i l l f o c u s only on n u t r i e n t limited growth of marine macrophytes, p a r t i c u l a r l y kelp. Studies of the n u t r i e n t requirements of seaweeds indicate t h a t while over 50 e l e m e n t s may be found in seaweeds (8) , l e s s than half of these may a c t u a l l y be r e q u i r e d f o r growth.

Most of t h o s e e l e m e n t s which

are

r e q u i r e d for growth are present in seawater in excess amounts; however, a few elements such as N, P, or Fe may l i m i t growth a t c e r t a i n times because they a r e present in low concentrations o r , as in the case of iron, are in a b i o l o g i c a l l y unavailable form.

These three elements may l i m i t the yield

(bicmass) as well as the growth r a t e (metabolic reactions) of the seaweed. This l a t t e r a s p e c t has been s t u d i e d q u a n t i t a t i v e l y by examining one component of i t , the r e l a t i o n s h i p between the ambient concentration and r a t e of uptake of the limiting n u t r i e n t . Nutrients generally enter plant c e l l s as e i t h e r cations or anions.

These

i o n s may move across the c e l l membrane by passive d i f f u s i o n ( i . e . down an electrochemical g r a d i e n t ) , or by a c t i v e t r a n s p o r t , a p r o c e s s r e q u i r i n g energy since movement i s against an electrochemical g r a d i e n t .

The l a t t e r

uptake mechanism i s thought t o be the most important f o r the l i m i t i n g n u t r i e n t s because the e x t e r n a l o r ambient c o n c e n t r a t i o n i s generally

© 1982 Walter de Gruyter & Co., Berlin • New York Synthetic and Degradative Processes in Marine Macrophytes

100 several orders of magnitude l e s s than the i n t r a c e l l u l a r

concentration.

This active uptake mechanism exhibits saturation k i n e t i c s ; a t low nutrient c o n c e n t r a t i o n s the uptake r a t e i n c r e a s e s l i n e a r l y with

increasing

concentration, however, at high nutrient concentrations a further increase in nutrient concentration does not r e s u l t in any further i n c r e a s e in the uptake r a t e .

The r e l a t i o n s h i p between uptake r a t e o f the l i m i t i n g

nutrient and i t s concentration i s g e n e r a l l y d e s c r i b e d by a r e c t a n g u l a r hyperbola, s i m i l a r to the Michaelis-Menten equation for enzyme k i n e t i c s (Fig. 1A).

The equation i s : V = Vmax

Kg

+

S

where V and V max are the uptake and maximal uptake r a t e s ,

respectively,

and S i s the substrate (limiting nutrient) c o n c e n t r a t i o n .

Kg i s c a l l e d

the half-saturation constant and i t i s the substrate concentration where V = Vmax / 2 . The lower the value o f Ks f o r a p a r t i c u l a r macrophyte, the higher i s the a f f i n i t y o f the c a r r i e r s i t e s for the nutrient. Ihe two uptake kinetic parameters in the above equation which are r o u t i n e l y used to describe the uptake c h a r a c t e r i s t i c s of a p a r t i c u l a r p l a n t , are Kg and ^max°

Recent

l Y ' i t has been suggested that the slope of the i n i t i a l

part

o f the hyperbola may be a more useful parameter for comparing competitive a b i l i t y for the limiting nutrient, rather than the Kg v a l u e , because in the l a t t e r case, when Vmax i n c r e a s e s or d e c r e a s e s the value o f the K s automatically changes also (24). The kinetic parameters may be calculated more accurately by rearranging the Michaelis-Menten equation above t o yield a s t r a i g h t l i n e from which Ks and max can be c a l c u l a t e d from the slope or a x i s i n t e r c e p t . The advantages and disadvantages of the three possible linear p l o t s have been examined by Dowd and Riggs (11) and the S

/ V vs. S or the

V

/ S v s . V plots are generally superior to the V s v s . V v

plots. Active uptake may not f o l l o w the simple s a t u r a t i o n k i n e t i c s d e s c r i b e d above.

Studies with higher p l a n t s have revealed t h a t the pattern of

uptake may be biphasic or multiphasic ( 1 3 , 3 2 ) . evidence for such a pattern in macrophytes.

Tb date t h e r e i s no good

Macrophytes may take up the

101

Fig. 1: Hypothetical plots of nutrient uptake rate (V) and concentration of the limiting nutrient ( S ) , showing: A) active uptake o n l y , where V i s the maximal uptake rate and K , the half-saturation constant, which i s the concentration where V = V / 2 and, B) a c t i v e uptake plus a l i n e a r d i f f u s i v e component ( s o l i d ^ i n e ) and only a diffusive component (dotted line). limiting nutrient by a secondary p r o c e s s , d i f f u s i o n .

Its

relationship

with the c o n c e n t r a t i o n of the limiting nutrient i s shown in Fig. IB.

In

the case of simple diffusion, the rate of uptake i s generally proportional to i t s e x t e r n a l c o n c e n t r a t i o n ( i . e . a l i n e a r relationship even at high nutrient c o n c e n t r a t i o n s ) .

D i f f u s i o n a c r o s s the c e l l membrane i s not

l i k e l y to be important, except at concentrations greatly exceeeding those found environmentally ( 9 , 1 7 ) .

I t may be important for the movement of the

n u t r i e n t ion i n t o the apparent f r e e space or apoplast ( 1 3 , 2 9 ) which includes the c e l l walls and the i n t e r c e l l u l a r spaces ( 1 6 , 1 8 ) .

The volume

of the apparent free space may be considerable, with values of up to 20 to 30% of the c e l l volume for the red alga, Porphyra p e r f o r a t a ( 1 2 ) .

When

an alga is placed in a nutrient median an i n i t i a l rapid uptake of ions may occur f o r a few minutes and t h i s uptake i s c o n s i d e r e d

t o o c c u r by

diffusion into the apparent free space. Of a l l the n u t r i e n t s required f o r growth o f macrophytes, n i t r o g e n

is

regarded as the most commonly limiting nutrient of coastal surface waters during the summer months ( 3 6 ) .

The a b i l i t y to procure or compete f o r the

l i m i t i n g n u t r i e n t or to m o b i l i z e i n t r a c e l l u l a r r e s e r v e s during

this

nitrogen limited summer period, may p a r t i a l l y determine the s u c c e s s o f a

102 macrophyte in the benthic community.

For this reason, there has been

considerable interest in determining the uptake kinetic parameters, V max and K g , for nitrogen in order to quantitatively assess the a b i l i t y of an alga to compete f o r t h i s l i m i t i n g

resource.

For example, a good

competitor would have a high Vmax . and a low Ks value. The number of s t u d i e s on n i t r o g e n uptake by macrophytes g r e a t l y lags those f o r phytoplankton. Cnly a few phaeophytes (5,17,21,44,50) and other macrophytes (9,19,20) have been studied, but already there is considerable variability in the methods of assessing uptake rates.

Uptake vs. Growth The specific uptake rate (V) is equal to the specific growth rate (u) when nutrients are not limiting, or during steady state growth under nutrient l i m i t i n g conditions.

From nimerous experiments with marine phytoplankton

(2,6), i t has been shown that under transient conditions when the limiting nutrient is provided to an alga growing under nutrient limitation, V > u until the amount of the limiting nutrient per c e l l , or the c e l l quota, Q, reaches i t s normal (maximal) concentration.

Therefore, during periods of

nutrient limitation, the transient maximal uptake r a t e ,' Vmax', which is ^ measured during short term n u t r i e n t uptake experiments, would over-estimate the maximal growth rate, u , by several times ( 9 ) . The max half-saturation constant, K g , determined during transient conditions of short term uptake experiments, may also be greater than that for growth. Ebr tvo red algae, DeBoer et a l . (7) found the Kg f o r ammonium limited growth was very low, ranging frcm 0.2 to 0.4 jaM.

Similarly, for some kelp

such as Laminaria saccharina the Kg for nitrate limited growth was 1.3 to 1.5_pM (5) while the Kg f o r uptake ranged from 4 to 6

nitrate for

Laminaria longicruris (21). Uptake rate is a good approximation of growth rate when nutrients do not l i m i t growth, or when steady state growth occurs under nutrient limiting conditions.

Under nutrient l i m i t e d

c o n d i t i o n s uptake r a t e

i s an

assessment of an a l g a ' s short term response under transient conditions,

103 while the growth rate represents a long term response and consequently i t i s more of

an integrating parameter.

Both measurements are r e a l l y

necessary to understand an a l g a ' s t o t a l response _in s i t u ; in the past there has been a bias towards measuring only nutrient uptake rates. Growth rate measurements w i l l not be covered in d e t a i l as these have recently been reviewed by Chapman ( 3 ) .

B r i e f l y these methods involve

destructive sampling f o r changes in dry weight, and plant carbon or nitrogen, or nondestructive sampling which include changes in wet weight, surface area or the movement of punched holes away from the meristem in the case of same kelp.

Et>r ecological or f i e l d purposes, non-destructive

sampling is preferable since the growth of the same plant can be followed over time and related to the l i m i t i n g nutrient concentration of the ambient water.

Ideally, one would like to relate growth rate to another

rate parameter, the specific flux of the limiting nutrient, rather than a standing stock parameter, nutrient concentration.

This relationship can

only be accurately determined in continuous culture in the laboratory and the thorough study by DsBoer et a l . (7) is a good example of how this i s achieved. The use of different methods of measuring uptake rates makes comparison of data d i f f i c u l t or in some cases impossible.

Therefore at this early phase

of studies with marine macrophytes i t would be desirable to adopt a more standard procedure for determining the nutrient uptake rate. w i l l consider

This review

various aspects of the methods f o r determining nutrient

uptake and make reccrmendations wherever possible.

Considerations While the reasons f o r making nutrient uptake measurements may vary, for the purposes of this review, the methods which are discussed w i l l be primarily for an ecological measurement of uptake rate, rather than solely for the elucidation of physiological mechanisms. thoroughly reviewed elsewhere (14,29,48).

The l a t t e r has been

104 I.

Experimental considerations

1.

Normalization of uptake r a t e s . Ihe four commonly used normalizations 1) area (pmol cm- 2 h- 1 ) , 2) wet weight (pmol g wet wt- 1 h - 1 ) , 3) dry

are:

weight (jumol g dry wt

h-"*"), 4) s p e c i f i c uptake (jjmol

nutrient in the plant h-"*", which simplifies to h-"*"). by a r e a ,

jamo!-"*" particulate Since normalization

w e i g h t and c o m p o s i t i o n o f p a r t i c u l a t e m a t t e r a r e

not

interconvertible, i t i s advisable to report in more than one u n i t or a t l e a s t g i v e conversion f a c t o r s , so t h a t data in the l i t e r a t u r e may be compared.

Normalization on an area basis i s a useful d e s c r i p t i o n o f the

uptake p r o c e s s s i n c e the use of these units makes some attempt to r e l a t e the rate at which ions are taken up a c r o s s a u n i t s u r f a c e area o f membrane.

cell

Dry or wet weights are used to normalize uptake rate on a

biomass b a s i s .

The s p e c i f i c uptake rate i s useful in allowing comparison

of rates between diverse groups of organisms and estimating the e f f e c t s of pre-history ( i . e . response of a plant from a nutrient depleted environment vs. one from a nutrient rich environment).

Since surface area and wet and

dry weights are constant throughout an experiment, a d i f f e r e n t pattern o f uptake vs. time or substrate will be obtained than when uptake i s divided by the amount of particulate nutrient in the plant, which increases during the uptake experiment as the a l g a t a k e s up t h e l i m i t i n g

nutrient.

Although in some c a s e s a constant amount of particulate nutrient in the p l a n t may be used in the c a l c u l a t i o n o f s p e c i f i c uptake r a t e i f

the

incubation period i s short, t h i s i s usually the amount a t the beginning o f the experiment.

At present i t i s unclear j u s t how quickly the uptake rate

o f the l i m i t i n g n u t r i e n t i s a f f e c t e d by the limiting nutrient taken up during the experiment. 2. Field v s . laboratory measurements.

Ideally one would l i k e to make the

uptake measurements for autecological studies in the f i e l d , since one does not have to contend with the d i f f i c u l t i e s of simulating important physical ( l i g h t quality and quantity, temperature, and water movement) and chemical (nutrient concentration and pH) f a c t o r s which a r e c h a r a c t e r i s t i c o f the field,

l b simulate f i e l d c o n d i t i o n s , s e v e r a l s t u d i e s have used an in

s i t u approach f o r carbon uptake o r oxygen e v o l u t i o n measurements o f primary productivity by enclosing the macrophyte in a p l a s t i c bag ( 2 7 , 4 5 ) , or large p l a s t i c cylinder ( 2 3 ) .

This approach i s most s u i t a b l e

for

105 isotopes (e.g. detected.

15

N,

32

P,

because small amounts of uptake can be

One of the problems in using isotopes is that different parts

of the thallus accumulate the isotope at different rates and samples from different parts of the thallus must be taken and averaged in order to obtain a whole-thallus uptake measurement (the most useful measurement for ecological purposes).

Nutrient uptake measurements can also be determined

by following the disappearance of the limiting nutrient from the medium, provided the ratio of plant biomass to medium volume is high enough to produce a relatively high rate of nutrient removal. The major disadvantages of field experiments are:

1) varying natural

conditions from day to day (e.g. insolation), and 2) the difficulty in producing a significant number of simultaneous replicates. These problems are overcome under laboratory conditions; however, extrapolation to the natural environment is often difficult. 3. Laboratory conditions.

If uptake experiments are conducted in the

laboratory, then every effort should be made to simulate the environmental conditions in the field.

Since it is difficult to reproduce the high

irradiance values obtained outdoors, it is preferable to conduct a nutrient uptake or photosynthetic rate vs. irradiance experiment to determine at what light levels these metabolic processes are saturated. Saturation generally varies between 30 and 100 pE m —2 —1 irradiance above 100

jjEb

S

-2

s

-1

, however, an

should be saturating for most species.

There have been no experiments to date testing the effect of light quality on nutrient uptake rates.

If fluorescent lights are used, daylight

fluorescent tubes are preferable to the more frequently used cool white tubes, because their spectrum simulates underwater light more closely. The spectral distribution can be corrected further to approximate the spectral distribution of 5 m underwater light (Jerlov Type 3), by inserting a sheet of 1/8" thick blue Plexiglas (Rohm and Haas #2069) between the lights and the plants (22). The photoperiod should also simulate natural conditions.

This is

important for longer term experiments where diel periodicity in uptake may influence results.

If the C/N ratio (atonic) of the macrophyte is 10, the uptake r a t e o f

limiting

nitrate

or

aitmonium during the day and night may be equal ( i . e . no p e r i o d i c i t y ) . D i f f e r e n c e s in water temperature between the l a b o r a t o r y and the f i e l d may g e n e r a l l y be c o r r e c t e d

by assuming

a Q^Q o f

t e m p e r a t u r e s l e s s than about 15 C ( 2 8 ) .

approximately

2 for

This may not be true f o r short

term transient changes in temperature which may o c c u r when s h o r t

term

uptake e x p e r i m e n t s are conducted a t a d i f f e r e n t temperature than those encountered in the f i e l d . 4.

Medium.

F i l t e r e d natural

phytoplankton),

seawater

( 0 . 4 5 jjm f i l t e r

to

remove

p r e f e r a b l y from the c o l l e c t i o n s i t e , should be used.

If

the nutrient under study i s present in v e r y low c o n c e n t r a t i o n s ,

then the

filtered

limiting

medium may be e n r i c h e d w i t h known amounts o f

nutrient f o r the uptake experiment. saturating

amounts o f

When the f i l t e r e d medium c o n t a i n s

the n u t r i e n t o f

"stripped o u t " by p u t t i n g

the

interest,

a l a r g e biomass o f

t h i s n u t r i e n t may be

the macrophyte

into

the

medium f o r s e v e r a l hours; these plants should not be used f o r the uptake experiments. since

I t should be noted that many Phaeophyceae may be u n s u i t a b l e

they o f t e n r e l e a s e

substances which d i s c o l o r the water during the

nutrient s t r i p p i n g p r o c e s s .

A l l o t h e r n u t r i e n t s , v i t a m i n s and

metals must remain saturating throughout the uptake experiment.

trace

These can

be added by making up an enrichment s o l u t i o n such as ES ( 3 5 ) minus the nutrient of

interest.

For n u t r i e n t s such as n i t r o g e n one must choose

which form ( e . g . n i t r a t e , compound) to add.

nitrite,

ammonium, o r some o r g a n i c

nitrogen

In the f i e l d under sunnier conditions when nitrogen may

t>e l i m i t i n g , uptake of ammonium, produced through r e g e n e r a t i o n

primarily

by h e r b i v o r e s , may be more important e c o l o g i c a l l y than n i t r a t e .

I f uptake

r a t e s of ammonium are studied, r a t e s f o r ammonium c o n c e n t r a t i o n s are not o f

ecological

>10 JJM

i n t e r e s t ; in f a c t , concentrations over 30 } M have

been reported to be t o x i c f o r some species o f macrophytes ( 4 7 ) .

One must

a l s o bear in mind that uptake r a t e s o f ammonium may be much g r e a t e r (up t o 50%) than n i t r a t e uptake r a t e s ( 9 ) and t h e r e f o r e caution must be exercised in extrapolating these high uptake r a t e s to other seasons o f the year when n i t r a t e may be the p r i n c i p l e form taken up.

107 Uptake studies conducted in the f a l l , winter or early spring months should use n i t r a t e as the most e c o l o g i c a l l y appropriate form of nitrogen; however, in the summer, pulses of n i t r a t e may also be added to the euphotic zone during periods of storm a c t i v i t y or t i d a l mixing (42). Concentrations of up to 30 jjM are of ecological interest. Organic nitrogen sources have seldom been examined.

Urea has been most

frequently studied and inferior growth was reported in most instances (7). The uptake of 17 d i f f e r e n t amino acids by the brown a l g a , G i f f o r d i a mitchellae was tested (37). had the highest r a t e .

Nine amino acids were taken up and leucine

Calculations revealed that at natural amino acid

concentrations (ca. 1 pM), uptake of amino acids, at l e a s t under the conditions used in the study, would only supply a few percent of that alga's nitrogen requirements.

Most studies to date have presented the

macrophyte with only one organic nitrogen source at a time. simulate natural conditions.

This does not

Further studies are required on mixed

substrates ( i . e . inorganic plus organic, or several organic compounds added t o g e t h e r ) .

Nutrient preconditioning of the alga is also v e r y

important in determining s u b s t r a t e preferences.

For example, in

experiments with phytoplankton, uptake of amino acids was enhanced i f

the

phytoplankton had been previously growing under nitrogen l i m i t i n g conditions (51). If uptake rates are determined by disappearance of nutrients, then enough biomass must be used to be able to measure a decrease in the nutrient concentration in the medium in a reasonable length of time.

However, too

much biomass may decrease the uptake rate due to decreased l i g h t absorption as a result of overlapping t h a l l i , or the rapid exhaustion of the l i m i t i n g

nutrient before the end of the experiment.

The above

criteria can be accommodated with a biomass of approximately 5 g wet wt l-"*" for most species, however, much higher concentrations may be used depending on the thickness and form of the thallus ( e . g . filamentous v s . blade).

Johnston (25) recommends a dry seaweed/water volune ratio of 0.1

to 0.3 for 24 h incubations to ensure no nutrient or CO^ deficiencies. 5.

Collection.

Collection of plants should be carried out c a r e f u l l y in

order to incur a minimum of s t r e s s , particularly light and temperature

108 shocks.

I t is preferable to c o l l e c t whole p l a n t s detached at

holdfast.

the

Storage in water in a covered ice chest and inmediate return to

the laboratory is desired.

If experiments are conducted over a series of

days, c o l l e c t i o n s should be made at the same time every day in order to minimize any diel periodicity e f f e c t s . c o l l e c t epiphyte-free t h a l l i .

Every attempt should be made to

If this is not possible then epiphytes may

be gently removed by wiping the t h a l l u s .

A compound microscope may be

useful to determine the completeness of removal.

The e f f e c t of the

epiphyte will vary with the species of epiphyte, and the type and l i f e history of the host.

Fbr example, the pennate diatom, Navicula incerta

adversely influences the growth and even the survival of germlings of Fucus spiralis (39). Bacterial contamination is more d i f f i c u l t to control.

Antibiotics such as

streptomycin and penicillin have been employed to inhibit bacterial uptake (21), but the concentration where there is no e f f e c t on a l g a l uptake may vary according to the age or nutrient past history of the alga.

I t is

unlikely that there i s an a n t i b i o t i c concentration which completely inhibits the bacteria but has no e f f e c t on the alga. 6.

Experimental design.

There are twa basic approaches to determine the

uptake k i n e t i c s of an alga.

The f i r s t and the most common is to monitor

the depletion of the nutrient from the a l g a ' s incubation medium. approach g i v e s an e s t i m a t e o f excretion).

This

the net uptake (gross uptake minus

However, this r e l i e s on the assumption that the excreted

nutrient i s in the same form as the monitored nutrient being taken up. The second approach, which to our knowledge has seldom been employed f o r seaweeds, is to monitor the isotopic nutrient accumulated by the plant. The disadvantage of this method r e l a t e s to the assumption that

the

isotope/nonisotope excreted nutrient w i l l have the same ratio (e.g. 15 14 N/ N) as the gross uptake ratio. This same problem plagues researchers 14 using C for productivity estimates. In this review we w i l l focus on monitoring the depletion of nonisotopic nutrients from the medium to estimate net uptake since i t i s the simplest method f o r studies.

ecological

However, studies using isotopes are required to enhance our

knowledge of nutrient excretion, residency time of the nutrient within the plant tissue and elucidation of physiological mechanisms.

109

Using the method of uptake of nonisotopic nutrient, there are two basic experimental designs in use.

The simplest arrangement is to place one or

several plants in high concentration of the limiting nutrient ( e . g . 30 jiM NCXj") and follow the decrease in the limiting nutrient with time ( F i g . 2A) .

This method has been frequently used to measure uptake rates for

phytoplankton and is referred to as the perturbation method ( 2 , 6 ) .

It

consists of removing small aliquots of medium on a time series basis until the nutrient has been exhausted or no more is taken up.

Nutrient samples

can be analyzed by hand (41), although an Autoanalyzer has the advantage of a smaller sample size and nearly real time read-out of results.

I t may

take many hours f o r the limiting nutrient to be taken up, and if the plants have been growing under a day/night c y c l e , interpretation of the pattern of uptake can be complicated by a diel periodicity in the uptake rate.

lb overcome this, i t is recommended that a higher biomass be used

to reduce the time necessary to complete the experiment.

Ifowever, one

must assure that carbon does not become limiting under the higher biomass conditions.

Time (h) Fig. 2: Hypothetical time s e r i e s of nutrient disappearance from the medium using: A) the perturbation method where saturated uptake is linear with time, B) the multiple container - constant incubation time method, and C) the perturbation method where saturated uptake is nonlinear with time. The second method of determining uptake rates involves the use of many containers

f i l l e d with f i l t e r e d medium, enriched with the l i m i t i n g

nutrient in a series of concentrations (e.g. 2, 4, 6, 8, 12, 16, 20, and 30 pM NCU~).

A d i f f e r e n t plant i s placed in each container and the

110 disappearance o f the l i m i t i n g n u t r i e n t i s determined a f t e r a defined period o f time ( F i g . 2B).

Generally, the incubation time i s short ( e . g .

10 to 60 min) and constant for each concentration.

An a l t e r n a t i v e to t h i s

method i s to use a long incubation time for the higher concentrations and a shorter time for the lower concentrations of the limiting nutrient.

The

v a r i a b l e incubation time has the serious disadvantage of overemphasizing the uptake rate at low nutrient concentrations i f the uptake r a t e i s not l i n e a r with time ( F i g . 2C).

This n o n l i n e a r i t y of uptake with time has

been observed f o r some phytoplankton ( 6 ) and in some h i g h e r (26,29).

The v a r i a b l e

incubation

plants

time method does not o f f e r any

advantages in assessing uptake r a t e and i t i s not recommended f o r use under any circumstances. In the Introduction of t h i s review the p o s s i b i l i t y of uptake occurring by p a s s i v e d i f f u s i o n , as opposed to active uptake, was discussed.

There are

several ways to eliminate or estimate the contribution of diffusion of the nutrient into the apparent free space or apoplast.

This i n i t i a l component

of uptake can be separated from active t r a n s p o r t o f the n u t r i e n t a c r o s s t h e plasmalemma by e s t i m a t i n g the time required f o r the n u t r i e n t to d i f f u s e i n t o and s a t u r a t e

the apparent

free

space. T h i s can be 45 accomplished by using a radioactive tracer such as Ca. The plant tissue i s placed in r a d i o a c t i v e l y labelled mediun for several hours and then i t i s placed in nonlabelled medium of the same c o n c e n t r a t i o n .

In order to

e s t i m a t e e f f l u x uncomplicated by plant reabsorption of the isotope, the e f f l u x medium i s replaced with f r e s h , n o n l a b e l l e d s o l u t i o n a t r e g u l a r intervals.

From t h i s time s e r i e s o f measurements, a pattern of e f f l u x can

be obtained (Fig. 3) in which apparent free space efflux can be separated from cytoplasmic and v a c u o l a r e f f l u x e s by i t s higher r a t e (37,48, see chap. 6 of 29).

Knowing the time required to saturate the apparent

free

space one can preincubate the experimental plants for t h i s length of time ( e . g . 5 min).

Alternatively, one could d i s r e g a r d the uptake during the

f i r s t 5 minutes in the n u t r i e n t disappearance studies.

This choice i s

l e s s desirable because i f the apparent f r e e space i s r e l a t i v e l y then

the concentration of

the l i m i t i n g

nutrient

at the

large, lowest

concentrations used in the experiment may be lowered c o n s i d e r a b l y b e f o r e the uptake component of i n t e r e s t i s observed.

111

0

4 8 12 16 Washing T i m c ( h )

Fig. 3: Hypothetical time series showing e f f l u x of isotope from plant tissue as a function of wash time. Region 1 represents the apparent free space, region 2 the cytoplasm and region 3 the vacuole. Radioactively labelled plants are placed in nonlabelled mediun and the amount of label appearing in the wash medium is measured (see text for d e t a i l s ) . Further tests to determine i f uptake is active or passive, include the use of metabolic inhibitors, uptake determinations at different temperatures and different substrate concentrations.

These tests have been used to

characterize the active uptake of leucine by the brown a l g a , G i f f o r d i a mitchellae (37) and this research o f f e r s a good example to follow. In both types of uptake experiments i t is necessary to s t i r the medium to prevent a small zone of nutrient depletion from forming around the thallus.

If this zone is allowed to develop, then the actual uptake rate

i s reduced because the nutrient ion must diffuse across this zone, giving rise to diffusion-limited uptake rates ( 3 3 ) .

In the kelp, Macrocystis

pyrifera, Wheeler (52) found that water speeds of approximately 4 cm s were necessary f o r maximum uptake rates.

Mixing may be achieved by

112 aerating the medium or by s t i r r i n g with a magnetic b a r .

In the former

c a s e f o r uptake of ammonium a t low c o n c e n t r a t i o n s , the a i r should be "scrubbed" free of aitmonia by passing i t through IN ZnClj and then through d i s t i l l e d water, to prevent ammonia in the a i r frcm being added during the aeration process.

If a s t i r r i n g bar i s used, i t should be placed

inside

some kind of mesh cage to prevent i t frcm injuring the t h a l l u s , or the thallus may be suspended above the s t i r r i n g bar. Each of the two b a s i c d e s i g n s f o r n o n i s o t o p i c n u t r i e n t u p t a k e measurements has s e v e r a l

rate

a d v a n t a g e s and d i s a d v a n t a g e s ( 4 6 ) .

The

perturbation method allows one to observe how the pattern of uptake r a t e changes with time.

This has been shown to be important in phytoplankton

(6,10) and in higher p l a n t s ( 2 6 , 2 9 ) .

This experimental design could

s i m u l a t e some environmental s i t u a t i o n s t h a t may be encountered by a macrophyte.

Fbr example, large amounts of n i t r a t e may be brought into the

euphotic zone by storm a c t i v i t y (49) or when an intrusion of high s a l i n i t y bottom water replaces lower s a l i n i t y i n t e r s t i t i a l water in the sediments, r e l e a s i n g l a r g e amounts of nutrients (40).

The f i e l d response to such a

l a r g e n u t r i e n t change can only be a s s e s s e d with the experiment.

perturbation

The d i s a d v a n t a g e of t h i s method i s that only one or a few

plants are used and i t i s d i f f i c u l t to a s s e s s whether these p l a n t s are typical of the f i e l d population under study.

Another disadvantage i s that

the nutrient past h i s t o r y i s changing during the experiment ( i . e . p l a n t continues to overcome i t s nutrient debt).

the

This r e s u l t s in reduced

uptake rate l a t e r in the experiment p o s s i b l y due to a p r o c e s s such a s feedback i n h i b i t i o n (6,26).

This i s not a serious disadvantage for t h i s

type of experiment, however, because such a p r o c e s s a l s o occurs in the field. The m u l t i p l e c o n t a i n e r - c o n s t a n t i n c u b a t i o n time e x p e r i m e n t

is

recommended for determining the nutrient uptake parameters, Vmax and Ks . Since a new or d i f f e r e n t p l a n t i s used for each concentration, and the incubation time i s constant, the past history i s the same for each uptake experiment,

resulting

measurements.

An additional advantage of this type of experiment i s t h a t

in no p a r t i c u l a r b i a s f o r a c e r t a i n s e t of

many d i f f e r e n t p l a n t s are used, which gives r i s e to more v a r i a b i l i t y in the uptake rate data (44), but actually provides a b e t t e r assessment of

113 the population response s i n c e i n t e r p l a n t v a r i a b i l i t y i s taken i n t o account. To r e d u c e the v a r i a b i l i t y in the d a t a , many more uptake measurements are required; each concentration should be run in t r i p l i c a t e at l e a s t . In most studies i t i s desirable to obtain both the uptake pattern and the uptake kinetic parameters v_ and K_s for the population by incorporating = max i n t e r p l a n t v a r i a b i l i t y into the l a t t e r measurements. Therefore, i t i s recommended t h a t both types of uptake experiments be c a r r i e d out i f possible. II

Biological considerations

1.

Prehistory or preconditioning.

In order to properly interpret uptake

measurements, the preconditioning or prehistory of the plant in the f i e l d should be known.

This can be accomplished by f r e q u e n t l y and r e g u l a r l y

monitoring the ambient limiting concentrations for at l e a s t several weeks in advance of the experiment, p r e f e r a b l y throughout the depth range encountered by the plants.

In the case of nitrogen, the limiting nutrient

vnuld include measuring at l e a s t nitrate and ammonium concentrations, but dissolved organic nitrogen may a l s o be important.

In an eleven year study

of the English Channel, Butler e t a l . (1) showed t h a t while n i t r a t e and ammonium decreased to very low l e v e l s during the summer months, dissolved organic nitrogen rose during t h i s period to s i g n i f i c a n t

levels

(approximately 6-8 pM), indicating that i t may be an important available source of nitrogen for some species. The prehistory of the plant in the f i e l d can a l s o be assessed by measuring the concentration of various internal components of the plant which change as the external nutrient concentration changes.

Bt>r nitrogen l i m i t a t i o n ,

the most sensitive parameter appears to be the internal nitrogen ( n i t r a t e , ammonium or amino acids) pools, which can be determined by hot ethanol extraction ( 4 ) .

In studies of Laminaria saccharina the i n t e r n a l

nitrate

pool increased a t external nitrate concentrations of about 10 >iM (5) and in Macrocystis pyrifera

changed s i g n i f i c a n t l y in a few days in response

to low external concentrations (53). The C/N r a t i o a l s o r e f l e c t s the nutrient prehistory.

In some continuous

114 culture experiments with tvro red algae, DeBoer e t a l . (7) showed t h a t C/N r a t i o s >10 i n d i c a t e d n i t r o g e n limitation and the r a t i o increased as the degree of limitation increased. in i n t e r p r e t i n g

However, some caution should be exercised

C/N r a t i o s ,

because

in e s t u a r i n e areas nitrogen

constituents have teen reported to increase as s a l i n i t y decreases (30) and the r a t i o may a l s o vary with the type o f alga (31); the Phaeophyta had higher r a t i o s than e i t h e r the Chlorophyta or Rhodophyta. Another condition that must be considered in nutrient uptake experiments i s the degree o f d e s i c c a t i o n o f the a l g a .

Recently, i t has been shown

that when sane i n t e r t i d a l algae are desiccated upon emergence ( a s during t h e d a y t i m e i n t h e summer a t low t i d e ) and then re-submerged, subsequent uptake rate i s enhanced due to a desiccation e f f e c t

(43).

the In

the high i n t e r t i d a l alga, Fucus d i s t i c h u s , the uptake rate was enhanced by a f a c t o r o f approximately two when the alga was desiccated by 30%.

Ft>r

low i n t e r t i d a l and subtidal algae there was l i t t l e or no enhancement o f nutrient

uptake upon d e s i c c a t i o n .

For a s t a n d a r d c o n d i t i o n

for

interspecies comparisons, i t i s recommended that a l l species be completely hydrated before the beginning of the experiment. 2.

Variation between plant p a r t s .

Uptake determinations on vrfiole t h a l l i

are p r e f e r a b l e f o r e c o l o g i c a l measurements, but i f the thallus i s too large then a consideration of which portion of the thallus to use becomes important.

Meristematic r e g i o n s o f the thallus are generally the most

active metabolically.

Therefore, i f t i s s u e from only t h i s p a r t i s used

f o r uptake measurements, extrapolation to whole plant uptake will r e s u l t in an overestimation. Cutting of the thallus to produce t i s s u e segments may a l t e r the uptake r a t e due to a wounding response resulting in increased respiration ( 2 3 ) , or elimination of the t r a n s p o r t system or a c t i v e sink region ( 3 4 ) .

A

comparison of excised sections of Macrocystis t i s s u e with whole b l a d e s , showed a decrease in uptake rate as high as 80% by the cut sections ( 5 0 ) . In addition, freshly cut discs of Laminaria longicruris have been reported to r e l e a s e brownish-colored substances and mucilage, and the water had to be changed before the experiment began ( 2 1 ) .

115

N i t r a t e Conc'n (pM)

Fig. 4: Nitrate uptake rate for the f i r s t ( a ) , second ( o ) , and t h i r d (•) year whole p l a n t s o f Laminaria g r o e n l a n d i c a as a f u n c t i o n o f n i t r a t e concentration. Rates were determined for whole plants except for third year plants where a l o n g i t u d i n a l s t r i p from the blade was used. The plants were incubated at an irradiance of 50 >iE m s for 1 h at 13 C in f i l t e r e d and enriched sea water to which d i f f e r e n t concentrations of n i t r a t e were added. About 5 g 1 of plant tissue was placed in a l a r g e p l e x i g l a s s tank and the sea water was f r e q u e n t l y s t i r r e d during the incubation. Disappearance of n i t r a t e from the medium (see r e f e r e n c e 41 f o r method) was used to calculate the uptake r a t e . Data points represent a single rate measurement and curves were f i t t e d visually. 3.

Variation as a function of plant age.

Many seaweeds are p e r e n n i a l ,

c r e a t i n g n a t u r a l populations c o n s i s t i n g o f s e v e r a l age c l a s s e s .

Any

extrapolation of nutrient studies to f i e l d conditions must recognize the p o s s i b i l i t y of d i f f e r e n t uptake k i n e t i c s for the resident age c l a s s e s . Our preliminary s t u d i e s on the n i t r a t e uptake k i n e t i c s o f t h r e e year c l a s s e s of Laminaria groenlandica have demonstrated a decreased uptake with i n c r e a s i n g age ( F i g . 4 ) .

F u r t h e r , the shape of the uptake curve

changed from the c l a s s i c a l r e c t a n g u l a r hyperbola f o r the f i r s t

year

p l a n t s , to a curve with enhanced uptake at higher n i t r a t e concentrations for second and third year plants.

116 The opposite trend of uptake rate with age of tissue has been observed f o r Macrocystis by Schmitz and Srivastava (38).

They found that punched discs

from mature blades took up more phosphate than similar discs from young meristematic regions. regions

In other experiments, they showed that the mature

took up and t r a n s p o r t e d

the phosphorus

to sink

regions

(meristems). 4.

I n t e r s p e c i f i c variation.

Comparative studies by Gagné and Mann (15)

of Laminaria l o n g i c r u r i s populations growing in areas of constant v s . seasonal nitrogen a v a i l a b i l i t y ,

indicated a possible d i f f e r e n t genetic

response to these d i f f e r e n t nutrient regimes.

This finding indicates the

n e c e s s i t y of caution in e x t r a p o l a t i n g r e s u l t s obtained f o r a s i n g l e population.

T£> our knowledge there are no studies elucidating the degree

of v a r i a b i l i t y in nutrient uptake within populations.

Summary

K e l p b i o l o g y has been expanding

rapidly

in the l a s t

few

years.

P h y s i o l o g i c a l aspects such as n u t r i e n t uptake have been seen t o be important

in u n d e r s t a n d i n g

their f i e l d biology.

Frequently

physiological uptake experiments have been conducted by f i e l d

these

ecologists

who have i n s u f f i c i e n t exposure to various phytoplankton uptake methods to be able to decide on the appropriate method f o r their s p e c i f i c s i t u a t i o n . We hope that t h i s review w i l l be useful f o r ecologists trying to choose the most appropriate procedures f o r their nutrient uptake experiments with macrophytes.

Acknowledgments T h i s research was supported by the Natural Sciences and Engineering Research Council of Canada.

We thank Drs. W.N. Wheeler and D.H. Turpin,

and T.E. Thomas f o r c r i t i c a l l y reviewing t h i s manuscript. assistance was provided by Ms. K. Lloyd.

Technical

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47.

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48.

Walker, N.A., Pitman, M.G.: _In Encyclopedia of Plant Physiology, New S e r i e s , V o l . 2, Transport in Plants I I , Part B, Tissues and Organs, (U. Duttge and M.G. Pitman, e d s . ) , Springer V e r l a g , New York, pp. 93-126 (1976).

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50.

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51.

Wheeler, P.A., North, B.B., Stephens, G . C . : 249-259 (1974).

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119 52.

Wheeler, W.N.:

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53.

Wheeler, W.N. : In S y n t h e t i c and Degradative Processes in Marine Macrophytes. (L.MT Srivastava, ed.) Walter de Gruyter, Berlin, 1981

DISCUSSION BIEWELL:

Were the curves in your figure 4 done by mathematical analysis,

or by a computer or did you draw them by eye? HARRISON:

They were drawn by eye.

More work needs to be done, e s p e c i a l l y

using higher concentrations, before these data becane meaningful. LUNING:

Gould i t be that t h i s enormous d i f f e r e n c e in n i t r a t e uptake by

d i f f e r e n t age group plants i s due to completely d i f f e r e n t growth a c t i v i t y ; that i s , that the f i r s t year plants are much more f a s t growing than the second and third year plants? DRUEHL:

The f i r s t two years' plants appeared to have the same growth rate

on both surface area and fresh weight b a s i s , in the third year the growth rate was greatly increased. LUNING:

Increased!

DRUEHL:

That i s c o r r e c t .

McLACHLAN:

I am s t i l l curious about t h i s d i f f e r e n c e between the

second and third year.

first,

Gould i t be that the second and third year plants

have more inactive (mature, Ed.) tissue and supply s u f f i c i e n t amounts o f n i t r o g e n to the growing p o r t i o n s o f the plants and that perhaps uptake curves are rather similar? HARRISON:

That i s a good idea and i t should be done — t h a t i s , to take

a c t i v e t i s s u e s from each one of these age classes and compare uptake in active t i s s u e s . SRIVASTAVA:

Might not the free space component i t s e l f be d i f f e r e n t in one

year vs. two year material?

120 HARRISON:

I t probably i s , but we could not detect any change in the

uptake rate over a 2 h period, sampling every 15 min.

NITROGEN NUTRITION OF MACROCYSTIS

W. N. Wheeler1 Marine Science Institute, University of California, Santa Barbara, USA

Introduction Early in this century, many of the larger kelps were harvested for potash. Later, other chemicals, iodine and alginic acid, were found to be present in the brown algae in commercially significant quantities.

The harvesting

and subsequent processing of kelps thus became the basis for an industry, which is now world-wide (5). Recently, interest has been diverted toward the utilization of brown algae, especially Macrocystis, as a renewable source of biomass for energy production.

Economically viable quantities

of the plant, however, can only be achieved under intensive cultivation, and nutrient fertilization of large kelp beds is likely to be an expensive necessity. Answers to questions of nitrogen nutrition of the large kelps, therefore, are paramount to a successful cultivation effort. Heretofore, little work has been done outside of growing laboratory-reared sporophytes in defined media and there is hardly any information on the nitrogen nutrition of adult sporophytes in the field.

Since any

cultivation effort must take place in the sea, with adult plants, a knowledge of the nutrient ecology of the adult sporophyte is fundamental to any fertilization strategy.

The emphasis of this paper, therefore, is

on the adult sporophyte and its response to its nitrogen environment. Although the data presented are predominently my own work, I have tried to set this paper within the context of the available literature on the nitrogen physiology of Macrocystis and other marine macroalgae.

1

Present Address:

Department of Biological Sciences, Simon Fraser

University, Bumaby, B.C. V5A 1S6, Canada

© 1982 Walter de Gruyter & Co., Berlin • New York Synthetic and Degradative Processes in Marine Macrophytes

122 Materials and Methods Whole surface and subsurface fronds of Macrocystis pyrifera were collected from kelp f o r e s t s near the University of California, Santa Barbara, USA. Ihey were transported to the laboratory within 1 h , and held in outdoor tanks with flowing seawater for a maxim an of 36 h before analysis. (8 an or 1 . 3 an in diameter) were cut from blades and cleaned o f epiphytes. 1 h.

Disks visible

The cut disks were then preincubated in experimental media for

Preincubation allowed the removal of unwanted wound e x u d a t e s , gave

time for the f i l l i n g of free space within the tissue with the experimental medium in use, and may have lessened the e f f e c t of any wound r e a c t i o n on the uptake process. Incubation media consisted of nitrogen-depleted seawater with added, known concentrations of KNO^ and/or NH^Cl.

Tissue disks were added to j a r s

(1

l i t e r or 20 ml capacity) with tissue to medium volume r a t i o of 3g l i t e r " " ' ' or l e s s .

Most experiments were run f o r 1 h, and the concentrations of

nitrogen in the water b e f o r e and a f t e r the incubation were measured. E a r l i e r experiments involving b r i e f treatments of disks with a variety of d i s i n f e c t a n t s , including formaldehyde, b l e a c h , and v a r i o u s

antibiotics,

showed t h a t uptake r a t e s were unaffected by b a c t e r i a l contaminants over the experimental period of 1 h. I r r a d i a n c e was provided by a t u n g s t e n - i o d i d e

lamp (GE Q u a r t z l i n e —? -1 Q150013/C4) which gave a maximum irradiance of 300 pE m s . Tfemperature was held constant at 15 C (+ 0 . 5 C) using e i t h e r a water bath or cold room.

Water motion in the incubation j a r was provided with synchronized

magnetic s t i r r e r s (see 2 9 , 3 0 ) o r with a s h a k e r .

N i t r a t e and ammonium

determinations were made according to Strickland and Parsons (25).

Tissue

n i t r a t e was determined according to Chapman and Craigie ( 3 ) .

Results and Discussion M u l t plants of Macrocystis pyrifera near Santa Barbara can have as many a s 100 f r o n d s .

Each frond may grow to a l e n g t h o f 20 m o r more and

produce hundreds of blades. Wheeler (29) has shown that s e l e c t i v e sampling

123 of blade tissue along a number of fronds of different ages gives a general picture of frond and plant development,

ihere is an age gradient among

the blades of a frond from the apical meristem to the base (holdfast).

As

in some t e r r e s t i a l grasses (21), these ontogenetic d i f f e r e n c e s are pronounced and appear to overshadow irradiance e f f e c t s on pigments and photosynthesis.

It is, thus, imperative to f i r s t focus on the ontogenetic

variability among blades. Nitrate uptake Ontogenetic e f f e c t s .

The blades of

pyrifera were shown to have only a

s l i g h t photosynthetic gradient on a surface area basis from the proximal to the distal end by Clendenning (6).

A similar shallow gradient seems to

prevail for nitrate uptake along the length of a blade (Fig. 1). ~

60

UJ ¡3 Q.

20

ZD

ro 10 DISTANCE

20 FROM

30

40

50

PNEUMAT0CYST (cm)

F i g . 1: N i t r a t e uptake o f blade d i s k s along a mature blade o f Macrocystis pyrifera. Blade disks sampled from each meter segment of subsurface fronds y i e l d uptake rates for NO^ (Fig. 2), which seem to f o l l o w the photosynthetic p a t t e r n described f o r blades along the length of a frond (29).

In

general, the disks from the embryonic blades near the meristem showed low uptake rates when measured under optimal uptake conditions (300 jjE m s-"*"; 25 }iM NCL; > 6 cm s""*" water speeds; 15 C).

The disks from the

124

E o

o

E UJ =) CO CO

LU

É

û. ro O

ro

o

2

DISTANCE FROM

MERISTEM

(m)

Fig. 2: Distribution of nitrate uptake and concentration of nitrate in the tissue along two juvenile fronds of Macrocystis pyrifera. Two small disks were cut fran blades about 30 cm from the pneimatocyst at increasing distances fran the apical meristem. Cpen circles represent disks used for uptake determinations; closed circles represent the nitrate concentrations of adjacent disks. The top figure shows a frond with high tissue nitrate, the bottom figure a frond where nitrate is low. The points at 3 m represent data for sporophylls.

125 mature blades showed the h i g h e s t uptake r a t e s .

Senescing blades a l s o

showed r e l a t i v e l y low uptake r a t e s . I t has been hypothesized (16) that sporophylls, because of t h e i r p o s i t i o n below the thermocline, may tie important centers of n i t r a t e uptake.

These

blades do not show signs of senescence so noticeable in other b l a d e s , and therefore p e r s i s t longer.

There i s , however, no evidence from my data

that their uptake rates are any higher than those of vegetative blades a t corresponding depths (Fig. 2 ) . Concentration of n i t r a t e in the tissue may also play a role in the n i t r a t e uptake c a p a c i t y o f t h a t t i s s u e . concentration

in the t i s s u e

Figure 2 shows t h a t i f the n i t r a t e is

high

the

uptake

rates

may be

correspondingly low (upper graph) and vice v e r s a (lower graph; see a l s o s e c t i o n on s t o r a g e ) .

The a b i l i t y o f a p a r t i c u l a r t i s s u e t o take up

n i t r a t e , thus, might be the r e s u l t o f a combination o f the past growth h i s t o r y arri feedback inhibition.

Experiments to separate the two e f f e c t s

are now under way. Light.

A high photon flux density may enhance the uptake o f n i t r a t e by

mature blades in

p y r i f e r a , although the d a t a p r e s e n t e d a r e

not

s t a t i s t i c a l l y s i g n i f i c a n t (Fig. 3).

These data, however, do indicate a —2 —1 substantial uptake of n i t r a t e a t 0 )aE m s (dark). Water motion.

Water motion, delivered e i t h e r as a defined uni-directional

c u r r e n t in a water t u n n e l , or as a g e n e r a l turbulence in incubation chambers, produces a s i g n i f i c a n t e f f e c t on the uptake of n i t r a t e by mature blade disks of M. pyrifera (Fig. 4 ) .

Nitrate uptake r a t e s in one s e t o f —2 —1 experiments rose from 9 to 44 nmol cm h , an enhancement o f 488%, as water motion increased from 0 to 3 cm s-"*". Concentration of n i t r a t e .

Older saturating water motion (6 cm s - ''') and - 2

photon flux densities (300 /jE m

- 1

s

) , d i s k s from mature blades o f M.

p y r i f e r a respond to changes in ambient n i t r a t e h y p e r b o l i c manner (Fig. 5 ) .

concentration

in a

Although the form i s Michaelis-Menten, t h i s

in no way implies a knowledge o f the mechanisms involved.

Using the

terminology o f Michaelis-Menten k i n e t i c s allows comparison of uptake curves between d i f f e r e n t species.

The V

i s the maximun uptake

126

TÖCT

200

300

PHOTON FLUX DENSITY (jjE M" 2 S"') F i g . 3: N i t r a t e and ammonium uptake r a t e s v s . i r r a d i a n c e f o r M a c r o c y s t i s p y r i f e r a mature blade d i s k s . Top c u r v e shows u p t a k e of ammonium; NH. c o n c e n t r a t i o n in the incubation medium 9 JuM. Bottom curve shows uptake of n i t r a t e ; NO, c o n c e n t r a t i o n in the median was 9 juM. Sample s i z e , 10 b l a d e disks; v e r t i c a l bars represent S.E..

CURRENT SPEED (CM S"1) F i g . 4 : N i t r a t e and ammonium u p t a k e r a t e s v s . w a t e r m o t i o n f o r Macrocystis p y r i f e r a mature blade d i s k s . Top c u r v e shows u p t a k e o f ammonium; NH. c o n c e n t r a t i o n in the i n c u b a t i o n medium was 3 . 5 jjM. Bottom curve shows uptake of n i t r a t e ; NO, c o n c e n t r a t i o n in the medium was 15 juM. Sample s i z e , 10 b l a d e d i s k s ; v e r t i c a l b a r s r e p r e s e n t S . E . . Horizontal b a r s r e p r e s e n t S . E . o f 50 w a t e r s p e e d m e a s u r e m e n t s ( t u r b u l e n t fluctuations).

127 rate, or the uptake rate at infinite ambient concentration of NO^; the Kg i s the ambient concentration at 1/2 the maximum uptake rate. Ft>r the — 2 —1 mature blade of M. pyrifera, the v is approximately 75 nmol cm h — max the K is approximately 13 jJM. For young sporophytes of M. p y r i f e r a , Haines and Wheeler (11) calculated a V o f 95 nmol cm- 2 h for - 2

n u t r i e n t - r i c h , deep-water-grown p l a n t s and 69 nmol cm nutrient-poor surface-water-grown p l a n t s .

The K s v a l u e s

accordingly, being 13.1 jjM f o r deep water plants and 8.7 surface water plants.

- 1

h

for varied

f o r the

The above numbers and others have been converted to

a fresh weight basis for comparison (Table I ) .

N CONCENTRATION

(jjMOL L"1)

Fig. 5: Nitrate and ammonium uptake r a t e s v s . c o n c e n t r a t i o n f o r Macrocystis pyrifera mature blade disks. Disks were incubated in varying ambient concentrations of NH. ( c i r c l e s ) and N03 ( s a u a r e s ) . During incubation saturating conditions of irradiance (300 pE/m /sec) and water motion (6 cm/sec) were maintained.

Ammonium uptake Ontogenetic e f f e c t s .

Wheeler (28) using methylamine as an analogue of the

ammoniim ion demonstrated that apical sections and juvenile sporophytes of M. pyrifera had much lower uptake rates than mature tissues.

But, using

128 Täble I: Nitrate uptake constants. Alga

Codium fragile

Fucus spiralis Laminaria longicruris Macrocystis pyrifera

Tfemp (C)

Ks

6 12 18 24

1.9 3.8 6.9 7.7

5 10 15 0 12 16 16 15

5.6 6.7 7.8 nd nd 13.1 8.7 13.0

26 20 20

4.9 2.5 2.4

Hypnea museiformis Gracilaria foliifera Neoagardhiella baileyi

(pM)

\Jnax (¿mol g fr wt

1

h

0.283 0.60a 1.09a 0.96a 3

- 48 b '39b 4.96 1.19C 1.63° 3.05^ 4

2.25f

Ref

(12) If

11 II

(27) If

ft

(13) tl

(11) H

3.43

(here)

2.85 0.97 1.16

(11) (8) (8)

a

Wet wt/dry wt = 10. Vfet wt/area = 0.046. ° Wet wt/dry wt = 5.9. Young, deep water sporophytes. | Young, surface water sporophytes. Mature blades, nd Not determined. cut disks frcm blades, apical sections were found to have higher uptake rates than whole blades. Light. Variation in photon flux density appears to have no effect on the rate of ammonium uptake in mature blades of M. pyrifera (Fig. 3). Water motion. Water motion, either as a uni-directional current in a water tunnel or as turbulent flow in an incubation chamber increases uptake of attmonium by M. pyrifera blade disks (Fig. 4).

For example,

under an ambient concentration of 3.5 JJM ammonium, the uptake rates -2

increased from 2 to 11 nmol cm

-1

h

when water speeds increased from 0 to

1

5 cm s" . Concentration of ammonium. Anmonium uptake kinetics appear complex. Et>r

129 l&ble I I :

Ammonium uptake constants.

Alga

Tfemp (C)

Ks (yM)

Unax (jimol g f r wt

Godium fragile

6 18 30

1.47 1.56 2.1

1.30a 2.78a 2.153

Fucus spiralis

h

)

Ref

(12) II ll

(27)

Macrocystis pyrifera Macrocystis pyrifera Macrocystis pyrifera

5 10 15 16 8 15

6.4 6.4 9.6 5.3 50 33

2.36^ 4.17

(11) (28) (here)

Gracilaria f o l i i f e r a Hypnea museiformis Neoagardhiella baileyi

20 26 20

1.6 16.6 4.9

2.38a not saturatable 1.19a

(8) (11) (8)

a

e

II II

Wet wt/dry wt = 10. Wet wt/area = 0.046. Elnbryonic sporophytes, below 22 /jM. Methylamine uptake in mature blades. Mature sporophyte blades; area/wtwt = 45.8.

j u v e n i l e M^ p y r i f e r a Haines and Wheeler ( 1 1 ) reported

a sharp

discontinuity in the uptake curve above 20 jjM ambient NH^ and interpreted that as being indicative of a 2 component uptake system.

Cne system was

believed to be composed of a high a f f i n i t y (or low concentration) uptake mechanism, the other of a low a f f i n i t y (or high concentration) mechanism. By contrast, Wheeler (28), using methylamine on mature blade disks showed saturation slightly above 100 /jM with a Kg of about 50 ]JM.

She claimed

that because these nunbers were 10 times higher than corresponding values for ammonium using juvenile sporophytes, the a f f i n i t y of the c a r r i e r system f o r methylamine was 1/10 that of ammonium.

However, the data

presented here for ammonium using mature blade disks agree well with the methylamine data of Wheeler (28).

The d i f f e r e n c e in uptake by young

sporophylls and mature tissues noted by Wheeler (28) may, t h e r e f o r e , be age related and not be due to a lesser a f f i n i t y for methylamine by the carrier system.

Cne can only wonder why the saturation levels are so high

when measured concentrations of ammonium in coastal waters rarely exceed

130

I

2 V/S

3

4

5

6

(cm h'1)

-6 -4 Fig. 6: Wbolf-Hofstee plot of ammonium uptake between 10 and 10 M. Data from figure 5 of mature blades of Macrocystis pyrifera are presented here. r= 0.727, n=21, y= -32.5x + 191. 5 JJM.

Table I I gives Vmax and Kg data for ammonium uptake for a number of

different marine algae. Only Macrocystis and perhaps Hypnea appear to have such low a f f i n i t y uptake mechanisms. Because the uptake of methylamine or ammonium has a pH optimum (pH 8.5) , i s temperature dependent, and i s competitively inhibited by analogues, Wheeler (28) has concluded that the uptake of ammonium (or methylamine) is active and brought about by an "ammonium permease."

The Wbolf-ftofstee

plot of ammonium uptake in mature blade disks shown in f i g u r e 6 also indicates an active uptake of ammonium. In a nunber of other plant systems, including phytoplankton and higher plants, ammonium uptake i s known to block the simultaneous uptake of nitrate, presumably

by a feed-back i n h i b i t i o n .

In many macroalgal

systems, however, both n i t r a t e and ammonium appear to be taken up simultaneously, ftnmonium may be taken up preferentially as in Gracilaria (8) or i t may have no e f f e c t whatsoever on nitrate uptake, as is the case with M. pyrifera (11).

131 Organic sources of nitrogen Urea is a common source of organic N used as f e r t i l i z e r for higher plants. North (22) has shown that gametophytes of M. pyrifera show positive growth in a medium containing less than 0.01 M urea as the sole nitrogen source. D i r e c t uptake s t u d i e s have not been done.

Urea,

supplied

at

concentrations of less than 10 ppm, has been shown to enhance the growth rate of Laminaria angustata and J^ r e l i g i o s a ( 1 4 ) .

Recently, Kirk and

Kirk (18) have shown that several members of the Chlorophyta are able to take up and utilize amino acids.

Schmitz and R i f f a r t h (24) have shown

active uptake and assimilation of a number of amino acids by G i f f o r d i a . They also showed that leucine had the highest uptake rate with a K of 30 -1

to 120 mM and a Vmax of 3 to 3.8 jJmol 100 g dry wt

-1

h

.

Storage Chapman and Craigie (3) demonstrated that Laminaria longicruris could store n i t r a t e in concentrations as high as 28,000 times over ambient. This nitrate would remain in the tissues f o r as long as 2 months a f t e r n i t r a t e had dissappeared from the surrounding water.

Buggeln (2) also

found that Alaria could take up and store n i t r a t e as much as 3000 times over ambient, but, contrary to Laminaria, Alaria stored n i t r a t e only f o r periods of a few days. Because a surplus of n i t r a t e appears in the tissues o f

Macrocystis

p y r i f e r a , i t appears that the uptake of n i t r a t e is f a s t e r than reduction (Fig. 2).

its

Concentration of nitrate in M. p y r i f e r a tissues can

be as high as 120 ^pmol g fresh w t - 1 although concentrations from 30-60 jjmol g fresh wt-"'" are more common.

These values are very similar to those

recorded f o r Laminaria saccharina in culture

( 4 ) , and f o r

natural

populations of Alaria (2) and Laminaria longicruris (3). Assimilation My unpublished observations indicate that Macrocystis p y r i f e r a , l i k e Alaria, can reduce up to 50 jjmol g fresh wt

of stored nitrate in 7 days,

an assimilation rate of approximately 0.3 ^jmol g fresh wt sporophytes of Laminaria

h

.

The

assimilated 50 jmol of stored n i t r a t e per gram

132 of fresh t i s s u e over 2 months, a reduction r a t e o f 0 . 0 3 4 pmol g f r e s h wt" 1 h - ^", which i s almost ten times slower than the rate estimated f o r M. pyrifera.

These values for the estimated l o s s r a t e o f n i t r a t e from the

t i s s u e and by i m p l i c a t i o n n i t r a t e reductase a c t i v i t y are not as high as those recorded for higher plants (see 20).

Nitrate reductase a c t i v i t y in

t e r r e s t r i a l p l a n t s grown with and without n i t r a t e f e r t i l i z e r range from 0 . 1 to 14.6 jmol n i t r i t e g fresh wt-"*" h-"*- formed; the computed values o f 0.034 to 0 . 3 /jmol g fresh w t - 1 h - 1 for kelps thus are on the low end. Nitrate r e d u c t a s e has been demonstrated in Undaria and

Alaria

(26).

F u r t h e r , the data o f Takagi and Murata (26) suggest that the enzyme has i t s highest a c t i v i t y in the stipes and holdfast regions,

followed by the

t r a n s i t i o n r e g i o n , the sporophyll and then the blade.

Some work with

young, cultivated sporophytes of Laminaria s a c c h a r i n a i n d i c a t e s t h a t in v i t r o n i t r a t e reductase a c t i v i t y , although present, i s weak (unpublished data).

N i t r a t e r e d u c t a s e has been c h a r a c t e r i z e d from the red a l g a ,

Forphyra ( 1 ) .

In Porphyra, the NAEH-dependent reduction has an apparent

Kg of 50 ^iM n i t r a t e . Hie assimilation of n i t r a t e through n i t r a t e reductase produces n i t r i t e and f i n a l l y ammonium.

From anmonium, the assimilation of nitrogen appears to

go predominantly through the glutamine s y n t h e t a s e pathway ( 1 5 ) . a c t i v i t i e s o f glutamine s y n t h e t a s e have been measured i n saccharina (unpublished data) and Giffordia ( 1 7 ) .

Using

15

High

Laminaria

N0 3 , Haxen (15)

demonstrated that in Macrocystis angustifolia the major portion of reduced N was incorporated i n t o glutamate via the glutamine synthetase pathway. Assimilation of nitrogen through a s p a r t a t e and a l a n i n e was very s m a l l . Feeding with "^N-glutamine i n d i c a t e d t h a t g l u t a m a t e was t h e m a j o r acceptor.

Inhibition of glutamine synthetase via methionine sulphoxamine

stopped a s s i m i l a t i o n o f n i t r o g e n and produced i n c r e a s e d ammonium.

levels

of

Common amino a c i d s found in the free s t a t e in the fronds and

s t i p e s of M. angustifolia from S. Africa were alanine (greatest in s t i p e ) , glutamine and glutamate (15).

133 Seasonal changes in nitrate concentration in seawater In order to understand the nitrogen ecology o f M a c r o c y s t i s ,

it

is

important to understand the seasonal v a r i a b i l i t y of nitrate in coastal waters. Typically in southern California, upwelling brings n i t r a t e into the surface waters during the months of March, April and May.

The nitrate

dissappears from the surface waters, except in trace amounts, a f t e r June. In many cases, this amount does not climb again until the following March. For almost 8 months, then, Macrocystis sp. must get i t s nutrients from transient l o c a l mixing, land r u n o f f , or u t i l i z e the s l i g h t l y higher concentrations available below the thermocline, a region usually no greater than 1 m above the bottom. Macrocystis pyrifera standing crops, as deduced from harvest records, have been shown to vary seasonally. seasonal growth variability surface irradiance.

Harger (10) calculated that half of the

was due to seasonally variable upwelling

and

Upwelling and water temperature were found to play

the major roles in determining M. pyrifera growth in another study ( 1 9 ) . Individual frond and plant growth rates of

pyrifera were higher during

the spring than in l a t e f a l l which corresponds well with high and low nitrate concentrations in surface waters (7).

Fertilizing M. p y r i f e r a in

situ with chemical inorganic nitrogen f e r t i l i z e r s or a r t i f i c a l l y upwelled "deep" water has resulted in increased growth rates (23).

Inorganic

nitrogen is probably the most limiting nutrient for growth of M^ p y r i f e r a in southern C a l i f o r n i a .

This i s not true of M. p y r i f e r a in central

C a l i f o r n i a (9), where substrate type, irradiance, and excess water motion (storms) seem to determine kelp growth and population dynamics.

Acknoledgements I would like to thank D. Ooon, P.J. Harrison, and J. Woessner f o r reading the manuscript.

Parts of this work were completed

in partial fulfillment

of the degree of Doctor of Philosophy at University of C a l i f o r n i a , Santa Barbara.

Parts of the vrork were supported by the Department of Commerce,

N.O.A.A. Office of Sea Grants - Grant No. 04-7-158-44121 and the State Resources Agency p r o j e c t s R/FA 10 and R/A 16A and National Science

134 Foundation Grants GA-27484 and ENG76-22720 to M. Neushul Charters.

and

A.C.

Equipment and supplies were supplied by Neushul Mariculture

Inc.

References 1.

Araki,S., Ikawa T., Ctohusa, T., Nisizawa, K. : B u l l . Jap. Soc. Fish. 45, 919-924 (1979).

2.

Büggeln, R.G.:

3.

Chapman, A.R.O., Craigie, J . S . :

4.

Chapman, A.R.O., Markham, J.W., LÜning, K.: (1978).

5.

Chapman, V.G.:

6.

Clendenning K.A.:

7.

Goon, D.A.: Bot. Mar. 24, 19-27 (1981).

8.

D'Elia, C.F., DsBoer, J.A.:

9.

Sei.

J. Phycol. 14, 156-160 (1978). Mar. Biol. 40, 197-205 (1977). J. Phycol. _14, 195-198

Seaweeds and their uses, ffethuen, London 1950 Proc. Int. Seaweed Symp. _4, 55-65 (1963).

J. Phycol. 14, 266-272 (1978).

Foster, M.S.: In Synthetic and degradative processes in marine macrophytes (LTFT. Srivastava, e d . ) , Walter de Gruyter, Berlin - New York 1981

10.

Harger, B.W.W.: Coastal Oceanography and hard substrate ecology in a C a l i f o r n i a n kelp f o r e s t . Ph.D. Thesis. University of California, Santa Barbara 1979

11.

Haines, K.C., Wheeler, P.A.:

12.

Hanisack, M.D., Harlin, M.M.:

13.

Harlin, M.M., Craigie, J.S.:

14.

Hasagawa, Y . , Komaki, S . , Honma, K.: 165-187 (1962).

15.

Haxen, P . C . : The major pathway of nitrogen assimilation in the marine kelp, Macrocystis a n g u s t i f o l i a Bory. B.S. Honors Thesis, University of Cape Tbwn, Cape Tbwn, South Africa 1978

16.

Jackson, G . A . : Nutrients and productivity of the g i a n t k e l p , Macrocystis pyrifera, in the near-shore. Ph.D. Thesis, C a l i f o r n i a Institute of Technology, Pasadena, California 1975

17.

Kiefer, H.: Untersuchungen zur Characterizierung und physiologischen Regulation der Nitratreductase und des Glutaminsynthetase bei der marinen Braunalge Giffordia mitchellae. Diplomarbeit, U n i v e r s i t ä t Köln, FRG 1980.

18.

Kirk, D.L., Kirk, M.M.:

19.

Kirkwood, P.D.:

J. Phycol. 14, 319-324 (1978). J. Phycol. 14, 450-454 (1978). J. Phycol. 14, 464-467 (1978). Hokkaido Fish. B u l l . 19 ,

J. Phycol. 14, 198-203 (1978).

Seasonal patterns in the growth of the giant kelp,

135 Macrocystis p y r i f e r a . Ph.D. T h e s i s , Technology, Pasadena, California 1977

California

Adv. Bot. Res.

Institute

of

20.

Lee, J.A., Stewart, G.R.:

1-43 (1978).

21.

Ludlow, M.M., Wilson, G . L . : (1971).

Aust. J. b i o l .

S e i . 24, 1077-1087

22.

North, W.J.: Kelp habitat improvement p r o j e c t . 1974 - June 1975

Annual r e p o r t , July

23.

North, W.J., Gerard, V.A.: In Synthetic and degradative processes in marine macrophytes (L.M. Srivastava, e d . ) , Walter de Gruyter, Berlin - New York 1981

24.

Schmitz, K., R i f f a r t h , W.:

25.

Strickland, J.D.H., Parsons, T.R. : 167, 1-310 (1972).

26.

T&gaki, M., Murata, K.: (1955).

27.

Tbpinka, J . A . :

Z. Pflanzenphysiol. 96, 311-324 (1980). B u l l . Fish. Res. Board Canada

B u l l . Fac. Fish. Hokkaido Univ. 6, 25-28

J. Phycol. 14, 241-247 (1978).

28.

Wheeler, P.A.:

J. Phycol. 15, 12-17 (1979).

29.

Wheeler, W.N.:

Mar. Biol. 56, 97-102 (1980).

30.

Wheeler, W.N.:

Mar. Biol. 56, 103-110 (1980).

DISCUSSION WILLENBRINK:

Did you do a time curve or the k i n e t i c s for the nitrate

uptake? WHEELER:

Yes, although not shown, uptake of n i t r a t e a p p e a r s t o be

unsaturable f o r at l e a s t 12 h.

The curves presented here were measured

while the uptake was linear and nonsaturated. BICWELL:

properly should be applied to single stage enzymatic systems

and I do not think you could honestly apply i t here.

Ft>r i f you do, you

are in great danger of drawing erroneous conclusions f o r a system such as uptake of an ion by an entire plant.

Ftor as soon as a l i t t l e b i t has been

taken up that would a f f e c t the uptake r a t e .

The diminution of r a t e which

you thought was the attainment of the maximum v e l o c i t y might in f a c t be due to removal or metabolism o f the ion a t the other s i d e .

The other

thing I was concerned about was that your data f o r nitrate uptake ( F i g . 5) do not show saturation, yet you calculate saturation values.

I think that

136 in both cases, nitrate and ammonium, you were pushing the data a b i t f a r . WHEELER:

You are q u i t e r i g h t about the d e f i n i t i o n of

problem arises here as in the previous question.

The same

I have previously shown

that the uptake in these kelp discs is linear over at least a 12 h period. Under these c o n d i t i o n s , s a t u r a t i o n o f the internal pools i s not found. Although in i t s s t r i c t e s t sense, the

can only be applied t o enzyme

systems, i t g i v e s us here a framework within which we can e f f e c t i v e l y compare interspecies d i f f e r e n c e s in uptake b e h a v i o r .

As t o your l a s t

comment, I would also agree perhaps there i s not enough data to s t r i c t l y calculate a saturation point.

The main purpose of the study was, however,

not to t o t a l l y d e f i n e the c h a r a c t e r i s t i c s of the curve, but to look at uptake behavior over a natural range of concentrations. ALBRIGHT:

Do you think that sane of the problems with uptake of materials

may be caused by epiphytes, particularly bacteria and perhaps microalgae, because they can operate at extremely low concentrations with v e r y high uptake rates. WHEELER:

I have done sane work with antibiotics and so have Pat Wheeler

and Marilyn Harlin.

What vie find i s that over short periods, one or tvro h

of incubation, the bacterial and microbial portion of the uptake i s v e r y snail and can usually be neglected. ALBRIGHT: WHEELER:

In what range is i t ? The highest rate found f o r microbial or other uptake i s about

20% of the total a f t e r an 8 h incubation.

Fbr short incubation periods of

one h or l e s s , i t is usually less than 10%. SCHMITZ:

Your measurements were made by noting the decrease of the ion in

the medium.

How much is the f r e e space in the discs?

Are a l l these data

corrected f o r uptake in the f r e e space? WHEELER:

A l l experiments wsre started a f t e r a one h preincubation period

during which the plant d i s c s recovered fron wounding, f i l l e d f r e e space and adapted to experimental conditions. McLACHLAN:

I am bothered by the uptake mechanism being proposed h e r e ,

137 which seems to disregard the dissociation of ammonium in sea water, especially when concentrations of 100 to 200 |iM are involved. WHEELER:

The ammonium uptake mechanism in Macrocystis is s t i l l under

debate. There are two lines of evidence that suggest that the uptake may be active, but whether there are one or two separate systems is under debate.

still

The data above 100 }JM simply extend measurements taken at

lower concentrations, and add l i t t l e to the explanation of the mechanism involved. McLACHLAN:

That s t i l l does not take into account the dissociation of

ammoniun in sea water. ALBRIGHT:

The use of a n t i b i o t i c s f o r inhibition of marine or other

bacteria has come under extreme criticism.

In many cases i t just does not

work. McLACHLAN:

Both the antibiotics are not e f f e c t i v e against gram negative

bacteria. ALBRIGHT:

Absolutely, and 75% of the bacteria isolated in sea water or

almost any water are gram negative.

UPTAKE OF INORGANIC IONS AND THEIR LONG DISTANCE TRANSPORT IN FUCALES AND LAMINARIALES

J . Y . Flex:'h Laboratoire de Physiologie végétale, Faculte'des S c i e n c e s , U n i v e r s i t é de Bretagne Occidentale, F-29283-BREST. Cedex, France.

Introduction As i s true f o r land p l a n t s , marine macrophytes a l s o r e q u i r e c e r t a i n e s s e n t i a l elements for t h e i r growth, d i f f e r e n t i a t i o n and r e p r o d u c t i o n . However, in contrast to the land plants, which absorb inorganic ions frcm s o i l by t h e i r r o o t s , the marine algae are able to take up t h e i r n u t r i e n t s d i r e c t l y frcm the ambient sea water through a l l parts of t h e i r t h a l l i . Mast of the mineral elements which are known to occur in sea water have a l s o been found in seaweeds.

Moreover, i t has been known from the

e a r l i e s t investigations that these plants have the a b i l i t y to accumulate in t h e i r tissues some of the elements which are present in sea water only in t r a c e amounts.

As such, t h e s e p l a n t s have s e r v e d a s

important

commercial sources f o r I , K, and Na (for review see 1 ) , and the mineral content of many of t h e s e s p e c i e s has been c a r e f u l l y determined

(2,3).

More r e c e n t s t u d i e s have given comparative values on the distribution of the various mineral elements in d i f f e r e n t species ( 4 - 8 ) , and in d i f f e r e n t morphological regions o f the t h a l l u s , i . e .

s t i p e and lamina

(9-12).

However, u n t i l r e c e n t l y , with a few e x c e p t i o n s ( 1 3 , 1 4 ) , no d e t a i l e d information was a v a i l a b l e on the v a r i a b i l i t y in the mineral content of d i f f e r e n t regions and tissues of the larger algae.

In the l a s t ten years,

v a r i o u s a n a l y s e s o f m i n e r a l e l e m e n t s have been performed in the Laminariales and Fucales, with regard to the age and function of d i f f e r e n t t i s s u e s ( 1 5 - 2 4 ) , and from these s t u d i e s i t has become c l e a r that the mineral composition of the larger algae i s a function not only of d i r e c t uptake o f elements from the sea w a t e r , but also of t h e i r long distance transport through the t h a l l u s .

© 1982 Walter de Gruyter & Co., Berlin • New York Synthetic and Degradative Processes in Marine Macrophytes

140

I f we exclude from c o n s i d e r a t i o n the v a s t l i t e r a t u r e

dealing

with

permeability of giant algal c e l l s , such as those of N i t e l l a and Chara, to v a r i o u s ions i t appears that much of the recent research on ion uptake by l a r g e r algae has been s t i m u l a t e d by one o r more o f t h e

following

considerations: - mass cultivation of algae including use of f e r t i l i z e r s ( 2 5 - 2 8 ) , - growth regulation ( 2 9 ) , - e f f e c t of pollution on algal growth ( 3 0 , 3 1 ) , - monitoring changes in sea water (32-34), - sewage cleaning ( 2 6 ) , - detoxification of animal feed ( 3 5 ) , - food chain ( 3 6 ) , and - human food(37). In the present paper, I will deal with uptake and long distance t r a n s p o r t o f inorganic ions in the l a r g e brown algae in as far as these processes r e s u l t in the accumulation of these ions in the t h a l l u s .

Uptake and Accumulation of Ions Concentration factors "Concentration factor" i s defined as the r a t i o of the c o n c e n t r a t i o n o f a mineral element in algal tissue to i t s concentration in sea water.

Table

I shows that concentration f a c t o r s d i f f e r widely f o r d i f f e r e n t mineral e l e m e n t s , and f o r d i f f e r e n t s p e c i e s taken from the same h a b i t a t .

They

d i f f e r also for d i f f e r e n t h a b i t a t s , and for d i f f e r e n t seasons ( 4 1 , 4 2 ) . Some o f the h i g h e s t c o n c e n t r a t i o n f a c t o r s a r e known f o r Mn, PO^, I ar NO^.

Other high concentration factors are known for T i , Fte, Cr, Cu and Pb

(10 4 - 10 6 )

(4,8,43).

Some authors have described an ascending order o f c o n c e n t r a t i o n f o r Na < F < Ca < Mo < K < Co < Cu < As < Ni < Mn < Zn < I

in various

algae, including the Fucales and Laminariales; the concentration ranging from 1 or l e s s for Na to 6 x 10

4

for I ( 9 ) .

Others have

factors factors

141 TSable I: Concentration factors (amount per unit fresh (f) or dry (d) weight of algae/ amount per unit sea water) for sane ions whose uptake or translocation is reported in large algae. Ions

Algae

Gone. Factor

m2+

Standard

iteference

(38)

Fucus

0.2 - 2.2 x 105

d

II

Sargassum

0.7 x 104

d

( 8)

II

Laminaria

0.8 x 104

d

( 8)

Laminaria

236

f

(39)

Fucus

1.7 - 2.5 x 104

d

(38)

II

Fucus

0.6 - 1.1 x 103

f

( 4)

II

Ascophyllun

1.4 x 103

f

( 4)

II

Laminaria

0.4

f

( 4)

II

Zn

2+

-

1

X

103

3

II

Laminaria

2.4 x 10

f

(39)

II

Laminaria

70 - 150

d

( 8)

Ca 2+

Fucus

3 - 4.5

f

(40)

II

Laminaria

4.5 - 7

f

(40)

II

Laminaria

5 or less

f

(16)

II

various algae

1 - 12

d

( 9)

Laminaria

1.2 or less

f

(16)

Mg 2+ +

various algae

10 - 30

d

( 9)

II

Laminaria

48 or less

f

(16) ( 9)

K

Na+

various algae

1 or less

d

II

Laminaria

0.7 or less

f

(16)

Cl~

Laminaria

less than 1

f

(16)

so42-

Laminaria

3.5 or less

f

(16)

NO ~

Laminaria

1.4 x 104 or less

f

(16)

5

f

(16)

4

d

( 9)

P0 4

Laminaria Laminaria

3 x 10 or less 6 x 10

classified the mineral elements into 3 groups on the basis of their accumulation coefficient (Ka) in marine algae: (1) elements with Ka < 1 x 102 comprised Na, Br, Sr, Se. (2) elements with Ka > 1 x 102 but < 1 x 104 comprised Rb, Cs, Ba, Sc, Go, Sb,flg,Th, U.

142 (3)

elements with Ka > 1 x 104 included Fe, Cr, Zn, Zr, Hf, As, Hg, Au, La, KH, Yb.

Such a wide range of concentration f a c t o r s and the f a c t that anions as w e l l as cations may be accumulated or excluded by algal tissues (Table I ) seems to indicate that d i f f e r e n t processes of uptake may be involved

for

d i f f e r e n t ions ( f o r a detailed review see 45). Ion competition and e f f e c t of external concentration on uptake Fran the classical experiments of Osterhout on e l e c t r i c a l resistance of Laminaria tissues under various external ionic conditions (46), i t is well known t h a t a s a l t competition occurs with respect to c e l l membrane permeability, e . g . , Ca^ and Na+ are antagonistic to each o t h e r , so are i|

Mg

and K .

Recent studies on Laminaria, Fucus, and Macrocystis have

shown t h a t , contrary to the situation in phytoplankton, the presence of NH^+ does not inhibit NO^- uptake by these algae, and that both the ions are used simultaneously (29,48,49). macrophytes, e . g . , Codium, a d d i t i o n , the uptake of

NH^"1"

NO^-

and

However, in some other marine

does a f f e c t NO^- uptake ( 4 7 ) . NH^+

In

by Fucus and Macrocystis

is

strongly affected by the external concentration of these ions (48,49).

At

r e l a t i v e l y lover concentrations, the relationship between nutrient uptake and nutrient concentration in the medium i s h y p e r b o l i c , and seems to follow Michaelis-Menten kinetics.

However, there were d e v i a t i o n s from

simple hyperbolic relations at r e l a t i v e l y high external concentrations of NO^- and NH^+, and the authors suggested that more than one process may be involved, for instance in the uptake of

by Macrocystis ( 4 9 ) , as

is the case in phytoplankton (50). Effect of l i g h t and darkness on uptake Several papers have been published on the e f f e c t of l i g h t on ion exchange in l a r g e algae (Table I I ) .

I t i s w e l l known that illunination has an

immediate and a strong p o s i t i v e e f f e c t on potassium uptake, and to a lesser extent, on sodium extrusion (51,52).

The e f f e c t of dark-light has

143 Table I I : E f f e c t of illumination on ion exchange in l a r g e a l g a e . positive e f f e c t , ( - ) no marked e f f e c t . Ion

(+)

Algae

Effect

Reference

K + , Na+

Ulva lactuca

+

(51,52)

K+,

Palmeria palmata

+

(53)

Fucus s p i r a l i s

+

(48)

Palmeria palmata

+

(54)

Ca 2+

Fucus vesiculosus

-

(55)

Zn 2+ 131,-

Laminaria digitata Ulva lactuca

+

(56)

NO3"

Enteromorpha sp.

+

(27)

N03

Laminaria longicruris

+

(29)

N03~

Fucus s p i r a l i s

-

(48)

Na+

NH + 13^2+

(39)

been interpreted in terms of a supply of K+ carriers during photosynthesis in the l i g h t and their removal by respiration in the dark ( 5 3 ) . uptake and

Na+

Both K +

extrusion are considered t o be regulated e i t h e r by two

active but separate mechanisms (52) or by Na + extrusion being dependent upon K+ accumulation (57).

In any case, such mechanisms r e q u i r e energy

and hence can be related to metabolism. E f f e c t of metabolic inhibitors on uptake Various metabolic i n h i b i t o r s , such as i o d o a c e t a t e , phenyl

urethane

d i n i t r o - o - c r e s o l , arsenate, potassium cyanide, and 2,4-dinitrophenol, have been reported to a f f e c t K + and Na + exchange in Ulva lactuca (51) and Hormosira banksii ( 5 8 ) , Rb+ uptake in Fucus serratus and 2— d i g i t a t a (59), and SO^

Laminaria

uptake in Fucus serratus (60).

E f f e c t of temperature on uptake As is true for l i g h t , the e f f e c t o f temperature on ion uptake by l a r g e algae i s ion s p e c i f i c and dependent on the species concerned (Table I I I ) . Thus, a strong negative e f f e c t in going from 15 C to 5 and 0 C has been

144 liable I I I : Effect of temperature on ion exchange in l a r g e a l g a e . positive e f f e c t , ( - ) no marked e f f e c t . Ion

Algae

Effect

Reference

NO3"

Enteromorpha sp.

+

(27)

NO3"

Laminaria longicruris

+

(29)

NO3-

Fucus spiralis

-

(48)

H 2 PO 4 "

Laminaria digitata

+

(23)

Zn2+

Fucus, Qiteromorpha

+

(61)

NH 4 +

Fucus vesiculosus

-

(55)

Ca 2+

Laminaria digitata

-

(23)

(+)

reported for NO^ uptake by Laminaria l o n g i c r u r i s ( 2 9 ) , in contrast to the results obtained with the same ion in Fucus spiralis (48). 32 of

By the use

P as a t r a c e r , i t has been shown that phosphate a b s o r p t i o n

in

Laminaria digitata is much slower at 0 C than at 12 C, and that no leakage occurs f o r hours when the tissues incubated at 12 C are subsequently immersed at 0 C in sea water free of tracer (Fig. 1). Similar experiments 45 ++ carried out by the use of Ca showed no s i g n i f i c a n t d i f f e r e n c e in Ca uptake in L. digitata at 12 C and 0 C, which suggests t h a t , contrary to the uptake of phosphorus, calcium uptake i s not d i r e c t l y r e l a t e d cellular metabolism.

to

Moreover, when the ^Ca labelled tissue was immersed

in sea water free of tracer, a large proportion of the radio-calciun, 80% in the f i r s t 4 hours, was rapidly released into the medium, suggesting that most of

i t was sequestered in the intercellular free-space (Fig. 2;

see also reference 23). Mechanisms of uptake by brown algae Fran the available data, twD kinds of mechanisms seem to be involved in ion uptake by large brown algae.

Cne is l i g h t and temperature dependent,

requires energy and is inhibited by metabolic inhibitors; the other seems to be governed by physical processes, and is apparently independent of metabolism.

145

hours Fig. 1: Time course of absorption and exsorption of phosphorus by d i s c s punched from the middle of the lamina of Laminaria d i g i t a t a . Absorption in f i l t e r e d sea water a t 0 and 12 C. Exsorption in f i l t e r e d sea water a t 0 C (arrow), imount of P taken up or retained by the tissues i s expressed as micrograms per 12 mm diameter d i s c . (From Floc'h and Penot [23])

Fig. 2: Time course of absorption and exsorption of calcium by Laminaria d i g i t a t a . Same conditions as in Fig. 1. (Fran Floc'h and Penot [23] )

146 I t i s v e i l known that two of the most c h a r a c t e r i s t i c f e a t u r e s o f brown algae are: (1)

the great amount of negatively charged polysaccharides in t h e i r

cell

walls; (2)

the occurence o f v a r i o u s types o f v e s i c l e s in the

protoplasm,

vacuoles or physodes, whose composition and role are not well known. In t h i s connection, some of the e a r l i e r work on zinc uptake in brown algae is relevant.

Gutknecht (62) studied zinc uptake in several marine algae,

including Fucus, and concluded that the main uptake mechanism for zinc was a simple ion-exchange reaction with the acidic polysaccharides of the c e l l wall.

The passive metal accumulation in brown algae, as suggested by seme

a u t h o r s ( 3 8 , 6 3 ) , would be c o n s i s t e n t with such a hypothesis.

However,

although there was no evidence t h a t Zn + + uptake in Laminaria could be r e g u l a t e d by metabolism, Bryan (39) suggested t h a t the polysaccharides vrere not much involved in Zn

++

binding.

intercellular

Skipnes e t

al.

(64) provided further support for t h i s idea: while strontium accumulation seemed to be an ion-exchange process involving the negatively charged wall polysaccharides, only a fraction of the gradual, long-term uptake o f by Ascophyllum seemed to be due to an ion-exchange p r o c e s s .

Zn + +

Skipnes e t

a l . considered that the [ | i n t e r c e l l u l a r charged polysaccharides acted as ion b u f f e r s , allowing Zn uptake into the c e l l a t a constant rate independent of tidal height.

They suggested t h a t Zn + + was bound t o

contained in membrane-bound v e s i c l e s , probably vacuoles.

substances

Interestingly, a

recent study (65) on metal chelation by p h l o r o g l u c i n o l s from

nodosum

and Fucus vesiculosus agrees with t h i s hypothesis, and the authors suggest t h a t physodes c o u l d be one o f the p o s s i b l e s i t e s f o r such n a t u r a l chelators of metals. o f Zn

I t must be pointed out, however, t h a t accumulation

in physodes would not occur without a prior entry of Zn

c e l l which may be dependent upon metabolism.

into the

The relationship between the

Zn + + uptake by J\_ v i r s o i d e s and the temperature-dependent m e t a b o l i c a c t i v i t y of the alga, as suggested by Munda ( 6 1 ) , i s consistent with t h i s idea. As regards sulfated polysaccharides of brown algae, i t has been suggested that fucoidan plays an important part in but the a c t u a l r o l e o f t h e c e l l

S uptake by Fucales

(60,66),

w a l l s o f brown a l g a e a s iri

situ

147 ion-exchangers is not known. Preferential distribution of ions and their uptake A nonhomogeneous distribution of phosphate (13), and potassium and iodine accumulation (14) has been reported previously in d i f f e r e n t parts of the Laminaria t h a l l u s .

A more d e t a i l e d

study showed a p r e f e r e n t i a l

d i s t r i b u t i o n of several mineral elements along the thallus of various members of the Laminariales, which was specific to the ions and related to the age of

the t i s s u e s and their function, e . g . , blade, h o l d f a s t ,

meristem, e t c .

( F i g s . 3,4; see also references 17,18).

A similar

distribution of phosphorus occurs along the thallus of Fucus vesiculosus (16).

Moreover, analysis of P, K, Na, Ca and Mg, performed from December

to April, in blades of growing L. hyperborea, showed that the distribution pattern of ions between the older and younger parts of the lamina changes during the growth of the alga and that this variation i s ion s p e c i f i c (24). Since uptake of mineral elements may continue throughout the l i f e of an algal tissue (38,39), this longer duration of uptake may explain the larger accunulation of ions, such as K and Ca Laminaria hyperborea (24).

in the older tissues of

By contrast, a lower concentration of ions in

those same tissues may be explained by reduced uptake of these ions by older fronds, as seems to occur f o r NH.+ and NO ~ in Fucus s p i r a l i s 32 (48), or by their greater extrusion. However, a comparison of P uptake by the younger and older laminae of the same thallus of L. hyperborea did not show any significant difference in the tissue's capacity for uptake or extrusion (Fig. 5).

A similar r e s u l t was reported f o r blade discs of

Macrocystis (67).

Therefore, i t appears that at least the heterogeneous

d i s t r i b u t i o n of

phosphorus along the thallus of the

Fucales and

Laminariales cannot be explained by a d i f f e r e n t i a l exchange ability of the d i f f e r e n t regions and tissues.

As a result, as postulated by Wille (13),

a translocation of minerals from one part of the thallus to another must be taken into account.

148

2.0

1.0

3.0 2.0 1.0

g 0.8

? cr CD

0.6

0.4

0.7 0.5 0.3

0

50

100

150

centimetres

Fig. 3: Distribution of potassium, sodium, magnesium and calcium along the thallus of Laminaria d i g i t a t a . Contents expressed in milliequivalents per gram of dry weight. Means of four s e r i e s of measurements and standard deviations are presented. Samples collected in September. Measurements by flame spectrometry (K,Na) and by ccmplexometry (Mg,Ca).

149

4.0 2.0

0.2

0.1

g t5 ? g

5

1.0

0.6 0.2

0.2 0.1

0

50

100

150

centimetres

Fig. 4: Distribution of chloride, phosphate, s u l f a t e and n i t r a t e along the thallus of Laminaria d i g i t a t a . Gontents expressed in milliequivalents per gram of dry weight. Means of four s e r i e s of measurements and standard deviations are shown. Samples collected in Semptember. Measurements made by colorimetrie methods.

150

Fig. 5: Comparative phosphorus uptake and release by an old and a very young lamina of the same thallus of Laminaria hyperborea in January. Uptake measured by the use of P in carrier-free sea water: ex sorption (arrow) in non-labelled sea water. T = 12 C. Results expressed in pg of P taken up per g dry weight of algal tissue.

Long Distance Transport of Ions Although translocation of inorganic substances in algae has been doubtful for a long time (68,69), a long distance transport of mineral elements has been demonstrated d i r e c t l y

in the l a s t ten years in many of the

Laminariales and Fucales (Table IV, see also references 15-24,67). Ion specificity By the use of various tracers

( 3 2 P , 86 Rb,

35

S,

99

MO,

4 5 Ca,

36

C1),

i t has

been shown that mineral elements do not move identically in the thallus of Laminaria digitata: phosphorus, sulfur and rubidium show directed long distance transport, whereas chloride, molybdenim and calcium do not seem to move (21). The need for phosphorus in growing or meristematic regions is well known, so is the f a c t that these regions are strong sinks. be reviewed in detail further on.

This transport w i l l

151

Table IV: Brown algae in which directed long distance transport of mineral elements has been reported. Algae

Translocated Isotope

Reference

FUCALES Fucaceae Fucus vesiculosus Fucus spiralis Fucus serratus

32

P,

86

Pb,

35

S

->2

P 32 p

(19,20) (Fig. 6) (16)

->2

Fucus ceranoides Cystose iraceae

P

(a)

12

Cystoseira baccata

P

(Fig. 6)

LAMINARIALES Laminariaceae Laminaria digitata

32

laminaria hyperborea Laminaria setchelli

32

Laminaria ochroleuca Laminaria saccharina

P,

86

Rb,

32P, P 32 P 32 P

86

Fb

35

S

(18,21,23) (19,24) (Fig. 7) (16) (16)

Chordaceae Chorda filum Phyllariaceae Saccorhiza polyschides

32

P

(Fig. 6)

32 P

(Fig. 6)

Alariaceae Alaria esculenta Alaria marginata Egregia menziesii

32

32P, P 32 P

Lessoniaceae Macrocystis integrifolia 32P 32 Nereocystis luetkeana P (a) = unpublished data

86

Rb

(19) (Fig. 7) (Fig. 7) (67) (Fig. 7)

152 Sulfur translocation occurs in Laminaria d i g i t a t a towards the meristem ( 2 1 ) , where chemical analysis also shows an accumulation of the cold 35 element (Fig. 4). S was also shown to move towards the apical meristem in Fucus vesiculosus (unpublished r e s u l t s ) .

There i s no d e t a i l e d

i n f o r m a t i o n in the l i t e r a t u r e on the d i s t r i b u t i o n of the sulfated polysaccharide fucoidan in the different regions of the thallus of brown algae. T h e r e f o r e , i t i s not possible to r e l a t e the long distance 35 transport of S to any region that may accumulate this polysaccharide. Sulfur translocation may also be related to protein synthesis which is usually high in meristems. 86 Translocation of fib has been shown to occur towards the growing young 86

tissues of Laminaria and Fucus. tracer for

K+

Whether

Rb can be considered as a

in algae, as i t is in higher plants (70), is a moot point.

Vfest and Pitman (71) compared

86Pb

and

42K

uptake by Ulva and concluded

that the two elements had d i f f e r e n t rates of uptake. A correspondence 86 + between Rb translocation and K accumulation was evident only f o r a short period (December to February) in the very young meristematic tissue QC

of the new lamina of

hyperborea (21) , and, therefore

considered as a tracer f o r

K+

in a limited way.

Rb could be

There is very l i t t l e

information, to date, on K+ translocation in algae, although this ion i s present in a high concentration in sieve-tube exudate of Macrocystis (9.2 to 12.6 mg ml-"'", see references 67,72), and in medulla exudate o f Nereocystis (7.7 mg ml-"'", see reference 73). The immobility of some of the mineral elements when fed to algae may result either

from a linkage to structural components, or from an

exclusion from the algae.

The l a t t e r phenomenon would e x p l a i n

the

immobility of "^Cl when injected into Laminaria t i s s u e , since there i s less chloride in the alga than in the sea water (concentration factor = 0.5 in the lamina, see reference 21). Molybdenum i s a cofactor of n i t r a t e reductase ( 7 4 ) .

Although NO^-

distribution is not homogeneous in the thallus of Laminaria ( F i g . 4; see also reference 75), l i t t l e is known about the relative activity of nitrate 99 reductase in different parts of the thallus (76,77). Since Mo did not move, i t is possible that the internal concentrations of this element are

153 sufficient for the enzyme activity in different parts of the thallus. Since calcium uptake does not seem to be directly controlled by c e l l u l a r 415 metabolism (Fig. 2), the immobility of Ca may be related to calcium binding to acidic wall polysaccharides in brown algae (21,24), especially in the older tissues of Laminaria which are known to be rich in guluronic acid fractions with a strong a f f i n i t y for calcium (78). Thus, i t appears that in brown algae, both uptake and translocation of mineral elements are ion s p e c i f i c .

Since most of the research on long

distance transport of mineral elements in algae has been carried out with 32 P, the following review w i l l deal with the c h a r a c t e r i s t i c s of i t s transport. Transport of

3? P

Directionality, "source to sink" r e l a t i o n s h i p . In a l l studies dealing with the Fucales and Laminariales, 32P translocation occurs from the older tissues toward the younger growing regions of the thallus. Whatever the 32 P moved in a basipetal

fed region in the laminae of the Laminariales,

d i r e c t i o n toward the meristematic transition zone, the holdfast, and the sporophylls, never toward the older tissues (Figs. 6,7). In Chorda filum 32 P transport is acropetal (Fig. 6) and, i n t e r e s t i n g l y , the menstem i s 3? subterminal in that alga (79); since P i s not translocated above the meristem, t h i s apparent exception in the

Laminariales i s in good

agreement with the general rule that transport is toward the young growing tissues. A comparison of

32 P accumulation along the t h a l l i of the Laminariales

( F i g . 7, see also reference 20) and the cold phosphorus accumulations as determined by chemical analyses (Fig. 4), indicates that translocation i s an important process which links the i n i t i a l site of phosphate entry to the final site of accunulation or consumption.

In the Laminariales the

main sinks or sites of accumulation seen to be the transition zone between the lamina and the stipe, the meristoderm of the s t i p e , the base of the s t i p e , the younger haptera of the holdfast, and the growing sporophylls (as in Alaria, Fig. 7).

154

VFI

•

1

Ä

&M

O ]

riïus

?

32r

Cystoseira baccata

5cm Fucus spiralis

m

Chorda filum

32,

J

•>

5cm

Saccorhiza polyschides

(

y

32PWÊ

->2

5cm

Fig. 6: ftutoradiographs of brown algae labelled with P. Uptake: 3 h (white arrows); migration: 15 days in s i t u (large arrows). Et>r Fucus and Sacchorhiza, the outlines of t h a l l i are delineated by dotted l i n e s . For Cystoseira and Chorda, (A) r e p r e s e n t s the whole t h a l l u s , (B) shows the autoradiograph a f t e r translocation of the radioisotope. For Chorda, me: meristem; the anall black arrows indicate an occasional s l i g h t generalized labelling which could be an a r t i f a c t .

Nereocystis

luetkeana

Laminaria setchellii 5 cm Scm

Egregia menziesii

Alaria marginata

5 cm

5 cm

Fig. 7: Autoradiographs of brown algae labelled with (white arrows); migration: 24 h in situ (large arrows).

32 P.

Uptake: 3 h

156 In Laminaria hyperborea the older parts of the blade serve as a source of phosphate for the growth of the younger parts, in much the same way as has been shown for carbon assimilates (80).

Specifically, for phosphorus, i t

has been shown that concentration of cold phosphorus increases rapidly in the young, fast-growing lamina, while i t declines in the older lamina 32 (24). There is no significant difference in P uptake by young and old tissues, and, f i n a l l y , there is no greater leakage of phosphorus from the older than from the younger tissues (Fig. 5). 32 Translocation of P occurs in the Fucales as well.

In Fucus vesiculosus

the tracer moved to the apical meristem and to the basal disc (Fig. 6), where the amounts of cold phosphorus also had been found to be the highest (17,19).

In these algae, the apical meristem and the basal disc seem to

be the main sinks for phosphorus.

Removal of the apical meristem in F.

vesiculosus and the holdfast of Laminaria d i g i t a t a slows down the ~ translocation of 32P, but this i s followed by wound h e a l i n g which stimulates the migration of phosphate (15). Moreover, an exceptional 32 acropetal translocation of P in L. d i g i t a t a , toward a region of the lamina which had been experimentally wounded, showed that regenerating tissues act as strong sinks. The above review indicates that phosphate translocation in brown algae i s regulated by a "source to sink" relationship, similar to the pattern of mineral translocation in higher plants. Pathway of transport.

As suggested by the intensity of l a b e l l i n g

in

autohistoradiograms, the midrib of many of the Fucales seems to be the main pathway of mineral

transport

(19).

If

the midrib o f

Fucus

v e s i c u l o s u s was removed, l e a v i n g o n l y the wings, there was no 32 translocation of P toward the apex of the treated branch; by contrast, 32 i f the midrib was l e f t intact and the wings removed, P moved through the treated branch as well as through the control. Et>r the Laminariales, an experiment, based on the classical "stripping" in 32 higher plants ( 8 1 ) , was carried out to d e f i n e the pathway o f P translocation.

The c o r t i c a l tissue in Laminaria d i g i t a t a stipe was

separated from the medulla by two impermeable sheets and the migration of

157 the radioisotope from the fed lamina through the different tissues of the stipe was followed.

Ihe results showed clearly that the medulla was the 32

pathway of the basipetal long distance transport of

P, and that a

secondary lateral transport occurred from the medulla to the meristoderm which was stopped by the impermeable sheets (Fig. 8).

Since the

meristoderm of the stipe is a well known meristematic tissue in the Laminariales, it is not surprising that it acts as a "sink" for mineral translocation.

Central Part7 Cortical / Part—4

ImpermeableSheets

1

samples

cortex

centre

A

520

1964

730

B

436

1337

245

C

7

1393

13

D

6

1280

7

E

8

1218

8

F

38

1141

22

G

570

1255

200

cortex

32p d p m / 1 0 0 s e c / 1 0 0 mg fresh wt Translocation: 6 days in situ

Uptake: 2 4 hours Detail of the stipe 32

Fig. 8: Distribution of P in different parts of the stipe of Laminaria digitata. Longitudinal incisions separated the medulla from the cortex and impermeable sheets were inserted between the two. (Adapted frcm [19])

We do not know which part of the tissue or cells in the medulla of the 32

Laminariales are involved in ion translocation, but since

P has been

found in the sieve tube sap of Macrocystis and shows the same velocity and

158 directionality of transport as "^C (67), i t most probably moves through 14 the sieve elements (for C translocation in the sieve elements of the Laminariales,

see Schmitz, t h i s v o l u m e ) . Further research 32 microlocalization of P is necessary to confirm this assunption. Velocity, rate, seasonality.

in

Since phosphate translocation occurs against

a concentration gradient in brown algae, this transport is not a case of mere d i f f u s i o n , as thought e a r l i e r metabolisn.

( 6 9 ) , but i s r e l a t e d

to

algal

Moreover, the velocities of transport, as measured in several

of the laminariales (2 to 45 an h - 1 , see references 19, 20,67), are a good deal faster than those measured in killed tissues (0.06 cm h-"*", reference 20), or those reported earlier by Parker (69) f o r Fucus vesiculosus and Laminaria agardhii (v < 0.08 am h-"*"). due to d i r e c t i o n a l

transport,

In addition to the heavy l a b e l l i n g

some autoradiograms show a l i g h t ,

homogeneous l a b e l l i n g of the thallus (Chorda filum, F i g . 6 ) .

Such a

l a b e l l i n g has been reported to go across the barrier between the host a l g a , Ascophyllum nodosum, and i t s epiphytes,

in both

directions,

i r r e s p e c t i v e of the radioisotopes used (82), and interpreted as a very fast transport — up to several meters h - ^ (83).

Such a high v e l o c i t y of

transport has been reported in land plants as well, though same authors consider i t an artifact (84). Measurement of the amounts of translocated

32 P in young t h a l l i (age < 1

year) of Laminaria digitata from the source (middle of the lamina) to the sinks (base of the lamina, stipe, holdfast), showed that less than 1% of the total radiophosphorus taken up was translocated in 24 h ( 1 6 ) .

This

r a t e , which was obtained during December, i s rather weak and probably larger amounts are translocated during the period of active growth.

Since

phosphate absorption presumably occurs over the whole lamina, the total amount of phosphate supplied to the growing tissues by the source through translocation does not seem to be so insignificant. Ihe relationship between source and sink v a r i e s during the year and i s closely related to the growth pattern of the a l g a . 32 digitata,

Thus, in Laminaria

P translocation starts in the f a l l when the growth of the alga

s t a r t s , and decreases sharply or stops in summer, when the growth slows down.

Moreover, i t has been shown that d i f f e r e n t parts in the growing

159 region of the lamina of IJ^ d i g i t a t a may accumulate d i f f e r e n t amounts of 32 P from long distance transport and thus may have d i f f e r e n t "sink" capacities (20). Effect of cycloheximide on long distance transport of

32 P. Cycloheximide,

an inhibitor of protein synthesis, was applied to three r e g i o n s of Laminaria digitata, the old part of the lamina (traditional "source"), the young meristematic region (traditional "sink") and the intermediate region ("pathway") and i t s e f f e c t on 32P transport was tested after supplying the radioisotope

to the o l d p a r t

The results showed that when cycloheximide was applied to the base of the lamina, there was no 32P translocation into this region.

(22).

In other wards, this part no longer acted

as a sink. When cycloheximide was applied between the base of the lamina 32 P - f e d r e g i o n , the r a d i o i s o t o p e did not move past the

and the

cycloheximide-treated region. Finally, when cycloheximide was applied to 32 the same region as P, either as a pretreatment or simultaneously, the r a d i o i s o t o p e was r e t a i n e d at the source.

Although the e f f e c t s of

cycloheximide on photosynthesizing tissues of brown algae are not well known, these results may indicate a dependence of 32P loading as well as i t s long distance transport on continued protein turnover. The lack of 32 translocation of P to the t r a d i t i o n a l sink area i s more r e a d i l y e x p l a i n e d by an i n h i b i t i o n o f

p r o t e i n synthesis and a consequent

inhibition of meristematic and growth activity. Possible forms of phosphate transport and accumulation. Floc'h and Penot 32 (23) studied the patterns of P labelling in pho sphoryl a ted compounds in regions of uptake, translocation and accumulation in Laminaria d i g i t a t a . In the uptake region (lamina), 3 h after the beginning of incubation, the bulk of radioactivity was incorporated into organic compounds, hexose monophosphates being most heavily labelled.

This result seems to confirm

the metabolic dependence of phosphorus uptake ( F i g . 1 ) . Subsequently there was a decrease in radioactivity in the organic phosphate fraction and a simultaneous 32P accumulation in the inorganic phosphate pool: 32 after a period of 13 days, 65-70% of the P taken up was found in the inorganic f r a c t i o n . In the medulla of the stipe (the conducting zone), 32 the pattern of P phosphorylated compounds was similar to that in the uptake region in the f i r s t few hours, i . e . hexose monophosphates were the

160 most labelled intermediates, and this pattern was maintained throughout the 13 day experiment. In the accumulating tissues (the s i n k ) , the op pattern of P- phosphorylated compounds was at f i r s t similar to that in the uptake and conducting zones, but subsequently there was an increase of labelling simultaneously in the inorganic f r a c t i o n and in the insoluble organic f r a c t i o n of phosphate.

Floc'h and Penot (23) suggested that,

since the ratio of P^/P ester remained constant in the conducting zone, while i t increased considerably in the region of uptake as well as in the sink, inorganic phosphate could be one of the possible translocating 32 forms. The slow transfer of P into the inorganic pool in the uptake region, as shown by Floc'h and penot (23) in 32 why no while

d i g i t a t a , could explain

P translocation was found in Alaria marginata 5 h after feed ing,

14 C,

applied simultaneously, was translocated during that time

(85).

32 P-

Mare recently, Schmitz and Srivastava (67) reported on the pattern of

phosphorylated intermediates in the fed region (blade) as well as in the sieve tube sap of Macrocystis i n t e g r i f o l i a . Their results are in good agreement with those obtained f o r Laminaria in terms of both the high 32 amount of P found in the inorganic form in the fed region and the high l e v e l s of l a b e l l i n g of hexose monophosphates in the sieve tube sap. Moreover, for the f i r s t time, these authors reported that double labelled and "^C hexose monophosphates occured in the sieve tube sap of M. integrifolia along with "^C l a b e l l e d mannitol and amino acids.

These

authors suggested that phosphorus was probably translocated in an organically bound form, but this does not exclude the p o s s i b i l i t y that phosphorylation may s t i l l occur in the sieve tube sap (86). The available data on phosphorus uptake, translocation and accumulation in brown algae show several parallels with the available data on phosphate 32 uptake and translocation in higher plants. The slow, but continuous P transfer from the soluble organic fraction to the inorganic fraction after uptake in Laminaria d i g i t a t a i s in good agreement with the obtained by Bieleski and Laties in potato tuber tissue (87).

results These

authors found that phosphorus went f i r s t to a metabolic pool and then to a nonmetabolic pool, with the time of equilibration being f a i r l y long.

In

addition, Bieleski (88) suggested that, since inorganic phosphorus i s the

161 f i r s t phosphate form to enter the phloem, i t seems to be the translocating form in land p l a n t s .

Many other s i m i l a r i t i e s have been found between

mineral translocation in algae and in higher plants (24,89). An attempt has been made t o e x p l a i n the mechanism o f

long

distance

transport in large algae by considering the mass-flow theory of Munch (see Schmitz, t h i s v o l u m e ) .

However, o t h e r mechanisms a r e

possible.

" A c t i v a t e d d i f f u s i o n , " which postulates that "the tissues receiving from and supplying the pathway play an a c t i v e part in the interchange

of

solutes" (90), either by means of a push at the source or a consumption at the sink, could also be applied to the algae as w e l l , especially since the source as w e l l as the sink t i s s u e s o f the Fucales and the Laminariales have been shown to play an active part in translocation. aspects of

Moreover, some

the electro-oanotic theory (91) may find support, as a r e l a y ,

in ion translocation in marine macrophytes since the c e l l w a l l s of

these

p l a n t s are p e c u l i a r l y r i c h in n e g a t i v e l y charged molecules and since enzymatic a c t i v i t y occurs in the s i e v e tube sap which i s r i c h in K + , features which are essential to this theory. Finally, the demonstration that a long d i s t a n c e transport o f mineral elements occurs in a l g a e (15-24,67) opens up many new questions.

Fbr

instance, in what chemical form(s) i s phosphate translocated or in what form(s)

a r e v a r i o u s o t h e r m i n e r a l elements loaded, unloaded, and

transported, and what is the s i t e of such transport and accumulation are questions for which vie s t i l l have no answers.

Acknowledgements I am v e r y grateful to Professor L.M. Srivastava, Simon Fraser university, f o r providing l a b o r a t o r y f a c i l i t i e s during my tenure as a

Visiting

Research Associate in the Eepartment of Biological Sciences, Simon Fraser University, Burnaby, B . C . , Canada, and f o r c r i t i c a l l y discussing manuscript, and helping to write i t in English.

the

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DISCUSSION WILLENBRINK:

From which r e s u l t s do you conclude that i t i s a c t i v e

transport of phosphate?

Of course, i t could be a c t i v e transport in an

indirect manner. FLOC'H:

Since the v e l o c i t i e s are higher than d i f f u s i o n , and since the

transport occurs against concentration, wg have to suppose that metabolism i s involved in that translocation.

I think i t could be r e l a t e d t o the

theory sometimes accepted in higher plants - the s o - c a l l e d d i f f u s i o n theory.

activated

Because the transport i s toward the younger parts o f

the p l a n t , where the phosphate is consumed, we can suppose that this i s one of the indirect e f f e c t s of metabolism on the transport. SRIVASTAVA:

I was i n t r i g u e d by the d i s t r i b u t i o n of Pi and UDPG and

hexose-phosphates and so on, in the three areas.

Even a f t e r 4 days and 13

days the r e l a t i v e concentrations of these things in the conducting

zone,

not in the fed or accumulating zone, remained almost the same! FLOC'H:

That was not the c o n c e n t r a t i o n

concentration. SRIVASTAVA: SCHMITZ:

--

I have no i d e a o f

the

I t was % of radioactivity in the d i f f e r e n t compartments.

And that does not change in the conducting zone f o r 13 days!

I f you looked only at % r a d i o a c t i v i t y , could i t be that you have

only a very tiny l i t t l e b i t of

P l e f t in the source of the uptake region

166 a f t e r 13 days and you compute that up to 100%. FLOC'H:

I doubt that.

I measured that l e s s than 1% of the r a d i o a c t i v i t y

taken up was translocated per 24 hours. SCHMITZ:

Were those p u l s e - l a b e l l i n g e x p e r i m e n t s , o r did you f e e d

continuously for 13 days? FLOC'H:

They were pulse-labelled, of course.

SRIVASTAVA:

Is i t possible that a l i t t l e b i t of the inorganic phosphate

is a l l the time being converted to organic form and being translocated. FLOC'H: Wall, what surprises me and what i s evident i s these l a r g e pools 32 of P in the uptake r e g i o n s . I t i s the same in higher p l a n t s , f o r example f o r phosphate uptake by potato tuber c e l l s .

Ihere i s a slow but

permanent transfer from the organic pool to the inorganic pool. SCHMITZ: FLOC'H:

Did you analyze for polyphosphates? No.

ISbt much i s known about the l o c a l i z a t i o n of these inorganic

pools — localization in the c e l l or even in which c e l l . DUNCAN:

Did you see any difference in uptake rate or t r a n s l o c a t i o n

rate

or in products between the high-growing and subtidal algae? FLOC'H:

I have not compared the rates of t r a n s l o c a t i o n in a l g a e in the

higher i n t e r t i d a l and s u b t i d a l .

But there i s translocation in algae of

each of these d i f f e r e n t l e v e l s — in Fucus, Cystoseira and in Laminaria. DUNCAN:

Is there any difference in the compounds, such as polyphosphates?

The reason I ask i s that the amount of acid phosphatase i s much higher in the high growing p l a n t s than in low growing p l a n t s , which should be r e l a t e d to the amount o f phosphates i n s i d e o r coming i n t o the plant. Perhaps t h i s might show in your work. FIOC'H:

Perhaps. I do not know.

TRANSLOCATION OF ORGANIC COMPOUNDS IN LAMINARIALES

K. Schmitz Botanisches I n s t i t u t der Universität zu Köln, Gyrhofstr. 15, D-5000 Köln 41, F.R. Germany

Introduction Two t r a n s p o r t pathways may t r a d i t i o n a l l y be distinguished in plants, the a p o p l a s t i c and the s y m p l a s t i c

(21).

Symplastic

transport

seems

advantageous f o r algae because i t i s more e f f i c i e n t and the translocate can b e t t e r be conserved and kept under c o n t r o l .

As f a r as we can s e e ,

only the symplastic pathway i s used f o r the d i s t r i b u t i o n of important nutrient materials in Laminariales. A cooperative medium-distance (parenchyma-transport) and a l o n g - d i s t a n c e t r a n s p o r t o f organic compounds are o p e r a t i o n a l in the sporophytes of Laminariales.

P h o t o s y n t h e t i c C02~fixation i s mainly confined t o the

meristoderm and the outer cortex of the thallus and the photoassimilate must move through the c o r t i c a l c e l l s b e f o r e e n t e r i n g the l o n g - d i s t a n c e t r a n s p o r t system in the medulla ( 1 4 , 4 1 ) . probably o c c u r s v i a c y t o p l a s m i c plasmodesmata.

This parenchyma t r a n s p o r t

intercellular

connections,

the

The d i s t r i b u t i o n and frequency of these connections was

r e c e n t l y studied f o r two s p e c i e s o f Laminaria ( 3 1 ) .

Although t h e s e

c o n n e c t i o n s are common and show up r e a d i l y between meristoderm and the inner c o r t i c a l c e l l s , t h e i r occurrence between sieve elements and c o r t i c a l c e l l s i s r a r e and so f a r has been seen only in the blades, not in the mature stipes of laminariales.

In s t i p e s , the sieve element system seems

t o be " i s o l a t e d " from o t h e r c e l l s .

I f the sieve elements and c o r t i c a l

c e l l s form a symplast, which seems to be the case in the b l a d e s , loading of

t h e s i e v e elements i s p o s s i b l e as long as t h e r e i s a continuous

concentration gradient from the meristoderm t o the s i e v e t u b e s . g r a d i e n t has not yet been measured.

This

The concentration difference between

the sieve elements and the adjacent c o r t i c a l c e l l s i s also not known.

© 1982 Walter de Gruyter & Co., Berlin • New York Synthetic and Degradative Processes in Marine Macrophytes

It

168 may well be that the concentration of solutes within the sieve elements, as indicated by an analysis of the sieve tube sap (25,37), is higher than in the c o r t i c a l c e l l s , which would necessitate an u p h i l l mechanism.

transport

Thus, although there are structural indications f o r a

symplastic transport of photosynthate, there are s t i l l no supporting physiological data.

Structural Aspects of the Long-Distance Transport System Long-distance transport of photoassimilate and inorganic ions seems to be a cannon feature of Laminariales (8,32,42).

All species investigated so

f a r have been shown to have a long-distance transport system in the medulla.

This system is composed of separate f i l e s of elongated sieve

elements, which are inter-connected by other cross-connecting elements.

sieve

Gompared to the inner cortical c e l l s , the sieve elements have

increased numbers and increased diameters of perforations in their cross w a l l s , an anatomical feature which should considerably f a c i l i t a t e a symplastic transport along these c e l l s . Various genera of Laminariales show d i f f e r e n t degrees of

specialization

with respect to their sieve elements, from the most "primitive" sieve elements having small but numerous pores as in Laminaria, to those of Macrocystis with relatively few but wide pores resembling the sieve tube members of Cucurbita.

While the sieve pores of Laminaria may s t i l l be

regarded as plasmodesmata (35,40,46), the sieve pores of Macrocystis closely resemble the sieve pores of higher plants (26/45). In cross and radial longitudinal sections of stipes and blades the sieve elements show three successive developmental stages: - their origin from innermost cortical c e l l s and differentiation as functional sieve elements, - the mature phase, when they function as transport c e l l s , - their structural and functional degeneration. A mature sieve element, therefore, is a c e l l structurally specialized to perform o p t i m a l l y as a transport c e l l .

The sieve elements of the

Laminariales are not associated with companion c e l l s .

Thus, there i s no

169 separation between a conducting compartment ( t h e f i l e

of

the

sieve

elements) and an energy-providing compartment (the companion c e l l ) , as in the phloem of angiospems.

Both functions are i n t e g r a t e d

element of the Laminariales. structure.

Mature s i e v e e l e m e n t s o f

characterized

L a m i n a r i a and A l a r i a

are

by numerous small vacuoles and the presence of

cell

o r g a n e l l e s and membrane systems t y p i c a l (35,36,40).

in each s i e v e

This integration i s r e f l e c t e d in their f i n e

o f most parenchyma

cells

The sieve elements of Macrocystis, however, r e v e a l a thin

p e r i p h e r a l l a y e r of cytoplasm with p l a s t i d s , v e s i c l e s , mitochondria, occasional dictyosomes and endoplasmic reticulum (26,45), with the central part of

the c e l l r e l a t i v e l y f r e e of cytoplasmic ccmponents (unpublished

observations).

According to Parker and Huber (26), however, the c e n t r a l

lumen i s f i l l e d with f i n e granular t o f i b r i l l a r material or masses of electron-dense aggregated or dispersed slime depending on the method of fixation.

In our own observations, P-protein has never been detected in

the s i e v e elements of the Laminariales.

Mature s i e v e e l e m e n t s

of

Macrocystis apparently lack a nucleus and a tonoplast as w e l l , and t h e i r sieve pores appear as open intercellular connections.

Physiology of Translocation The most s p e c i a l i z e d long-distance transport system in the Laminariales seems to be the one in M a c r o c y s t i s .

The f o l l o w i n g

t h e r e f o r e , w i l l mainly focus on this genus.

presentation,

The working hypothesis f o r

the long-distance transport of organic compounds in Macrocystis i s the osmotic pressure flow hypothesis o r i g i n a l l y proposed by Munch (21).

The

conditions for an osmotic pressure flow are: - the existence of separate "source" and "sink" areas, - the linkage of source and sink by a continuous f i l e of conductive sieve elements with open sieve pores and unobstructed lumina, - an enclosure of the transport compartment by a semipermeable membrane, - a gradient of osmolarity between source and sink which must c r e a t e a parallel pressure gradient high enough to drive a solution flow, - removal of osmotically active translocate from the transport compartment at the sink,

170 - efflux of water from the transport system, mainly a t the sink. The consequences of such transport a r e : - a s t r i c t l y directed transport from source to sink, - a mass stream of dissolved matter and the solvent water, - movement o f d i f f e r e n t

s u b s t a n c e s a t t h e same t i m e i n t h e same

direction, - movement of d i f f e r e n t substances a t the same speed unless e l e c t r o s t a t i c or metabolic factors are involved. The e x i s t e n c e o f s o u r c e and s i n k a r e a s

in the L a m i n a r i a l e s

was

convincingly demonstrated by experiments with intact and amputated plants and by m e a s u r e m e n t s

of

growth

and r a t e s

of

carbon

fixation

(2,3,7,17,18,22,30).

These experiments showed that growing regions were

carbon d e f i c i e n t ( s i n k s ) and dependent, f o r normal growth, on carbon supply frcm surplus producing areas (sources) such as mature blades. That organic compounds a r e t r a n s l o c a t e d

in t h e L a m i n a r i a l e s was 14 demonstrated by use of radioisotopes, mainly C. But dyes ( 2 4 , 2 5 ) and o t h e r r a d i o i s o t o p e s , o r g a n i c a l l y bound or as inorganic ions, have also been shown to be mobile in these plants (Table I ) .

The transport of these

i s o t o p e s and dyes i s always directed toward the sink areas.

Mare recent

experiments using "^C t o l a b e l the photosynthate in the blades have 14 confirmed that photoassimilate moves frcm the C-exposed blades along the stipe of Macrocystis to the growing regions ( 1 5 , 1 6 , 2 4 , 3 2 , 3 7 , 3 8 ) . ¡Ybvement 14 of C along a stipe occurs in i n t a c t p l a n t s as well as in exuding c u t fronds which release the radiocarbon with the sieve tube sap ( 2 5 , 3 7 , 3 8 ) . Radioactive translocate has been localized by histoautoradiography within the s i e v e elements along the t r a n s p o r t pathway ( 3 8 , 4 1 ) .

Moreover, cut

s t i p e s of Macrocystis exude a c l e a r fluid at a r a t e o f 0 . 5 - 2 ml h-"*" or more, and in experiments using o r g a n i c compounds ( 6 , 2 5 , 3 4 , 3 7 , 3 8 ) .

the exudate c o n t a i n s

"^C-labelled

The exudate i s released by the mature

sieve tubes at the periphery of the medulla, c e l l s vAiich were

identified

by histoautoradiography to be the translocating sieve elements (38).

Fran

t h e s e f a c t s , we conclude t h a t the exudate i s the s i e v e tube sap and r e p r e s e n t s the t r a n s l o c a t e , and furthermore, t h a t the source and sink regions are connected by a continuous conductive long-distance transport

171

T&ble I : S p e c i e s o f brown a l g a e t h a t have been shown to t r a n s l o c a t e various radioisotopes. Plant species Chordaceae Chorda filum

Radioisotope

Reference

14,

(42)

Laminariaceae Laminaria d i g i t a t a

14

C

32

P,

86

Rb, 35,S (9,10,11,14)

L. hyperborea

14

C

32

P,

86

Rb

L. japonica

14

L. saccharina

(12,19,33,41)

C 14 c

(42) (19,20,33)

L. s e t c h e l l i i

14

C

(32)

L. s i n c l a i r i i

14

C

(32)

Pleurophycus gardneri

14

C

(32)

Cymathere t r i p l i c a t a

14

C

(32)

Costaria costata

14

C

(32)

Agarum fimbriatum

14

C 14 r

(32)

C 14,

(32)

Hedophyllum s e s s i l e

(32)

Dessoniaceae Fostelsia palmaeformis Nereocystis luetkeana

14

Macrocystis i n t e g r i f o l i a

14

M. pyrifera

14

Lessoniopsis l i t t o r a l i s

C,

(23,32) 32

P , 3H

(16,32,37,38,39)

C 14~

(15,24,25) (32)

Alariaceae Alaria esculenta

14

C,

A. marginata

14

C

Egregia laevigata

14

Eisenia arborea

32

P,

86

lfc

(2,10)

(32,36)

C 14„

(32) (32)

Fucaceae Fucus vesiculosus

32

P,

86

Rb

Ascophyllun nodosum

32

P,

86

K>

45

Ca,

Sargassaceae Sargassum pallidum

14,

36

C1,

(10)

"to

(29) (42)

172 system,

these f a c t s also suggest that the structure of the sieve tubes of

Macrocystis i s conducive to a s o l u t i o n flow, which would probably not occur i f the sieve pores were occluded or the lirruna of the sieve elements f i l l e d with dense cytoplasmic components. A comparative analysis of the photosynthate and translocate revealed

that

n e a r l y t h e same compounds which a r e r a d i o a c t i v e l y l a b e l l e d during photosynthesis are subject to long-distance transport. carbohydrate

is mannitol.

In a d d i t i o n ,

Ihe main l a b e l l e d

h e x o s e - monophosphates,

-diphosphates, PGA, and several amino a c i d s a r e a l s o h e a v i l y

labelled.

The p e r c e n t a g e o f r a d i o a c t i v i t y confined t o the amino a c i d s in the translocate may be as high as 40%.

The chemical a n a l y s i s o f the s i e v e

tube sap shows that mannitol concentration may be as high as 10% (w/v) or 67% (w/w) on a dry weight b a s i s .

Amino a c i d s comprise 15% (w/w) and

potassium roughly 8% (w/w) on a dry weight b a s i s .

Sodiun, on the other

hand, may be as low as 0.75 to 0.8 n*g ml-"'' o f the s i e v e tube sap. These data i n d i c a t e t h a t the most h e a v i l y l a b e l l e d substances, mannitol and amino acids, are also q u a n t i t a t i v e l y the p r i n c i p a l substances t h a t a r e translocated.

In a d d i t i o n , the concentration of potassiun in the sieve

tube sap i s much higher than in the sea water, whereas the reverse i s true f o r sodium.

This ionic s e l e c t i v i t y c l e a r l y suggests that the plaanalemma

of the transport compartment ( s i e v e tubes)

i s a s e l e c t i v e l y permeable

barrier. The release of appreciable amounts of exudate for several hours from c u t ends of Macrocystis s t i p e s can only be explained i f i t i s assumed that the s i e v e elements are under high hydrostatic pressure.

A continuous loading

of osmotically active substances i n t o the s i e v e elements in the source region would cause water to be attracted to the sieve elements and r e s u l t in the build-up of pressure high enough to d r i v e the s o l u t i o n along the s i e v e tubes to the c u t end o f s t i p e ( s i n k ) .

This phenomenon can be

demonstrated by using frond c u t t i n g s , 60 t o 100 cm long with 3 t o 6 a t t a c h e d b l a d e s , which a r e s t i l l able to release exudate with a uniform rate for several hours.

I f radiocarbon i s c o n t i n u o u s l y supplied to a

mature blade under c o n d i t i o n s o f p h o t o s y n t h e s i s , l ^ c w i l l normally be 14 released with the exudate. The time interval between the feeding o f C to the blade and i t s appearance in the sieve tube sap a t the cut end of

173 the stipe i s a rough measure of the t r a n s l o c a t i o n v e l o c i t y . Similar 32 3 experiments may be performed using the r a d i o i s o t o p e s P and H, fed singly or in combination with 14 C to the same blade. The d a t a from such 14 an experiment are presented in figure 1 and T&ble II and show that C and 32 P moved upward and downward along the s t i p e . Most r a d i o a c t i v i t y was, however,

translocated

toward t h e b a s a l c u t end of the

stipe.

Radioactivity f i r s t appeared in the sieve tube sap 90 to 100 minutes a f t e r the experiment was started and steadily increased up to 240 minutes when the experiment was stopped.

Since the fed blade and the s i t e of s i e v e

tube sap c o l l e c t i o n were 680 imi apart, transport velocity must have been a t l e a s t 41 to 45 cm h-^".

This experiment a l s o shows t h a t "^C and

^P

when fed simultaneously are translocated together in the same direction. I t a l s o suggests that the radioactively l a b e l l e d compounds in the s i e v e tube s a p , i d e n t i f i e d by t h i n - l a y e r chromatography, move in a solution stream (see a l s o references 37,38). If translocation of photoassimilate in Macrocystis i s a s o l u t i o n stream, the main t r a n s l o c a t e should be the solvent water.

Provided there i s no

channelized recirculation of water, water should enter the s i e v e element system in the blade (source) following the import of osnotic substances and i t should leave the sieve tubes mainly a t the sink.

Several r e s e a r c h

groups have t r i e d to demonstrate the transport of water in the phloem of vascular plants using t r i t i a t e d water (THO), but the r e s u l t s have been mostly c o n t r a d i c t o r y (1,4,5,13,27,28,43,44).

Vfe have tried to trace the

movement of THO in M. i n t e g r i f o l i a by applying THO to a mature blade under c o n d i t i o n s of p h o t o s y n t h e s i s . Such an a p p l i c a t i o n of THO has the 3 3 advantage that i t yields H-labelled water and H-labelled photosynthate, both of which can be analyzed separately.

The experiments were done with

intact fronds a s well a s frond segments which r e l e a s e d exudate. movement of THO and systems.

H - l a b e l l e d o r g a n i c compounds o c c u r r e d

The

in both

An experiment with an intact frond i s presented in figure 2.

A

blade close to the growing apex was exposed to a THO-seawater medium for 3 h.

The distribution of THO and

H-labelled o r g a n i c compounds along the

s t i p e a f t e r 3 h shows that radioactivity moved nearly exclusively toward the apical growing region where a s l i g h t accumulation of (39).

H-label occurred 3 Experiments with frond segments a l s o revealed a movement of H

174

stipe length

A" dpm/10mm stipe 32 p

14C

1271

2969

1833

3918

2395

5022

12194

19987

8626

13139

300mm

9329

14700

30 30 30 70 30 30 50 30 70 30 30 30 70 "30

8208

13018

30

5907

10138

100 "100

30

40

3542

6435

60 '30

t I

- cut o

-O -O

-o -O -O

-o o

- cut

2 0 5 0 mm

collecting s i t e for S T S A"

Fig. 1: Translocation of C and P along a stipe of a frond cutting of Macrocystis i n t e g r i f o l i a . The indicated blade qf the intact frond was exposed for 4 h to a sea water medium containing C- sodium bicarbonate and P- orthophosphate under conditions of photosynthesis. Fifteen minutes a f t e r the experiment was started, the apical and basal parts of the frond were cut o f f . Sieve tube sap was collected from the basal end in 1Q min batches for the duration of the experiment and analysed f o r C and p (see T&ble I I ) . At the end of the experiment, 30 mm pieces of stipe were sampled both apically and basally fran the fed blade, extracted and counted for radioactivity. The dpm/lOmm stipe for both C and P in the stipe segments are listed.

175

dpm / 10mm T-photosynthate

stipe jHo

stipe length

9543

10815

1888

3758

2179

3096

5337

2542

8913

2904

15366 1738

7188 2838

951

1135

75

0

Fig. 2: Translocation of H in an i n t a c t frond of Macrocystis integrifolia. A mature blade was exposed to a THO-seawater medium for 3 h in the light. The growing apex was enclosed in a translucent plastic bag with 200 ml of sea water enriched with carbonate. At the end of the experiment 50 mm pieces of stipe were rapidly cut at 100 itm intervals and immediately deep frozen. These samples were lyophilized, the condensate from each sample was collected separately and the radioactivity, confined to THO only, was measured. From the dried tissue samples, H-labelled organic compounds were extracted and their radioactivity measured. The data are presented in the figure. A water sample was withdrawn from the plastic bag enclosing the apex and lyophilized. The condensate revealed appreciable radioactivity of THO (for details see text).

176 Table II: Release of C and P with sieve tube sap (STS) collected fran a frond segment shown in figure 1. STS was collected in 10-min fractions. Experiment

dpn/ 25 pi STS 14 X> ^C E

tune (min) 90 100 110 170 130 140 150 160 170 180 190 200 210 220 230

-

100 110 120 130 140 150 160 170 180 190 200 210 220 230 240

27 48 148 210 221 254 479 528 1086 1254 1558 1750 1792 2034 2059

along the stipe towards the basal cut end.

0 10 72 131 204 363 467 817 1119 2135 1973 1954 2129 2739 3732

Both THO and ^H-labelled

compounds were detectable and showed a clear gradient in extracts of stipe sections cut at regular intervals along the transport pathway at the end of an experiment.

Tritiated label was also released with the exudate

collected at the cut end of the stipe; the ^H-radioactivity in the exudate steadily increased with time when THO was fed continuously to a mature blade about 50 cm away. Nearly all the radioactivity in the sieve tube sap was confined to organic compounds; the radioactivity of THO was always low and did not change significantly with time, which suggests that THO rapidly exchanges with water in the surrounding tissue and perhaps with the sea water outside the plant (39, but cf. 4,27,28,44). indeed transported along with

If THO is

H-labelled organic compounds, there should

be a release of THO at the sink area (growing apex) as well.

The apex of

a NL integrifolia frond, therefore, was enclosed in a plastic bag with 100 to 200 ml of sea water enriched with carbonate and optimized in oxygen (see Fig. 2). At the end of the experiment, after 3 h, the sea water in 3 -1 the bag was analysed for THO and revealed more than 2,500 dpm of H ml

177 of d i s t i l l a t e which c l e a r l y indicates that THO i s released from the transport compartment at the sink.

Ooncluding Remarks The Laminariales have evolved an e f f e c t i v e long-distance transport system for inorganic and organic compounds.

The anatomy and f i n e structure of

sieve elements in d i f f e r e n t species shows that they have reached d i f f e r e n t l e v e l s of specialization, sane clearly more advanced than the others.

In

an evolutionary l i n e , Laminaria vvould perhaps represent the most primitive and Macrocystis the most specialized sieve tube system. several

structural

There are also

s i m i l a r i t i e s between the sieve elements of the

Laminariales, especially Macrocystis, and those of vascular plants, but a number of unique features such as the presence of vesicles and organelles, including a nucleus, underscore the suggestion that the sieve elements of the Laminariales have evolved p a r a l l e l to those of higher plants.

The

experimental data on the physiology of translocation in Macrocystis, which a p p a r e n t l y has the most s p e c i a l i z e d

t r a n s p o r t system, support a

pressure-flow mechanism for the movement of water (solvent) and dissolved organic and inorganic m a t e r i a l s .

This solution moves in a s t r i c t l y

directed manner in the sieve elements which connect source and sink areas. The transport system i s loaded with osmotic substances (assimilates) at the source region or mature blades, which causes water to enter the s i e v e elements and l a t e r both, the water and the dissolved osmotic materials, are removed from the translocation pathway at the sink (growing regions) . We assume that the driving force for the long-distance transport through the s i e v e elements might be an osmotic gradient along the transport pathway.

The existence of such an osmotic gradient s t i l l remains to be

shown.

References 1.

Biddulph, 0 . , Gory, R.:

2.

Buggeln, R.G.:

Plant Physiol. 32, 608-619 (1957).

J. Phycol. 10, 283-288 (1974).

178 3. 4. 5.

Büggeln, R.G.: J. Phycol. 13, 212-218 (1977). Cataldo, D.A., Christy, A.L., Coulson, C.L.: Plant Physiol. 49, 690-695 (1972). Choi, I.C., Aronoff, S.: Plant Physiol. 41, 1119-1129 (1965).

6.

Crafts, A.S.: im. J. Bot. 26, 172-176 (1939).

7.

Duncan, M.J.: Helgolander wiss. Meeresunters. 24, 510-525 (1973).

8.

Floc'h, J.Y., Penot, M.: (1970).

C.R. Acad. Sei., Paris 271 , 288-291

9.

Floc'h, J.Y., Penot, M.: (1971).

C.R. Acad. Sei., Paris 273, 1100-1103

10. Floc'h, J.Y., Penot, M.: Physiol. Veg. 10, 677-686 (1972). 11. Floc'h, J.Y., Penot, M.: C.R. Acad. Sei., Paris 282, 989-992 (1976). 12. Floc'h, J.Y., Penot, M.: Z. Pflanzenphysiol. 96, 377-385 (1980). 13. Gage, R.S., Aronoff, S.: Plant Physiol. 35, 53-64 (1960). 14. Hellebust, J.A., Hang, A.: Can. J. Bot. 50, 169-176 (1972). 15. LDbban, C.S.: Plant Physiol. 61, 585-589 (1978). 16. Lobban, C.S.: J. Phycol. 14, 178-182 (1978). 17. Lüning, K.: Mar. Biol. 2, 218-223 (1969). 18. Lüning, K.: Helgolander wiss. Meeresunters. 20, 79-88 (1970). 19. Lüning, K., Schmitz, K., Willenbrink, J.: Proc. Int. Seaweed Symp. 7, 420-425 (1972). 20. Lüning, K., Schmitz, K., Willenbrink, J. : Mar. Biol. Z3, 275-281 (1973). 21. Münch, E.: Die Stoffbewegungen in der Pflanze. Verlag G. Fisher, Jena 1930 22. Nicholson, N.L.: J. Phycol. 6, 177-182 (1970). 23. Nicholson, N.L., Briggs, W.R.: Am. J. Bot. 59, 97-106 (1972). 24. Parker, B.C.: J. Phycol. 1, 41-46 (1965). 25. Parker, B.C.: J. Phycol. 2, 38-41 (1966). 26. Parker, B.C., Huber, J.: J. Phycol. 1, 172-179 (1965). 27. Peel, A.J.: Physiol. Plant. 23, 667-672 (1970). 28. Peel, A.J., Field, R.J., Ooulson, C.L. , Gardner, D.J.C.: Plant. 22, 768-775 (1969). 29. Penot, M., Penot, M.: Phycologia 16, 339-347 (1977).

Physiol.

30. Sargent, M.C., Lantrip, L.W.: Am. J. Bot. 39. 99-107 (1952). 31. Schmitz, K., Kuhn, R.: Planta (in press). 32. Schmitz, K., lobban, C.S.: Mar. Biol. 36, 207-216 (1976).

179 33. Schmitz, K., Lüning, K., Willenbrink, J. : 418-429 (1972).

Z. Pf lanzenphysiol. 67,

34.

Schmitz, K., Srivastava, L.M.

Planta 116, 85-89 (1974).

35.

Schmitz, K., Srivastava, L.M.

Cytobiology 10, 66-87 (1974).

36.

Schmitz, K., Srivastava, L.M.

Can. J. Bot. 53, 861-876 (1975).

37.

Schmitz, K., Srivastava, L.M.

Plant Physiol. 63, 995-1002 (1979).

38.

Schmitz, K., Srivastava, L.M.

Plant Physiol. 63, 1003-1009 (1979).

39. Schmitz, K., Srivastava, L.M. 40. Sideman, E.J., Schreier, D.C.

Plant Physiol. 66, 66-69 (1980). An. J. Bot. 64, 649-657 (1977).

41.

Steinbiss, H.H., Schmitz, K.:

42.

Titlyanov, E.A., Peshekhodko, V.M.: Trudy Biologo-pocvennogo instituta Novaja serija 20, 137-140 (1973).

43. Trip, P., Gorham, P.R.:

Planta 112, 253-263 (1973).

Plant Physiol. 43, 1845-1849 (1968).

44. lyree, M.T., Zimmerman, M.H.: Jta Encyclopedia of Plant Physiology, New Ser. 1, (M.H. Zimmermann, J7A. Milburn, eds.), Springer, Berlin, pp. 478-479 1975 45.

Ziegler, H.:

Protoplasma 57, 786-799 (1963).

46.

Ziegler, H., Ruck, I.: Planta 73, 62-73 (1967).

DISCUSSION DRUEHL: Have you looked at day vs. night translocation? SCHMITZ: No. DRUEHL:

Is there any possibility of an experiment like that?

It might

give you an idea of the osmotic potential as a mechanism for translocation. SCHMITZ:

My ideas about finding out whether there is osmotic potential go

in another direction.

I think one has to prepare a plant, just leave one

blade, cut the others off, and then analyze stipe sections for a gradient in radioactivity.

I have looked at the sieve tube sap; if you leave the

other blades on you will not be able to pick up a clear gradient. DRUEHL:

In what stage of development is the nucleus lost and is the

nucleus absent for most of the species for which there is translocation?

180 SCHMITZ:

I am pretty sure that Macrocystis sieve elements do not c o n t a i n

a nucleus a t maturity.

We have t r i e d

Feulgen s t a i n i n g and s e r i a l

sections and we have not found any nuclei in mature e l e m e n t s . nucleus i s l o s t , the c e n t r a l part o'f the sieve

When the

element i s nearly empty

and the sieve pores have reached t h e i r l a r g e s t s i z e , a l s o they are open. That i s in Macrocystis. s i e v e elements — vacuoles s t i l l , contain n u c l e i .

In Laminaria we are looking at t o t a l l y d i f f e r e n t

they are not so well developed, they contain l o t s of

they c o n t a i n c h l o r o p l a s t s and mitochondria and t h e y So you have a developmental l i n e in an evolutionary sense

from l e t us say Hedophyllum, which looks even more p r i m i t i v e

than

Laminaria, to M a c r o c y s t i s .

in a

You w i l l find t h e same s i t u a t i o n

Macrocystis plant a l s o , i f you look a t the sieve elements, from the inner c o r t e x toward the medulla; you have the whole s e r i e s , which you have in the phylogenetic sense, in one and the same plant.

Macrocystis j u s t goes

further than Laminaria in modification of the sieve element protoplast. SRIVASTAVA:

Cn the question of the nucleus, Mao Lien, a graduate student

o f mine, has sane stages in the dissolution of the nucleus in Macrocystis. So most l i k e l y i t does go. FLOC'H:

I have a comment on uptake and our controversy about the form in

which phosphorus i s t r a n s l o c a t e d . apparent.

A c t u a l l y , the c o n t r o v e r s y i s only

Our r e s u l t s a r e q u i t e s i m i l a r , but we have two

interpretations.

possible

We have no d e f i n i t e c o n c l u s i o n s or proof yet as to

whether p h o s p h a t e

is

hexose-monophosphate.

translocated

as i n o r g a n i c phosphate or

as

I think i t would be important to find out which

substance enters f i r s t into the t r a n s l o c a t i n g c e l l s , but t h a t would be v e r y d i f f i c u l t to s o l v e , because o f the very f a s t turnover o f t h e s e phosphorylating compounds,

ihe turnover of ADP i s about one minute, and

t h a t o f the hexose-monophosphates i s about 5-6 minutes.

So I think the

problem i s very hard. SCHMITZ:

I t h i n k we were very c a r e f u l in p o i n t i n g out t h a t our data

suggest t h a t phosphorus i s being t r a n s l o c a t e d in the organically-bound form, but we do not want to exclude the p o s s i b i l i t y t h a t i t may a l s o be translocated as phosphate.

You know that we did in v i t r o i n c u b a t i o n o f

181 sieve tube sap with radioactive phosphate and found a rapid incorporation of phosphate into many substances. enzymes for these syntheses.

So the sieve tube sap does contain the

I also made it clear that the sieve element

in the Laminariales are different frcm those of vascular plants because we have here everything in one cell.

Here, it appears, we do not have the

separation of the translocating part from the metabolically active part, the companion cells which are nucleate and packed with mitochondria and riboscmes, as in vascular plants. FLOC'H:

That was what I meant.

direction of translocation.

I have another question.

It is about the

Have you found a competition between the hold-

fast and scimitar in Macrocystis? SCHMITZ: Yes, there is competition along the stipe.

If the blade is

closer to the tip, most of the translocate will move to the tip, and if the blade is closer to the holdfast, most of the translocate will move to the holdfast.

If there are young fronds at the holdfast, they will

attract even more translocate.

If the blade is in an intermediate

position, you will get transport up and down. We do not exactly know how this wDrks, but it does. BIDWELL:

I was very interested in your data where the plant was

translocating towards the base, after it had been cut off.

If the system

was working exclusively with a Munch type pressure flow system, of course as soon as you cut it off, the translocation ought to stop, but this does not happen.

It is the same in higher plants; you can cut the plant off

from its supposed sink and the translocation will still continue. Ihe inference is, that there may be unloading mechanisms involved that are affected other than just the avidity of the sink for the solutes that are present. SCHMITZ:

I think that the fact of cutting creates an enormous sink and

the solution just flows out, and that is the unloading process.

But if

the whole system is closed, we have to postulate that the translocate as well as the water have bo move out, otherwise, the osmotic capacity will build up to the point that the whole thing comes to a stop. water has to came out.

Also, the

182

BIDWELL: Well, if you had an unloading at the sink, then the water would follow the unloading by oanosis, would it not? SCHMITZ:

Yes.

SRIVASTAVA:

It will follow passively.

Going back to the question of directionality, there may be

something to the concept that Klaus does not mention, that is of "imprinting."

The conduits get imprinted toward flow in a certain

direction for sometime, and then something else has to happen, which is probably metabolically controlled, to change the directionality.

Even in

these plants, the apical blades, which are for a long time transporting upwards only, towards the close of the fall when the scimitar has matured, the photoassimilate travels primarily downwards.

So there is a change in

directionality there. BIDWELL:

This business of the upper fronds translocating upwards and the

lower fronds translocating downwards that is all fact.

The question has

been examined, and if the system is an absolutely open tube pressure flow system the proportion going to any sink should be the same for every leaf from the system, irrespective of the distance, because the proportional removal from different sinks is the same.

You find exactly the same

situation in virtually all higher plants where the flagleaf feeds the seed and in wheat where the lower leaves feed the root.

This is presumably

controlled by sane additional mechanisms. SCHMITZ:

I suppose if we could perform an experiment, collecting from the

basal and the apical cuts simultaneously and labelling the blade exactly in the middle, we would obtain the same radioactivity.

I do not know? We

32

expect it! As to the directionality of against a concentration gradient.

P transport, it is a transport

I think the directionality in these

plants is given by the overall concentration gradient in the sieve tube system and that will probably direct the flow.

Part I I I .

Marine macrophytes in coastal

ecosystems

THE REGULATION OF MACROAIGAL ASSOCIATIONS IN KELP FORESTS

M.S. Faster Landing Marine Laboratories P.O. Box 223, Mass Landing, CA 95039, U.S.A.

MDSS

Introduction What processes control or regulate macroalgal species composition and abundance in kelp forests?

The answer to t h i s q u e s t i o n has been

approached f o r other communities ( 7 ) , particularly the rocky intertidal zone, where the organisms are accessible, well described and r e l a t i v e l y easy to manipulate

(6).

In contrast, kelp f o r e s t s are r e l a t i v e l y

inaccessible, d i f f i c u l t to manipulate and most studies on the P a c i f i c coast of

North America have been confined to southern California

(5,10,16,30,32).

Moreover, much of this work involved

qualitative

descriptions made during the calmer simmer months (for an exception, see Rosenthal e t a l .

[32]).

These and other studies (3,12,25,26,31) do,

however, document seasonal fluctuations in a l g a l abundance within a particular forest, as well as dramatic spatial changes in abundance and species ccmposition under the same canopy type. A number of factors have been suggested as important in regulating these algal associations on a l o c a l scale (eliminating geographic variation). Sand may prevent settlement or destroy established plants by burial and abrasion (3,17,32). hardness.

Detachment mortality may be dependent on substratum

Light affects a variety of plant processes and can vary with

t u r b i d i t y , d e p t h , season and o v e r s t o r y canopy development a l g a l - a l g a l competition f o r l i g h t

[5,10,17,27,31,32]).

undoubtedly a f f e c t growth as w e l l

(21).

(e.g.,

Nutrients

Water motion can a f f e c t

settlement (28), remove adults (3,5,32,33) and alter nutrient availability (21,28).

Grazing

can m o d i f y or c o m p l e t e l y remove

vegetation

(3,5,10,14,17,23,31), and competition with other algae (11,16,17) and s e s s i l e invertebrates (15,17,29) for space can a f f e c t species ccmposition

© 1982 Walter de Gruyter & Co., Berlin • New York Synthetic and Degradative Processes in Marine Macrophytes

186 and distribution. added.

Many o f these f a c t o r s i n t e r a c t and o t h e r s could be

Their r e l a t i v e importance varies from s i t e to s i t e , but at any one

s i t e probably several factors play a r o l e . What are the dominant regulatory processes f o r k e l p f o r e s t s and how do they vary frcm one forest to another?

This paper attempts to answer these

questions f o r three k e l p f o r e s t s in c e n t r a l C a l i f o r n i a .

Long-term

seasonal changes in algal species composition and abundance are correlated with s i t e - s p e c i f i c physical and b i o l o g i c a l c h a r a c t e r i s t i c s of importance to the organization of algal associations.

apparent

These correlations

are explained by hypotheses that are p a r t i a l l y tested by manipulative f i e l d experiments and s i t e contrasts.

Study Areas Six kelp f o r e s t s were i n v e s t i g a t e d over the past three years, but only three are discussed in d e t a i l here.

A complete discussion of a l l

and methods i s a v a i l a b l e in Foster e t a l .

(18).

These three

sites

forests,

designated as S t i l l w a t e r Cove, Sandhill B l u f f and Greyhound RDck, are located between Carmel Bay and Ano Nuevo Island in c e n t r a l (Fig.

1).

California

The c o a s t a l climate in t h i s region is highly seasonal, with

most large swells associated with winter storms.

Upwelling i s common in

the area arri nearshore nutrient l e v e l s are generally high ( 4 ) .

Nearshore

temperatures (4) and on-site temperature measurements using d i v e r - h e l d thermometers r e v e a l

no s i g n i f i c a n t d i f f e r e n c e s between s i t e s , and

s a l i n i t i e s are also similar ( 4 ) . The physical and biological characteristics of the s i t e s are summarized in Table I .

S t i l l w a t e r Gove is r e l a t i v e l y protected from the predominantly

northwest winter swell; i t i s inside Carmel Bay and opens t o the south. The substratum in both the kelp f o r e s t and the intertidal zone i s hard rock of moderate r e l i e f .

Diver o b s e r v a t i o n s and secchi d i s c

indicate long periods of r e l a t i v e l y c l e a r w a t e r .

readings

Macrocystis p y r i f e r a

( g i a n t kelp) is the most common surface canopy kelp and grows from depths below 30 m.

187

The shore north of Santa Cruz i s g e n e r a l l y sandy beach backed by high cliffs.

The substratum i s sand and/or Santa Cruz mudstone with some shale

to depths of 20 m.

This rock varies in hardness, but i s g e n e r a l l y s o f t

and e a s i l y eroded.

Ihe subtidal substratum a t Sandhill Bluff i s generally

f l a t , while Greyhound RDck i s composed of moderate-relief p a r a l l e l ridges. The c o a s t

in t h i s r e g i o n f a c e s southwest and, except f o r the area

immediately southeast of Ano Nuevo Island, i s f u l l y exposed to northwest swells.

These swells frequently exceeded a height of 4 m between October

and A p r i l , 1 9 7 7 - 7 8 , and breaking s u r f was observed o v e r s i t e anchored a t 10 m depth.

buoys

Swell during the winter o f 1978-79 was more

moderate (generally l e s s than 3 m).

The Sandhill Bluff s i t e i s p a r t i a l l y

protected by a gnall point and faces more to the southwest so water motion i s s l i g h t l y reduced r e l a t i v e to Greyhound Rock.

High w a t e r motion

d i s t u r b s the nearshore sediments and, combined with run-off frcm numerous small creeks, produces generally turbid water frcm l a t e f a l l through early spring at these northern s i t e s .

Macrocystis pyrifera forms the surface

188 Table I: physical and biological characteristics of the survey sites. Character

Stillwater Gove

Depth 3

Substratum

Greyhound Itock

9-14 m

6-9 m

6-12 m

sandstone

mud stone

mudstone

low

moderate

Typographic Belief^ moderate 0

Sandhill Bluff

Water Clarity

clear

turbid

turbid

Swell Exposure

low

moderate

high

Grazers^

anall snails

urchins

and crabs

approx. 1/m

Macrocystis

Macrocystis

Nereocystis

pyrifera

pyrifera

luetkeana

Canopy Kelp

to 30 m Understorye

# of species^

2

urchins approx. 3/m'

to 12 m

to 12 m

Pterygophora

fleshy red

fleshy red

californica

algae and

algae and

and coralline

Desmarestia

Desmarestia

algae

ligulata

ligulata

14 (13-15)

31 (29-33)

30 (24-34)

a

Stillwater Cove also has seme conglomerate and lava. Qualitative estimates from surveys; low 1-2 m; moderate 2-5 m.

c

Average qualitative estimates made during all surveys based on diver estimates and secchi disc measurements (1-5 m - turbid; >8 m - clear).

^Composition and abundance determined by counts of plants and in quadrats. e

Cnly the most common species and types listed.

^Some groups, such as encrusting corallines, not determined to species. Data are means for surmer surveys (range).

canopy at Sandhill Bluff, but does not grow below 12 m, even though suitable substratum is available. luetkeana forest.

Greyhound Rock is a Nereocystis

This canopy kelp is also rarely found below 12 m.

189 Methods Permanent, long-term survey areas were established a t Sandhill Bluff and Greyhound Rock in summer ( J u l y ) 1977 and a t S t i l l w a t e r Cove in e a r l y spring (March) 1978.

These areas have been surveyed in spring (March-May)

and summer (July-September) s i n c e t h a t t i m e .

The s u r v e y a r e a s

at

Greyhound Rock and S a n d h i l l B l u f f are c i r c u l a r (40 m r a d i u s ) , each 2

encompassing 5026 m .

The centers of these areas were o r i g i n a l l y placed

a t the seaward edge of the kelp surface canopies so that one-half of the survey s i t e vrould be inside the canopy and one-half o u t s i d e .

A f t e r the

i n i t i a l s u r v e y s , i t was apparent t h a t the canopy edge v a r i e d due t o continued growth or removal by s t o r m s .

However, a r e a s toward shore do

have g e n e r a l l y more adult kelp plants.

Since giant kelp grows to depths

below 30 m in Stillwater Gove, surveys there were done entirely within the forest.

The d a t a were c o l l e c t e d in e i t h e r one 40-m r a d i u s a r e a of

moderate depth (10-13 m) or twD 20-m radius a r e a s , one in shallow water (9-12 m) and one in deep water (12-14 m).

The vegetation i s similar over

t h i s e n t i r e d e p t h r a n g e , so the d i f f e r e n t s u r v e y a r e a s a r e n o t distinguished below. Seasonal changes in s u r f a c e canopy a r e a s and d e n s i t y a t the two Macrocystis p y r i f e r a s i t e s were d e t e r m i n e d from i n f r a r e d

aerial

photographs taken by the C a l i f o r n i a Department of Fish and Game.

The

t o t a l canopy cover in fixed locations, which included the survey a r e a s , was determined from the projected transparencies.

Subtidal plant cover

was estimated by divers using a modification of the point c o n t a c t method (16) employed by t e r r e s t r i a l plant e c o l o g i s t s (20).

The sampling device

consisted of a 1.4 m long metal bar with a 1.8 m long s t r i n g attached to the bar l i k e a l o o s e string on a bow. the s t r i n g .

Five knots were tied a t random on

The bars were positioned a t random within each study a r e a .

By moving the s t r i n g from one s i d e of the bar to the other, ten points were sampled per bar location.

A point i s determined by grasping a knot,

p u l l i n g the s t r i n g t a u t and p r e s s i n g the knot to the substratum.

The

diver then notes the presence of a l l organisms i n t e r s e c t i n g an imaginary l i n e extending through the knot p e r p e n d i c u l a r to the substratum to a height of 2 m.

More than one alga can occur over and/or under one p o i n t ,

190 so algal cover can exceed 100%.

Tbtal cover of a particular species was

calculated by dividing the sum of a l l contacts with the species (total nuriber of points at which the species was found) by the t o t a l number of points surveyed.

Algal species were identified using the monograph by

Abbott and Ifollenberg (1). The abundance and d i s t r i b u t i o n of large i n v e r t e b r a t e g r a z e r s were 2 determined at a l l three s i t e s by counts in randomly-placed 1 or 5 m quadrats

and on M a c r o c y s t i s

pyrifera

plants.

Sea

urchin

(Strongylocentrotus franciscanus) removal experiments were performed at two of the s i t e s .

The methods and results of these experiments are

available elsewhere ( 9 , 1 8 ) , but some results w i l l be mentioned in the following.

Results The most abundant species throughout the year in the algal association at Stillwater Cove are Macrocystis p y r i f e r a , a subcanopy of Pterygophora californica and a bottom cover of Calliarthron tuberculosum and various encrusting corallines.

Cystoseira osmundacea is also common, e s p e c i a l l y

in summer, when reproductive P. c a l i f o r n i c a plants are large (1-1.5 m t a l l ) and, if cortical rings are annual (19), over ten years old.

The C.

tuberculosum plants are also generally large (5-15 cm t a l l ) and, given their slow growth rates (22), probably quite old. Sandhill Bluff also has a surface canopy of Macrocystis pyrifera, but the subsurface canopy of Pterygophora californica i s very sparse and plants are r a r e l y over 0.5 m t a l l .

Suimer bottom cover is made up of fleshy red

algae, e s p e c i a l l y Polyneura l a t i s s i m a , Plocamium c a r t i l a g i n e u m and Phycodrys s e t c h e l l i i .

Desmarestia ligulata var. ligulata is occasionally

very abundant and encrusting corallines are common. Subcanopy kelps and articulated c o r a l l i n e s are a l s o sparse in the Nereocystis luetkeana forest at Greyhound FDck.

Bottom cover at this site

is similar to Sandhill Bluff, with Polyneura latissima most abundant in summer, followed by Botryoglossum farlowianum v a r .

farlowianum.

191 Desmarestia l i g u l a t a var. l i g u l a t a

i s abundant e v e r y summer, and

encrusting corallines are camion at a l l times of the year.

The number of

algal species within the survey areas i s similar at the s i t e s north of Santa Cruz.

The number i s consistently lower at Stillwater Gove (Table

I). While the patterns described above are generally true, there can be large seasonal and year-to-year variations in species ccmposition and especially abundance.

The summer of 1977 followed twa years of very reduced winter

storms and the Macrocystis p y r i f e r a canopies at that time probably represent near-maximum development ( F i g . 2).

Canopy cover declined to

near zero at both M^ pyrifera sites by spring 1978/ after a rather severe winter in 1977-78.

The canopy at Stillwater Gove recovered more rapidly

than that at Sandhill Bluff ( F i g . 2 ) , and although good quality a e r i a l photographs were not available f o r spring 1979» d i r e c t observations indicated that minimum cover followed the winter storm period and maximum cover occured prior to the f i r s t storms in early f a l l .

Our observations

of other M^ pyrifera canopies indicate this seasonal pattern i s typical f o r central C a l i f o r n i a .

The canopy at Sandhill Bluff recovered more

slowly fran the winter storms of 1977-78» and was s t i l l absent over the survey area in summer 1978.

Although the canopy at Sandhill Bluff

exhibited a general spring minimum, f a l l maximum trend, the year-to-year differences were more pronounced at this site than at Stillwater Cove. The Nereocystis luetkeana canopy at Greyhound Rock i s thin and patchy, even at maximum development, and d i f f i c u l t to assess with a e r i a l photography.

Qualitative observations from sea and shore indicate i t also

reaches maximun cover in f a l l , although usually a month or two later than the Macrocystis pyrifera maximum. in f a l l 1978.

Maximun cover so far observed occurred

Minimum cover in this annual plant also occurs in spring.

Upright algal cover follows seasonal trends similar to the surface canopy kelps (Fig. 2).

At Stillwater Cove, the cycle has been consistent, with a

minimum of around 100% cover and a maximum of about 150%.

In contrast,

year-to-year understory cover at Sandhill Bluff has been highly

192

S T I L L W A T E R

250

C 0 V E

\

200 _

200

150 150

100 o: LU > -50 O o

I00 • 50 SU'77 2 5 0

IO X

SP'78

SU178

SP'79

O I

SU'79 75

1 S A N D H I L L BLUFF/

60 45

I

£ I

30 15

77

SP'78

—i SP'79

1 SU'79

SU'78 SP'79 S U R V E Y DATE

SU'79

SU'78

5

I

GREYHOUND ROCK

SU'77

SP'78

Fig. 2: Macrocystis pyrifera and upright (nonencrusting) algal cover at Stillwater Cove, Sandhill Bluff and Greyhound Rock. Mean percentage cover of upright algae and canopy cover (m2) of M^ pyrifera are shown. For upright algae, numbers indicate the nunber of groups of :en points in each sample; * = cover only approximate due to poor quality photo; ? = no photo available; su = sutmer; sp = spring.

193 variable with a maximum of 270% in the summer of 1978, about three times greater than in summer 1977.

Similar year-to-year differences have been

observed in the two other Macrocystis pyrifera forests surveyed north of Santa Cruz.

Greyhound Bock is most similar to Sandhill Bluff in species

composition, but understory algal abundance has not undergone the large year-to-year variation found at the latter site (Fig. 2).

The variation

in abundance at Greyhound Rock is more similar to Stillwater Cove, and a similar trend has been observed in the one other Nereocystis luetkeana forest surveyed about 2 km south of Greyhound Hock. Figure 3 shows the variation in abundance of understory algae which are most common in at l e a s t one of the survey areas.

Calliarthron spp.

(primarily C. tuberculosum) and Bossiella spp. (B. c a l i f o r n i c a and B. orbigniana) have been combined to represent the most abundant articulated corallines.

The figure illustrates the differences in species composition

between sites discussed above, as well as the r e l a t i v e contribution of these species to the seasonal and year-to-year variability.

At Stillwater

Gove, seasonal changes are largely the result of changes in growth of the perennnial algae, Pterygophora c a l i f o r n i c a , Cystoseira osmundacea and articulated corallines, which a l l exhibit maximum cover in simmer.

These

plants are rare at the sites north of Santa Cruz. The annual sporophytes of Desmarestia l i g u l a t a var. l i g u l a t a are a consistent, abundant part of the summer flora only at Greyhound Rack (Fig. 3).

This alga was uncommon at Stillwater Cove and Sandhill Bluff except

during the summer 1978 surveys.

At that time, i t covered more area than

any other alga at Sandhill Bluff.

Polyneura latissima also has an annual

c y c l e at the northern s i t e s and also reached i t s peak abundance at Sandhill Bluff during summer 1978.

This alga does not occur at Stillwater

cove. Large sea urchins (Strongylocentrotus franciscanus and S^ purpuratus) are extremely rare at S t i l l w a t e r Cove and small individuals (1-3 cm test diameter) are found only occasionally beneath mats of corallines.

articulated

Sea urchins were probably abundant prior to the early 1960s

(2,24), but sea otter (Enhydra lutris) predation since that time removed

194 Pterygophora cali fornica

80

60 •

STILLWATER COVE • SANDHILL BLUFF o GREYHOUND ROCK"

40 • 20 •

0

80

Cystoseira osmundacea

Calliarthron

Desmarestia ligulata f\va r. ligulata

Polyneura

Bossiei/a

spp. + spp.

60 \ 40 20 0 80

60 "

40 • 20

/atissima

I \ I \ I \ I'

^

A / \ / \

/ Av *\

//

v

0 SU 77

Fig. 3: sites, spring.

SP SU SP s u 78 78 79 79 SURVEY DATE

SU 77

' Y , ' v -I SP SU SP s u 78 78 79 79 SURVEY DATE

(Dover of common understory algal species at the three survey Data are means (sample sizes in Figure 2 ) ; su = summer; sp =

almost all large individuals other than those in inaccessible crevices. Both abalone (Haliotis spp.) and sea urchins are found in areas inhabited by sea o t t e r s ,

if

s u i t a b l e c r e v i c e s are available (24), but such

microhabitats are rare in Stillwater Cove.

Grazing snails (Tegula spp.)

and crabs (primarily Pugettia producta) are common at Stillwater Cove, but

195 no obvious e f f e c t s of their grazing have been noted on the algae. The northern front of the expanding sea otter population in California has not progressed beyond Santa Cruz, and Strongylocentrotus franciscanus is common at Sandhill Bluff (Table I ) .

Highest densities occur at the outer

edge of the kelp forest, with animals clumped in depressions or along the few available ledges.

S. franciscanus is very abundant at Greyhound Rack, 2 with highest densities (4-5/ta ) on the walls and valleys between the rock ridges.

Bicrusting c o r a l l i n e s are the most common in these habitats.

Nereocystis luetkeana and foliose understory algae are most abundant on the ridge tops where sea urchin densities are low.

Grazing snails and

crabs are extremely rare at both northern sites.

Discussion The results of the survey, when combined with the physical characteristics of

the s i t e s and o t h e r i n f o r m a t i o n discussed below, suggest that

substratum composition, water motion and grazing are of primary direct and indirect importance in regulating the algal associations, but the relative influence of these f a c t o r s varies between s i t e s .

Figure 4 l i s t s the

f a c t o r s f o r each s i t e and integrates them into regulatory pathways which attempt to explain the observed species composition and abundance. The substratum c h a r a c t e r i s t i c s and r e l a t i v e l y low water motion at S t i l l w a t e r Cove are responsible in part for the clear water, resulting in enough l i g h t f o r Macrocystis p y r i f e r a to grow in over 30 m of water. Winter storms remove canopy fronds, but usually not e n t i r e plants, producing a spring minimum, but allowing rapid recovery to a f a l l maximum (Fig. 2).

Pterygophora c a l i f o r n i c a can p e r s i s t attached to the hard

substratum under the low water motion conditions, and occurs in long-lived stands.

The changes in cover of this and the other perennial species

( F i g . 3) are primarily due to d i f f e r e n t i a l blade growth and loss, not recruitment.

Sialler perennial understory algae can persist without being

removed by storms or buried by sand, and low l i g h t produced by the combined canopies of M. p y r i f e r a and P. c a l i f o r n i c a apparently favors perennial corallines. Rosenthal et a l . (32) found that M. pyrifera

196

UJ > O

LU Ct

O

WALLS AND VALLEYS

HIGH DENSITIES AND DISPERSED IN PROTECTED LOCATIONS - *" ALGAE REDUCED TO ENCRUSTING CORALLINES

Fig. 4: Factors, factor interactions and regulatory pathways a f f e c t i n g the algal associations in the three survey sites. (A) Stillwater Gove, (B) Sandhill Bluff, (C) Greyhound Rock. Primary f a c t o r s are shown in boxes, f a c t o r interactions by joining lines and regulatory pathways by arrows pointing from cause to e f f e c t . recruitment occurs in patches where groups of adult plants are removed occasionally by storms.

This mechanism may be important f o r M^ p y r i f e r a

as well as for P. californica in Stillwater Gove. Other evidence suggests that recruitment and growth of at l e a s t brown algae are controlled by competition f o r l i g h t within S t i l l w a t e r Cove. Desmarestia ligulata var. l i g u l a t a was only abundant there during the summer of 1978 (Fig. 3), following a large reduction of the Macrocystis p y r i f e r a canopy ( F i g . 2 ) .

Moreover, the experimental

removal o f

Pterygophora californica results in a dramatic recruitment of M. pyrifera,

198 p. c a l i f o r n i c a and D^ l i g u l a t a v a r . communication).

ligulata

(D.

Reed,

personal

The g e n e r a l absence o f f l e s h y red a l g a e may also be

related to competition for l i g h t , but t h i s has not been investigated.

Sea

urchins are r a r e a t S t i l l w a t e r Cove, and other grazers do not appear to have s i g n i f i c a n t e f f e c t s . At both Sandhill Bluff and Greyhound Rock, the low l i g h t l e v e l s produced by r e s u s p e n d e d

s e d i m e n t s and t e r r e s t r i a l r u n - o f f probably reduce

successful recruitment and growth of surface canopy kelps below 12 m (Fig• 4).

Sea u r c h i n s c o u l d s e t t h i s

lower l i m i t , but evidence from a

physically similar area suggests t h a t p l a n t s would not grow in deeper water even i f sea urchins were removed (31). The s o f t rock and high water motion a t Sandhill B l u f f i n t e r a c t to cause variations in the Macrocystis pyrifera canopy, e i t h e r by removing fronds or e n t i r e p l a n t s .

Entire plants were removed during the large swells of

winter 1977-78, and the canopy did not recover over the survey area summer 1979 ( F i g . 2 ) .

More complete counts of plants and stipes before

and a f t e r p e r i o d s o f extreme water motion a r e needed t o c l a r i f y interaction.

until this

Pterygophora c a l i f o r n i c a does not form stands of large (old)

p l a n t s a t t h e two n o r t h e r n s i t e s .

Perhaps l a r g e r p l a n t s are more

susceptible to removal from the s o f t rock by water motion. Our observations suggest t h a t , a t both northern s i t e s , the understory a l g a e must be adapted f o r rapid growth during the periods of abundant l i g h t ( l a t e spring-summer).

In addition, because the combined e f f e c t s o f

reduced l i g h t , b u r i a l , abrasion and removal by storms reduce the chances of winter survival, these plants must be a b l e t o p e r s i s t in a r e s i s t e n t v e g e t a t i v e stage (spores or h o l d f a s t s ) .

Articulated c o r a l l i n e s and other

upright perennial p l a n t s may be u n s u c c e s s f u l because t h e i r

relatively

tough t h a l l i are more l i k e l y to be completely removed along with portions o f the substratum during storms. The great increase in understory cover a t Sandhill Bluff during the summer o f 1978 ( F i g . 2) was probably a r e s u l t of increased l i g h t caused by the removal o f Macrocystis p y r i f e r a during the preceding w i n t e r .

Other

o b s e r v a t i o n s i n d i c a t e competition f o r space was not important.

In the

199 summer of 1977, Desmarestia 1 igulata var. l i g u l a t a was most common just outside the canopy, and there was no reason to assutie that space was less available inside the canopy.

In addition, as discussed above, D.

ligulata var. l i g u l a t a increased at S t i l l w a t e r Cove when M. p y r i f e r a canopy was greatly reduced, and became abundant when the Pterygophora californica canopy was removed.

Pearse and Hines (31) also found that the

experimental removal of portions of the

pyrifera canopy at Point Santa

Cruz resulted in an increase in understory algae. The moderate densities of sea urchins at Sandhill Bluff appear to have l i t t l e negative e f f e c t on the vegetation, and may actually increase the abundance and diversity of low-growing algae by removing overstory plants (9).

Paint Santa Cruz has a Macrocystis p y r i f e r a f o r e s t in a habitat

similar to that at Sandhill B l u f f , but without sea urchins (31). surveys at Point Santa Cruz showed the understory a l g a e

CUr

underwent

fluctuations similar to Sandhill Bluff, suggesting that if there are sea urchin grazing e f f e c t s at the l a t t e r s i t e , they are subtle r e l a t i v e to those associated with storms and surface canopy cover. Nereocystis luetkeana is distributed in patches on the tops of ridges at Greyhound Rock, and our sea urchin removal experiments indicated that i t is restricted to these areas by grazing. the winter of

The increase of this plant after

1977-78 may have been due t o increased substratum

availability, a reduction in sea urchins, or seme other factor or factors. Understory algae are distributed like due to sea urchin grazing.

luetkeana, and this may also be

The regulatory model (Fig. 4) indicates two

possible reasons why understory a l g a l cover did not undergo the large year-to-year variation observed at Sandhill Bluff and other northern Macrocystis pyrifera s i t e s .

F i r s t , the N. luetkeana canopy i s not as

dense as that of giant kelp, and reaches its f u l l e s t development later in the f a l l . growth.

I t has less e f f e c t on light and l e s s influence on understory Second, our data show that sea urchins keep large areas of

Greyhound Ffcick relatively free of upright algae at a l l times of the year, regardless of t u r b i d i t y , wave action or canopy growth.

These areas are

thus "stabilized" and act to reduce the overall variation when lumped with areas where algal cover i s high and more v a r i a b l e .

Sea urchins are

probably reduced on the ridge tops at Greyhound Itock because of the high

200 water motion there r e l a t i v e to the w a l l s and v a l l e y s .

This hypothesis

a l s o suggests that sea urchin d e n s i t i e s are low a t Sandhill Bluff because the r e l a t i v e l y f l a t topography provides few refuges frcm water motion. Algal d i v e r s i t y , as measured by the number of species, a t the twD northern s i t e s i s about twice t h a t a t S t i l l w a t e r Cove (Table I ) .

A number of

investigators have found that competition can lead to dominance o f a few s p e c i e s , and that competitive exclusion often occurs unless some physical or biological disturbance acts to prevent i t ( s e e 7 and 8 f o r

reviews).

Both l a r g e s w e l l s and grazing appear to provide such disturbances at Greyhound Rock and S a n d h i l l B l u f f ,

while

the l a c k of grazing

and

r e l a t i v e l y benign physical conditions a t S t i l l w a t e r Gove have apparently favored the establishment o f a few l o n g - l i v e d a l g a e which e f f e c t i v e l y exclude others by shading. die f i n a l question not addressed in figure 4 i s : o f s u r f a c e canopy k e l p a t a p a r t i c u l a r s i t e ?

What determines the type The d i s t r i b u t i o n s

N e r e o c y s t i s luetkeana and Macrocystis p y r i f e r a o v e r l a p i n

of

central

C a l i f o r n i a and h i s t o r i c a l and r e c e n t comparisons at various locations indicate that canopy type i s correlated with water motion and sea urchin grazing ( 3 1 ) .

Our observations a l s o suggest t h a t N. luetkeana i s more

r e s i s t a n t to water motion and more tolerant of sea urchin grazing than M. pyr i f e r a ; however,

pyr i f era may outccmpete

luetkeana for l i g h t .

Much of the above discussion and many o f the proposed i n t e r a c t i o n s i n f e r r e d from natural history observations.

are

Quantitative measurements of

water motion and l i g h t quantity and q u a l i t y need to be c o r r e l a t e d with a l g a l abundance and species composition, and the e f f e c t s of these factors tested in the f i e l d , perhaps with transplant e x p e r i m e n t s .

In a d d i t i o n ,

f u r t h e r r e s e a r c h i s n e c e s s a r y t o c l a r i f y the relationships between the l i f e h i s t o r i e s , morphology and growth c h a r a c t e r i s t i c s o f the a l g a e and t h e i r respective environments.

These data suggest, as proposed by Cbnnell

(7) for other communities, that the algal association in k e l p f o r e s t s

is

an expression of the e f f e c t s of predation and competition, and that these b i o l o g i c a l p r o c e s s e s a r e moderated by p h y s i c a l d i s t u r b a n c e .

Further

comparative, long-term surveys and f i e l d experiments are needed bo t e s t t h i s conceptual framework so t h a t the g r e a t d i f f e r e n c e s in k e l p f o r e s t

201 algal associations can be understood in terms of general ecological processes.

Acknowledgements I am especially grateful to C. Agegian, R. Oowen, D. Reed, D. Rose and R. VanWagenen for their generous assistance and for sharing their ideas and unpublished results.

V. Breda, J. Heine and J. Oliver provided helpful

comnents on the manuscript.

Ihis work was supported by contracts with the

United States Marine Mammal Commission and the California Department of Fish and Game.

References 1.

Abbott, I., Hollenberg, G.: Marine Algae of California. Stanford University Press, Stanford 1976

2.

Andrews, H.:

3.

Barrales, H., Dobban, C.:

4.

Broenkow, W., Smethie, W.: 583-603 (1978).

5.

Clarke, W., Neushul, M.: In Pollution and Marine Ecology (T. Olsen, F. Burgess, eds.) J. Wiley, New York, pp 29-42 1967

6.

Oonnell, J.:

7.

Connell, J.: In Ecology and Evolution of Communities (M. Cody, J. Diamond, eds.) Belknap Press, Harvard, pp 460-490 1975

8.

Oonnell, J.:

9.

Oowen, R.: Trophic interactions within a Central California kelp forest. M.S. thesis, California State university, Hayward, 1979

10.

Dawson, E., Neushul, M., Wildman, R.:

Ecology 26, 24-37 (1945). J. Ecology 63, 657-677 (1975). Estuarine and Coastal Mar. Sci. 6 r

Ann. Itev. Bool. Syst. 3, 169-192 (1972).

Science 199, 1302-1310 (1978).

11.

Dayton, P.:

12.

Devinny, J., Kirkwood, P.:

13.

Dsvinny, J., Molse, L.:

Pac. Nat.

1-80 (1960).

Fish. Bull. 73, 230-237 (1975). Bot. Mar. 17, 100-106 (1974).

Mar. Biol. 48, 343-348 (1978).

14.

Foreman, R.:

15.

Fbster, M.:

Proc. Int. Seawsed Symp. ]_, 55-60 (1972).

Helgolander wiss. Meeresunters. 30, 468-484 (1977).

16.

Poster, M.:

Mar. Biol. 32, 313-329 (1975).

17.

Fbster, M.:

Mar. Biol. 32, 331-342 (1975).

202 18.

Poster, M., Agegian, C., Gowen, R., VanWagenen, R., Rose, D., Hurley, A.: National Technical Information Service (N.T.I.S.) # 293891, Springfield 1979

19.

Frye, T.:

20.

Greig-Snith, P.: 1957

21.

Jackson, G.:

22.

Jöhansen, H., Austin, L.:

23.

Ißighton, D.L.:

Publ. Puget Sound Biol. Sta. 2, 65-71 (1918). Quantitative Plant Ecology.

Buttervrorths, London,

Limnol. Oceanogr. 22, 979-995 (1977). Can. J. Bot. 48, 125-132 (1970).

Nova Hedwigia 32, 421-453 (1971).

24.

Dowry, L., Psarse, J.:

25.

ffclean, J.:

Mar. Biol. 23, 213-219 (1973).

26.

Neushul, M.:

Ecology 48, 83-94 (1967).

27.

Neushul, M.:

Nova Hedwigia 32, 241-254 (1971).

28.

Neushul, M.: In Contributions to the Systematics of Benthic Marine Algae of the "North Pacific (I. Abbott, M. Kurogi, eds.), Japanese Society of Phycology, Kobe, pp. 47-74 1972

29.

Neushul, M., Bbster, M. , Coon, D. , Wöessner, J., Harger, B. : Phycol. 12, 397-408 (1976).

30.

North, W.J.:

31.

Pearse, J., Hines, A.:

32.

Hosenthal, R., Clarke, W., Dayton, P.: (1974).

33.

ZoBell, C.E.:

Biol. Bull. 122, 95-114 (1962).

J.

Nova Hedwigia 32, 1-97 (1971). Mar. Biol. 51, 83-91 (1979). Fish. Bull. J2, 670-684

Nova Hedwigia 32, 269-314 (1971).

DISCUSSION FOREMAN:

Mike, what are your present thoughts about such things as,

recruitment, the time and capabilities of certain species to come in and one year graphs, particularly in reference to Desmarestia vs.

foliose

reds, in these northern sites? FOSTER:

T£> me, the most interesting aspect of these sites is how things

like Polyneura, Phycodrys and other annuals keep coming back year after year, are they coming back frcm spores or are they coming back fran little fragments?

Same for Desmarestia.

It did not occur in any great abundance

inside those kelp forests for three years previous to that storm. the storm it was abundant.

So, how did it get there?

After

Is it a persistent

203 gametophyte or is i t new settlement from patches on the outside?

These

s i t e s are a l s o q u i t e d i f f e r e n t from S t i l l w a t e r Cove or Ooches Prietos Cove, where I did my e a r l i e r work, - they are under continual disturbance. At Stillwater Gove, I would probably get results similar to those I got at Goches Prietos Cove. i t means.

But, i f

As to d i v e r s i t y , i t is very hard to figure out what it

i s taken t o mean number of species, the typical

nunber of s p e c i e s at S t i l l w a t e r Cove i s in the order o f t e n ;

typical

numbers at these exposed s i t e s , which are unstable, i s in the order of 30. So, there may be an inverse relationship between d i v e r s i t y and s t a b i l i t y in these two systems, but there is a l o t of f r e e space available, i f you are ready to colonize, at these northern s i t e s and that could be a more important phenomenon. FOREMAN:

Do you have any evidence of Desmarestia being a preferred urchin

food? FOSTER:

The only evidence we have i s that during that summer in which

Desmarestia was v e r y dense we had another s e t o f sea urchin removal experiments a t Sandhill B l u f f , which I did not describe.

At that time,

they had a l o t of choices, Desmarestia, f o l i o s e red a l g a e ,

Macrocystis,

b u t , by t u r n i n g

them o v e r t o see what was stuck t o t h e i r jaws, we

invariably found Desmarestia, and, although at that time Desmarestia was very abundant, what they had in their stomachs was disproportionate to the abundance in the environment. not, I do not know.

NDW whether that i s a true p r e f e r e n c e or

I should have s a i d , they e a t Desmarestia.

I was

under the impression that i t ate their teeth away! NORTH:

Back in the days when the urchins were doing the scraping and I

was doing the burning with quick lime, we would go f o r six miles in the P t . Ioma kelp bed, or what used t o be the k e l p bed, and there would be almost no algae.

Ihe urchins would be completely dominant.

Vfe WDuld wipe

out the urchins from a s e c t i o n and the f i r s t thing that would come in would be diatoms, they would disappear very quickly, then Ectocarpus, that would disappear very quickly, and then i t was often Desmarestia. t r i e d doing

some p r e f e r e n c e e x p e r i m e n t s

Leighton

in the l a b o r a t o r y

with

Desmarestia and the urchins in the l a b o r a t o r y would not touch i t when other algae were o f f e r e d .

But in the f i e l d , i t was as you said, they just

204 gobbled it up. When it is there, they will eat it.

In the laboratory

they will eat something else. FOSTER:

There is a real danger in extrapolating laboratory food

preference experiments to the field.

There are a number of contradictory

examples, not only for sea urchins, but for other kinds of grazing animals. DRUEHL: We did a Macrocystis harvest here in Bamfield. We looked at the understory and changes in it with complete Macrocystis removal and just canopy removal twice a year. Desmarestia.

We got the very same story you got with

Our other Laminariales did not respond.

Partial harvest

—

canopy removal, not plant removal — , gave intermediate results between complete harvest and control. FOSTER:

If you keep that area harvested, do the Laminaria eventually

respond to the point that Desmarestia cannot invade that community? DRUEHL: Vfe kept it harvested for eighteen months and Desmarestia remained dominant. NORTH:

I have seen young plants of Macrocystis come in months after

transplants were removed, so that the gametophytes must last a long time. But these were areas where nothing had grown for many years, except Desmarestia was always there, and I think that is a very interesting question.

Is it all over the ocean, or is sitting there as a gametophyte

or what? FOREMAN:

How do you handle variable quadrat size? As you said you took

one to five metre quadrats. FOSTER: What we are doing is comparing dispersion estimates based on the different quadrat sizes, to see if we can interconvert.

If we cannot we

will probably express the data in one case as per metre squared, in the other case as per five metre squared and let the reader decide. FOREMAN: My studies have shown that a large percentage, roughly thirty percent, of all species occuring within the nearshore seaweed beds are sporadic and transient; they may occur one year and never be seen again

205 for five.

Some w i l l o c c u r on b a r e s u b s t r a t e o r a l g a l c o v e r ,

i t does

not

make any d i f f e r e n c e . FOSTER:

That i s i n t e r e s t i n g because even in t h e s e n o r t h e r n a r e a s

although same.

the s p e c i e s composition

where,

f l u c t u a t e s widely, i t s t i l l stays the

In S t i l l w a t e r Cove, I could t e l l you a t a l m o s t any time i n the y e a r

n o t o n l y what s p e c i e s you would f i n d t h e r e , b u t what abundance t h e y vrould be

in.

CABOT:

Did you e v e r measure l i g h t ?

FOSTER:

We b e g a n t o do t h a t l a s t

submersible meters.

year.

We had o n e o f

these

Licor

The p r o b l e m with a l l l i g h t measurements along

this

whole c o a s t i s t h a t , j u s t from my o b s e r v a t i o n s , you m i g h t go o u t o n e d a y and i t

is

really

very variable.

t u r b i d and the n e x t day i t w i l l be r e a l l y c l e a r . We c a n p e r h a p s t a l k a b o u t r e l a t i v e

light

Pterygophora canopy.

But a s t o a b s o l u t e v a l u e s , I do n o t know what

mean b e c a u s e o f

i n h e r e n t v a r i a b i l i t y from day t o d a y .

n o t h i n g about how p l a n t s respond. h i g h l i g h t o r do they average i t ? take a secchi

disc.

is

reduction,

c e r t a i n l y in the Pterygophora canopy i t i s much h i g h e r t h a n o u t s i d e the

It

the they

Vfe a l s o know

DD t h e y respond t o two o r t h r e e days o f So what we do e v e r y time we go o u t , we

ROLES FOR DETRITUS IN COMPLEMENTING PRODUCTIVITY OF COASTAL SYSTEMS

J.R.

Sibert

Pacific Biological Station, Nanaimo, B.C. V9R 5K6 Canada

In t h i s paper I w i l l broadly discuss c o a s t a l ecosystem p r o d u c t i v i t y and a s s e s s the r o l e o f macrophytes in the system.

An a l t e r n a t i v e approach to

primary production w i l l be proposed and applied to the analysis of c o a s t a l systems. There are two rather d i s t i n c t ways o f viewing primary p r o d u c t i v i t y . first

i s the c l a s s i c a l

The

trophodynamic concept a r t i c u l a t e d f i r s t by Elton

(2) and l a t e r c r y s t a l l i z e d by Lindeman ( 4 ) .

In a s t r i c t l y

Lindemanian

food c h a i n , t h e r e a r e e x a c t l y f o u r t r o p h i c l e v e l s with no p l a c e omnivores.

for

Emphasis i s given t o the f i x a t i o n o f e n e r g y by p r i m a r y

production and i t s subsequent degradation as material passes up the food chain.

Photosynthesis i s the only process by which primary production may

occur and the two terms have cane to be near synonyms.

The study o f plant

growth from t h i s p o i n t o f view i s s e l f - j u s t i f y i n g

and t h e r e

is

no

compulsion t o c o n s i d e r the f a t e o f o r g a n i c m a t t e r f i x e d in the f i r s t trophic l e v e l . An a l t e r n a t i v e p o i n t o f view i s t o e n l a r g e

t h e meaning o f

primary

p r o d u c t i o n t o " p r o d u c t i o n o f n u t r i t i o u s p a r t i c l e s f o r consuners." d e f i n i t i o n o b v i o u s l y i n c l u d e s p h o t o s y n t h e s i s but a l s o a d m i t s processes.

This other

Conversion o f d i s s o l v e d o r g a n i c m a t t e r i n t o p a r t i c l e s by

b a c t e r i a l a c t i v i t y and transformation o f nonnutritious d e t r i t a l

particles

into n u t r i t i o u s ones by microbial conditioning may a l s o be included. point of view requires consideration o f the f a t e o f organic matter a t f i r s t trophic l e v e l and i s d i r e c t l y relevant to f i s h e r i e s s t u d i e s .

© 1982 Walter de Gruyter & Co., Berlin • New York Synthetic and Degradative Processes in Marine Macrophytes

This the

208 Table I : R e l a t i v e importance o f imported c a r b o n f o r two c o a s t a l ecosystems of d i f f e r e n t s i z e . Figures recalculated frcm various sources as shown. System Nanaimo Delta Surface area (km2) Carbon source (gC m Fluvial DOC Phytoplankton Macroalgae

yr

)

S t r a i t of Georgia

6.5a

6900 b

2000 a 7.5a , 0.9-7.5 , e

130 a 120 e f 2.7-6.7

Naiman and Sibert (6)_give the average DOC concentration in the Nanaimo River as 6.44 mg CI . This estimate i s m u l t i p l i e d by the volume of fresh water e n t e r i n g the S t r a i t (145 x 10 m ) and divided by the , surface area. a

c(15) d(8) . . Foreman, R.E., personal communication. |(7) Foreman e s t i m a t e s t o t a l a l g a l biomass in S t r a i t of Georgia a t 27-67 * 10 grams dry weight. This estimate was increased by 2.3 to c o r r e c t f o r m o r t a l i t y and exudation and a carbon content of 30% was assumed (personal ccnimunication). Applicability of this enlarged productivity definition depends on both the boundaries of the systen arri i t s s i z e or scale (11).

In a very l a r g e , or

global ecosystem, or one with boundaries closed to material imports, i t i s o b v i o u s t h a t the o n l y p r o c e s s c a p a b l e o f photosynthesis.

introducing energy i s

In much analler systems such as river d e l t a s or segments

of the c o a s t a l zone, l a r g e amounts of material are imported from r i v e r s and adjacent areas by longshore t r a n s p o r t .

Some e f f e c t s of s c a l e are

i l l u s t r a t e d in Table I , for two coastal systems in British Columbia.

The

estimates in the table were compiled from v a r i o u s s o u r c e s and r e p r e s e n t a v e r a g e s f o r the e n t i r e system, t h a t i s the t o t a l input of m a t e r i a l divided by the surface area of the system.

The large amount of d i s s o l v e d

o r g a n i c carbon (DOC) introduced by the Nanaimo River overwhelms a l l other sources of carbon, a conclusion p r e v i o u s l y reached by Seki e t a l .

(9).

This e f f e c t i s diminished in the S t r a i t of Georgia by.the much greater

209 surface a r e a . I do not mean to suggest t h a t s i z e i s t h e o n l y important d i f f e r e n c e between the Nanaimo Delta and the S t r a i t of Georgia. The point i s t h a t the r e l a t i v e importance of imports depends on size of the system. The c a l c u l a t i o n s in Table I assume t h a t the e n t i r e production of seaweeds along the rock margins of the S t r a i t of Georgia i s uniformly d i s t r i b u t e d throughout the S t r a i t .

While there are few d a t a on t h i s s u b j e c t , Smith

(12) suggests t h a t most kelp d e t r i t u s remains in the v i c i n i t y of the beds where i t was produced.

The c a p a c i t y t o r e t a i n m a t e r i a l s may be a v e r y

i m p o r t a n t f e a t u r e of open systems and p r o b a b l y c o n t r i b u t e s t o t h e maintenance of high productivity of coastal ecosystems (14,16). Detritus may a l s o provide a p r o d u c t i v e b a s i s f o r c o a s t a l ecosystems a t times of t h e year when p h o t o s y n t h e s i s i s low.

Figure 1 shows abundance

and productivity c y c l e s f o r s e v e r a l i m p o r t a n t components of e s t u a r i n e systems in the S t r a i t of Georgia and elsewhere.

Dissolved organic carbon

(DOC) i s r e l a t i v e l y high and c o n s t a n t t h r o u g h o u t t h e y e a r and has a maximum in A p r i l ,

phytoplankton productivity i s low during most of the

year with an e a r l y summer maximum.

Sedge biomass i s low in w i n t e r and

shows a prolonged l a t e summer maximum.

Benthic microalgae productivity on

sand f l a t s i s lowest in autumn and e a r l y w i n t e r with a g r a d u a l

increase

b e g i n n i n g in J a n u a r y and r e a c h i n g a maximum in mid summer.

Benthic

macroalgae, primarily Ulothrix f l a c c a and P y l a i e l l a l i t t o r a l i s growing in the marsh, a t t a i n maximun biomass in autumn and mid-winter. Secondary production, i l l u s t r a t e d by meiofaunal abundance, seems t o have two p e a k s , one in s p r i n g and a second l a t e r in the summer.

The sunmer

maximum may be a s s o c i a t e d with blooms of b e n t h i c m i c r o a l g a e o r w i t h d e t r i t u s d e r i v e d from o t h e r summer productivity sources. the spring secondary production i s l e s s o b v i o u s .

The b a s i s f o r

F i n a l l y , j u v e n i l e s of

two commercially i m p o r t a n t s p e c i e s of f i s h , P a c i f i c h e r r i n g and chum salmon, a t t a i n t h e i r maximum numbers in c o a s t a l n u r s e r y a r e a s in e a r l y spring.

Since both of these species s u f f e r l a r g e juvenile m o r t a l i t y , the

b a s i s of t h e i r p r o d u c t i v i t y i s of some p r a c t i c a l i n t e r e s t .

Data on

h e r r i n g feeding and growth in the coastal zone are scarce but chim f r y in the Nanaimo e s t u a r y are known t o depend h e a v i l y on a s i n g l e s p e c i e s of harpacticoid copepod which also a t t a i n s i t s maximum biomass in e a r l y

210

Mar

Apr

May

June

July

Aug

Sept

Oct

Nov

Dec

Jan

Feb

Fig. 1: Abundance and productivity cycle for important components of sane estuarine systems. Data taken from various sources: frati top to bottom the references are 3 , 1 3 , 1 0 , 7 , Pomeroy, W . M . , personal communication, 5,1,1.

211 spring ( 3 , 1 0 ) .

The r e l a t i v e t i m i n g o f t h e s e e v e n t s i s such t h a t

the

p r o d u c t i v i t y o f the j u v e n i l e f i s h i s more l i k e l y t o depend on d e t r i t u s and winter a l g a l production than on the customary summer p r o d u c t i v i t y

cycles.

D e g r a d a t i o n o f marine macrophytes i s an o b v i o u s source o f d e t r i t u s t o d r i v e secondary production in the c o a s t a l zone. In summary, the concept o f primary p r o d u c t i v i t y may be expanded t o include a l l p r o c e s s e s which g e n e r a t e n u t r i t i o u s p a r t i c l e s f o r consumers. definition

is

heterotrophic

particularly processing

important

to the c o a s t a l

zone

Ihis where

o f imported d e t r i t u s may g r e a t l y augment

photosynthetic processes in the production o f n u t r i t i o u s p a r t i c l e s . t h i s p o i n t o f v i e w , d e t r i t u s has two f u n c t i o n s :

From

1) to increase t o t a l

system p r o d u c t i v i t y in the summer; 2) t o provide a productive base d u r i n g t i m e s o f t h e y e a r when p h o t o s y n t h e s i s

i s low. I h i s l a t t e r function may

p a r t i c u l a r l y be important for s u r v i v a l o f j u v e n i l e s o f commercial

fish

which occupy the c o a s t a l zone p r i o r t o the major b u r s t s o f photosynthetic productivity.

References 1.

Brown, T . J . , Stephens, K.V.: (1976).

F i s h . Mar. Serv. Data Rep. 2 0 , 146 pp.

2.

Elton, C.:

3.

Healey, M.C.:

Animal Ecology. Sidgwick and J a c k s o n , London,

1927

4.

Lindeman, R . L . :

5.

Levings, C.D., Maody, A . I . : 606, 51 pp.

F i s h . Mar. S e r v . R e s . Dev. T e c h .

6.

Naiman, R . J . , S i b e r t , J . R . :

Limnol. Oceanogr. 23, 1183-1193 ( 1 9 7 8 ) .

7.

Naiman, R . J . , S i b e r t , J . R . : (1979).

J . F i s h . Res. Board Canada 3 6 ,

8.

Parsons, T . R . , LeBrasseur, R . J . , B a r r a c l o u g h , W . E . : Board Canada 2 7 , 1251-1264 ( 1 9 7 0 ) .

9.

S e k i , H., Stephens, K . V . , Parsons, T . R . : (1969).

10.

Sibert, J . R . :

11.

S i b e r t , J . R . , Naiman, R . J . : In Marine B e n t h i c Dynamics, ( K . R . T e n o r e , B . C . C o u l l , e d s . ) , Univ. South Carolina P r e s s , pp. 311-323 1980

J . F i s h . Res. Board Canada 36, 488-496 ( 1 9 7 9 ) . Ecology 23, 399-418 ( 1 9 4 2 ) .

J.

Rep.

504-520

Fish.

Arch. Hydrobiol. ^ 6 ,

Res. 37-47

J . F i s h . Rss. Board Canada 36, 497-503 ( 1 9 7 9 ) .

212

12. 3nith, B.D. : A qualitative and quantitative assessment of seaweed decomposition in the Strait of Georgia. M.Sc. thesis, Department of Botany, University of British Columbia. 168 pp. 1979 13.

Stevenson, J.C.:

J. Fish. Res. Board Canada 19, 735-810 (1962).

14. Valiela, I., Tfeal, J.M., \folkmann, S., Shafer, D. , Carpenter, E. J. : Limnol. Oceanogr. 23, 798-812 (1978). 15. Waldichuk, M. : J. Fish. Res. Board Canada 14, 321-486 (1957). 16. Wbodwell, G.M., Whitney, D.E., Hall, C.A.S., Houghton, R.A.: Limnol. Oceanogr. 22, 833-838 (1977).

DISCUSSION DRUEHL:

Could you test possible contributions of run off from rivers in

an area where you have different seasons of run off, but maybe the same types of early season fish stock? SIBERT: Yes, I think that would be an interesting thing to do.

We could

easily compare Nanaimo, which has a fall-winter run off, with a place like Squamish which has glacial run off. COON:

Dd you have any information on effects of logging on stream run off

and its subsequent effect on estuaries? SIBERT: Well, there is a lot of work at Carnation Creek on the detrital production in the stream in relation to fresheting and logging events, but they are not looking at the estuary. DUNCAN:

Even though the greatest amount of carton is coming down from a

river, the contributions from the other carbon producers in the estuary may be significant at seme times. SIBERT: Well, the point is that you get carbon ccming down the river, say at Nanaimo, at a time of the year when other sources of production are very low, photosynthesis is low, so it has the effect of smoothing out the curve for total carbon.

The problem is we do not know if the dissolved

carbon coming down the river in the fall is susceptible to microbial attack, and also, we do not know how much of it is retained.

It could

213 j u s t go gushing right through. CABOT:

Did you find any u t i l i z a t i o n of Pylaiella?

SIBERT:

That i s apparently utilized by gammarids.

f a v o r i t e , they prefer something e l s e .

I do not think i t i s a

DEGRADATION OF THE KELPS MACROCYSTIS INTEGRIFOLIA AND NEREOCYSTIS LUETKEANA IN BRITISH COLUMBIA COASTAL WATERS1

L.J. Albright, J . Chocair, K. Masuda and M. Valdes Department of Biological Sciences, Simon Fraser University Burnaby, B.C. V5A 1S6, Canada

Introduction A s i g n i f i c a n t p r o p o r t i o n of the s u b - t i d a l rocky s h o r e l i n e of coastal B r i t i s h Cblunbia i s colonized by r e l a t i v e l y extensive beds of m a c r o a l g a e , including those of M a c r o c y s t i s i n t e g r i f o l i a and N e r e o c y s t i s l u e t k e a n a (5,6,21).

Where present, these large kelps are believed to s i g n i f i c a n t l y

contribute t o marine food webs by s e v e r a l means.

These i n c l u d e :

1)

e x c r e t i o n of d i s s o l v e d organic m a t t e r , which i s subsequently metabolized by f r e e - l i v i n g and attached heterotrophic b a c t e r i a (10) and a n i m a l s (7) and 2) sloughing of kelp t i s s u e s which are r a p i d l y colonized and degraded by microorganisms.

Oolonized d e t r i t u s p a r t i c l e s such a s t h e s e may in

t u r n be a s i g n i f i c a n t food source for members of higher trophic l e v e l s of c o a s t a l marine food chains (22). This i n v e s t i g a t i o n was undertaken t o e v a l u a t e the i m p o r t a n c e of M. i n t e g r i f o l i a and N^ luetkeana to the microbial and animal p o r t i o n of t h e marine food web in and adjacent t o k e l p beds by 1) d e t e r m i n i n g

situ

r a t e s and e x t e n t s of k e l p d e g r a d a t i o n , 2) o b s e r v i n g the succession of microbial species and numbers a s s o c i a t e d with k e l p d e t r i t u s a t v a r i o u s s t a g e s of d e g r a d a t i o n and 3) e v a l u a t i n g the n u t r i t i v e q u a l i t y of decomposing kelp t i s s u e s .

1

Portions of t h i s paper have been p r e v i o u s l y p u b l i s h e d in N a t u r a l i s t e

Canadien 107: 3-10 ( 1 9 8 0 ) .

We thank the publishers of t h i s journal f o r

permission to copy p a r t s of t h a t manuscript in t h i s work.

© 1982 Walter de Gruyter & Co., Berlin • New York Synthetic and Degradative Processes in Marine Macrophytes

216 Materials and Msthods Collection and incubation of k e l p .

M. i n t e g r i f o l i a was c o l l e c t e d

(5

August 1977) and incubated at a sheltered bay a t Dixon Island (48° 51' 16" N; 125° 6' 36" W) approximately 2 km from the community of Bamfield, British Golunbia.

N. luetkeana

was harvested and placed for degradation

(28 August 1976) a t Brockton Point (49° 17' 50" N; 123° 6' 54" W) within the harbour l i m i t s of Vancouver, British Oolumbia. Immediately a f t e r h a r v e s t , 100 g p o r t i o n s (wet weight) of laminae and s t i p e s of M. i n t e g r i f o l i a and laminae and pneumatocysts of each were placed in l i t t e r bags of 1.5 mm mesh s i z e .

luetkeana

These bags were

incubated both in the water column at 10 m depth, approximately 50 m from s h o r e , and on the substratum of the i n t e r t i d a l region a t each location. L i t t e r bags were removed a t 5-8 day i n t e r v a l s and t h e i r contents were assayed as described below. In s i t u degradation analyses.

The degrading kelp retained in each l i t t e r

b a g , was weighed a t s p e c i f i e d

time i n t e r v a l s .

Each wet weight

determination was in duplicate and the values wsre averaged. Scanning e l e c t r o n m i c r o s c o p y . glutaraldehyde environment.

(pH 7 . 0 )

K e l p s a m p l e s were p l a c e d

in 2.5%

immediately a f t e r removal from the marine

Each sample was then c r i t i c a l point dried and very l i g h t l y

sprayed with a 1% s o l u t i o n of Kodak P h o t o - f l o 200.

Following these

treatments, samples were examined using an ETEC autoscan scanning electron microscope. Enumeration of viable bacteria.

Viable b a c t e r i a were determined by the

method d e s c r i b e d by Laycock ( 1 3 ) .

In e s s e n c e , kelp t i s s u e s were

cycle-comminuted using a cooled Waring blender and a p p r o p r i a t e d i l u t i o n s of each slurry were spread and plate counted on 2216E marine agar (Difco). Plates wsre incubated f o r 15 days a t 15 C b e f o r e being counted.

The

v i a b l e h e t e r o t r o p h i c b a c t e r i a / g of the contents of each l i t t e r bag were then calculated. Total bacterial numbers wsre determined by f l u o r e s c e n t microscopy using the acridine orange d i r e c t count (AODC) technique as d e s c r i b e d by Hobbie

217 et aK

(12).

ATP analyses.

Ihe material cycle-blended as described above was analyzed

for ATP by the method of Bancroft e t a l . Chlorophyll a a n a l y s e s .

(1).

Chlorophyll £ was analyzed as described by

Strickland and Parsons (25). Crude f i b r e , carbon, n i t r o g e n and ash d e t e r m i n a t i o n s .

Crude

fibre

a n a l y s e s were performed by the method described in Strickland and Parsons (25, pp. 235-236).

Tbtal carbon and nitrogen were assayed with the use of

a CHN analyzer (Beckman), using the technique described by Menzel and Vaccaro ( 1 6 ) . Kelp samples of known wet weight, previously blotted to remove sea water, were heated to 95 C f o r 3 days t i l l c o n s t a n t weight, to determine dry weights.

These dried tissues were then f i r e d a t 500 C f o r 2 h and the

r e s i d u e s weighed.

These values were used to calculate ash weight/g dry

weight kelp.

Results arri Discussion When 80 g portions of N. luetkeana laminae are cycle-comminuted f o r 30 s , placed in 920 ml of sea water in a 2 1 Erlenmeyer flask and incubated a t 15 C in the dark, the numbers of bacteria increase dramatically ( F i g .

1).

Within 4 days the numbers reach lO^/ml of kelp suspension when assayed by the p l a t e count technique and approximately 5 x l O ^ / m l by the AODC —13 technique. Assuming each b a c t e r i a l c e l l has a mass o f 1 x 10 g then the 80 g of laminae produced 1 g (plate count) , or 50 g (AODC technique) o f b a c t e r i a l biomass for b a c t e r i a l production e f f i c i e n c i e s from k e l p o f approximately 1 or 62%, r e s p e c t i v e l y .

Since d i r e c t counts are a b e t t e r

assay o f b a c t e r i a l numbers than p l a t e c o u n t s , the l a t t e r probably the more accurate of the two.

figure

is

I t also compares favourably with

the approximately 41% e f f i c i e n c y for conversion of Laminaria d e t r i t u s to b a c t e r i a l biomass noted by Robinson e t a l . efficient

(19).

These r a p i d

and

c o n v e r s i o n s o f kelp to b a c t e r i a l biomass i l l u s t r a t e

the

218 potential f o r degrading kelp to influence bacterial growth rates and activities in coastal ecosystems.

1013

E < GC LU

I-

o < CD

2

6

10

14

18

22

26

INCUBATION TIME (DAYS) Fig. 1: Growth of bacteria in a comminuted kelp-seawater system when assayed by the plate count (o) and direct count techniques ( • ) . In late summer and early f a l l (August-October) , j j i situ degradation of tissues of M. i n t e g r i f o l i a and N. luetkeana w i t h i n l i t t e r

bags to

mineralized components (e.g. carbon dioxide and water), dissolved organic matter and particulate organic matter (1.5 mm or less in s i z e )

proceeds

r e l a t i v e l y rapidly in both the sub- ard intertidal portions of the marine environment of southern coastal British Golumbia.

Times of degradation to

residues of approximately 1.5 mm or less varied from 5 to 8 weeks (Fig. 2).

Other investigators have reported that within an 8 week period the

salt marsh plants Juneus, Distichlis, Spartina and Salicornia spp. decayed

219 to approximately 70, 50, 45 and 10% o f

their original

weights

r e s p e c t i v e l y , when contained within 2.5 mm mesh size l i t t e r bags and placed in a marine marsh ( 1 7 ) .

Morrison e t a l . (15) found that leaf

samples of the oak Quercus virginiana and the pine Pinus e l i o t t i i

lost

approximately 15 and 40% of their o r i g i n a l weights 6 weeks after being placed in a marine estuary.

Thus, the rates of kelp degradation in

coastal British Cblumbia waters are probably among the greatest for plant material decaying in the temperate marine environment.

Indeed, these

degradation rates would probably have been greater i f the kelp tissues were not contained within l i t t e r bags.

Physical stresses due to wave

action and water current movements WDuld probably increase the degradation rates (8,18).

Fig. 2: Rates of degradation of M. integrifolia (laminae and stipes) and N. luetkeana (laminae and pneumatocysts) incubated at Dixon Island and Brockton Point f respectively• Ttie symbols ( o ) and ( • ) denote samples incubated inter- and subtidally, respectively.

220

221 Fig. 3: Scanning electron micrographs o f c o l o n i z a t i o n o f N. luetkeana laminae and pneimatocysts during incubation for various lengtEs of time in l i t t e r bags suspended in the water column. (A) lamina surface, day 0; (B) lamina s u r f a c e , day 3 ; (C) lamina s u r f a c e , day 13; (D) pneumatocyst surface, day 0; (E) pneumatocyst surface, day 4; (F) pneumatocyst surface, day 22. The m i c r o b i a l p o p u l a t i o n s , as observed with t h e s c a n n i n g

electron

microscope, associated with healthy and decaying N. luetkeana t i s s u e s , are displayed in F i g . 3.

In g e n e r a l , ws noted that the surfaces of healthy

t i s s u e s o f t h i s k e l p were s p a r s e l y p o p u l a t e d by p e n n a t e

diatoms,

y e a s t - l i k e c e l l s , single or diploid rods, or more canmonly, microcolonies o f end-attached b a c t e r i a which appeared t o be v e r y s i m i l a r t o

the

epiphytic bacterium Leucothrix mucor d e s c r i b e d by Bland and Brock

(2).

Ihe presence of small d e t r i t u s p a r t i c l e s on healthy tissues was common. Within 3 days of placement of healthy kelp tissues in l i t t e r bags in the marine environment s i g n i f i c a n t quantities of both detritus p a r t i c l e s and bacteria appeared on the k e l p ( F i g . 3 ) . pennate diatoms a l s o s e t t l e d .

By 5 days, l a r g e numbers o f

As degradation proceeded other microbial

types such as centric diatoms, y e a s t - l i k e c e l l s and protozoa colonized the k e l p t i s s u e s as w e l l .

By approximately day 14 a complex matrix o f

d e t r i t u s , b a c t e r i a , algae, fungi and protozoa coated the decaying k e l p (Fig. 3).

The approximate time order of settlement and colonization was

d e t r i t u s , b a c t e r i a , followed by algae, fungi, protozoa. Cundell and M i t c h e l l (4) noted t h a t the sequence o f m i c r o o r g a n i s m s c o l o n i z i n g a wood surface immersed in sea water was: bacteria followed by d e t r i t u s , pennate diatoms, s t a l k e d d i a t o m s , p r o t o z o a , barnacles and isopods, in that o r d e r .

Morrison e t a l .

macroalgae,

(15) a l s o noted

t h a t the i n i t i a l c o l o n i z e r s o f both oak and pine leaves placed in the marine environment were bacteria followed by a variety of eucaryotes, both algae and f u n g i .

Other workers have found similar patterns of microbial

colonization on materials placed in the marine environment ( 9 , 2 3 , 2 6 ) . The rapid colonization of decaying kelp tissues by b a c t e r i a , as observed by scanning electron microscopy, was also noted upon assay of t o t a l viable heterotrophic b a c t e r i a l numbers (colony forming u n i t s - CFU) using the 5 spread plate technique. These numbers increased from approximately 10 / g

222 of healthy kelp t i s s u e , harvested in l a t e summer, to approximately lO^Vg of decaying t i s s u e within 1.5 to 5 weeks of placing the material in l i t t e r bags (Fig. 4 ) .

In a l l samples s t u d i e d , the k e l p t i s s u e s placed in the

i n t e r t i d a l environment more q u i c k l y attained greater numbers of viable heterotrophic bacteria than those placed a t 10 m depth in the water column (Fig. 4).

Since the b a c t e r i a l contents of s u r f a c e sediments usually

exceed those of overlying waters by at l e a s t tvra orders of magnitude and the l i t t e r bags (with c o n t e n t s ) r e s t e d on sediments a t low t i d e s , the greater i n i t i a l colonization rates of the intertidal incubated samples are understandable.

However, the reason f o r the g r e a t e r

(approximately

ten-fold) extent of bacterial colonization of intertidal incubated samples i s not known with c e r t a i n t y .

Two p o s s i b l e e x p l a n a t i o n s f o r t h i s

enhancement a r e : 1) n u t r i e n t s from sediments enhanced the growth of b a c t e r i a c o l o n i z i n g the i n t e r t i d a l l y decaying kelp; and 2) many of the bacteria were adventitious species washed onto the i n t e r t i d a l l y incubated kelp from sediments.

The numbers of viable heterotrophic bacteria/g of

healthy k e l p t i s s u e s are approximately the same a s those reported by Laycock (13) f o r Laminaria l o n g i c r u r i s and Chan and McManus (3) f o r Polysiphonia lanosa and Ascophyllum nodosum. On a per u n i t wet w e i g h t b a s i s ,

the ATP c o n t e n t o f h e a l t h y M.

i n t e g r i f o l i a laminae i s very much greater than t h a t of s t i p e s ( F i g .

5).

However, upon p l a c i n g these t i s s u e s in l i t t e r bags within the marine environment, ATP l e v e l s of both t i s s u e s approached minimal v a l u e s within a p p r o x i m a t e l y one week.

If ATP content i s an index of kelp t i s s u e

v i a b i l i t y i t vould appear that the k e l p fronds and laminae were "dead" within one week of placement within l i t t e r bags.

The subsequent gradual

increases in ATP content of both decaying laminae and s t i p e s were probably due t o m i c r o b i a l c o l o n i z a t i o n and growth on these t i s s u e s .

Scanning

electron microscopy i n d i c a t e d m i c r o b i a l c o l o n i z a t i o n within 3 days of placement in the s e a ; however, the microbial bicmasses were probably not s u f f i c i e n t l y l a r g e within the f i r s t week o f

incubation to

i n f l u e n c e t o t a l ATP content of the decaying m a t t e r .

greatly

Relatively large

i n c r e a s e s in ATP l e v e l s occurred only a f t e r a p p r o x i m a t e l y 3 weeks incubation _in s i t u .

This was probably due to m i c r o b i a l attachment and

growth with a r e l a t i v e l y large contribution from the heterotrophic

223

INCUBATION TIME (WEEKS) F i g . 4: V i a b l e h e t e r o t r o p h i c b a c t e r i a l c o u n t s o f d e c a y i n g M. i n t e g r i f o l i a laminae and N. luetkeana pneumatocysts. (o) and (•) denote samples incubated i n t e r - and subtidally, respectively. Rates and extents of b a c t e r i a l c o l o n i z a t i o n o f M^ i n t e g r i f o l i a s t i p e s and N^ luetkeana laminae were similar.

224

2

3

4

5

6

INCUBATION TIME (WEEKS) F i g . 5 : ATP c o n t e n t o f decaying M. i n t e g r i f o l i a laminae and s t i p e s incubated i n t e r - (o) and subtidally [ • ) .

225 bacteria (see Figs. 3 and 4 ) .

Since heterotrophic bacterial numbers

reached maximal values in 4-5 weeks, increased ATP concentrations observed at the end of 3 weeks were probably due to increases in numbers of other microorganisms, e.g. algae, fungi and protozoa (see Fig. 3).

Cn the basis

of ATP data alone, however, one cannot determine the relative contribution of each type of microbe to the total microflora at any one time. Chlorophyll a content of NU integrifolia laminae is much greater than that of stipes ( F i g . 6 ) , probably because the laminae are the major area for photosynthesis of this marine plant.

When both laminae and s t i p e s ,

contained in l i t t e r bags, were placed in the inter- and subtidal marine environment the chlorophyll a content decreased, probably due to microbial degradation.

However, within 1-3 weeks chlorophyll a l e v e l s increased.

This is indirect evidence that the majority of the i n i t i a l colonies were probably heterotrophic microorganisms with a greater proportion of the later-colonizing ones being microalgae. Both healthy and decaying tissues of M. integrifolia and N. luetkeana are believed to be food sources for microorganisms and animals (see Figs. 3 and 4; [14], and L. Druehl, personal communication).

Therefore, to assess

the relative value of M^ integrifolia as an animal food, analyses of

this

kelp, both healthy and at various stages of decomposition, were done for crude fibre, percent carbon and nitrogen as v e i l as ash weight. Along with the loss of organic matter in decaying macrophytes in the marine environment, there are apparent changes in n u t r i t i v e content. Crude fibre content of M. integrifolia laminae marginally decreased with time, whereas that of stipes remained relatively constant in both interand subtidal environments ( F i g . 7 ) .

These r e s u l t s are in g e n e r a l

agreement with those of other investigators who have noted that crude fibre content of Spartina derived detritus remained r e l a t i v e l y constant (17) and that of decaying red mangrove leaves showed a slight increase (11). The carbon content of both healthy laminae and stipes of M. i n t e g r i f o l i a was approximately 30%, whereas the nitrogen content of laminae was approximately twice that of stipes (Table I ) .

As degradation of these

226

1

2

3

4

5

6

7

INCUBATION TIME (WEEKS) F i g . 6: Chlorophyll a content of decaying M^ i n t e g r i f o l i a laminae and s t i p e s incubated i n t e r - (o) and s u b t i d a l l y ( • ) .

227

1

2

3

4

5

6

7

8

INCUBATION TIME (WEEKS) F i g . 7: Crude f i b r e c o n t e n t of d e c a y i n g M. i n t e g r i f o l i a l a m i n a e and s t i p e s incubated i n t e r - (o) and s u b t i d a l l y

228 tissues within l i t t e r bags proceeded , the carbon c o n t e n t decreased to approximately 24-26%, whereas that of nitrogen increased (see Table I ) .

TSble I : Ttital carbon and nitrogen c o n t e n t o f integrifolia tissues degraded in l i t t e r bags s u b t i d a l l y 3 a t 10 m depth in the water column. Material was harvested in l a t e sutimer. Incubation Time (weeks) 0 1 2 3 4 5 6 7 7.8 a

%C 30.0 29.1 29.0 29.8 29.8 26.9 26.5 26 26.1

Laminae %N 2.1 2.1 2.3 2.2 2.2 2.5 2.5 2.5 2.5

C/N 14.3 13.9 12.6 13.6 13.6 10.8 10.6 10.4 10.4

%C 29.5 24.6 25.8 25.8 27.2 30.1 28.0 24.1 —

Stipes %N

C/N

1.0 0.9 0.9 1.2 1.8 1.6 1.3 1.5

29.5 27.3 28.7 21.5 15.1 18.8 21.5 16.1

—

Data for samples incubated i n t e r t i d a l l y showed similar patterns.

In l a t e summer, ash content of healthy M^ i n t e g r i f o l i a s t i p e s , on a dry weight b a s i s , was somewhat g r e a t e r than that of the laminae.

However,

upon degradation within l i t t e r bags, ash content of the laminae increased, whereas t h a t o f the s t i p e s remained r e l a t i v e l y constant (Fig. 8 ) .

The

samples incubated i n t e r t i d a l l y tended to have a g r e a t e r ash c o n t e n t than the ones incubated in the s u b t i d a l water column.

Since the i n t e r t i d a l

samples were a s s o c i a t e d with sediment, t h e y may more r e a d i l y

have

accumulated same inorganic elements. I t has been suggested (20) that the required C:N r a t i o f o r marine animal food, as d e t r i t u s should be at l e a s t 1 7 : 1 .

Diets with greater C:N r a t i o s

may be d e f i c i e n t in nitrogen compounds, p a r t i c u l a r l y protein. indicate that healthy

i n t e g r i f o l i a laminae may be o f

b e t t e r q u a l i t y in t h i s sense than s t i p e s .

Our r e s u l t s

nutritionally

Ihe laminae showed C:N r a t i o s

o f approximately 1 4 . 3 : 1 vs. 2 9 . 5 : 1 , for s t i p e s in m a t e r i a l harvested in l a t e summer (Table I ) .

Moreover, the C:N r a t i o s of both tissues

229

CD X

INCUBATION TIME (WEEKS) Fig. 8: Ash content of decaying M. integrifolia laminae and stipes incubated inter- (o) and subtidally {•).

230 decreased as degradation proceeded until the ratio for laminae detritus approached 10:1 and that for stipes was < 17:1.

Thus, degradation of

these tissues in litter bags, appears to increase their nutritive value. Other studies of decomposing macrophytes in the marine environment have shown similar patterns of relative nitrogen increases (11,17,24).

Acknowledgements Vfe acknowledge the financial support of Simon Fraser University for this project through its Programs of Excellence funding.

Technical assistance

of Caroline Grant is appreciated.

References 1.

Bancroft, K., Paul, E.A., Wiebe, W.J.:

2.

Bland, J.A., Brock, T.D.:

Limnol. Oceanogr. 21, 473-480

(1976). Mar. Biol. 23, 283-292 (1973).

3.

Chan, E.C.S., ffcManus, E.A.:

4.

Cundell, A.M., Mitchell, R.:

Can. J. Microbiol. 15, 409-420 (1969). Int. Biodeterior. Bull. 13, 67-73

(1977). 5.

Druehl, L.D.:

Phycologia 9, 237-247 (1970).

6.

Druehl, L.D.:

Can. J. Bot. 56, 69-79 (1978).

7. 8.

Fankboner, P.V., Druehl, L.D.: Experientia 32, 1391-1392 (1976). Fenchel, T.M., Jorgensen, B.B.: Bi Advances in Microbial Ecology. \AD1. I. (M. Alexander, ed.), Plenum, New York, pp. 1-58 1977 Floodgate, G.D.: In Marine Borers, Fungi and Fouling Organisms of Wood (E.B.G. Jones~T~S.K. Eltringham, eds.), Organization for Economic Cooperation and Developnent, Paris, pp. 117-123 1971

9.

10.

Ftgg, G.E.:

Oceanogr. Mar. Biol. Ann. Rev. _4, 195-212 (1966).

11.

Heald, E.J.: The production of organic detritus in a south Florida estuary. Ph.D. dissertation, Uhiv. of Miami, Florida 1969

12.

Hobbie, J.E., Daley, R.J., Jasper, S.: 1225-1228 (1977).

13.

Iaycock, R.A.:

14.

Leighton, D.L.:

15.

Harrison, S.J., King, J.D., Bobbie, R.J., Bechtold, R.E., White, D.C.: Mar. Biol. 41, 229-240 (1977).

Appl. Environ. Microbiol. 33,

Mar. Biol. 25, 223-231 (1974). ttova Hedwigia 32, 420-453 (1971).

231 16.

Menzel, D.W., Vaccaro, R.F.:

17.

Cdutt, E.P., de la Cruz, A.A.: In Estuaries, (G.H. lauff, ed.), £mer. Assoc. Adv. Sei., Washington, D.C., pp. 383-388 1967

Limnol. Oceanogr. 9, 138-142 (1964).

18.

Rau, G.H.:

19.

Robinson, J.D., Mann, K.H., Novitsky, J.A.: Amer. Soc. Limnol. Oceanogr., 2nd Winter meeting, los Angeles, Calif., abstract (1980).

20.

Russell-Hunter, W.D.: Aquatic Productivity: An Introduction to some Basic A s p e c t s of B i o l o g i c a l Oceanography and Limnology. Collier-Macmillan, London 1970

21.

Scagel, R.F.:

22.

Sibert, J., Brown, T.J., Healey, M.C., Kask, B.A., Naiman, R.J.: Science 177, 649-650 (1977).

23.

Skerman, T.M.:

24.

Sr\ith, B.D.: A qualitative and quantitative assessment of seaweed decomposition in the Strait of Georgia. M.Sc. thesis, University of British Golunbia, Vancouver, B.C. 1979

25.

Strickland, J. D.H., Parsons, T.R.: A Practical Handbook of Seawater Analysis. 2nd ed., Bull. Fish. Res. Board Canada 1972

26.

Wbod, E.J.F.:

Limnol. Oceanogr. Z3, 356-358 (1978).

Pac. Sei. 15, 494-539 (1961).

N.Z. J. Sei. Ofechnol. B. 38, 44-57 (1956).

Marine Microbial Ecology. Reinhold, New York,

1965

DISCUSSION WHEELER:

Have you looked at degradation on the plant itself rather than

in litter bags? ALBRIGHT:

Nd. You can get a whole time series of decay using plants, which you

WHEELER:

may be able to compare with data frcm litter bags. DRUEHL:

A graduate student of mine, Bill Roland, looked at the

colonization of everything from early bacteria to bryozoa on Macrocystis blades at different heights as a seasonal phenomenon and you get a classic pattern —

great differences between plants at different times of the year

and with age. SIBERT:

Your 60% efficiency of conversion of kelp to bacterial biomass

sounds pretty good to me.

It agrees well with the laboratory data.

232 ALBRIGHT:

It worries me a bit, 62.5% seems awfully high for efficiency of

degradatious material. SIBERT:

It does, but that is the way bacteria are!

FOSTER:

Oould you explain the decrease in C/N ratio simply by a drop in

carbon? ALBRIGHT:

The data show some drop in carbon and some increase in

nitrogen, but not very much.

The thing is if you get colonization by

microorganisms I would expect them to bring a lot more nitrogen onto the plants colonized; probably non-protein nitrogen, such as in the cell wall of the bacterial cell, acetylglucoseamine for example.

What I was

wondering about is whether this nitrogen is available for nutrition, as sane people working with seagrasses claim. SCHMITZ: What do you understand by "crude fiber"? ALBRIGHT:

This is the assay of Strickland and Parsons (ref. 25) for crude

fiber and it is pretty well cellulose. SCHMITZ:

Did you ever try to extract specifically for alginate?

ALBRIGHT: ISb. SRIVASTAVA:

You are using 1.5 nm hole bags, those are big holes for all

kinds of things to be ccming in and going out and you have no control over that. What is left inside may not be a true reflection of the trend from one week to the next.

I have no solution for the problem, but this is a

major factor of error. Maybe you should try to have bags of different pore sizes. ALBRIGHT: Vfe used litter bags because, it was convenient to do it that way, to get something going on the project.

Eventually, we hope to use

the Cepex-type bags, and not have to worry about losing detrital material. With Cepex-type bags we will try to retain everything to get an appreciation of kinetics of degredation. CABOT:

Did you look at any sectioned material to see whether bacteria

233 ware inside the cells? ALBRIGHT:

No. We looked at it with SEM. Bacteria would probably be one

of the first things to invade the tissue as a primary colonizer. Probably, it would not be fungi.

We did not notice particularly high

levels of fungi, ever, in any of our degrading material. CABOT: Would you say that they were all healthy blades; do you not have to cut them? ALBRIGHT: They are healthy blades, but we have to cut them. another problem with this type of experiment.

This is

Tb get enough material of

100 grams into those bags, you have to cut small portions and you lose many things, excretions, photosynthate, initially that way. ffcCCNNAUGHEY:

I would think that those holes in the bags would be large

enough for harpacticoids to get in. Do you see any harpacticoids creeping around in your kelp? ALBRIGHT:

Tbwards the end of the experiment we did, particularly on the

samples that were incubated in the intertidal region close to the sed iment. McCCNNAUGHEY:

There are harpacticoids which appear to be specifically

adapted for invading seagrasses.

You can see tham actually burrow into

the seagrass blades. CABOT:

In some of our studies, we have seen gammarid amphipods sometimes

associated with decaying Nereocystis.

Part IV.

Polysaccharides, kelp farming and harvest

TOWARD IMPROVED UNDERSTANDING OF POLYSACCHARIDE SYNTHESIS AND STORAGE IN MARINE ADSAE

R.G.S. Bidwell Genu Products Canada L t d . , and A t l a n t i c Regional L a b o r a t o r y , Halifax, N.S. ( Address : Wallace RR #1, Nova S c o t i a , Canada, BOK 1YO)

N.R.C.,

Introduction The most important commercial compounds in marine algae a t the present time are p o l y s a c c h a r i d e s .

The many i n d i v i d u a l a p p l i c a t i o n s o f

carbohydrates depend on t h e i r very s p e c i f i c chemical p r o p e r t i e s .

these

However,

our present knowledge o f the c h e m i s t r y and p h y s i c a l c h e m i s t r y o f

algal

p o l y s a c c h a r i d e s i s inadequate t o d e f i n e the c h e m i c a l parameters that underlie s p e c i f i c desirable or undesirable

industrial

properties.

F u r t h e r m o r e , we have o n l y l i m i t e d knowledge o f the b i o l o g y o f

algal

polysaccharide production ( 1 8 , 2 1 ) : when they are made (both seasonally and d i u r n a l l y ) , where in the p l a n t , from what precursors, and how influenced (both q u a l i t a t i v e l y and q u a n t i t a t i v e l y ) by what chemical or environmental factors.

I n f o r m a t i o n on a l l t h e s e p o i n t s i s v i t a l l y n e c e s s a r y

developing the h a r v e s t s t r a t e g y o f a f i n a n c i a l l y s u c c e s s f u l industry.

for

seaweed

The r e c o r d o f f a i l u r e s t o d a t e on both c o a s t s o f Canada and

elsewhere a t t e s t s to the danger o f ignoring the need for b a s i c r e s e a r c h . I wish to s t a t e f i r s t that I do n o t have the answers t o q u e s t i o n s must be a s k e d .

that

However, in my e x p e r i e n c e with commercial seaweed

c u l t i v a t i o n during the past three years I have had an o p p o r t u n i t y t o s e e what i s needed in the way o f b a s i c research for a successful industry, and how i t should be tackled. r e w a r d s come o n l y l a t e

Since sane o f the research i s expensive and the i n t h e game, i t i s necessary to

establish

p r i o r i t i e s and a s t r a t e g y f o r financing and a c t u a l l y doing t h e r e q u i r e d research.

My purpose in t h i s paper i s t o provide an account o f how I

think t h i s should b e s t be done.

© 1982 Walter de Gruyter & Co., Berlin • New York Synthetic and Degradative Processes in Marine Macrophytes

238 Good harvest or c u l t u r e s t r a t e g y r e q u i r e s methods t h a t g i v e the b e s t s u s t a i n e d y i e l d of the most d e s i r a b l e p o l y s a c c h a r i d e s . c o s t - b e n e f i t a n a l y s i s i s n e c e s s a r y to d e t e r m i n e the b e s t

Continuous possible

s t r a t e g y , r a t h e r than seeking the l a r g e s t return frcm any given harvest tactic.

This in turn requires knowledge about the range of biological and

biochemical aspects of plant growth and development, including a number of questions about the polysaccharides.

These f a l l into ten main categories,

which a r e : ( i ) s y n t h e t i c pathways, ( i i ) mechanisms of glycosylation and s u l f a t i o n , ( i i i ) s t o r a g e s i t e s , ( i v ) d i u r n a l and a n n u a l t i m i n g o f synthesis

and s t o r a g e ,

(v) e f f e c t s of n a t u r a l or

controllable

environmental f a c t o r s , ( v i ) c o n s e q u e n c e s o f the p h y s i o l o g i c a l

or

developmental s t a t e of the p l a n t , ( v i i ) c o n t r o l by chemicals of the physiological and biochemical a s p e c t s of p o l y s a c c h a r i d e s y n t h e s i s and s t o r a g e , ( v i i i ) e f f e c t s of h a r v e s t damage, ( i x ) post-harvest ripening techniques, and (x) s e l e c t i o n of n a t u r a l l y or g e n e t i c a l l y synthesized high-yielding v a r i e t i e s . Biological Research Program The following s e c t i o n will provide a brief summary of the present s t a t u s and future requirements of the ten questions mentioned above. Synthetic pathways.

At present quite a l o t i s known about the s y n t h e t i c

pathways and precursors of alginate and fucoidan, although the problem of the multiple i n v e r s i o n s required t o convert mannitol or g l u c o s e i n t o fucose i s s t i l l unresolved (Fig. 1).

L-Fucose

D-Galactose

Research in several laboratories

D-Mannose, Mannitol

D-Glucose

Fig. Is Planes of symmetry of a l g a l monosaccharides. Note t h a t two, three or four inversions are necessary to convert D-sugars into L-fucose.

239 has shown that photosynthetic carbon i s transported mainly as mannitol in brown

algae,

and t h a t m a n n i t o l

polysaccharide s y n t h e s i s

i s the major s u b s t r a t e both

and o f r e s p i r a t i o n

biochemical focus, however, i s lacking.

(2,3,19,22).

of

Finer

Although t h i s information i s not

of the highest p r i o r i t y , i t w i l l lead to a b e t t e r understanding o f the biochemical pathways to individual polysaccharides and the f a c t o r s , both b i o l o g i c a l and environmental, t h a t a f f e c t t h e s e pathways.

The same

s t a r t i n g m a t e r i a l i s used to produce d i f f e r e n t end products having d i f f e r e n t l e v e l s of commercial d e s i r a b i l i t y .

Understanding the switch or

branch p o i n t s may permit t h e i r c o n t r o l , as suggested in figure 2, with consequent improvement of the industrial value of the harvest.

alginate

mannitol

photosynthate

Fig. 2: Pathways of polysaccharide synthesis in brown a l g a e ( s e e r e f s . 6,10,14). Metabolic c o n t r o l a t the switch or branch points (a,b) might lead to commercial p o s s i b i l i t i e s for increasing the r e l a t i v e production of more valuable types of carbohydrate. Mechanism of glycosylation and s u l f a t i o n .

While research in t h i s area

is

n o t o f the h i g h e s t p r i o r i t y , some u s e f u l l e a d s may come from b e t t e r understanding o f these p r o c e s s e s .

Recent work on t r a n s g l y c o s y l a t i o n

suggests that the process in marine algae i s not very d i f f e r e n t frcm that of higher plants ( 8 ) .

Work on the incorporation of sulfate suggests t h a t

t h i s s t e p follows p o l y s a c c h a r i d e formation, and interest (4,16). is

so may be o f g r e a t e r

Because sulfation requires ATP, and hence l i g h t

(which

t h e l i m i t i n g f a c t o r f o r most a l g a l p h o t o s y n t h e s i s ) , and because

sulfation greatly a f f e c t s the properties of polysaccharides, i t may be o f considerable i n t e r e s t to understand the process.

As has been suggested by

Hirst, sulfation may r e l a t e to dehydration o r i n v e r s i o n in pre-formed p o l y s a c c h a r i d e s such as f u c o i d a n , carrageenan or agar ( 1 2 ) . therefore be of i n t e r e s t to determine p o s s i b l e e f f e c t s o f

I t will

short-term,

perhaps i n t e n s i v e , treatment with sulfate or other chemicals designed to a f f e c t the sulfation of polysaccharides prior to harvesting.

240 Storage s i t e s .

Research in t h i s category has much higher p r i o r i t y than in

the previous ones because i t r e l a t e s to harvest strategy for continuous production.

By and l a r g e , p o l y s a c c h a r i d e s are h i g h e s t in the young

sections of the plant ( 1 3 ) .

Ft>r plants l i k e Laminaria t h a t grow from a

b a s a l meristem, t h i s i s not so good.

However, i t i s most helpful for

rockweeds and other plants that grow from the t i p .

I t may be unimportant

f o r a t o t a l - h a r v e s t technology, but i t i s of considerable concern where p a r t i a l or sequential harvesting i s appropriate. Timing.

Clearly, harvest timing depends p r i m a r i l y on p l a n t growth and

seasonal convenience.

However, there are strong seasonal variations in

the content and q u a l i t i e s of p o l y s a c c h a r i d e s .

For example, Stewart e t

a l . (20) found t h a t the carbohydrate c o n t e n t o f Ecklonia r a d i a t a was higher in l a t e sutrmer than in spring by a factor of nearly two. Seasonal e f f e c t s also occur because d i f f e r e n t reproductive s t a g e s , which may be strongly seasonal, may make d i f f e r e n t types of polysaccharide ( 1 8 ) . Ft>r example in the Gigartinaceae, as exemplified by Chondrus crispus, Chen e t a l . (7) showed t h a t the t e t r a s p o r o p h y t e makes lambda-carrageenan, whereas

t h e g a m e t o p h y t e makes t h e c o m m e r c i a l l y more

kappa-carrageenan.

desirable

I t would be of value to analyse both the q u a l i t a t i v e

and q u a n t i t a t i v e a s p e c t s o f polysaccharides frcm brown algae during the seasonal growth of algal beds. Environmental f a c t o r s .

The e f f e c t s o f n a t u r a l o r o f

controllable

environmental f a c t o r s have high p r i o r i t y f o r i n v e s t i g a t i o n . variations may be of great i n t e r e s t .

P h o t o s y n t h e s i s adds c o n s i d e r a b l e

amounts of carbon to the plant each day. converted into polysaccharide?

Diurnal

At what time of the day i s t h i s

I f t h i s occurs immediately, as suggested

by Hellebust and Haug for alginate ( 1 1 ) , then harvesting i s best done l a t e in the day.

I f s y n t h e s i s o c c u r s o v e r n i g h t , as may be f o r some o t h e r

polysaccharides ( 1 1 , 1 4 ) , then continuous daytime harvesting i s acceptable. Cn the longer term the e f f e c t s o f , for example, a week of sunless weather or an unnaturally warm spell are disastrous in terms of yield of cultured Chondrus (unpublished d a t a ) . Cbmparable information on wild stands would be o f v a l u e .

Harvesting should c l e a r l y be t i e d t o the weather.

In

241 addition, there i s the p o s s i b i l i t y of affecting the q u a l i t y and q u a n t i t y of p o l y s a c c h a r i d e s by f e r t i l i z a t i o n . added bo the sea need not tie wasted.

Surprisingly, chemical f e r t i l i z e r s Chinese commercial seaweed growers

have experienced over 80% recovery o f chemicals added to dense seaweed beds, which is b e t t e r than most flatland agriculture (C.K. TSeng, personal communication). Physiological or developmental s t a t e of plants.

These e f f e c t s may not be

so great with browns, although they are known to be of great importance in red algae ( 7 , 1 8 ) .

However, 1 i t t l e information i s available about ways in

which r e l a t i v e q u a n t i t i e s o f various polysaccharides in brown algae may change with development, or how the q u a n t i t y or q u a l i t y o f any given polysaccharide

is affected.

R e s e a r c h on t h e s e p o i n t s may be o f

considerable value in determining overall harvest strategy. Chemical c o n t r o l .

I f t h e r e i s inherent v a r i a t i o n in the q u a l i t y and

quantity of polysaccharides, then i t should be possible to control t h i s to the harvester's advantage by the use o f c h e m i c a l s . asparagine

As an example, the

c o n t e n t o f wheat l e a v e s can be increased many-fold by

appropriate b r i e f treatment with nitrogen and the phytohormone, auxin.

Vfe

used t h i s chance observation as the basis of a canmercial biosynthesis of labelled asparagine in the 1960's ( 1 7 ) .

Now, auxin and the o t h e r higher

plant hormones may not work in brown algae.

In f a c t , no one seems to know

what are the chemical hormone systems of seaweeds. physiology

i s wide open.

This f i e l d o f p l a n t

Marine a l g a e a r e so p r i m i t i v e t h a t

their

developmental control systems may be quite d i f f e r e n t from those o f higher plants.

But control mechanisms they must have.

s h a l l be in a very powerful p o s i t i o n physiology. costly.

When ws discover them we

to c o n t r o l

a l g a l growth and

C e r t a i n l y , the research needed will be b a s i c , extensive and

Clearly, some way needs to be found to develop a research program

to investigate these problems. Harvest damage.

This question i s simple but important.

Do the e f f e c t s of

p a r t i a l h a r v e s t i n g increase or decrease polysaccharide production in the remaining p o r t i o n s o f the p l a n t ? damage to the plants?

What are the e f f e c t s o f mechanical

242 Post-harvesting

ripening.

These

techniques

have

been

applied

to

c u l t i v a t e d seaweeds in the p a s t , but they could e q u a l l y be a p p l i e d t o wild harvest.

Some red a l g a e

s u c h a s Chondrus c r i s p u s c a n , b y

appropriate

t r e a t m e n t , be made t o i n c r e a s e t h e i r c o n t e n t o f d e s i r a b l e c a r b o h y d r a t e s by a s much a s

50% w h i l e

(unpublished d a t a ) .

suffering

a net weight l o s s of o n l y

held under s p e c i a l c o n d i t i o n s ( u s u a l l y n o t v e r y demanding) container

f o r a p e r i o d o f about one week.

10%

in a

simple

The t e c h n i q u e could e a s i l y be

a p p l i e d t o brown a l g a e in holding tanks o r p o n d s . the p h y s i o l o g i c a l

about

Such t r e a t m e n t s u s u a l l y r e q u i r e t h a t the p l a n t s be

Studies

to

determine

and c h e m i c a l t r e a t m e n t o f brown a l g a e f o r t h i s purpose

should be done. S e l e c t i o n o f productive v a r i e t i e s . yielded

some

very

An e x t e n s i v e program t h a t has

interesting

results

is

now i n

progress

c a r r a g e e n o p h y t e s a t Genu Canada L t d . ' s Nova S c o t i a l a b o r a t o r y success of prediction

o f growth p o t e n t i a l

"artificial

l e a f " technique d e v e l o p e d

algae

(1,9).

(5).

carrageenan

content.

permit

for chloroplasts

and

facilities

S i n c e a number o f

success of

accurate the

unicellular in

growth

productivity

and

for the

the h a r v e s t or

cultivation

s p e c i e s h a v e now become s u s c e p t i b l e

l a b o r a t o r y c u l t i v a t i o n throughout t h e i r programs

for

The

A s e l e c t i o n program f o r brown a l g a e i s c l e a r l y o f

g r e a t importance f o r t h e f u t u r e

genetic

that

for

screening

T h e s e m e t h o d s a r e b a s e d on

C l o n e s from p r o m i s i n g p l a n t s a r e e v a l u a t e d

c h a m b e r s and o u t d o o r c u l t i v a t i o n

industry.

(15).

t h e program depends on new t e c h n i q u e s f o r the r a p i d

o f c a r b o n a s s i m i l a t i o n r a t e s under c o n d i t i o n s

already

life

cycle,

of

the development

improvement o f g r o w t h r a t e s and

of

commercial

d e s i r a b i l i t y o f t h e s e s p e c i e s i s a l o g i c a l n e x t s t e p with high p r i o r i t y . Chemical r e s e a r c h program. chemistry needs (i)

to r e l a t e

application,

B e s i d e b i o l o g i c a l r e s e a r c h , much c a r b o h y d r a t e

t o be done.

TVJO

requirements are o f s p e c i a l

chemical or physical-chemical and

(ii)

structure

to

industrial

t o develop r a p i d , simple t e c h n i q u e s f o r e v a l u a t i n g

l a r g e numbers o f samples in a simple l a b o r a t o r y b y t e c h n i c a l Quantitative

importance:

analytical

personnel.

p r o c e d u r e s o f t h i s s o r t have been developed

for

c a r r a g e e n a n s by Genu Products Canada L t d . , and vrork i s now p r o c e e d i n g

on

the rapid a n a l y s i s o f

industrial

qualities.

chemical r e s e a r c h i s s t i l l needed in t h i s

field.

Itowever, much s o l i d b a s i c

243

Operation and funding.

In the previous sections I have recommended much

b a s i c and expensive r e s e a r c h for an industry that can never be large or l u c r a t i v e and which i s known f o r i t s many p a s t f i n a n c i a l

failures.

C l e a r l y a company devoted to harvesting kelps or cultivating agarophytes cannot r e a l i s t i c a l l y be expected to have the resources to set up programs for a l l

the r e s e a r c h

I have o u t l i n e d .

The c o s t a l o n e would be

prohibitive, aside from the d i f f i c u l t i e s of adequate s t a f f i n g .

Yet the

s u c c e s s of the venture may depend upon the outcome o f these research programs.

The s o l u t i o n to t h i s dilemma depends upon the approach to

research programs and the nature of t h e i r d i r e c t i o n . F i r s t , there are government and university l a b o r a t o r i e s f u l l y s e t up and c o m p e t e n t t o do the required r e s e a r c h , so no c a p i t a l c o s t s need be considered.

However, i t has been found unproductive merely to c o n t r a c t

out research projects to such laboratories.

The reason i s that university

and government s c i e n t i s t s , by and l a r g e , do not a p p r e c i a t e the s p e c i a l problems and research needs of industry. timing are quite d i f f e r e n t .

Their p r i o r i t i e s and concepts of

They a r e , in essence, ivory tower s c i e n t i s t s ,

not i n d u s t r i a l i s t s . The obvious alternative of hiring young s c i e n t i s t s has been t r i e d many times by anall industrial concerns and found counterproductive.

The newly

graduated tend to be too concerned with their s c i e n t i f i c r e p u t a t i o n , and furthermore they lack the broad, comprehensive "synthetic" experience that comes fran long years of practising research science.

But h i r i n g

senior

s c i e n t i s t s on a full-time basis i s obviously much too expensive. The successful compromise i s to g e n e r a t e t h i n k - t a n k s by forming l o o s e , s y n t h e t i c groups o f s e n i o r s c i e n t i s t s ,

together with engineers, market

analysts and industrial leadership, on a consulting b a s i s .

The c o s t s a r e

r e l a t i v e l y low, but the "brainstorm" e f f e c t o f a group of experienced s c i e n t i s t s in v a r i o u s f i e l d s

t o g e t h e r with d e s i g n e n g i n e e r s

and

i n d u s t r i a l i s t s i s very g r e a t .

The output, per unit c o s t , exceeds by one

to several orders of magnitude the usual output of e i t h e r a conventional government or u n i v e r s i t y l a b o r a t o r y or an i n d u s t r i a l r e s e a r c h

unit.

Actual research i s done by technicians housed in appropriate government or

244 university l a b o r a t o r i e s ,

and c o s t s c a n u s u a l l y b e l a r g e l y c o v e r e d

by

g r a n t s frcm v a r i o u s government s o u r c e s . The i m p o r t a n t d i s t i n c t i o n i n t h i s system i s t h a t t h e o v e r a l l d i r e c t i o n t h e r e s e a r c h p r o g r a m ccmes from i n d u s t r y . of

t h e s c i e n t i f i c program a r e made by t h e

l e a d e r s h i p of

of

Final d e c i s i o n s in a l l phases engineering-industrial

t h e t h i n k - t a n k , n o t by t h e s c i e n t i s t s .

This i s

essential,

because the i n d u s t r i a l i s t s are u l t i m a t e l y r e s p o n s i b l e f o r t h e s u c c e s s f a i l u r e of

the p r o j e c t ,

and b e c a u s e s c i e n t i s t s

n o r m a l l y do n o t e a s i l y

g r a s p t h e i n d u s t r i a l and e n g i n e e r i n g r e q u i r e m e n t s . is

the s c i e n t i s t s

that establish

Cn t h e o t h e r h a n d ,

t h e v a l i d i t y and v a l u e o f

research p r i o r i t i e s f o r the i n d u s t r i a l

or

leadership.

it

specific

T h i s way, t h e most

u s e f u l and i m p o r t a n t r e s e a r c h g e t s done f i r s t , and r e s e a r c h l i n e s , however interesting,

that are not leading

to advantageous cost b e n e f i t s

can

q u i c k l y be dropped o r s h e l v e d .

References 1.

Bidwell, R.G.S.:

2.

Bidwell, R.G.S., 581-590 ( 1 9 5 8 ) .

Can J . B o t . 55, 809-818 ( 1 9 7 7 ) .

3.

B i d w e l l , R . G . S . , Ghosh, N . R . :

Can. J . B o t . 40, 803-807

(1962).

4.

B i d w e l l , R . G . S . , Ghosh, N . R . :

Can. J . B o t . 41, 209-220

(1963).

5.

B i d w e l l , R . G . S . , McLachlan, J . L . , L l o y d , N . D . H . : P l a n t P h y s i o l . 2Q ( i n p r e s s ) .

6.

Bidwell, R.G.S., P e r c i v a l , E., Snestad, B.: (1972).

7.

Chen, L.C-M., JVfcLachlan, J . , N e i s h , A . C . , S h a c k l o c k , P . F . : b i o l . A s s . UK 53, 11-16 ( 1 9 7 3 ) .

8.

Cbughlan, S . , Evans, L . V . :

9.

EDdd, W.A., B i d w e l l , R . G . S . :

10.

H e l l e b u s t , J . A . , Haug, A . :

Can. J . B o t . 50, 169-176

(1972).

11.

H e l l e b u s t , J . A . , Haug, A . :

Can. J. B o t . 50, 177-184

(1972).

12.

Hirst, E.L.:

13.

Kremer, B . P . :

14.

Lsppard, G.G.:

15.

Lloyd, N.D.H., M c l a c h l a n , J . L . , B i d w e l l , R . G . S . :

Craigie,

J.S.,

Krotkov, G.:

J . Exp. B o t . 29,

Can. J .

Proc.

Bot.

Can.

36,

Soc.

Can. J . B o t . 50, 191-197 J.

mar.

55-68 ( 1 9 7 8 ) .

N a t u r e 234, 45-47 ( 1 9 7 1 ) .

P r o c . Chem. S o c . , J u l y 1958, p p . 177-187 Helgolander w i s s . Pfeeresunters.

(1958).

27, 115-127 ( 1 9 7 5 ) .

Can. J . B o t . 52, 773-781 ( 1 9 7 4 ) . Proc. I n t .

Seaweed

245 Symp. 10, (in press). 16.

loewus, F., Wagner, G., Schiff, J.A., Weistrop, J . : 48, 373-375 (1971).

17.

Maxwell, M.A.B., Bidwell, R.G.S.:

18.

McCandless, E.L., Craigie, J.S.: Ann. Rev. Plant Physiol. 41-53 (1979). Nicholson, N.L., Briggs, W.R.: Am. J. Bot. 59, 97-106 (1972).

19.

Plant Physiol.

Can. J. Bot. 48, 923-928 (1970).

20.

Stewart, C.M., Higgins, H.G., Austin, S.:

21.

Harvey, J . R . : Manners, e d . ) 1978

22.

Yamagouchi, T., Ikawa, T . , Nisizawa, K. : 217-229 (1966).

Nature 192, 1208 (1961).

Biochemistry of Carbohydrates, Vol. I I , (D.J. pp. 151-177. University Park Press, Baltimore, MD Plant Cell Physiol.

DISCUSSION DRUEHL:

There i s one aspect that you did not mention, that is, industry

helping industry.

I am now alluding to the f a c t of p o s i t i v e exchange

between various companies, and how the industry is going to grow, as a group?

Or does the industry want everybody to reproduce everything over

and over? BIDWELL:

First, I must mention that the post-harvesting ripening system

was patented by a government laboratory, by the NRC, not by any industrial concern.

In fact, there are two companies in Nova Scotia now that are

using the c u l t i v a t i o n and various other aspects of the system that Neish established and both of these have bought rights on the patent.

Now as

f a r as the industry helping industry, there are seme reasons why they do not tend to be quite so friendly about cooperating as you might think they should be.

Cbviously, i f you can get there six months ahead of the other

fellow you are quite a bit ahead in terms of your development.

Also,

if

you spend your investor's money on doing some research, you really do not want to give i t to others who have not spent that money.

That is the kind

of attitude you face in industry and i t is one that seme of us at Genu are trying to break down.

I t has now got to the stage, as you say, that

anybody's f a i l u r e i s everybody's f a i l u r e , and anybody's success i s everybody's success.

So I think i t i s important that the industrial

246 concerns should get together as far as possible. WILLENBRINK:

You mentioned in the f i r s t p a r t of your l e c t u r e t h a t you

could hopefully change the direction of the overall biosynthesis.

Did you

mean by changing the genes, by using r e c e n t techniques in g e n e t i c s and other ways? BIDWELL: quite

F i r s t , I must s t r e s s the word ' h o p e f u l l y . 1

Second, I am not

s u r e why, but t h e r e are ways, many s i t u a t i o n s t h a t r e l a t e

environmental factors which will cause a massive s h i f t in metabolism.

to An

example i s the asparagine synthesis system in wheat leaves where sucrose i s converted into asparagine.

There may be o t h e r kinds o f e v e n t s ,

seem t o be m e d i a t e d by e n v i r o n m e n t a l

factors,

probably

hormone-type mechanisms, which in turn a c t to c o n t r o l systems.

There are any number of possible mechanians:

i n d u c t i o n o r by enzyme c o n t r o l ,

that

through

these

enzyme

e i t h e r by enzyme

o r even by t h e s h o r t

distance

translocation or transport of substrates to make them a c c e s s i b l e , say from one compartment to another, to make them accessible to d i f f e r e n t synthetic systems.

I was r e a l l y thinking of that sort of control.

I do not know

how i t would work or what could be done, but sane experiments might g i v e us sane leads. SRIVASTAVA: (Fig. 2 ) . BIDWELL:

You have the arrows going both ways, mannitol to a l g i n a t e Do you have any basis for that?

Scnie time ago, we did some pulse chase experiments with

14

C and

found t h a t the a l g i n a t e f r a c t i o n was actually turning over, whereas the fucoidan and laminaran, at l e a s t in the r e l a t i v e l y short term experiments, that i s hours or days, that we did, seemed to be end products. WILLENBRINK:

I am going to add another q u e s t i o n .

I s i t not

true,that

only the l a s t step for the mannitol biosynthesis i s completely r e s t r i c t e d to mannitol and the s t e p b e f o r e l e a d s a l s o to a l g i n a t e ?

I t h i n k the

alginate synthesis is not via mannitol. BIDWELL:

I am not q u i t e s u r e .

But, we did find t h a t m a n n i t o l

alginate were being interconverted rather readily.

and

FARMING MACROCYSTIS AT COASTAL AND OCEANIC SITES

W. North, V. Gerard, and J . Kuwabara California I n s t i t u t e of Technology, Kerckhoff Marine Laboratory 101 Dahlia S t r e e t , Cbrona del Mar, C a l i f o r n i a , USA

Since California kelp beds f i r s t a t t r a c t e d a t t e n t i o n as a source of potash in 1910, commercial i n t e r e s t in the giant kelp, Microcystis, has expanded considerably.

Tbday, approximately 150,000 wet t o n s of k e l p ,

primarily

M a c r o c y s t i s , a r e h a r v e s t e d a n n u a l l y in C a l i f o r n i a and u t i l i z e d p r o d u c t i o n of a l g i n a t e s and o t h e r l e s s e r p r o d u c t s .

for

Actual crop s i z e

v a r i e s from y e a r t o y e a r , b u t even t h e l a r g e s t c r o p s do n o t p r o v i d e s u f f i c i e n t raw material to meet current demands of industry.

Furthermore,

i n t e r e s t has recently arisen in possible usage of Macrocystis biomass as a f e e d s t o c k f o r methane p r o d u c t i o n .

There a r e t h u s needs n o t only f o r

enhancing productivity of existing Macrocystis p o p u l a t i o n s , b u t a l s o f o r expanding the resource by developnent of oceanic farms.

Evidence suggests

t h a t both of these o b j e c t i v e s hinge on maintenance of o p t i m a l n u t r i e n t conditions f o r support of maximim growth.

Kelp n u t r i t i o n a l requirements v s . n u t r i e n t a v a i l a b i l i t y In order to meet t h i s o b j e c t i v e , e i t h e r in coastal kelp f o r e s t s or oceanic farms, n u t r i t i o n a l requirements of the plant must f i r s t be d e f i n e d .

The

elemental requirements f o r development and growth of Macrocystis p y r i f e r a from s e t t l e d

s p o r e s t h r o u g h t h e g a m e t o p h y t i c s t a g e t o embryonic

sporophytes have recently been determined (1).

Seven m i c r o n u t r i e n t s and

two m a c r o n u t r i e n t s were n e c e s s a r y a d d i t i o n s to the a r t i f i c i a l seawater medium (Aquil) f o r successful developnent (Table I ) . m i c r o n u t r i e n t a v a i l a b i l i t y in coastal waters.

L i t t l e i s known of

However, addition of the

two macronutrients, n i t r a t e and phosphate, to surface water collected from n e a r s h o r e s i t e s u s u a l l y s t i m u l a t e d growth of j u v e n i l e sporophytes in the laboratory (North, unpublished d a t a ) .

© 1982 Walter de Gruyter & Co., Berlin • New York Synthetic and Degradative Processes in Marine Macrophytes

Macrocystis

Plots of

248 liable I : Nannomoles of nine nutrients and of EDTA added to the completely d e f i n e d a r t i f i c i a l sea water Aquil to y i e l d a medium t h a t s u s t a i n e d development by Macrocystis zoospores completely through the gametophyte stage to m u l t i c e l l u l a r embryonic sporophytes in 12 culturing days. Nutrient used

nM added

Free ion conc. (nM)

Major species present and (%)

Fe+3

3.5 x 10 2

7 x 10-11

FeEDTA

(100)

Mn

30

MnEDTA

(65)

+2

3

MnCl

(23)

Co + 2

70

7 x 10~ 2

CoEDTA

(99)

Cu

10

4 x 10

-5

CuEDTA

(99)

ZnEDTA

(100)

CaEDTA

(89)

FeEDTA

(6)

+

+2

Zn +2

1.7 x 10 2

7 x 10" 2

Md0 4 -2

1.0 x 10 2

1 x 10 2

EDTA-2

6.0 x 10 3

7 x 10~ 5

N0

2.0 x 10

2 x 10

3-1

P0 4 ~ 3

4

2.0 x 10 3

4

3 x 10"1

h

P04~2

(51)

MgHPC^

-1

I

1

1.0 x 10 2

(47)

1 x 10 2

phosphate vs. n i t r a t e (in t h i s paper " n i t r a t e " i s used to denote n i t r a t e (NO^) and n i t r i t e (NC^), e d . ) c o n c e n t r a t i o n s from inshore sea water samples show t h a t small amounts o f phosphate remain even when n i t r a t e f a l l s to near u n d e t e c t a b l e l e v e l s ( F i g . 1 ) , i n d i c a t i n g t h a t n i t r o g e n becomes limiting to growth before phosphorus. Macronutrient concentrations in the coastal waters of Southern C a l i f o r n i a fluctuate in a seasonal pattern.

Natural upwelling of nutrient-rich water

from depths of 100-300 m or more occurs t y p i c a l l y during the spring and e a r l y summer.

During the upwelling s e a s o n , n i t r a t e l e v e l s are high

throughout most o f the o f f s h o r e water column ( F i g . 2 ) .

Significant

upwelling i s r a r e during the l a t e summer, f a l l , and winter, and n i t r a t e concentrations are t y p i c a l l y low, e s p e c i a l l y in o f f s h o r e s u r f a c e w a t e r . Temporal fluctuations in dissolved phosphate c l o s e l y follow those of

249 I.Oi—

0.5—

•

O £L

3"

+

n ' -h

® « 2I I JUL

AUG

SEP

JUN

Inner

E

hHi +i

JUN

b.

"h :

Early

JUL

AUG

SEP

JUN

JUL

Middle

AUG

SEP

Outer

Harvest

5

. 4£ t » 3è

2.

! e

'

0

ft 1

-fi

JUN

JUL

m

AUG

Inner

SEP

u y JUN

JUL

AUG

SEP

Middle

JUN

JUL

AUG

SEP

Outer

Fig. 3: Mean standard growth rates of Macrocystis fronds at the inner (-0.7 m depth), middle (-1.5 m depth) and outer (-2.3 m depth) stations in the kelp bed between early June and mid September, 1974. (a) control; (b) early harvest at 1.6 m below MWL. Dashed lines indicate new fronds tagged about July 26. Vertical lines at the top of the bars denote 95% confidence interval about the mean. growing near the outer edge of the bed displayed greater frond elongation rates than those growing near the inner edge, and that the growth rates decreased after July.

However, for the shorter fronds tagged on July 26,

this decline in late summer was not as marked.

The harvested plants

showsd similar trends and furthermore show that harvesting resulted in reduced frond elongation rates, especially at the inner site, and that this rate did not recover over the rest of the experimental period.

271

M

INNER

MIDDLE

OUTER

GROWTH PERIODS

I"2

Fronds >

50 cm long

Fronds

50 cm long


50 cm) on a c t u a l growth r a t e s of f r o n d s .

Mean growth r a t e s of f r o n d s < 50 cm were a l w a y s

272 s i g n i f i c a n t l y l e s s (PCO.OOl) than those recorded for fronds >50 cm for any comparable treatment or time period (see also 6 ) .

The e f f e c t of station

position (or depth) on growth rates was s i g n i f i c a n t

(P