Plant physiology: physiology of plant tolerance: educational manual 9786010428805

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AL-FARABI KAZAKH NATIONAL UNIVERSITY

S. D. Atabayeva

PLANT PHYSIOLOGY: PHYSIOLOGY OF PLANT TOLERANCE Educational manual

Almaty «Qazaq university» 2017

UDC 58 (075) LBC 28.57 я 73 А 90 Recommended for publication by the decision of the Faculty of Biolology and Biotechnology Academic Council, RISO of the Kazakh National University named after Al-Farabi (Protocol №5 dated 11.07.2017); Educational and methodical association on groups of specialties «Natural sciences», «Humanities», «Social sciences, economics and business», «Engineering and technology» and «Arts» of Republican educational and methodological council of higher and postgraduate education of the Ministry of Education and Science of the Republic of Kazakhstan on the basis of the Kazakh National University named after al-Farabi (protocol No.2 from June 29, 2017 y.)

Reviewers: doctor of Biological Sciences, professor A.A. Nurzhanova doctor of Biological Sciences, professor B.N. Mynbayeva doctor of Biological Sciences, professor I.S. Savitskaya

А 90

Atabayeva S.D. Plant physiology: physiology of plant tolerance: educational manual / S.D. Atabayevа. – Almaty: Qazaq university, 2017. – 190 p. ISBN 978-601-04-2880-5 In the study guide the current state of knowledge about the physiological mechanisms of plant resistance tounfavorable environmental conditions, mechanisms of plant adaptations and regulation of plants under adverse stressstress conditions on which environmental biotechnology is based are analyzed. The study guide describes all types of regulation, functioning in the plant organism as a membrane, genetic, hormonal, electrophysiological, metabolic, and trophic regulation. The guide describes physiological basis of plant tolerance to heavy metals, defense mechanisms against heavy metals like as synthesis of metallothioneins, phytochelatins and etc. The guideincludes the glossary, tests, tasks forself study works. The study guide is intended for university students studying in the field of «Biotechnology», «Biology», «Ecology» as well as for teachers, undergraduate, gradute students and researchers. Publishing in authorial release.

UDC 58 (075) LBC 28.57 я 73 ISBN 978-601-04-2880-5

© Atabayeva S.D., 2017 © Al-Farabi KazNU, 2017

CONTENTS LIST OF ACRONYMS ............................................................................. 5 INTRODUCTION ..................................................................................... 6 1.

PLANTS IN UNFAVORABLE ENVIRONMENTAL CONDITIONS ............................................. 7 1.1. Mechanisms of stress ....................................................................... 9 1.2. Plant adaptation mechanisms to stress ............................................ 14 1.3. Regulation mechanisms under stress conditions ............................. 17 1.3.1. Plants signal systems in stress conditions ........................................ 19 1.3.2. The components of signal transduction ........................................... 20 1.3.3. Regulation at the membrane level .................................................... 28 1.3.3.1 The role of membranes in regulation process ............................... 28 1.3.3.2. The types of membrane receptors ................................................. 35 1.3.4. Regulation at the genetic level ........................................................ 39 1.3.5. Regulation at the metabolic level ..................................................... 53 1.3.6. Regulation at the hormone level ...................................................... 61 1.3.7. Trophic regulation system ............................................................... 72 1.3.8. Electrophysiological regulation ....................................................... 76 2.

PHYSIOLOGY OF PLANT RESISTANCE TO HEAVY METALS .................................................................... 83 2.1. General characteristics of heavy metals ........................................... 85 2.2. Mechanisms of heavy metals uptake by plants ................................ 88 2.3. Accumulation and distribution of heavy metals in plants. Intracellular localization .................................................................. 97 2.4. Toxic effects of heavy metals for plants .......................................... 105 2.4.1. The effect of heavy metals on the properties of cell membranes ..... 105 2.4.2. The impact of heavy metals on the activity of enzymes. Oxidative stress ................................................................................ 106 2.4.3. Heavy metals effect on photosynthesis and respiration of plants ................................................................... 113 2.4.4. Heavy metals effect on cell division and nuclear apparatus ............. 117 2.4.5. The impact of heavy metals on mineral nutrition of plants .............. 119 2.4.6. Heavy metals effect on growth and development of plants.............. 121 2.5. Mechanisms of resistance of plants to heavy metals ........................ 125 2.5.1. Heat shock proteins ......................................................................... 125 2.5.2. Polyamines....................................................................................... 128

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2.5.3. Effect of heavy metals on organic acids and thiols content in plant cell ...................................................................................... 138 2.5.4. Meatallothioneins ............................................................................ 142 CONCLUSION.......................................................................................... 154 GLOSSARY .............................................................................................. 156 TEST TASKS ............................................................................................ 159 SELF STUDY WORK............................................................................... 177 LAB WORKS ............................................................................................ 180 REFERENCES .......................................................................................... 184 RECOMMENDED LITERATURE ........................................................... 188

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LIST OF ACRONYMS АA – аminoacid АBA – abscisic acid АDC – arginine decraboxylase АDH – аlcoholdehydrogenase АGC – аscorbicgluthathione cycle АFO – active forms of oxygen АОS – аntioxidant system AP – action potential АPО – аscorbate peroxidase АTP – adenosine triphosphate CAT – catalase DNA – deoxyribonucleic acid EP – excitation potentials EPR – endoplasmic reticulum HSP – heat shock proteins HB – gibberellin GDP – guanosinediphosphate Glu – glutathione GS – glutathionesynthetase GTP – guanosine triphosphate FA – fatty acids IAA – indolacetic acid IP3 – inositol triphosphate LDG – lactate dehydrogenase LP – lipid peroxidation MDA – malonicdialdehyde MDG – malate dehydrogenase MT – metallothioneins ODC – ornitine decarboxylase PА – polyamines PO – peroxidase RP – resting potential PPP – penthose phosphate pathway Putr – putrescine RNA – ribonucleic acid ROS – reactive oxygen substances Spd – spermidine Spm – spermine 5

INTRODUCTION In recent years, climatic conditions and environmental conditions become less favorable due to the cyclic changes in climate and atmospheric pollution, water and soil ecosystems manmade pollutants. Therefore, issues related to improving plant resistance, are of great importance. To crop the defining feature is the ability to tolerate the adverse effects of environment without sharp decline in growth processes and productivity. Recently, intensive work has been underway to obtain high-yielding resistant varieties using methods of conventional breeding and genetic engineering techniques. The ability to protect the organism against the adverse effect of abiotic and biotic environmental factors is a required property of any organism as nutrition, movement, reproduction and etc. Knowledge of the physiological bases of plant resistance allows to develop methods of assessment according to this characteristic as well as techniques for its increase Consideration of the total number of adaptive processes developing in plants in response to any damaging effect, allows to identify common non-specific physiological and biochemical defense reactions. Examination and identification of the mechanisms of impact of stress factors on plants can reveal the patterns of manifestation of plant responses to stress and help to identify the mechanisms of plant resistance. The purpose of the study guide is to make students get acquainted with modern ideas about the physiology of plant resistance under the influence of various stressors, mechanisms of stress regulation, specific and nonspecific reactions. This tutorial presents the description of mechanisms of stress reactions, coping strategies of plants to the influence of stressors, the system of regulation under stress conditions (genetic, membrane, hormonal, trophic, etc.).The possibility of the using of plant tolerance mechanisms in biotechnology is described. 6

I. PLANTS IN UNFAVORABLE ENVIRONMENTAL CONDITIONS In the second half of 30-th of the last century the Canadian scientist H. Selye introduced the concept of «stress» in medicine. According to Selye, stress – is the set of all non-specific changes occurring in a body of an animal under the influence of any strong influences (stressors), including the rearrangement of the body's defenses. According to Selye, stress as the body's response to an adverse effect goes through three phases: alarm, resistance and exhaustion. With regard to the plants the first phase of the stress response can not be called the alarm phase. As for plants, we can talk about the following three phases: primary stress response, adaptation, resource depletion reliability. For example, the leaves of seedlings of beans, hairdryer blown through for 12 – 30 minutes, fall due to wilting (phase primary reaction) but then rise again (adaptation phase), despite the continuing «dry wind» effect. Secondly, vegetable organisms, unlike animals, in most cases do not respond to stressor activation of metabolism, and, conversely, decrease its functional activity. In this regard, during stress in plant tissues increases concentration of hormones that inhibit the metabolism – ethylene and ABA (Polevoy, 1986; Alehina et al., 2007). Stressors are also divided into biotic and abiotic stressors by origin. The biotic stressors include pathogens – pathogenic fungi, bacteria and viruses, and herbivorous insects. Abiotic stressors have lack of moisture (drought), extreme temperatures (high and low), high content of ions in soil (soil salinity), hypoxia (lack of oxygen), very high or very low light, ultraviolet radiation, high concentrations of toxic gases (SO2, N02, O3) in the atmosphere and a number of others. Factors that cause stress in plant organisms can also be divided into three groups: 7

a) physical: insufficient or excess lighting or temperature humidity, radioactive radiation, mechanical impact; b) chemical, salts, gases, xenobiotics (herbicides, insecticides, fungicides, industrial waste, heavy metals, etc.). c) biological (defeat pathogens or pests, competition from other plants, animals influence, flowering, fruit ripening). If metabolic and functional activity is defined as «failure», in the physiology of plants, you can use the technical term «reliability», implying on the complete functioning of plant organism under normal conditions as well as under stress conditions. The reliability of a plant organism is determined by its ability to prevent or eliminate failures at different levels: molecular, subcellular, cellular, tissue, organ, organism and population. To prevent failure the stabilization system is used: redundancy principle, the principle of heterogeneity equivalent components, mechanisms of homeostasis. To eliminate any failures there are repair systems (recovery). At every level biological organization has its own mechanisms. On molecular level the redundancy principle is expressed in polyploidy, at the level of the organism – in the formation of a large number of gametes and seeds. Examples of the reducing activity on the molecular level is an enzymatic repair of DNA damage, on the organism level – the awakening of axillary buds in the damaged apical meristem, regeneration, etc. Adaptation of organism is understood as the process of adaptation of its structure and function to environmental conditions. Adaptation is achieved through a variety of mechanisms: genetic, biochemical, physiological, morphological and anatomical and etc. Resistance – the ability of plants to maintain a constant internal environment (maintain homeostasis) and to implement life cycle under the action of stressors. Plant resistance to stresses depends on the phase of ontogenesis. The most resistant plants are those that are in the resting state (in the form of seeds, bulbs, and etc.). The most sensitive plants are young ones, during germination, since under stress are primarily affected those links of metabolism that are associated with active growth. With the growth and development of plants their resistance to stresses gradually increases up to 8

the maturation of seeds. However, the period during the formation of gametes is also critical, because at this time plants are highly sensitive to stress and react to the impact of stressors by lower productivity (Alehina et al., 2007). Stress is a general nonspecific adaptive response of the organism to the action of any adverse factors.

1.1. Mechanisms of stress The concept of complex plant cell nonspecific reactions to various external influences (acid, alkali, high blood pressure, heavy metals) was introduced by D.N. Nasonov and V.Y. Alexandrov in 1940. According to their observations, there is an increase in the viscosity of the cytoplasm, a rise of acidity, protein denaturation, and others. This complex was named paranecrosis reactions. Later, when the idea of stress was expanded and movedbeyond the hormonal metabolism changes, the existence of non-specific reactions in plants caused no objections. It was formed a separate branch of science – phytostressology. The interest in the study of reactivity of plants is due to the fact that they are in a constantly changing environment. Furthermore, in agrophytocenosis plants are affected by unusual plant compounds such as xenobiotics. When comparing the phases of the triad (anxiety, adaptation, depletion) in plants and animals most doubts arose in the identity of the first phase. The first phase of the plant is proposed to refer to the primary inductive stress response. The following primary nonspecific reactions are includedin a cascade form and are involved in the integral processes: the functions of membranes, energy, growth, the ratio of fusion reactions and decay (Chirkova, 2002). The primary non-specific processes occurring in plant cells with a strong and rapidly rising effect of the stressors include: 1. The increase in membrane permeability by changing the molecular structure of the components leads to a reversible exit of potassium ions from the cell and the entry of calcium ions from the cell wall, vacuole, endoplasmic reticulum, mitochondria. Membrane depolarization, inhibition of H+-ATPase leads to acidification of the cytoplasm. 9

2. The entry of Ca2+ ions in the cytoplasm (from the cell walls and intracellular compartments: vacuoles, endoplasmic reticulum, mitochondria). 3. The decrease in pH of the cytoplasm, thereby activating hydrolases, most of which has an optimum pH in an acidic medium. The result – the strengthening of the processes of degradation of the polymers. 4. The increase of the activity of H-pump in the plasmalemma (and possibly in the tonoplast), which prevents unfavorable shifts of ion homeostasis. 5. The inhibition of transcription and replication, inhibition of protein synthesis, altering the conformation of the protein molecules. 6. The disintegration of polysomes, hydrolysis of mRNA of proteins existing before stress or their interaction with specific proteins, forming a «stress granules» in the cytoplasm. 7. The expression of repressed genes and the synthesis of a number of stress proteins. 8. The activation of the assembly of actin microfilaments and cytoskeletal networks, resulting in increased viscosity and lightscattering of cytoplasm. 9. The increase in uptake of O2, accelerated spending of ATP, the development of free radical reactions. 10. The reduction of the rate of photosynthesis due to changes in the structure of proteins and lipids of thylakoid membranes. 11. The inhibition of respiration,changes in the structure of mitochondria, reductionof ATP level. 12. The activation of free radical processes. 13. The increase in the synthesis of ethylene and ABA, inhibition of growth and division, and other physiological and metabolic processes that take place under normal conditions. Inhibition of the functional activity of cells occurs by the effect of inhibitors and switching energy for overcoming adverse changes. 14. The dominance of catabolic processes, i.e accumulation of degradation products. Their role is diverse, which is particularly evident in the next phase of the triad (Polevoy, 1986). These stress responses are observed under the effect of any stressors. They aim to protect intracellular structures and the removal 10

of adverse changes in cells. Firstly, they can play the role of the correction factor, because during the degradation processes the elimination of polymers with erroneous or defective structure is provided.Secondly, the monomeric compound may serve as a substrate for synthesis of stress proteins, plant hormones. Thirdly, the monomers as respiratory substrates are used as energy source. Fourthly, monomers such as mono- and oligosaccharides, amino acids, particularly – proline, betaine, bind water, which is especially important for the preservation of intracellular water by increasing the permeability of membranes and facilitating the release of water from the cell. Degradation products of proteins and lipids have properties of activators and inhibitors of metabolic processes by influencing the growth and morphogenesis of plants. I.A. Tarchevsky in 1991 suggested the concept of the signaling properties of oligomeric catabolism intermediates realized by the effect on transcription, translation, or the activity of the previously formed enzyme molecules. These stress metabolites are physiologically active decay products, like animals hormones are capable to perform a regulatory function in the subsequent restructuring of the metabolism of cells and a whole organism to a new mode of existence in extreme conditions. Thus, in the first stage of the Selye triad in plants, unlike animals, inhibition of hormone metabolism rather than activation is detected. In plants was found a signal transduction similar to that existing in animals under stress conditions. Bioelectric pulses are generated similar to the action of potential of nerve cells, which may serve as a signal of change in external conditions. It is assumed that acetylcholine and biogenic amines are involved in this process as the mediators. The data transmission method is very similar to the mechanisms of intercellular transfer of excitation in the synapse. Thus, similarity in the implementation phase of the first triad of animals and plants is observed. The described changes are interrelated and they are a starting point for the subsequent switching circuit exchange reactions, the purpose of which is not only to restore the original state of the cell, but also to activate metabolism process (Alehinа et al., 2007). 11

All these phenomena of adaptation syndrome (stress) are interrelated and develop as cascade processes. Currently, efforts are directed to the full transcript of mechanisms of stress at molecular and cellular levels. However, it is necessary to remember that all stressors along with the nonspecific effect have a specific effect on cells and tissues. In the second phase of the triad Selye – adaptation phase – in plants on the basis of changes that occurred during the first phase, the main adaptation mechanisms include reduction in the activity of hydrolytic and catabolic processes of synthesis and amplification. On this phase the decomposition products formed at the beginning of stress impact contribute to the «readiness» of metabolism to change. Proline accumulated as a result of hydrolysis of the protein interacts with the surface hydrophilic residues of proteins and increases their solubility, protecting against denaturation. As a result, the cell retains more water, which increases the vitality of plants to drought, salinity, high temperature. On this phase the decomposition products formed at the beginning of stress impact contribute to the «readiness» of metabolism to change. Degradation products of hemicellulose, pectin – oligoglycosides induce the synthesis of phytoalexins, which protect plant during infection. Resulting from degradation of organic nitrogen compounds polyamines help to reduce membrane permeability, inhibition of protease activity, reducton of lipid peroxidation (LPO), regulation of pH (Alehina et al., 2007).The second phase includes a protective response that is non-specific. It contributes to more intensive synthesis of proteins and nucleic acids due to formation of stress proteins – isozymes and the capacity of the enzyme systems is amplified. There is a stabilization of membranes, resulting in restored ion transport. It increases the activity of the functioning of mitochondria, chloroplasts, and accordingly, the level of energy. It reduces production of reactive oxygen species and inhibits lipid peroxidation. The role of compensating shunt mechanisms, such as enhanced activity of the pentose phosphate pathway is to be a respiration provider of reductant (NADPH) and pentoses required for synthesis (in particular, nucleic acids). At the level of the whole organism adaptation mechanisms inherent in the cell are complemented by new reactions. They are 12

based on the competitive relationship between the organs for the physiologically active substances and nutrients and are built on the principle of attracting centers. This mechanism allows the plant under stress to form the minimum number of generative organs (attracting centers), which can be provided with the necessary nutrients for maturing. Due to the transfer of nutrients from the lower leaves remain viable upper younger leaves (Chirkova, 2002). At the population level in the stress response is activated natural selection. As a result of natural selection more adapted organisms and new species appear. At this level the adaptation only means the preservation of those individuals who have a wide range of responses to extreme factors and, being genetically more resistant, are able to produce offspring. Plants that are not genetically adapted to stressors die or are eliminated, that results in the increase in overall stability of the population. The prerequisite for thismechanism of adaptation is intrapopulation variability of resistance level to the different factors or their complexes. Under of increasing stress conditions and effect of gradual exhaustion of the defense capabilities of organism also dominate nonspecific reactions. Under the effect of various agents the cell structure is destroyed. There is a destruction of the nucleus, a decomposition of chloroplasts grana, decrease in the number of mitochondria cristae. There appear additional vacuoles, where toxicsubstances resulting from changes of metabolism under stressful conditions are neutralized.Violation of ultrastructure of the main energy generators – mitochondria and chloroplasts leads to the depletion of energy of in cells, which entails changes of physical and chemical state of the cytoplasm. These changes indicate a strong, irreversible damage in the cells and mean the last stage of the Selye scheme – the phase of exhaustion. Some researchers propose to supplement the Selye triad another phase – fourth, calling it a phase of regeneration (restitution), the occurrence of which is possible after removal of the stressor. Resistance of plants may vary in ontogeny: particularly sensitive to stressors plants in juvenile age (during germination), as well as flowering and fruiting, and the most stable – at rest phase (seeds). Stressors usually act in complex. For example, it accompanied by 13

drought, flooding appears when oxygen deficiency and toxicity, toxic compounds, low temperature accompanied by weak illumination and excess moisture, etc. In response to stressors occur reactions specific to the effects of stress. These include an increase in the concentration of ions under salinity, metallothionein synthesis under the influence of heavy metals, leaf yellowing (chlorosis) in unbalanced mineral nutrition, growth of root collar by flooding, increased transpiration during drought, the synthesis of certain stress proteins, etc. But exactly these non-specific defense reactions are the most «economic» and universal means for the maintainingof biological systems balance with the environment, ensuring their reliability in a rapidly changing environment. After initial, short-term adaptation is achieved and the nature of the stressor to the organism is clear, mechanisms of the main phase of adaptation are triggered, where along with nonspecific reactions, nonspecific response are detected (Polevoy, 1986; Yakushkina, 2005; Alehina, 2007). Test questions 1. What is a stress? 2. What types of stressors do you know? 3. What is a paranecrosis? 4. What does a phytostressology study? 5. What is a nonspecific reaction to stress? 6. Name the nonspecific reaction of plants to stress.

1.2. Plant adaptation mechanisms to stress The most common manifestation of the effect of stressors is the suppression of plant growth and development, and at the level of phytocenosis – the decrease of plant productivity. Stressors lead to a decrease in the growth rate to a level lower than the level resulting from the genetic potential of the plant. Force of the harmful effects of various stressors can be assessed by comparing the record harvests of crops with average yields, calculated for many years. Sometimes several stressors act in combination with each other, and then their 14

harmful effect is amplified. For example, the effect of drought is often combined with high temperatures. Phenomena that occur in plants under the influence of stressors can be divided into two categories: 1) damage, manifested on different levels of structural and functional organization of the plant, for example, denaturation of protein molecules, metabolic disorders and reduction of elongation at cell dehydration under drought or soil salinity; 2) responses, that allow plants to adapt to the new stress conditions; they are affected by changes in gene expression, metabolism and physiological functions and homeostasis. The totality of such reactions is called acclimation. During the acclimation plants acquire resistance to the stressor. Acclimation occurs during the life of the organism and it is not inherited. However, it is based on the opportunities inherent in the genotype, i.e. within normal limits of plants – hereditarily determined amplitude of possible changes in realization of genotype. An example of the acclimation of plants is hardening. Some plants, winter cereals in particular, acquire the ability to survive at low negative temperature in winter, as autumn undergo hardening – the effect of low positive and close to 0°C freezing temperatures. Biochemical changes in the tissues, occurring in autumn, winter cereals give the ability to survive frost. If the fall was warm, the winter cereals freeze in winter. The adaptation plays an important role in plant resistance to the effect of stressors. Unlike acclimation, adaptation – hereditarily fixed constitutive feature, presents in a plant no matter independently of whether it is under stress or not. Adaptations do populations of organisms adapted to the respective conditions of surrounding environment. An example of adaptation to drought – morphological features of succulents, especially cacti. Fleshy stem, leaves, made into needles, a small number of stomata, deeply embedded in the fabric, thick cuticle, and a number of other features allow cacti implement life cycle in an economy of moisture and thus survive in arid climates. Adaptations are also manifested at the biochemical level.This is, for example, the biosynthesis of steroid pseudoalkaloids (glycoalka15

loids) in some species of Solanaceae, particularly in potatoes, toxic to herbivores and phytophagous insects. The biosynthesis of glycoalkaloids is a constitutive feature of these plants, which was formed in the process of evolution as protection from being eaten. Protective mechanisms as constitutive (adaptation – hereditarily fixed constitutive features present in the plant regardless of whether it is under stress or not) and that which forms during acclimation (responses that allow plants to adapt to the new stress conditions; they affect changes in gene expression, metabolism and physiological functions and homeostasis) can be divided into two main categories. Avoidance mechanism. They enable plants to avoid the effect of stressors. Example – the absorption of water from the soil by deeply penetrating root system of plants. Some xerophytes (plants arid habitats), such as the black haloxylon, have the length of root system that reaches several meters. This allows the plant to use ground water and not to feel lack of moisture in the soil and atmospheric drought conditions. This category also includes the mechanisms of ion homeostasis in the cytoplasm of plants resistant to soil salinization. The ability to maintain low concentrations of Na and Cl in the cytoplasm when the soil salinity allows these plants to avoid the toxic effect of ions on the cytoplasmic biopolymers. Mechanisms of resistance (endurance). Through these mechanisms plants survive, not avoiding the effect of stressors under stressful conditions. Such mechanisms include, in particular, biosynthesis of several isozymes performing catalysis the same reactions. Wherein each isoform possesses the necessary catalytic properties in a relatively narrow range of some parameter of the environment, such as temperature. In general, the entire set of isozymes allows the plant to carry out the reaction in a much wider temperature range compared with only one isozyme, therefore, to adapt to changing temperature conditions. The overwhelming majority of cellular resistance mechanisms is formed in the early stages of evolution, so the protective system, manifested at the cellular level in higher plants, and protective systems more primitive organisms have a common basis.In this regard, the study of mechanisms of resistance to stressors in bacteria, yeast and unicellular reveals the cellular mechanisms of resistance in higher plants. Much of the knowledge 16

about the reception of the signal and its transmission, as well as the tolerance of the cellular mechanisms in general, researchers have received, based on the basic works performed on E. coli, bacteria and yeast Saccharomyces cerevisiae. Mechanisms of reception and signal transduction pathways in plants begin directly studied until now. It was shown, that in the regulation of plant response to stress hormones, especially abscisic acid (ABA), ethylene and jasmonic acid are involved. In response to stressors the expression of some genes is enhanced, whereas the expression of other genes is suppressed. The new proteins are formed, which are not found in the cells in unstressed conditions. Although the main part of studies is focused on transcriptional activation of genes, data recently obtained indicate that degree of stress genes products and the activity of these products is regulated at posttranscriptional level. Such mechanisms include the activation of translation, posttranslational stabilization, the change of the enzymatic activity already synthesized proteins, and others. Test questions 1. What characterizes the damage? 2. What are the responses of plants to stress? 3. What is an acclimation? 4. What is an adaptation? 5. What does the term «norm of reaction» mean? 5. Describe the mechanisms of avoidance. 6. What characterizes the mechanisms of resistance? 7. What is a plants tolerance?

1.3. Regulation mechanisms under stress conditions The integrity of the organism, including plant is provided by the system of regulation, control and integration. Management – is the process of transferring the system from one state to another by moving the system from one state to another by acting on its variables. The term «regulation» in a broader meaning includes control. The regulation ensures the homeostasis of the organism, i.e. the constancy of parametersof the internal environment. Intracellular 17

regulation systems must have appeared during evolution. This includes the regulationon the enzymes level, genetic, membrane regulation. When multicellular organisms appear the intercellular regulation systems develop. This includes trophic, hormonal, electrophysiological systems (Chirkova, 2002) (Figure 1).

Figure 1. Systems of plant regulation (T.V. Chirkova, 2002)

In stressful conditions there is a set of defense reactions in any plant, regardless of its adaptability. However, the degree of resistance of plants exerts a decisive factor in their stress response. In unstable plants such reactions, quickly came into force, are shortlived and unable to protect the organisms from death (Figure 2). Particularly sensitive organisms may die even at the beginning of the adverse effects, in the first phases of the triad, before the adaptation phase. The transition to the new regime of resistant objects, as opposed to sensitive, occurs gradually, but offers more long-term maintenance of the equilibrium state of metabolism. Plant, different in resistance can respond to the impact of the same type, but the speed and amplitude of physiological transformations may be differ. As a result, with organisms resistant to stressors, when the homeostasis is stable and the reparation of changes is possible after returning to normal conditions the length of 18

the adaptation phase is longer than that with the sensitive plants. In unadapted plants much sooner exhausted adaptive capacities and irreversible changes occur. During switching metabolism to the new regime under stress reserve possibilities of the organism are united through a system of regulation (Figure 2).

Figure 2. The degree ofresistance ofthe tolerant and sensitive plantsto the action ofthe stressor

1.3.1. Plants signal systems in stress conditions Changes in metabolism, physiological functions and growth processes in stress associated with changes in gene expression. A plant responds to stress, if «recognizes» stressor on the cellular level. Recognition of the stressor, i.e. reception signal leads to the activation of a signal transduction pathway. The latter enters into the genome of inducing or suppressing the synthesis of certain proteins. Related gene expression responses of cells to the effect of the stressor integrated to the response of the whole plant, expressed in the most general case as the inhibition of growth and development of the plant and simultaneously increasing its resistance to a stressor (Chirkova 2002). 19

Changes in metabolism, physiological functions and growth processes in stress are associated with changes in gene expression. Response to the stressor occurs as follows: «recognition» of the stressor – reception of signal → activation of signal transduction pathways → the induction or suppression of gene of synthesis of certain proteins → response of the whole plant (inhibition of plant growth and development) → increased resistance to the effect of stressor (Chirkova, 2002). The prerequisite of regulation operation systemsis the perception (reception or perception), transmission and transformation (transduction) of external signal that is mediated by specific receptors of protein nature. «Cell signaling» – a new field of biochemistry which studies the mechanisms of transmission of external signals, or signal transduction. It includes the study of the molecular mechanisms of regulation of cellular metabolism by external signals. The concept of «cell signaling» refers not only to the transmission of signals to the genetic apparatus of cells, but also the whole complex of events, coupled with it (gain, attenuation, suppression of signals). For example, the interaction of a signaling molecule to a receptor may lead to millions of molecules, which recognize the response of the cell (Chirkova 2002). 1.3.2. The components of signal transduction Signaling cascade is a set of transmission and conversion of signals from the receptors to intracellular targets, originating from a few protein components. In addition to protein mediators involved in the transmission of signals and relatively small molecules which serve as secondary signals. These are calledthesecondaryintermediaries or messengers. Difference in the features of protein and non-protein mediators is that proteins form a kind of molecular machine, which, on the one hand, is sensitive to an external signal, and on the other hand has an enzymatic or other activity modulating this signal, whereas small molecules (e.g. calcium ions) actually serve as messengers (messengers) between different proteins, multienzyme complexes or cell structures (Chirkova, 2002). 20

G-proteins. Perceived by GPCR signal is transmitted to the G-protein. This can be a GTP-dependent activation of Gα-phospholipase C. Phospholipase (A1, A2, C, D) varies depending on where their action is directed in the phospholipid molecule. When phospholipase C is activated, calcium is released as a mediator of signal transduction (Figure 3). The bond of Gα to GTP within minutes and GDP is hydrolyzed by GTP-ase, resulting in changing the conformational state of the protein and its activator function is lost. However, Gα may again become part of the trimer, and the cycle is repeated. Despite the short duration, the life of the signal, and the intermediaries involved in its further transmission, the process of transduction is very effective. When signal in cascade transmits from the receptor through to G-protein to effector protein (e.g., an enzyme) the external signal is magnified because one molecule of receptor while being converted into an activated form is capable to transfer several molecules of G-protein into activated. Eukaryotes have also small G-proteins (Ras-proteins), that consist of one subunit and act as molecular switches, like the large G-protein (Chirkova 2002; Alehina et al., 2007). GPCR The signal G-protein Activation of GTP-dependent α-phospholipase С Releasing of calcium /messenger of signal transduction/ The hydrolysis of the link between Gα and GTP to GDP by GTP-ase Gα Figure 3. GTP-dependent activation of Gα-phospholipase 21

Secondary messengers. Calcium ions. The characteristic property of the second messengers, the calcium ions – is small compared to biopolymers having a molecular weight that is necessary for highspeed diffusing into the cytoplasm. In addition, the messenger must quickly split or deleted. Otherwise, an alarm system can remain in the «On» state after the action of an external signal stopped. Signals induced the opening of calcium channels, the calcium concentration in the cytosol strongly increased, which stimulates the activity of almost all involved in the regulation enzymes. Reducing the concentration of Ca2+ in the cytosol is provided by the work of the ATP-dependent calcium pump (Fig. 4), which contributes to the accumulation of calcium in the vacuoles or transport across the plasma membrane in the cell wall.

Figure 4. Effect of ATP – translocator of Ca2+ ions (T.V. Chirkova, 2002)

The phosphoinositol pathway. Calcium channels are controlled by the phosphoinositol signal transduction cascade. A phosphatidylinositol – a component of membrane in animal cells comprises two fatty acids. They are stearic and arachidic acids. The kinase phosphorylates inositol residues at the hydroxyl groups at positions 4 and 5. Phospholipase, C stimulated by G-protein, decomposes the lipid to 22

1,4,5-triphosphate (IP3) and diacylglycerol (DAG). These compounds are also involved in signal transduction; IP3 opens calcium channels (Figure 5), while DAG activates the Ca-dependent protein kinase. IP3 induces release of Ca2+, another secondary messenger, from vacuoles, endoplasmic reticulum into the cytosol, and thus the concentration of Ca2+ in cytosol increases. The concentration of Ca in the cytosol may also increase due to its admission into the cell through electrochemical potential dependent Ca2+ -channels of plasma membrane at depolarization of this membrane, induced by stressor. Increased concentrations of Ca2+ activate Ca2+-calmodulin-dependent protein kinase (SDPK), which in turn stimulates the biosynthesis of stress proteins. Calmodulin. Calcium acts as a mediator only after interaction with calmodulin (Figure 6). Calmodulin – is a soluble inositol protein (17 kDa), which is found in animals and plants. It consists of two domains linked by flexible α-helix. Each domain contains two Ca binding sites (Figure 6). Interaction of Ca2+ to all four Ca-binding centers leads to change in the conformation of calmodulin. As a result, it forms a complex with protein kinases, which, in turn, are activated. Phosphorylation of proteins. Protein kinases and phosphatases are essential elements of regulation of intracellular processes, since the proteins alter the conformation depending on phosphorylation or dephosphorylation. The efficiency and functional role of many protein kinases are also dependent on phosphorylation. In biology the cascades of enzymatic reactions are widespread. They consist of similar reactions. Their substrate – the result of reaction is a protein transformed into the active enzyme. This enzyme converts another protein into an active enzyme. This process is repeated several times. In eukaryotes, the majority of protein kinase phosphorylates OH-group of serine or threonine, and tyrosine residues in proteins. In protein kinase regulatory processes form a cascade of interrelated reactions. They are known as protein kinases A – cAMP-dependent, which belong to the adenylcyclase signaling cascade; protein kinase C – Ca2+ – phospholipid dependent pyruvate kinase, activated by DAG, Ca2+ and phospholipids, and included in the phosphoinositol 23

cascade of protein kinases, G – cGMP-dependent protein kinases and Ca2+-calmodulin-dependent protein kinases.

A

B Figure 5. The phosphoinositol pathway (http://www.picscience.net)

MAP (mitogen activated proteins) is a kinases cascade. It consists of three protein kinases. Protein kinases are enzymes capable of 24

catalyzing the reaction of addition of phosphate to serine, threonine or tyrosine residues in the protein molecule to form a phosphorylated form of the protein. These changes in conformation of the molecule cause an activation or inhibition of protein. Phosphorylation is a convenient way to control the activity of proteins. It is widely distributed in the cell. Phosphorylation is one of the main ways of regulation of intracellular processes, and especially the regulation of the process of the reading of genetic information.

Activeproteinkinase

Inactiveproteinkinase

Calmodulin

Calmodulin + protein kinase

Figure 6. Role of calmodulin in activation of protein kinases (T.V. Chirkova, 2002)

The last stage of the cascade is the formation of active kinase, called MAP kinase. MAP kinase phosphorylates and thereby alters the activity of several protein targets. MAP kinase becomes active only after binding with phosphate To activate MAP kinase in the cell there is a specialized kinase that can phosphorylate only MAP kinase: a kinase of MAP kinase. Initially, the enzyme is inactive as MAP kinase and also is activated by phosphate. Kinases of MAP cascade work as follows: MAPKK-dependent pyruvate kinase (МАРККК) ↓ Activation МАРК-dependent protein kinase (МАРКК) ↓ Activation Mitogen-activated protein kinase (МАРК) 25

For this process in the cell there is another protein kinase – a kinase of kinase of MAP kinase (Figures 7, 8). It is also initially inactive, but is activated differently. It is activated by a signal from the outside, passing through a series of intracellular signaling associated with secondary messengers. MAP kinase cascade is used by cells to regulate gene transcription in response to changes in the environment

Figure 7. The kinases of MAP cascade (http://www.studentpulse.com/articles/901/the-role-of-hydrogen-peroxide)

All cascades are characterized by a common structure. The cascade have the input – a signal, activating the first enzyme of cascade, and there is an output – the concentration of the active form of a pro26

tein,mostly also of an enzyme. It is interesting to know why the input of signal does not act directly on the enzyme activity of the output? It occurs because the cascade of reactions is needed to amplify the signal. The appearance of one of the active enzyme molecules leads to the formation of a plurality of product molecules. Thus, the result of cascade reactions in response to a single molecule of the input signal is the formation of a number of molecules of «signal» product, the concentration of which can be regarded as an output signal. Levels of perception and signal transduction. Multicellular organisms have two types of perception and signal transduction. The first is a level of the whole organism, which receives information from the environment. The second is the level of "communication" of cells within a multicellular organism.

Figure 8. The MAP cascade (http://cc.scu.edu.cn)

Their behavior may be regulated by cell-cell interactions that are mediated by integrating into the outer cell membrane receptors. In 27

general, in a multicellular organism exists a balance between the processes of cell proliferation and natural death – apoptosis. Under stress this balance can be disrupted, leading to a predominance of apoptosis and tissue degeneration. Test questions: 1. What is the signal reception? 2. What was the schemeof response to a stressor? 3. What is studying the field of biochemistry, called «cell signaling»? 4. What refers to the second messenger? 5. What are the components of the signal transduction. 6. What are the functions of G-proteins? 7. What refers to the second messenger? 8. What are the functions of phosphoinositol pathway, phosphorylation of proteins in signal transduction 9. Describe the work of MAP cascade.

1.3.3. Regulation at the membrane level 1.3.3.1. The role of membranes in regulation process Membrane regulation is realized through changes in membrane transport, binding, or release of enzymes and regulatory proteins, and by changing the activity of membrane enzymes (Polevoy, 1997). All the functions of membranes – barrier, transport, osmosis, energy, receptor-regulatory and etc. are different sides of the mechanism of regulation of intracellular metabolism (Figures 9-11). And of particular importance in all of these mechanisms is a system of membrane chemo-, photo- and mechanoreceptors that allows cells to assess qualitative and quantitative changes in the external and internal environment and in accordance with this change the functional activity of the cells. Shifts in the functional activity of the membrane accompanied by re-construction sites in their structure that contribute to the initial stage to increase their resistance until the effect of the stressor does not reach its maximum voltage. Structural changes in the membrane greatly affecting the lipids, especially the fatty acids as the most labile components. Under the action of the stressor may be a shift in the ratio of different groups of fatty acids changes the degree of saturation/unsaturation. 28

Figure 9. Structure of cell membrane (http://www.apsubiology.org/anatomy/2010/Exam_Reviews/Exam_1_Review/Ch03 _The_Cell_and_Membrane_Structure.htm)

Figure 10. Fluid mosaic structure of plasma membrane (http://www.rsc.org/Education/Teachers/Resources/cfb/cells.htm)

Possible changes chain length fatty acids, a positional arrangement of double bonds, the amount of polar groups. Due to the close 29

interaction and protein components in the membrane properties of lipid changes inevitably affect the function of membrane proteins. For the proper functioning of enzyme proteins it is necessary liquid state membranes, therefore under the influence of lipid membrane rearrangements catalytic function of proteins is altered. According to the liquid-mosaic model, the cell membrane is compared with the «lipid sea», in which at various level of immersion, like «icebergs» float proteins.

Figure 11. Functions of membrane proteins (http://www.slideshare.net/WongSookYen/stpm-form-6-biology-cell-membrane)

The membrane’s proteins play a regulatory role in cellular metabolism. In addition to receptor function they perform a regulation of conformational changes of membranes. The interphase restructuring linked with the interaction of the components of membrane systems – lipid-protein interactions that provide largely the necessary intensity of cell metabolism and control of (Chirkova, 2002). Structural changes in the membranes under the influence of adverse effects are related with the release of bounding forms of Ca2+, a 30

bridge between the carboxyl groups of the protein and the polar heads of phospholipids. In the nucleus, Ca2+ ions are involved in maintaining the structure of chromatin, mitochondria and chloroplasts, and play an important role in the regulation of enzyme activity. Basically Ca2+ is localized in the cell wall, where it binds to the carboxyl groups of uronic acids. In the cytosol, as it is known, the concentration of Ca2+ is low (10-5-10-8mol/L), whereas in the apoplast and in organelles it is 103-104 times greater. The flow of calcium from the apoplast into the cytoplasm increases dramatically when the degradation of cellular components as a result of stress is occurred. The excess of calcium is derived from the cytoplasm. However, even a momentary increase its concentration is enough to start the specific membrane channels and transport system, and also cause structural changes in the cell. One of the manifestations of plant response to stress effect is to alter the permeability of the outer membrane of the cells and increase the diffusion of organic and inorganic substances in the rooting medium. Changes in membrane permeability under stress suggests restructuring of membranes, which largely determines the potentially possible mechanisms of plants to withstand environmental stress. Permeability of plasma membrane for electrolytes is an integral indicator of the functional state of the plant. The increase of exoosmosis of electrolytes in stressful conditions reflects many processes, such as increasing the desorption of membrane electrolytes and the release them after degradation of labile biological complexes and increase the sorption capacity of the protoplasm. The measurement of plant’s tissues exudate conductivity many researchers used as an indicator of membrane damage by low and high temperatures and dehydration. There is a definite sequence of changes in cell membranes under the influence of low temperature phase transitions of membrane lipids, the violation of the membrane structure in the areas of interfaces that increases membrane permeability within the defective areas. The increase in membrane permeability is directly influenced by the processes occurring under the action of low temperatures. These in31

clude the inhibition of the functions of membrane-bound enzymes, including transport ATPases, pH change, increased lipid peroxidation, activation of membrane phospholipases (Chirkova, 2002). Plasmalemma and membranes of cell organelles (mitochondria, chloroplasts) of resistant plants are characterized by increased resistance and preserve the integrity under stress. The persistence of membranes is determined by the state of their components. Greater stability of membranes of adapted plants linked in particular to the quality and quantitative features of lipids. Thus, the increase in the content or maintenance at a level appropriate to the conditions of the norms of unsaturated fatty acids in the membranes of mitochondria under different treatments (cooling, oxygen deficiency, drought, infection, ethanol) promotes stability of the membranes. This is due to looser packing of polyene fatty acids than saturated in the bilayer and in the area of contact of phospholipids with proteins, which gives a large membrane plasticity, fluidity, flexibility. It is clear that these changes in the physical properties of membranes create better conditions for their functioning. Since the increase in the degree of unsaturation of fatty acids is observed in many external influences, it is, apparently, nonspecific reaction of plants to have long-lasting objects with the greatest stability (Chirkova, 2002). Greater stability of membranes of resistant plants is also associated with the quantitative changes in the composition of lipids, in particular with a high content of lipids and phospholipids. Stability of characteristic of the protein complex of membrane components of plants resistant to various impacts is determined by the maintaining of the structure of macromolecules in a high conformational flexibility. Long-term preservation of the integrity of membranes promotes the collapse of the braking components, lipids and proteins, which may be associated with the effective effect of the mechanisms of antioxidant protection, with inhibition of enzyme protein breakdown. Under various stress conditions there is an increase in membrane permeability, which entails a violation of cellular homeostasis. For resistant plants permeability of membranes is expressed as least as inhibition of H+ -pump is inhibited in comparison with the sensitive plants. This contributes to long-term maintenance of their energy 32

supplies necessary for the working of pumps in a stressful environment. Calcium Ca2+ -ions stabilize cell membranes. In the presence of calcium electrical resistance of membranes increases. It affects membrane permeability for other ions, is involved in the regulation of water transportation. The stabilizing effect of Ca2+ can also occur indirectly, for example through the contents of polyamines in the cell required to restore the permeability of membranes. Accumulation of polyamines is correlated with plant resistance. The polyamines play an important role in the homeostatic regulation of cell pH and stabilize the cell membranes. Recently, much attention is paid to the study of changes in the content of polyamines in plants exposed to different kinds of stress. The result of the stressors is the increase or decrease in the concentration of polyamines, depending on the type of stress, exposure time, type of plant. Polyamines are low molecular weight polycations and are present in all living organisms. The diamine putrescine and polyamines – spermidine and spermine are the low molecular weight aliphatic amines. Putrescine (NH2 (CH2) 3NH2) is a precursor of spermidine (NH2 (CH2)3 NH (CH2) 4NH2) and spermine (NH2 (CH2)3 NH (CH2)4 NH(CH2)3 NH2) (Figures 12, 13).

Figure 12. The polyamines structure (http://www.mdpi.com)

Positive charge and conformational flexibility provide the unique properties of polyamines to interact with cell membranes and macromolecules. Polyamines bound to negatively charged groups on the phospholipid membranes, thereby enhancing the stability and 33

membrane permeability. In this context, the physiological concentrations of spermidine and spermine as well as Ca, needed for stabilization of protoplast cells from lysis. Polyamines may affect the fluidity of the membrane to modulate the activity of enzymes (indirectly) (Galston, 2001; Alehina et al., 2007). Thus, the increase in the permeability of membranes under stress conditions is associated with impaired calcium metabolism, so it should help normalize the regulation of permeability.

A

B Figure 13. The polyamine biosynthetic pathway (http://www.mdpi.com)

34

So, among the causes of greater stability of cell membranes of resistant plants may include: 1) the adaptive adjustment of the membranes, 2) inhibition of the decay of their components, and 3) the ability to maintain the regulation of calcium mode of cell. In all probability, these reactions are interconnected through the membrane system of regulation. Because the membranes of less resistant plants are damaged by the action of stressors, it can be expected that the system of regulation of the permeability and maintain homeostasis operate them more efficiently than in intolerant plants. Therefore, the membrane permeability of plant cells is an indicator of stability in developing plants rapid diagnostic techniques, such as determining the intensity of the output from the tissues of electrolytes. The use the substances, stabilizing the membrane and prevent their decay reduces the permeability of membranes and enhances the resistance of plants. Such membrane compounds include calcium salts, antioxidants (vitamin E). Consequently, the membrane system of regulation, a component of the complex regulatory systems of the organism, contributes to the coordination of metabolism under stress conditions (Chirkova, 2002). 1.3.3.2. The types of membrane receptors. On the base of all forms of intracellular regulation is a single primary receptor-conformational principle: protein molecule – receptor «recognizes» specific factor for it and interacting with it, changes its configuration. There are three major types of receptors that are integrated into the outer cell membrane: – receptors, coupled with G-proteins; – receptors–ion channels; – receptors, associated with enzymes. These processes are as follows: substances that initiate transmembrane signaling receptor activation → → signaling to intracellular targets (Chirkova, 2002). If the target or effector protein is an enzyme, the signal modulates (increases or decreases) its catalytic activity. If the effector protein is an ion channel, the conductance of channel is modulated. 35

A

A

B Figure 14. G-protein-linked receptors (http://philschatz.com/biologybook/contents/m44451.html, https://flochalmers.wordpress.com) 36

Receptors coupled to G-proteins (such receptors designated as GPCR – G-protein coupled receptors), a signal is transmitted to the internal target using a cascade GPCR → G-protein → effector protein (Figure 3A, 3B). These GTP-binding proteins change their conformation upon binding to GTP or GDP. They have been studied mainly in animals. They are heterotrimeric proteins composed of three different subunits: Gα (45-55 kDa), Gβ (35 kD) and Gγ (8 kD) with the primary signal is localized at the outer side of the membrane and the portion contacting the G-protein at its cytoplasmic side (Figure 14). Trimers can interact with the receptor. Gα subunit has binding sites with GTP and GDP. The binding of GTP alters the conformation of the Gα subunit and the separation of the trimer. Associated with GTP the Gα-subunit functions as an activator of enzymes it plays an intermediary role in the transmission of signals. In animal cells Gα-GTF stimulates adenylatecyclase, which catalyzes the synthesis of cAMP and ATP. However, participation of cAMP of plants in signal transduction is not established yet. Primary signals for these receptors are diverse molecules including hormones acting generally at very low concentrations, on the order of 10-8 M/L or below. GPCR represents monomeric integral membrane proteins, the polypeptide chain which repeatedly crosses the cell membrane. In all cases, the portion of the receptor is responsible for the interaction with the primary signal is localized at the outer side of the membrane and the portion contacting with the G-protein is localized at its cytoplasmic side. Receptors – ion channels are integral membrane proteins consisting of multiple subunits, the polypeptide chain of which is the same as in the related G-proteins repeatedly crosses the membrane (Chirkova 2002).They, acting both as ion channels and receptors, are capable of specifically binding with the outer side of the primary signals and change their ionic (cationic or anionic depending on the type of receptor) conductivity (Fig 15A, 15B). In the absence of the signal the channel is closed, it is opened while its binding to the receptor. The binding site of the primary signal receptors associated with the enzyme is on the side which faces the extracellular space. 37

Cytoplasm

А

B

Figure 15. Ion-channel-linked receptors (http://bioserv.fiu.edu) С1 – an external signal, Р – a receptor protein: an asterisk denotes the components of the signaling system in the «on» state

A

B Figure 16. Enzyme-linked receptors (https://flochalmers.wordpress.com) С1 – an external signal, Р – a receptor protein: an asterisk denotes the components of the signaling system in the «on» state 38

In the mechanism of interaction with cytoplasmic target these receptors are divided into two groups: 1. The catalytic part, activated by the effect of an external signal, is on the cytoplasmic side (Figure 16 A, left). Receptors of this type are involved in the regulation of water and salt response. 2. The receptors do not possess intrinsic enzymatic activity (Fig. 16A, right; 16B). However, under the influence of an external signal, as shown for animals, they acquire the ability to bind the cytoplasmic (non-receptor) protein tyrosine kinase, which in the free state are inactive but they in combination with thereceptor are activated and phosphorylate proteins. The inclusion of phosphate residues in a receptor – «anchor» creates conditions for binding them to other target proteins which also phosphorylate and thereby transmit the signal transduction within the system. Test questions: 1. What is the signal reception? 2. What was the schemeof response to a stressor? 3. What is studying the field of biochemistry, called «cell signaling»? 4. What are the main functions of biological membranes? 5. Why does the membrane react quickly to changing environmental conditions? 6. What determines the receptor-regulatory function of membranes? 7. How does the structure of the membrane react to the action of stressors? 8. How do the stress factors influence the function of membrane proteins? 9. What is the role of calcium ions in the regulation of membrane? 10. What are the properties of membranes modified by the action of stressors? 11. What characterizes plasmalemma membrane and cell organelles in stable and unstable plants? 12. How do polyamines stabilize membranes? 13. What are the reasons for greater stability of cell membranes in resistant plants. 14. What types of membrane receptors are known?

1.3.4. Regulation at the genetic level Genetic regulation is carried out during the synthesis of new proteins, including enzymes, at the of transcription, translation and processing level (Polevoy, 1997). The role of genes is the storage 39

and transmission of genetic information. Information is recorded in the chromosomal DNA using the nucleotide triplet code (Figure 17).

Figure 17. Gene regulation in plant cell (http://www.slideshare.net/bindu567/194022023signaltransductionandgeneregulationinplant.development

Information is transmitted in the cells due to the synthesis of RNA on the DNA template (transcription) and the synthesis of specialized proteins on the mRNA template with ribosomes containing rRNA and ribosomal proteins and tRNA (translation). During and after transcription or translation modification occurs (processing) biopolymers are transported to the destination (Figure 18). Differential gene activity depends on various factors. For example, the synthesis of nitrate reductase enzymatic complex in cells, reducing nitrates to NH3, is induced by the substrate (nitrate) and one of phytohormones – cytokinin. It is known that auxin and cytokinins are required for induction of plant cell division. Excess of auxin in this pair of phytohormones includes genetic program of rooting and excess of cytokinin – a program of the development of shoots. For realization of the genetic information stored in the chromosomal DNA, the cell has a complex regulatory system, not all the sides of which are currently known. 40

Figure 18. Positive and negative regulation (http://chemistry.umeche.maine.edu/CHY431/Genome2.html)

Signal, perceived by the cell, is transmitted to the nucleus. Several distinct levels of regulation of the cellular response (Chirkova, 2002): 1. The level of transcription is a regulated transcription and subsequent processing (maturation) of the precursor mRNA, as well as degradation of mRNA precursor. 2. The level of translation, regulation may be subjected to the protein synthesis, its subsequent processing or degradation of the precursor out of protein after processing. 3. The level of mature proteins: regulation can be implemented in processes phosphorylation – dephosphorylation of proteins and change their properties in the shifts of the catalytic activity under the action of second messenger, modulation of proteins properties as a result of protein-protein interactions – the activation of protein kinase and changes of compartmentation of protein molecules during 41

the transition from cytoplasm to membrane leading to disruption of the properties of proteins, which are essential for the signal function. The most commonmechanism oftranscriptional regulationaspecific interactionof proteintranscription factors of cytoplasmto regulatory regionsof DNA. It were identifiedthreemain options forthis interaction (Figure 19, 20).

Figure 19. The main signal transduction pathway from the cytosol to the nucleus (V.I. Kulinskiy, 1997) T – transcription factor, PK – proteinkinase, P – the phosphate residue, I – inhibitor, ТК – tyrosinkinase; the full line – translocation of signal molecule into nucleus, the dashed line – the other variants of signal transduction.

In the first type of these interactions (Figure 20A) cytosolic prtein kinase penetrate to the nucleus, for example, MAP-kinase or a catalytic subunit of protein kinase A. In the nucleus they phosphorylate one (or more) of the intranuclear transcription factor (regulatory protein) that alters its affinity for DNA and /or its degree of activity. For example, protein kinase A is involved in cell development, synthesis of hormones and maintaining the circadian rhythm. In the second variant (Fig. 20B) a signal transmits to the nucleus protein phosphorylated by it. Prior to this process it was a latent transcriptional factor, and after the phosphorylation it becomes active, enters the nucleus and binds specifically to DNA. 42

In the third variant (Fig. 20C) an inhibitor or «anchor» subunit is phosphorylated in the protein complex and as a result of it is cleaved.After releasing from the complex it becomes an active transcription factor, which enters into the nucleus and binds to DNA. All three types of transmission of signal to the nucleus are associated with protein kinase phosphorylation of regulatory proteins – transcription factors and their precursors. The binding of active transcription factors to regulatory regions of DNA occurs rapidly and starts or enhances the process of transcription of «early» genes, i.e. genes responsible for rapid (within 15 minutes) cells responses. Emerging mRNA causes synthesis of protein products of «early» genes, which become the new transcription factors. The latter stimulate the «late» genes whose activity is realized within several hours or days.

Figure 20A. The first type of interaction of transcription factors with DNA

Figure 20B. The second type of interaction of transcription factors with DNA 43

Figure 20C. The third type of interaction of transcription factors with DNA

Figure 21. Role of perception and transduction of stress signal in the genome activation (T.V. Chirkova, 2002)

Discussed ways of perception and transduction of signals are used under the influence of stress factors. Perception and transmission of stress signal (drought) in the kernel are as follows: the receptor is localized on the plasma membrane, receives the signal and transmits it via intermediates – a signal transducer. Protein kinases and phosphatases either phosphorylate transcription factors in the 44

nucleus or their phosphorylated proteins enter into the nucleus and interact with transcription factors. This results in activation of stressinducible gene and as a consequence, in the synthesis of mRNA and stress proteins such as chaperones, ubiquitin, aquaporins, that enhance the plant tolerance (Figure 21). Chaperones and protease inhibitors.The proteins are called chaperones which bind polypeptides during their folding, or during the formation of tertiary structure, and assembly of the subunits of the protein molecule, i.e. the formation of quaternary structure. Interacting with the polypeptides, chaperones prevent mistakes in folding and assembly, and this prevents the aggregation of polypeptide chains (Figure 22).

Figure 22. Role of chaperones (http://www.lysosomalstorageresearch.ca/Fabry_eClinic/iv-chaperone-therapy.html) 45

Some chaperones play the role of «repair stations», correcting the incorrect folding. One of the main functions of chaperones are folding and unfolding and the assembly and disassembly of protein in their transport through the membrane. Polypeptide chain can pass through the pore in the membrane only in the expanded form. In cytosol some chaperones interact with the newly synthesized polypeptide chains and maintain their linear structure so that the polypeptide chain may be directly transported to the desired cell compartment. Other chaperones bind to amino acids of the polypeptide chain, as it will appear on the other side of the membrane and perform the folding.

Figure 23. Protease inhibitor (http://www.wjgnet.com/1949-8454/full/v2/i3/48.htm) 46

In the case of dehydration the tendency to damage cells and denaturation of proteins increases, so the protective role of chaperones in these conditions increases too. Stressful conditions activate the biosynthesis of chaperones in cells. When the osmotic stress occurs, the biosynthesis of inhibitors of proteases is induced, that prevent proteolytic degradation of proteins and cells retain their structure and functional properties (Figure 23) (Alehina et al., 2007). Proteases and ubiquitins. During cell dehydration, despite the effect of tread compounds and chaperones, some cellular proteins were denaturated. Denatured proteins must be hydrolysed. This function is performed by a protease and ubiquitin genes expression which is also induced by stress conditions. Ubiquitin is a low molecular weight (8.5 kDa) and highly conservative proteins. and highly conservative proteins.Binding to N-terminus of the denatured protein, they make it available to the action of protein proteases. In this way, the selective degradation of denatured proteins occur (Figure 24).

Figure 24. Activation of ubiquitines (http://physrev.physiology.org/content/82/2/373) 47

Osmolytes. Under salt stress and during denaturation regulation of osmotic pressure in the cytoplasm of cells is predominantly due to the biosynthesis of low molecular weight organic compounds, which are called osmolytes. This is a relatively small group of chemically different low molecular weight organic compounds (Figure 25). They are well soluble in water, non-toxic and, unlike inorganic ions do not cause changes in the metabolism, and they got their second name «compatible» solutes. Compatible substances tend to be neutral at physiological pH. In cytoplasm they are in undissociated form or in the form of zwitterions, i.e. molecules bearing positive and negative charges that are spatially separated. Some osmolytes are amphiphilic compounds. Molecules of amphiphilic substances have nonpolar (hydrophobic) and polar (hydrophilic) groups. The osmolytes includes also some polyhydroxylic compounds. To osmolytes belong proline, glycine betaine, mannitol and others (Alehina et al., 2007).

Figure 25. Osmolytes (http://www.intechopen.com/books/abiotic-stress-plant-responses-and-applicationsin-agriculture/abiotic-stress-adaptation-protein-folding-stability-and-dynamics)

The overall function of osmolytes is the participation in osmoregulation. Many of the inorganic ions, such as Na+ and Cl- in the 48

high concentrations are toxic, so they can not be used in the plant cell regulation of the osmotic pressure of the cytoplasm. At the same time, compatible with the biopolymers, osmolytes can be accumulated in the cytoplasm to several hundred macromoles per gram concentration without that toxic effect. Consequently, it is osmolytes rather than inorganic ions cell that is used for regulating the osmotic pressure of the cytoplasm. The role of osmolytes is especially important in conditions of drought and salinity, when it is necessary to concentrate in cells osmotically active substances. Differences in resistance of plants to dehydration are related to the degree of efficiency of system of osmolytes biosynthesis (Figure 26). Xerophytes and halophytes are plants living respectively with a low moisture content in the medium and on saline soils, osmolytes are synthesized with higher speed and accumulate them in large amounts compared to the plants growing under normal conditions in the absence ofsoil salinity effect.

Figure 26. Osmolytes functions (http://jeb.biologists.org/content/210/9/1622/F1.expansion.html)

Along with osmoregulation the compatible substances perform another very important function in dehydration – protective function 49

with respect to the cytoplasmic biopolymer. Therefore they are called osmoprotectants. It is believed that osmolytes do not destroy the hydration shells of biopolymers. Unlike Na+ and Cl- ions osmolytes, such as proline and glycine betaine do not penetrate through the hydration shell, and do not come into direct contact with the protein, but they prevent destruction by ions the hydration shell of the protein and its denaturation. Aquaporins. Transmembrane movement of water occurs mainly through water channels formed by aquaporin proteins. Due to changes in the number of water channels in the membrane and their conductivity allows for quick regulation of transmembrane fluxes of water, this is especially important when water deficit occurred. It was shown that the water uptake by cells in response to increase of intracellular concentrations of the osmolytes, accompanied by reduction of RWC (relative water content) during drought and turgor occurs through water channels. In A. thaliana water deficit induced expression of gene RD28, which encodes aquaporins localized in the plasma membrane. Genes encoding aquaporins are identified in M. cristallinum. It was shown that the number of transcripts of these genes correlates with turgor pressure in leaf cells of M. cristallinum under the action of the plant at high salt concentrations (Alehina et al., 2007).

Figure 27. Aquaporins (http://cstl-csm.semo.edu) 50

After treatment of the plant with sodium chloride in the period of the lowering of turgor pressure the number of transcripts decreased. Subsequently, the osmolytes accumulation increased and turgor recovered (Figures 27, 28). The increased concentration of transcripts in cells activated the translation process. The increased concentration of transcripts in cells activated the translation process. Increase of aquaporins in membrane and its subsequent activation, leads to increase in the water conductivity in plasmalemma, consequently, to increase of the water flow into the cell during recovery of turgor. During the drought the content of aquaporins increases not only in the PM, but also in the tonoplast. This increases the water conductivity in tonoplast, which also seems necessary to restore the RWC and turgor pressure (Figure 29). Changes in the activity of existing water channels of the membrane play an important role in the regulating of water conductivity in membrane under stress. One of the mechanisms of such regulation is the phosphorylation and dephosphorylation of aquaporins.

Figure 28. Water movement across membrane (http://plantphys.info)

51

Figure 29. Membrane transport systems in plant cells (http://www.cosmobio.co.jpg)

The phosphorylation results in the activation and dephosphorylation, consequently, it reduces the activity of water channels. The increase of aquaporins occurs as following: NaCl → reduction → number of transcripts rise with increasing content of osmolytes (the group of chemically different low molecular weight organic compounds, in contrast to inorganic ions are nontoxic and do not cause changes in the metabolism and are synthesized in response to water deficit – proline, glycine betaine and etc.) → activation of translation → increase of aquaporins → increase of their activation → increase water conductivity of plasma membrane → increase the flow of water into the cell. The set of proteins appeared in the plant cells due the water deficiency. Some of them are involved in the formation of resistance mechanisms directly, while others are involved in the regulation of gene expression induced by a stressor. The genes are expressed in plants at water stress are divided into 2 groups: functional and regulatory. The first group, the functional genes, includes genes that are directly responsible for the formation of resistance mechanisms, i.e. biosynthesis of aquaporins forming aqueous channels and enzyme 52

required for the biosynthesis of osmolytes (proline, glycine betaine, polyalcohols and etc.), proteins which protect the membranes and macromolecules (LEA proteins, chaperones and etc.), proteases and ubiquitins, accelerating protein metabolism in stressful conditions, and enzymes involved in detoxification (SOD, ascorbate peroxidase, glutathione-S-transferase and etc.). The second group, the regulatory genes, contains genes of proteins that are involved in signal transduction by expression of other genes forming mechanisms of resistance, such as genes of kinases, phospholipase C. This group includes genes of transcriptional factors that «recognize» the DNA elements in the genes expressed in stress. Test questions: 1. What are the levels of regulation of cellular response? 2. What characterizes the level of transcription? 3. What characterizes the level of translation? 4. What are the characteristics of mature protein level? 5. What are the three main options for the interaction of transcription factors with the cytoplasmic regulatory regions of DNA ? 6. What is the function of chaperones, inhibitors of proteases, osmolytes? 7. What are the safety and regulatory functions of the proteins induced by water deficit?

1.3.5. Regulation at the metabolic level The metabolic regulation system is based on the change in the functional activity of the enzymes. In living cells there are several ways to affect the enzymatic activity (Black, 1986; Field, 1997). Among these ways the most common is regulation by acting on enzymes such factors of intracellular environment as ionic strength, pH, temperature, pressure, and others. In this nonspecific regulation H+ ions play a special role. Most enzymes have a well-defined peak of activity in certain pH range (T.V. Chirkova, 2002). Isosteric regulation (regulation by substrates, cofactors and reaction products) enzyme activity is carried out at the level of their catalytic centers. Reactivity and orientation of the catalytic center of the enzyme depend on the amount of substrate (the law of mass effect). The intensity of the enzyme is defined as the presence of cofactors, coenzymes for two-component enzymes (nicotinamide adenine dinu53

cleotide for alcohol dehydrogenase), the specific effect of divalent metal ions (Mg2+, Mn2+, Zn2+), as well as inhibitors. The activity of these enzymes or others may be related to the competition for the common substrates and coenzymes, which is one of the modes of interaction of different metabolic cycles. Some enzymes except the catalytic (isosteric) centers have also allosteric centers, i.e. located elsewhere receptor sites which serve for binding of allosteric effectors (regulators). Typically, allosteric enzymes have catalytic and regulatory subunits (Figure 30).

. Figure 30. Allosteric enzymes (http://academic.pgcc.edu/~kroberts/Lecture /Chapter%205/enzymes.html)

As effectors may act certain metabolites, hormones or substrate molecule. As a result of binding of a positive or negative allosteric effector to the active center the change in the whole enzyme structure (conformation) is occurred, which leads respectively to activation or inhibition of the functional activity of the catalytic center. An exam54

ple of allosteric regulation is a regulation of the activity of phosphofructokinase – the key enzyme of glycolysis (anaerobic phase of biological oxidation of glucose). This enzyme carries out the transfer of a phosphate group from ATP to fructose-6-phosphate. It is allosterically inhibited by phosphorenolpyruvate, ATP, citric acid. When the concentration of these compounds is high (cell energy-rich), the oxidation of glucose via glycolysis is inhibited. On the contrary, with a lack of energy in the cell an orthophosphate is accumulated, which is an allosteric activator of phosphofructokinase. Consequently, the rate of glycolysis and ATP synthesis increases. An important method of regulating enzymatic activity is the transformation of latent enzyme (zymogen) into an active form. This is achieved by the destruction of certain covalent bonds in the molecule of polypeptide by proteases. During the limited proteolysis from the zymogen is separated a certain part of a polypeptide that converts the enzyme into an active form. Modification of the enzyme structure is another effective way of regulating their activity (T.V. Chirkova, 2002). An activation of many enzymes or an inactivation of them depends on the phosphorylation or dephosphorylation of protein kinases by protein phosphatases. There are other ways of modifying the structure of the enzymes. Potentially active enzymes may not function because their compartmentation (i.e., location in the special «compartments» cells), such as lysosomes, where an acidic pH, free radical oxidation of membrane lipids and some fat-soluble vitamins and steroids contribute to the release of lysosomal hydrolases. Inactivation of the enzyme may be due to their binding to specific inhibitors of protein nature, as well as their total destruction by proteases. Metabolic system of regulation that is based on the change in the activity of enzymes is very important under stress conditions. Since the enzyme activity is pH dependent, and the impact of stressors usually leads to the decrease in the pH of the cytoplasm, regulation of intracellular pH in stress conditions is very important. In plants for this regulation are required two mechanisms: biophysical – electrogenic ATP-dependent proton pump, whereby the hydrogen ions through the membranes are derived outwards against an electrochem55

ical gradient and biochemical – pH-sensitive processes of carboxylation and decarboxylation of organic acids during which is produced or consumed proton. Primary active transport is linked to ATP hydrolysis or redox reactons in the electron transport chain in chloroplasts and mitochondria (Figure 31). An example of the latter is the direct use of energy of respiration in ion transport against a concentration gradient without the prior accumulation of ATP. The mechanism of this phenomenon is that as a result of respiration on one side of the membrane (in outer side hydrogen ions are accumulated and the inner side of the membrane negatively charged) the cations enter inside, attracting to the negatively charged inner side of the membrane (T.V. Chirkova, 2002). There is another mechanism of active transport of substances, which is called the secondary active transport. Specific proteinsfunction as transporters of ions and the energy of ATP is released by using ATPase and spent on their movement through the membrane (Figure 32).

Figure 31. Primary and secondary active transport (https://kaiserscience.wordpress.com/biology-the-living-environment/cells/activetransport-across-cell-membranes/ 56

Figure 32. Primary and secondary active transport (http://www.slideshare.net/mrtangextrahelp /tang-06-transport-across-membranes)

Due to H-ATPase protons exit from the cell occurs and on the membrane electrochemical potential difference arises (ΔμH+). It is used for the transport of other ions with the participation of transporters of ions. Since the primary active transport of H+ against the electrochemical potential gradient mediates the transport of another ion by a gradient of electrochemical potential, this type of transport is called the secondary active transport (Field, 1986; Yakushkina, 2005). The gradient of pH and membrane potential generated by the H+-electrogenic (generates an ions gradient) pump is the driving force of the secondary active transport, such as H +-substrate symport and H+ -substrate antiport. Since this secondary transport transports the protons to the cytoplasm, there is always the danger of potential acidification. The acidification of the cytoplasm may not only under stress conditions, which inhibits the proton pump, but also in normal physiological conditions, when the input of protons predominates over their releasing. Possibly, therefore plant cells acquired unique 57

biochemical pH-stat to maintain proton homeostasis (Chirkova 2002). Classic pH-state consists of a complex carboxylates (phospoenolpyruvate (PEP)-carboxylase) and decarboxylated (NAD-malic enzyme) enzymes, which differ in pH optimum. At an alkaline pH cytoplasm PEP-carboxylase activates (optimum pH 8), resulting in increased production of oxaloacetate (OA) which then is reduced to malate by malate dehydrogenase (MDH). Malate as a strong acid neutralizes the pH. At acidic pH NAD-malate enzyme (ME), the optimum pH 6, decarboxylated malate and pH are shifted to the alkaline side (Chirkova 2002) (Figure 33). PEP- carboxylase /рН 8/ Oxaloacetate (ОА) Malate dehydrogenase (МDH) Malate NAD-malic enzyme (МE), /рН 6/, Decarboxylation of malate Shift of рН to alkaline side Figure 33. Metabolic regulation, PH-stat

Thus, there is a regulation of the pH of the cytoplasm by the synthesis or degradation of malate by coordinating the work of the two enzymes (Figure 20). In addition to the normal for all organisms the way of glycolysis via pyruvate- kinase (PK) – the only way for non-plant organisms, plants have an alternative route via PEP carboxylase, MDH and malic enzyme.One of the physiological functions of this pathway is to substitute the reaction of pyruvate kinase in the case of inorganic phosphorus (Pin) deficiency. 58

Another unique feature of glycolysis in plants is the way of its control. If in non-plant systems the glycolytic flux is controlled by activating or inhibiting the first key enzyme – phosphofructokinase (PFK) through a series of effectors, whereas in plants feedback regulation occurs: the consumption of PEP by pyruvate kinase or PEP carboxylase leads to the inhibition of reactions in the main enzyme glycolysis – PFK (Figure 34). Inorganic phosphorus (Pin) is another product of PEP- carboxylase reaction allosterically (regulation by non-covalent attachment to the enzymes of modulators molecules) activates PFK. But Pin facilitates PEP-inhibition, as it is involved in reaction of use of PEP (Figure 20). With the help of feedback a removal of protons excess is controlled. The protonogenic glycolysis can proceed only if the cytoplasm makes alkaline that activates PEP carboxylase. Such situation occurs under aerobic conditions in the case where the exudation of protons is increased by H+-pump which activates due to increasing of potassium ions concentration from in the outside. Pyruvate kinase is activated at low pH, for example, under anaerobic conditions, or when the ionic transport is active. Probably when there is a special need for energy plant cells are ready to go to the acidification of the cytoplasm, which can be compensated "for opening the barrier" for glycolysis through PEP carboxylase. Further oxaloacetate (OA) which MDH reaction is reduced to malate. When decarboxylation of malate by NAD-malic enzyme occurs, pyruvate, NADH and carbon dioxide are formed. These products serve as regulators of the respiratory tract of feedback. Pyruvate has to be oxidized in the Krebs cycle, but it has not only the function of the respiratory intermediate, but its role is much wider. It allosterically regulates the distribution of electrons between the cytochrome and alternative way, which not sensitive to cyanide. NADH is transferred to basic electronic transport chain of respiration, and to the alternative path with the alternative oxidase (AO). However, carbon dioxide – a product of the same malic enzyme reaction at low concentrations inhibits the cytochrome pathway. Therefore, in the alternative path more electrons and protons enter (T.V. Chirkova, 2002). 59

Figure 34. The structure and function of biochemical рН-state (T.V. Chirkova, 2002) HK – hexokinase, PFK – phosphofructokinase, GAPD – glyceraldehyde phosphate dehydrogenase, CH – carboanhydrase, PK – pyruvate kinase, АА – acetaldehyde, АО – alternative oxidase, OA-oxaloacetate, PA-piruvic acid, CytО – cytochrome oxidase, LDG – lactate dehydrogenase, PD – pyruvate decarboxylase, ADH – alcohol dehydrogenase, PEP – phosphoenolpyruvate, МE – malic enzyme, H+

– release of protons;

H+

– formation of protons.

Thus, in the case when an alternative path is connected to the malic enzyme reaction, it acquires advantages in consumption of protons. As a result of it the alternative path is close related to malic enzyme: low pH activates the malic enzyme, the pH shifts to the alkaline side. An alternative way reserves the products of malic enzyme for oxidation. Independent on control of the energy charge the alternative way is able in a short time to respond to shifts in pH, which is 60

quite important because in situations requiring pH-regulation, a quick response of the cell is necessary. It is assumed that the unique (only in plants) alternative pathway respiration is associated with it in a pH-stat. This way of electrons are not coupled with the formation of energy, usually activated by stressful conditions. Under anaerobic conditions pyruvate can be used in the reactions of lactate dehydrogenase (LDG) or alcohol dehydrogenase (ADG) to form a lactate or ethanol. Thus there the regeneration of NAD + and H+ is formed. In the synthesis of lactate from glucose via glycolysis, pyruvate is produced by one proton per molecule lactate. The formation of ethanol and glucose in the same manner is not associated with the release or consumption of protons. If the lactate and ethanol are formed from malate through malic enzyme reaction, the protons are consumed more (one proton H+ per molecule of lactate and two protons H+ per molecule of ethanol) (T.V. Chirkova, 2002). Thus, the malic enzyme is regarded as a pH-sensitive trigger of system switching the production of protons (during glycolysis) on the consumption of H+: in an alternative way in aerobic respiration conditions and the formation of lactate or ethanol in anaerobic conditions. Test questions 1. What is the basis of the metabolic system of regulation? 2. What are the components of the metabolic system of regulation? 3. What characterizes the biophysical components of the metabolic system of regulation? 4. What characterizes the biochemical components of the metabolic system of regulation? 5. What is the pH-stat classic? 6. What is the function of PEP carboxylase in maintaining the pH of the cell? 7. What is the function of the malic enzyme in the regulation of the pH?

1.3.6. Regulation at the hormone level Hormonal system is a key factor in the regulation and management of plants. Phytohormones auxin (indole-3-acetic acid), cytokinins 61

(zeatin, izopenteniladenin), gibberellins, abscisic acid, ethylene are relatively low molecular weight organic substances with high physiological activity present in the tissue in very low concentrations (picograms and nanograms per 1 g wet weight) by which the cells, tissues and organs interact (Figure 35).

Figure 35. The structure of plant hormones (http://www.tcichemicals.com)

Typically, plant hormones are produced in one tissue and act in others, but in some cases they function in the same cells where they are formed. A characteristic feature of phytohormones, distinguishing them from other physiologically active substances (vitamins, trace elements) is that they include physiological and morphogenetic programs such as rooting, ripening, etc. (Polevoy, 1986, 1997; Kulaev, 1995). The place of the synthesis of indole-3-acetic acid (IAA) is developing buds and young growing leaves. Hence the polar auxin moves from living cells of vascular bundles to the tips of the roots with speed of 0.5-1.5 cm/h. Cytokinins are formed in the apex of the root and xylem vessels passively transport them to all parts of the plant. Synthesis of gibberellins and abscisic acid (ABA) occurs in the leaves from which they are transferred to other parts of the plant according to the phloem sieve tubes. Both of these plant hormones are produced in the root tips. 62

Synthesis of ethylene in the greatest quantity occurs where there is a high concentration of IAA. In addition, a large number of both ABA and ethylene are accumulated in any organism in a state of stress. Therefore, these plant hormones are often called stress hormones. In particular, the shortage of water in the guard cells of stomata rapidly increases content of ABA, which induces stomatal closing gaps, thus reducing the rate of transpiration (Figures 36, 37).

Figure 36. The role of ABA in signal transduction (http://mol-biol4masters.masters.grkraj.org/html)

Each of phytohormones is the basis of these systems including enzymes of synthesis, binding (conjugation) and release of hormone from bound state, the ways of membrane transport, mechanisms of action that are defined by the presence of receptors and their localization, and finally enzymes cofactors and inhibitors of destruction phytohormone. In turn, the system of separate classes of phytohormones is linked into a single hormonal system. This communication is carried out at both the metabolism of plant hormones and their mechanism of action. 63

Active forms of phytohormones act only on cells competent of these phytohormones, i.e. cells, membranes and cytoplasm which contain specific receptors for these phytohormones. Phytohormone interaction with its receptor triggers a chain of reactions converting hormonal signal in the functional response of the cell. These responses may vary depending on the type of receptor, the concentration of the phytohormone concentrations and ratios of the level of other plant hormones, as well as the relationship with the receptor.

Figure 37. ABA signal under stress conditions. (http://plantcellbiology.masters.grkraj.org/html/Plant_Growth_And_Development6Plant_Hormones-Abscissins.htm)

Hormonal system is one of the most important regulatory system that controls the life of each plant at all stages of its development, not only in the normal conditions of existence, but at different stress conditions (Figure 38). Typically under the influence of stressors the growth of plants is inhibited, the content of indolylacetic acid (IAA), gibberellins and cytokinins reduces, but the number of inhibitors – abscisic acid (ABA), ethylene, jasmonic acid increases. 64

ABA and ethylene are even called stress hormones. Reduction of the level of hormones-stimulants and accumulation of growth inhibitors during stress have an important adaptive role because they lead to decrease in the intensity of metabolic processes, stop of cell growth and division, the transition of the organism to rest. All this results in economical consumption of energy resources. The plant gets more opportunities to direct them to maintaining the structure of the cell (Chirkova, 2002). Interacting with the receptor triggers a cascade of transduction of ABA reactions leads to the accumulation of calcium and alkalinization of cytoplasm. This in turn activates a number of enzymes transduction: Ca-dependent protein kinase, Mgdependent protein phosphatase, MAP kinase cascade. As a result of cytoplasmic processes phosphorylation and dephosphorylation are enhanced. Thus, regulation of the activity of both, the key enzymes of different metabolic pathways and transcription factors, is performed.

Figure 38. Hormonal system of regulation controls the plant life (http://journal.frontiersin.org/article/10.3389/fpls.2013.00155/full). 65

The latter enter the nucleus, bind to the promoters of various genes and lead to their expression or repression.ABA plays an important role in the response of plants to dehydration, salinity, the effect of low temperatures, hypo- and anoxia (Chirkova, 2002). Thus, the following occurs: Hydrolysis of bound forms of ABA ↓ The increase of ABA content in cell ↓ The increase of ABA synthesis in plastids and roots of plants ↓ Transport of ABA to the shoots of plants ↓ Change of expression of genetic programs in cells (the inhibition of mRNA synthesis and related proteins, induction of expression of genes of specific proteins called «proteins responsible to ABA»)

All proteins are gene products induced by ABA and can be divided into two groups. The first consists of regulatory proteins, various transcription factors and transduction enzymes. Regulatory proteins – the products of «early» genes that are expressed in the same first moments of the action of the stressor. They typically control the further expression of stress-activated genes, the products of which include a variety of functional proteins. Stressful functional ABA-dependent proteins or Rab proteins (responsible to ABA) – are, for example, a large group of LEA proteins that protect cells from death in a deep dehydration (Figure 39). The part of the water deficit induced cytoplasmic proteins protects biopolymers and cellular structures formed by them against deterioration caused by dehydration. LEA (1ate embryogenesis abundant) – proteins, which were first identified as the gene products of genes LEA, expressed in the seed phase of their maturation and drying. Later, some LEA proteins were detected in vegetative tissues of 66

plants during their loss of water during the water, salinity and low temperature stress.

Figure 39. Induction of LEA proteins synthesis (https://www.jircas.affrc.go.jp/kankoubutsu/highlight/highlights2009/2009_08.html)

LEA proteins are mainly hydrophilic, and this is consistent with their cytoplasmic localization. Many of them are enriched with alanine and glycine and lack cysteine and tryptophan. LEA proteins in accordance with their amino acid sequences and structure are combined into five groups (1-5). There are assumptions about the specific functions of the proteins in each group (Alehina et al., 2007). Group 1 – characterized by a high content of charged amino acids and glycine, this allows them to bind water effectively . The presence of LEA proteins in the cytoplasm of the group gives it a high water retention capacity. Group 2 – proteins act as chaperones. Forming complexes with other proteins, they prevent the latter from damage in a cell dehydration. Some LEA proteins (3-rd and 5-th group) are involved in the binding of the ions concentrated in the cytoplasm when cells lose water. The presence of the hydrophobic region of these proteins 67

leads to the formation of a homodimer with hydrophobic sequences, facing each other, whereas the charged region on the outer surfaces of the protein is involved in binding the ions (Alehina et al., 2007). LEA proteins of 4-th group can replace the water in the membrane region and maintain the structure of these membranes during dehydration. LEA proteins play an important role in plant resistance to water deficit. A number of plant species show the correlation between survival under water deficit and the accumulation of cells in their LEA proteins. For example, overexpression of genes encoding LEA proteins in the transformant rice (Oryza sativa), is correlated with the high resistance of the plants to water deficit. In the normal development of plants LEA proteins are synthesized during late embryogenesis, when there is a natural seed dehydration. The synthesis of these proteins in late embryogenesis also induced by ABA that accumulates in seeds before starting their dehydration. If the leaves are under normal conditions, these proteins are not detected. Drought causes the accumulation of ABA in leaves that induces the synthesis of LEA proteins and late embryogenesis required for cell survival during leaf water deficit. The study of these proteins and their genes is crucial for the creation of droughtresistant crop varieties. The genetic engineering, which allows to transform plants by relevant genes, i.e. to introduce these genes into the DNA of plants and create a new genotype. The inducible proteins also include biosynthetic enzymes of osmotics, transport proteins – aquaporins, ion channels, transporters of lipids, antioxidant enzymes, enzymes C4 and CAM – photosynthetic proteins, pathogenesis. The effect of ABA on membrane transport is on the basis of such rapid hormonal reactions like closing of stomata. The worsening of gas exchange, while drought or flooding, contributes to the stabilization of the water regime, ABA inhibits the activity of H+-ATPase, which leads to lowering of the pH of the cytoplasm and increased hydrolytic processes. Ethylene is synthesized in response to various stressors: root hypoxia, fungal pathogens, bacterial and viral origin, drought, adverse temperature conditions, mechanical damage, contamination with heavy metals. Ethylene freely diffuses through the cells and quickly disappears. Up to 90% of the synthesized hormone plant leaves for 68

1 min. Nevertheless, it manages to contact with receptor located in the plasma membrane. The system of signal transduction involves GTP binding proteins, protein kinases and calcium. Ethylene is a less potent inhibitor than the ABA. At hypoxia ethylene induces an epinastia of petioles, stimulates aging and abscission of leaves, in adapted plants accelerates the growth of shoots during the flooding that is necessary for them to reach the surface of water; activates the enzymes involved in the lysis of cell walls and the formation of aerenchyma and thus makes the plant less protected from death due to oxygen starvation (Figure 40).

Figure 40. The role of phytohormones under stress conditions (https://secure.jbs.elsevierhealth.com) 69

At pathogenesis the plant receives a signal from pathogen and involves the synthesis of ethylene response, and that, in turn, triggers a complex program of chemical plant protection, which, in particular, includes the synthesis of phytoalexins, which play the role of antidotes against parasites. Ethylene also affects the content, transportation, education, or the degradation of auxin, cytokinin, ABA. Thus, the action of ethylene is associated with regulation of the processes occurring in the cell wall, gene expression of apoptosis, stress proteins, the interaction with other phytohormones. Perceptions of hormonal signal. In the study of the mechanism of phytohormones effect it is necessary to consider the perception of the hormonal signal of cell and transfer it to the genetic apparatus, the role of hormones in the inclusion, as the suppression of switching genetic programs. Receptors recognize a hormonal signal and form a hormone-receptor complex, which in turn determines the chain of processes in the cell required for its highly specific response to a plant hormone (Chirkova 2002). The role of the receptor in the perception and transmission of the hormonal signal can be represented as follows: Hormone + receptor ↕ Hormone receptor complex ↓ System of transformation and transduction of in cells ↓ Induction of physiological programs The animal hormone receptors are installed in two basic types. The first type includes hormone receptors, not penetrating into cells, which are located in the membrane. They recognize hormone molecule on the outer surface of the membrane and the interaction with them change their conformational state. Further the transfer signal through G-proteins to enzymes involved in the synthesis of secondary mediators is occurred. Intermediaries also contribute to en70

hancement of the signal and its transmission to the various components of the cell, in particular, protein kinases phosphorylate cellular proteins, and thus changes their properties. Shifts in the activity of enzymes, regulatory proteins, structural cells are transferred to the genetic apparatus, it is turned on (or off) the action of programs, determining the effect of hormones. Receptors of the second type, which include steroid receptors, interact with hormones in the cytoplasm or nucleus, and the hormone-receptor complex is directly involved in the regulation of genetic programs inducing or repressing gene expression (Chirkova, 2002). The success of genetic engineering allows by genetic probes to detect in the genome of the plant genes responsible for all main elements of the hormonal signal of the first type. Localized in the membrane the hormone-binding proteins are candidates for the role of receptors of the first type. They were found for auxin, ethylene, gibberellin. The protein with properties of auxin receptor was sequenced (amino acid sequence set). Collective efforts of scientists from different countries were able to establish that this protein functions as type of membrane receptors of hormones of animals using secondary mediators. Hormonal signal transduction. According to two types of receptors, there are two major mechanisms of hormonal signal transduction in the cell. The first hormone-receptor complex is formed on the outer surface of the plasma membrane. This causes a rapid opening of the ion channel and the entrance of ions into the cell, or the inclusion of systems of second messengers – protein kinases that leads to slower opening. Both mechanisms can lead to later effects – changes in processes that are regulated by the nucleus of the cell (Chirkova, 2002). The stimulation of cell division in the nucleus increases the concentration of Ca2+ ions. Consequently, to penetrate into the nucleus can not only protein kinases and modified by them their transcription factors, but the secondary mediators. However, the signal transduction of Ca2+ in the nucleus is not clear yet. Cytosolic second messengers and protein kinases may 71

regulate the expression of genes and post-transcriptional levels, but the specific mechanisms of signaling to the nucleus is not installed yet. In recent years, it has been definitively proven that there are multiple regulation by hormones and second messengers all the basic functions of mitochondria, the activity of enzymes, including the Krebs citric acid cycle, the work of the respiratory chain, oxidative phosphorylation and energy processes. Thus, in each cell there is a complex signal transduction systems converting the external signals into intracellular and then into signals organelles. The signals of the majority of hormones from the plasma membrane receptors in the cytosol are transferred through the transmission system of secondary mediators (mostly protein kinases), but phosphorylation of the proteins alter their activity. Test questions 1. What kind of hormones are called stress hormones? 2. What is the role of ABA under stress? 3. Which proteins are functional stress ABA-dependent proteins and what is their role? 4. What is the function of LEA-proteins? 5. How does the perception of the hormonal signal occur? 6. How is the hormonal signal transduction performed?

1.3.7. Trophic regulation system Trophic regulation, interaction with the help of nutrients, is the easiest way of communication between the cells, tissues and organs. In plants, roots and other heterotrophic organs depend on the receipt of assimilates – products formed in the leaves during photosynthesis. In turn, the aerial parts need minerals and the water absorbed by the roots from the soil. The roots use assimilates coming from escaping on their own needs, and part of the transformed organic matter moves in the opposite direction. Trophic regulation is more quantitative than qualitative in nature. Thus, assimilates communicate between their suppliers and customers (Polevoy, 1997; Chirkova, 2002). 72

73

Figure 41. Regulation of genomes by stressors and sugars as signal molecules (Sheen et al., 1999)

Sugars not only perform the role of substrates for growth of heterotrophic organs but they are also signaling molecules that control the expression of genes that regulate the production of sugar in the leaves, and their consumption by other organs of plants.The sensor of hexose sensor signal may be a hexokinase, and sensor of sucrose signal is an extracellular or membrane-bound invertase. Entering in cell, hexoses is involved in the repression of the genes encoding the synthesis of a number of photosynthetic enzymes (photosynthesisdependent genes) that leads to the inhibition of photosynthesis (Figures 41, 42). However, there is a transduction of specific gene of consumption, i.e. genes encoding extracellular invertase which hydrolyzes the sucrose coming from sieve tubes into glucose and fructose, and contributes to supply carbohydrates consuming tissues (Chirkova, 2002). When comparing the effect on the genome of various stressors and carbohydrates there is identified a common origin of the different signals and similarities of impact on the relationship between production and consumption of sugars. Activated by stressor and sugar signaling pathways work independently from each other, but may communicate during transduction (Figure 42). Abiotic stressors (drought, salinity, low temperature) lead to increased hydrolytic processes, in particular starch hydrolysis. As a result – the accumulation of sugars occurs, which is accompanied by inhibition of their formation in the process of photosynthesis. In the case of infection (biotic stressors) pathogens released substances, which include high glucans there are oligoglycosides which «recognize» membrane receptors of plants. Thus, elicitors of carbohydrate nature induce expression of the genes that encode the synthesis of the compounds that play a protective role. All of these effects, as well as plant hormones (auxin, ethylene, gibberellin, cytokinin) are involved in the regulation of synthesis of extracellular invertase. In all likelihood, this may indicate its important role in strengthening the regulation of carbohydrate synthesis signals and carbohydrate intake. Carbohydrate signal transduction cascade comprises a protein kinase phosphatase. In transduction involving MAP kinases, 74

calcium ions, as intermediates, they can probably be included in the trophic system of education and the regulation of carbohydrate consumption.

Figure 42. Sucrose movement and it’s metabolism (http://journal.frontiersin.org/article/10.3389/fpls.2013.00201/full)

In yeast, phosphorylated protein Ssk1p activates various signal transduction pathways, including the MAP (mitogen activated protein) kinase cascade consisting of three sequentially phosphorylated in the presence of ATP, protein kinases: Ssk2p (MAPKKK) – MAPKK kinase, Rbs2r (MAPKK) – MAPK – kinase and Nog1r (MAPK) – MAP kinase (Chirkova, 2002; Alehina et al., 2007). Activated by phosphorylation MAP kinase induces the expression of many genes of yeast cells in aqueous and salt stresses, in particular of the genes encoding enzymes of glycerol biosynthesis – main osmolyte in yeast. Homologues of some components of MAP kinase cascade yeast cells are found in plants. Gene expression of these components is induced by stress conditions. Changes in the content of various nutrients have effects on metabolism and morphogenetic physiological processes in plants. 75

Under stressthe competition of various organs for nutrients intensifies, but usually plant development continues, it mayeven increase the speed of it, thus the formation of seeds accelerates. However,the formed organs are smaller and the number of leaves, fruits, seeds decreases. Nevertheless, the final seed value is a little different from normal. This indicates the regulation of trophic interactions with other systems that provide the link all parts of the organism. 1.3.8. Electrophysiological regulation Electrophysiological regulation system in plants involves the occurrence of gradients of bioelectric potentials (BEP) between different parts and generation of propagating potentials in plants (action potential and variable potential) (Polevoy, 1997). BEP gradients arise due to differences in the values of the membrane potential (MP) in the cells of different tissues and organs of vegetation zones of the organism. These gradients are not constant, and make slow periodic fluctuations due to changes in the conditions of internal and external environment. The difference of potentials between any parts of the plants do not exceed 100-200 mV, since these values correspond to the maximum value of membrane potential of plant cells. Action potentials (AP) represent electrical pulses depolarization of MP with a duration of 1-60 seconds and are distributed on the plasma membrane via the plasmadesmata in cells of the cell at a speed of 0.1-1.0 cm/s. Action potential is induced onlywhen the depolarization of the plasma membrane reaches a critical level and moves along the living cells of vascular bundles (Polevoy, 1997). Bioelectrogenesis, i.e the ability to generate electric potentials is one of the universal and essential properties for the life of the of living systems. Bioelectrogenesisof plants is important for the coordination of their functional activity and morphogenesis (Chirkova, 2002). As a result of bioelectrogenesis the bioelectric potentials occur. There are a resting potential (RP), the potential difference 76

across the membrane in the resting state, and excitation potentials (EP), the change of the RP in the excitation. The resting potential is composed of two components: the diffusion and metabolic. The diffusion component appears in the passive redistribution of ions across the membrane, respectively, previously formed by ion gradients. It is particularly important for two factors: the value of ion concentration gradient and the permeability coefficient of the membrane. The metabolic component is generated by electrogenic pump represented in plants predominantly H+ pump – H+ -ATPase. Excitation potentials are of two types: a variable potential (VP) and the action potential (AP). The first arises under the influence of strong stimuli (burns, mechanical tissue damage). The second corresponds to that in animals, but the duration of AP in plants is longer. The basis of the AP (local and disseminated) constitute passive ion fluxes. All these kinds of electrical activity are included in the system of electrophysiological regulation (Chirkova, 2002). Functions of bioelectrogenesis in plants are divided into control (mainly resting potential RP) and signal (action potential AP). To manage functions energy, regulatory and adaptive are included. Bioelectrogenesis on the surface of membrane (on intracellular membranes arises a potential difference, but the electrical properties of the cells are determined primarily by electrogenesis on plasmalemma) is as following. The potential difference is of ionic nature, i.e. due to the occurrence of ionic asymmetry uneven distribution on both sides of the membrane of cations and anions. The magnitude of the membrane potential is 100-300 mV. Electrical energy stored in the membrane can move to other forms of energy. When the membrane thickness of 100 A, the electric field strength on it is 10 V/cm, it can not affect its phase-structural state. The molecules of membrane proteins change orientation or conformational state, in the lipid matrix microviscosity changes occur, as well as the phenomenon of electrostriction (electromechanical com77

pression). Changes in the potential difference across the membrane under the influence of different factors affect the work of membrane proteins – enzymes, receptors, channels, systems, primary and secondary active transport. Thus, the regulation is carried by RP of cell activity (Chirkova, 2002). Membrane potentials are very labile and their changes under the action of various external conditions may have adaptive significance. In the first stage of the stress response occurs primary depolarization – a sharp drop in the membrane potential, which is accompanied by a decrease in the intensity of many membrane dependent processes. This contributes to «avoidance» cells from the negative effects of the stressor. In the next phase of the adaptive depolarization a membrane potential increases, membrane-bound system starts to work at a higher level, and cell activity is restored. Changes in membrane potentials are, apparently, the earliest in the development of a general adaptation syndrome and may serve as a stimulus for the emergence of adaptive responses at other levels of the organism. In plants among all types of electrical signals the main attention is focused on AP because their generation and distribution are one of the universal methods of transmitting of information about external influences, i.e. the main function of AP is a function as a signal. Unlike the action potential of animals Na+ and K+ and Cl- and K+ are not involved (Chirkova, 2002). Initially, under the influence of external stimuli the permeability for calcium ions in membranes increases that results from the opening of calcium channels. The concentration of calcium ions is much higher in the external environment, so they get inside the conducting action potential AP cells along the gradient. Entering inside the excited cells, Ca2+ activated chloride channels, which are also open. This makes it possible to get out of chlorine ions, which are more within the cells. Stream of negatively charged chloride ions outward leads to depolarization of the membrane, as its external side is positively charged and the inside negatively, this raises the ascending branch of action potential.

78

External stimulus

↓

The increase of membrane permeability for Са2+

↓

Entry into the cell of Са2+ions

↓

Activation of chloric channels by Са2+ ions

↓

Release of Cl-

↓

Depolarization of the membrane, the occurrence of the ascending branch of AP

↓

The opening of potassiumchannels

↓

Repolarization of membrane

↓

Restore the original values of the potential Figure 43. Electrophysiological regulation

Membrane depolarization facilitates the opening of potassium channels and an outward flow of potassium ions, which, as well as more chlorine ions within the cell than in the external medium. This thread has on membrane potential repolarization effect, i.e. leads to a recovery of its original value. Spread throughout the plant and reaching certain organs, action potential significantly alter the ionic composition of cells, especially the content of potassium and chlorine ions. Since the level of metabolic processes in the tissues is strongly dependent on the ionic composition, action potential may cause them a response. Thus the occurrence of AP in response to an external stimulus is nonspecific (Figures 43, 44).

79

80

Figure 44. Basic scheme of anticipated changes of ion transport in yeast cell under salinity stress (http:// journal.frontiersin.org/article/10.3389/fpls.2015.00425/ full)

The signal (information) role of action potential in normal processes of growth and development is manifested, for example, in the spread of electrical impulses toward the ovaries to prepare it for the perception of pollen and fertilize the plants or to motor responses: mimosa, sundew, venus, flytrap, and so on. Furthermore, the generation of action potential in plants occurs at moderate changes in the environment, even when the temperature difference is 1-2 °C. In all likelihood, this is a kind of «warning» for organs and tissues of a possible impending noticeable decrease in temperature. «Warning» role of action potential is reduced to a temporary increase in the stability of organs and tissues of plants to adverse effects, which is nonspecific (Chirkova, 2002). Thus, at weak effects associated with the function of action potential anticipatory reflection of reality, while strong stimuli – with the performance of the primary emergency communication signal, it allows the plant to begin quickly rebuilding vital functions. All intercellular regulatory system are closely linked. Plant hormones influence the activity of membrane transport and trophic factors. Electrical signals operate the transport of ions, metabolites, and so on. However, intracellular regulation systems at the cellular level are only through intracellular systems. In other words, the whole body while maintaining the principle of hierarchy of systems implemented interconnection of all regulatory mechanisms. Regulation system works in sensitive and tolerant plants. However, the first switching to a new level of metabolism is very fast, making it difficult regulatory association of defense reactions. Therefore the balance of individual protective reactions is soon violated and the disorder of metabolism occurs. In adapted plants during the long evolution period the necessity in a gradual and sequential interaction of different systems of regulation is formed for plant survival. Only concerted effort ensemble regulation systems are needed to coordinate the complex components of protective reactions, to contribute to the prolonged existence of the organism in adverse environmental conditions (Chirkova, 2002). Distributing of action potential do not carry specific information, and represents only a signal of external influences. However in tissues and organs AD may cause with nonspecific responses change in 81

some specific processes specific to the organ (e.g., the leaves change photosynthesis, roots increased absorption of substances and etc.). Test questions 1. What is the role of sugar in the signaling system? 2. What is the role of hexokinase and invertase in a signaling system? 3. What is bioelectrogenesis? 4. What do the terms «resting potential» and «potential field» mean? 5. What are the components of «resting potential»? 6. What are the types of excitation potentials? 7. What are the functions of bioelectrogenesis? 8. How does the membrane potential act during stress? 9. How are intercellular regulation systems in the plant organism interconnected?

82

2. PHYSIOLOGY OF PLANT RESISTANCE TO HEAVY METALS The development of modern technologies in industry and agriculture leads to an intense increase in the quantities greater than background concentrations of heavy metals in the environment. Constantly growing volumes of industrial wastes form new technological landscapes. They become a source of intense dust and extend for hundreds of kilometers, polluting the environment and posing a threat to public health and biodiversity in the region.

Figure 45. The sources of heavy metal contamination of the environment (http://thewatchers.adorraeli.com/2011/08/19/heavy-metal-in-and-around-thelakes/metals)

The source of anthropogenic inputs of heavy metals in the soil are industrial emissions due to the mining, metallurgical and chemi83

cal industry (Figures 45, 46). Heavy metals entering the atmosphere and, accordingly, to the soil from industrial emissions actively affect the vegetation and the ecosystem as a whole. Contamination of soil, plants and water with heavy metals in the vicinity of large industrial centers has become one of the most pressing environmental problems. Heavy metals are contaminants of anthropogenic origin which are characterized by high toxicity, mutagenic and carcinogenic effects. An important feature of heavy metals is that they belong to a class of non-specific substances that are «normally» present in the biosphere as opposed to specific contaminants such as pesticides. Another difference of heavy metals from other contaminants – heavy metals in principle does not apply the concept of «self-cleaning». As a result, all the processes of migration and scattering cause an irreversible increase in the concentration in water, soil, air, food, i.e. the pollution of the environment and biota (Panin, 1995).

Figure 46. The effect of heavy metals to human (http://www.slideshare.net/tutan2009/heavy-metal-pollution-in-soil-and-itsmitigation-aspect-by-dr-tarik-mitran)

84

2.1. General characteristics of heavy metals The term «heavy metal» characterized by a wide group of metals. The criteria used supplies numerous characteristics: atomic mass, density, toxicity, prevalence in the environment, the degree of involvement in the natural and technological cycles. In the works devoted to the problems of environmental pollution and environmental monitoring, to date, heavy metals include more than 40 metals of the Mendeleyev periodic system with atomic mass over 50 atomic units and a density more than 5 g/cm3. By classifying of N.Reymers, to heavy metals must be belong metals with a density of more than 8 g/cm3: V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Mo, Cd, Sn, Hg, Pb, Bi, and others. The important role in the definition of the term "heavy metal" played the following features of metals: high toxicity to living organisms at relatively low concentrations, as well as the potential of bioaccumulation. Almost all heavy metals (with the exception of lead, mercury, cadmium and bismuth, the biological role of which at the moment is not revealed), are actively involved in the biological processes. They are a part of many enzymes.

Figure 47. Heavy metals (http://www.phadjustment.com/TArticles/Heavy_ Metal_ Reduction.html)

By the definition of N. Reymers, separately from the heavy metals are considered precious and rare metals, respectively, and only 85

Pb, Cu, Zn, Ni, Cd, Co, Sb, Sn, Bi, Hg belong to heavy metals. In applied studies as heavy metals most commonly considered the following metals -Pt, Ag, W, Fe, Au, Mn (Figure 47). First of all, there are of interest those metals that most pollute the atmosphere due to the use of them in large amounts in industrial activity. And as a result of accumulation of heavy metals in the environment they pose a serious threat in terms of their biological activity and toxic properties. The most common metals, polluting the area around the metallurgic factories are zinc (Zn), copper (Cu), lead (Pb) and cadmium (Cd). Since many heavy metals are micronutrients the contamination of soils by heavy metals is a substantially accumulation of large amounts of essential trace elements (Zn, Mn, Cu, Ni) or metal, which may act as their analogs (Cd, Pb, Hg). Biophilicity and toxicity of chemical elements – the two sides of the same phenomenon: if the large amounts of elements it is required for living organisms, so they are less toxic. It follows from this fact that the trace elements are strong toxicants. Heavy metals such as copper (Cu) and zinc (Zn) are essential elements for plant growth, as a part of many enzymes and other proteins. Copper is a key component of several enzymes such as cytochrome oxidase, ascorbate оxidase, as well as a number of nonenzymatic nature proteins. Copper is a part of plastocyanin– component of electric transport chain of photosynthesis. It plays an important role in the life of organisms, increases oxidative processes, promotes the formation of chlorophyll. Zinc is essential for normal growth and development of most organisms. It is an important component of protoplasm, as associated with enzymes, regulators of cellular metabolism. Zinc is involved in the synthesis of chlorophyll, protects it from the destruction, affects the assimilation of nitrogen by plants, stimulates enzymes in carbohydrate and energy metabolism, participates in the construction of a number of enzymes (some phosphatase). The trace elements at high concentrations are very toxic to plants. Excessive amounts of them lead to the symptoms of the toxicity and suppressing plant growth by binding them to sulfhydryl groups of proteins, which leads to destruction and inhibition of their structure. 86

Cadmium and lead (Pb) are the most common environmental pollutants. Cadmium is a heavy metal normally found in the soil in trace amounts. However, the industrial activity and human agricultural practices increase the levels of cadmium in the soil. Throughout used fertilizers and pesticides can contain large amounts of this metal, which for a long time enters the soil with fertilizers. Most Cd, contained in the soil, available for plants, because the it’s soluble fraction is up to 35% of the total content. Cadmium is highly toxic, and has a high mobility. It is also noted the greater availability of Cd in comparison with other heavy metals such as Zn, Cu, Pb, which has a higher coefficient of biological absorption. Cadmium remains in the human body for many years, so the consumption of food with a content of the metal can induce chronic toxicity (Figure 48). Cadmium is a calcium antagonist. Increased accumulation of cadmium in the human body causes disease Itai-Itai, which reduce the content of calcium in the bones, which causes them to soften. The World Health Organization (WHO) has set a maximum limit of Cd in food – 60-70 mg per day, and the International Codex on Food Commission had set a limit of 0.1 mg/kg for cereals and oilseeds in international markets. Even plants growing soils that are deemed uncontaminated or slightly contaminated, as a result of cadmium contamination coming from the fertilizer or the atmosphere, some crops like durum wheat, flax, sunflower, potato can accumulate Cd in quantities exceeding the current maximum level for consumption (Kvesitadze et al., 2005). Lead (Pb) is one of the most dangerous pollutants. The main source of contamination of the environment – anthropogenic pollution. Extensive using of lead as an anti-knock fuel oil is one of the main reasons for the increase in the content of terrestrial and aquatic ecosystems. In the presence of urban wastewater detergents these substances dissolve lead compounds (polyphosphates, aminopolycarboxylic acids). Especially toxic lead compounds characterized by containing toxic anions, such orthoarsenates, chromate and azide. Biocidal propertiesof different organic lead compounds, particularly tetraethyl lead, which is used to increase the octane number of gasoline. Flying tetraethyl spreads rapidly through the air and by the action of UV rays is split into radicals. Triethyl radical reacts 87

with different substances having acceptor properties. As a result the ion of triethyl lead (Pb (C2 H5)3+ exhibits hydrophilic properties, and the presence of ethyl groups gives to the ion a lipophilic character, where by the ion of tetraethyl lead readily penetrate cell membrane and binds to the sulfur atom, peptides and proteins, causing changes in the their structure. It should be noted that in view of the high toxicity the use of the ethyled petrol in many countries have banned or restricted (Kvesitadze et al., 2005).

Figure 48. Cadmium and lead toxicity (http://www.mdpi.com/2072-6643/7/1/552)

Thus, the heavy metals are the most toxic pollutants and the study of the effect of heavy metals on plants, searching for ways to prevent environmental pollution with toxic elements is one of the priorities.

2.2. Mechanisms of heavy metals uptake by plants Heavy metals enter the plants through the soil and atmosphere. Uptake of heavy metals by plants depends on different factors as 88

plant species, properties of the medium, chemical properties of the metal and etc. (Figure 49). Plants absorb metals from soil through the root system. When an excessive amount of heavy metals in the soil the plants accumulate them in the aerial parts and roots. Heavy metals from the air may penetrate through stomata, but mostly dust particles are firmly fixed on the wax structure of the epidermis. From an aqueous medium heavy metals can enter through the stomata and the cuticle, including passive diffusion and active transport.

Figure 49. Factors affecting the uptake of metals in plants (http://www.hindawi.com/journals/ijce/2011/939161/fig3/)

The concentration of heavy metals in the soil solution is an important factor for phytoavailability of metals. Transport of ions through the membranes is carried out in different ways depending on the chemical properties of elements and their biological significance. The study of cadmium absorption by plants showed that the transport through the membrane is a passive process at high concentrations 89

and active, requiring energy, at low concentrations of Cd in the environment. For example, most of the work with lettuce and lupine showed that metabolically active component and H+-ATPase take part in the absorption of Cd. An electrochemical membrane potential plays an important role in the admission of ions into the cell, although the mechanism of transport across the plasma membrane (PM) of root cells is not entirely clear. Determination of potential difference has shown that cytosol is negatively charged (100-150 mV), and the diffusion of Cd into the cell energetically preferred. Small, neutral molecules can permeate through the membrane by the conventional diffusion. The diffusion rate of the substance is determined by its solubility in the membrane, by the diffusion coefficient and the concentration gradient inside and outside the cell. For charged particles it is of great importance an electric potential difference on the outer and inner sides of the membrane. It is assumed that the surface charge of the plasma membrane has a huge impact on the transportation of ions into the cell and highly charged ions with high valence, and the change in charge of plasma membrane plays an important role in the regulation of the transmembrane passage of metals ions. The cations are transported across the membrane by the existence of negative charge on its surface, generated either metabolically dependent proton transfer H+ -ATPase (primary transport systems) or passive removal of H+ (secondary transport systems) (Salt et al., 1993) (Figure 50). For example, delivery into the cytoplasm of Ni is more dependent on its activity on the membrane surface, rather than on the concentration of the solution, i.e. ions flow into the cell depends more on the electric gradient, rather than on the concentration. This transport mechanism is characteristic of many divalent cations. Proof of Cd2+/H+-antiporter in the roots of oats in tonoplast vesicles it was represented by Salt and Wagner (1993). The V-type (vacuolar) ATPase provides Cd 2+/H+-antiport, leading to the accumulation of Cd in the vacuole, accompanied by the release of protons. Cd2+/H+antiport mechanism is non-specific, able to transport calcium and other cations. 90

Figure 50. Three classes of membrane transport proteins: channels, carriers, and pumps (http://www.tankonyvtar.hu/hu/tartalom/tamop425/0010_ 1A_Book_angol_01_novenyelettan/ch02s02.html)

Transport systems of biological membranes can be divided into a system of ion channels and transporters. Ion channels are selective. Transporter proteins are not highly selective. In this process the substance is connected with the carrier in the binding site and transported across the membrane, which is released as a result of conformational changes of transporter protein. Ion transport is often carried out by a system of successive carriers. In the case of reversible binding of heavy metal ions through the cell wall components exchange physico-chemical adsorption, they can passively diffuse into symplast in the presence of a concentration gradient, which provides ion transport in plants (Figure 49). Sites involved in the exchange adsorption are non-selective. With their participation Cd, Zn, Cu, Hg and other metals enter to plants. The accumulation of metals in the headspace of the cell wall is determined by the ion exchange ratio, which depends largely on the amount of histidine protein groups, and carboxyl groups located on the surface of pectins (Figures 51, 52). 91

Figure 51. Cell wall barrier against cadmium ions (http://journal.frontiersin.org/article/10.3389/fpls.2015.00133/full)

Exchange-bound fraction of metal ions readily washed out of the root systems of plants after replacing the nutrient solution to which does not contain this element. Penetrating through the cell wall, some metal ions associated with the reactive components of the apoplast, the others – enter to the cytosol. 1. Extracellular avoidance of metal buildup through immobilization by root exudates. 2. Ectomycorrhizal association restricts metal movement to roots. 3. Metal ions are taken up by plant roots through channel proteins and/or H+-coupled carrier proteins. 4. From cytosol metals are transported and accumulated in vacuoles, the events are aided by vacuolar electrogenic proton fluxes. 5. Glandular trichomes 92

and epidermal structures (hydropotes) sequester metals in leaves. 6. Metal ions in cytosol can be detoxified via these routes. 7. Mechanisms involved in Cd chelation and compartmentalization in the vacuole. 8. Metal ions escaped from the complexation damage cellular macromolecules via the production of ROS.

Figure 52. Cellular mechanisms proposed to be involved in metal uptake, sequestration and detoxification in plants. ROS (http://scialert.net/fulltext/?doi=ijar.2006.122.141&org=10).

There is evidence that biphasic intake of metal ions such as cadmium, to the root systems reflects two above mentioned process. Wherein the linear part of the dependence of absorption of the metal ions on the time (for the roots of wheat – 75 min) corresponds to the binding of cadmium to the apoplast components and plateau (saturation phase) is the result of cadmium transport through the plasma membrane to the root cells. Microquantities of cadmium and, apparently, other heavy metals can be effectively delayed by cell wall or by apoplast reactive centers. These mechanisms prevent the delivery of excessive amounts of heavy metals in the cell and restrict the movement of metal ions in 93

the tissues of plants. The immobilization of heavy metals in the root system may be due to non-metabolic irreversible binding of the limited number of sites located on the surface of the cell wall and along symplast roots. On the one hand, the irreversible binding prevents the movement of frequently toxic ions in cells and tissues of the plants, on the other hands, it is a process that promotes the establishment of a concentration gradient and subsequently allows the accumulation of elements by diffusion.

Figure 53. Transport through plant roots (http://www.scoop.it/t/plant-roots-by-christophe-jacquet) 94

When entering into the roots the metals are accumulated in them, or are transported to the aerial organs. Metal are transported by the xylem, if metal ions cross the endoderm of roots (Figure 53). Endoderm cell walls are impregnated with suberin, a waxy substance that creates an impermeable water barrier, called Caspari belts (Figure 54).

Figure 54. Heavy metals radial transport (hys.org/news/2012-09-identification-rice-manganese-cadmium-uptake.html)

To cross this layer the metal ions have to move simplistically as apoplast trucks blocked. The passage of ions through the endoderm is a limiting factor in the translocation of metals to the aerial organs. Most of the heavy metals are usually localized in the cortex and rizoderm is and at relatively low concentrations metals not pass through the endodermal barrier. At high concentrations, this barrier is broken and the flow of heavy metal enters the tissue stele. Xylem loading is a complex process involved an energy dependent membrane transport processes. The mechanisms of penetration to the metal xylem vessels are not fully understood. It is assumed that the H+-ATPase in the plasma membrane of the xylem parenchyma cells actively promotes the secretion of protons. This proton gradient is then used for the transport of cations in the xylem against an electro95

chemical gradient through the mechanism of the cationically proton antiporter. It is also assumed that the ions can penetrate through the membrane channels xylem using thermodynamically passive process. These two mechanisms may include the specialized membrane ion transporters to facilitate loading ions into the xylem. The root cell walls bind metal ions from the soil and then are located with high affinity on sites of plasma membrane transport systems; metal ions are moved across plasmalemma. Uptake of metals occurs using secondary carriers such as channel proteins and /or H+-coupled carrier proteins. Uptake of cations through secondary transporters is facilitated by the membrane potential of the plasma membrane, which is negative charged on the inner side of the membrane. A number of different types of metal-transporters involved in the transport of metal in the plants (Figure 55). The ionic environment significantly affects on the movement of metal ions in the plant. The elements analogs and homologs thereof the greatest antagonism display and also the same valence cations capable of forming similar complexes.

Figure 55. Transporters of heavy metals Cd, As(V), As(III), Ni and Zn (http://scialert.net/fulltext/?doi=ijar.2006.122.141&org=10) 96

Thus, transportation of cadmium in the root systems is reduced by adding to a solution of calcium, zinc or manganese. It is known that cadmium competitively inhibits delivery of zinc in the cell, carried by a vector system. There are instances of synergy with the receipt of ions. The interaction between the heavy metals and other elements may indicate the existence of a common transport mechanism. It was observed an inhibition of absorption and transport to the aerial organs cadmium, copper, iron, manganese, zinc in pea seedlings. Kinetic analysis showed competitive inhibition of absorption of Cd and transport of these elements. There are common transporters for Cu, Fe, Zn, and possibly for Mn. Test questions 1. What is an anthropogenic source of heavy metals contamination of the environment? 2. Which regions of Kazakhstan the most contaminated with heavy metals? 3. What are the symptoms of heavy metal toxicity to humans? 4. What is meant by the term "heavy metals"? 5. What are trace elements of heavy metal? 6. What is the physiological role of trace elements – heavy metals such as copper and zinc in plants? 7. What is the effect on plants of heavy metals – trace elements when excessive content in the growth medium? 8. What is the physiological role of heavy metals such as lead and cadmium? 9. What are the sources of pollution cadmium ions? 10. What has been the toxicity of cadmium? 11. What are the main sources of lead in the environment? 12. What are the toxic compounds of lead?

2.3. Accumulation and distribution of heavy metals in plants. Intracellular localization In plants the roots contain the greatest amount of heavy metals. Lower amounts of heavy metals are accumulated in the stems and leaves, less – in the grain. The concentration of heavy metals in the grain and aboveground organs is mainly due to «the effect of detention» in their roots, which have more tolerant as compared to sensitive plant species. The existence of an effective mechanism for re97

tarding the heavy metals in the roots, no doubt. Comparison of heavy metals contents in soils and plant parts showed that the dependence of content in plants by soil concentration increases in the following order: Cd> Zn> Cu>Pb> Cr for monocots and depends on the mobility of the metal in the soil. For dicots, this pattern is less pronounced. As mentioned above, the absorption of ions of heavy metals by the root system from the soil and nutrient solution is carried out in various ways, on which the likelihood of intake of ions directly into the cytoplasm of cells and the rate of movement of the tissues and plants organs depend. The character of metal accumulation in organs of plants depends on the plant species and a metal nature (Table 1). Table 1 Intercellular localization of Cd and Pb in plants (I.V. Seregin &V.B. Ivanov, 2001). Plant species, Metal, tissue 1 Zeamays (Cd) Differentiated cells (Cd – 2х10-1 mMol; 3х10-3 mMol) Mature cortexandstelecells Rhizodermis Endodermis Pericycle Xylemparenchyma Rhytidiadelhus squarrosus (Pb – 4,8 mMol) Lemna minor (Pb – 3х10-3mMol) The outer layer of root cap The other cell layers of root caps Epidermis Cortex The basalpart of the root

Cell wall 2

Vacuole Golgiap- Endopla Nucleus Researparatus smicreti chmethod culum 3 4 5 6 7 +

+

+

The X-raymicroanalysis Electronic microscopy Histochemistry

+

Electronic microscopy

+

+ + +/+ +

+

+ + +

+ +

+

98

+

+

+ +

+ +

Electronic microscopy Histochemistry

1 Allium cepa (Pb-7,5х 10-3 mMol) Zea mays (Pb – 8х10-1 mMol; 7,5х10-3 mMol) Glycine soya (Pb)

2 +

3 +

4 +

+

+

+

+

+

+

5

Lupinus luteum (Pb – 4,3х10-2 mMol)

6

7 Autoradiography Electronic microscopy The X-ray microanalysis Electronic microscopy

Thecortexparenchyma

+

+

+

Histochemistry

Stele

+/-

+/-

+/-

Electronic microscopy

Pisumsativum (Pb)

+

Raphanussativus (Pb – 5 mMol)

+

+

+

Electronic microscopy

+

Electronic microscopy

Zinc at high concentration (25 mg/kg) was accumulated in significant amounts in the aerial parts of wheat and beans. The protective function of the roots towards cadmium was more expressed than for Zn, which is not accumulated in the roots, and moved in the stems and leaves (Figure 56). This is probably due to unequal role of these elements in the plant metabolism. Translocation rate of cadmium and lead to the aboveground part as compared to Zn was much lower. Most of the Pb is retained in the root system. Localization of the metal in parts of the plant is dependent on its mobility. According to the researchers, Pb in plants of lupine was contained mostly in the tips of the roots, less – in the basal part, and hypocotyls. The regularities of the distribution of Pb, Cd and Zn in the root tissues are not well understood. The apical root sections on metal content may vary from basal sections. Many authors have noted that at high concentrations of metals in the environment the basal part of the roots accumulate significantly more Pb, Cd and Zn, than apical one, especially in resistant populations. Other authors suggest that most of the metal accumulates in meristematic parts of the roots (Ivanov, Seregin, 2001). 99

Seed accumulation

Shot sequestration

Phloem loading and transport

Root uptake

Xylem loading

Figure 56. Localization of heavy metals in plants parts (http://plantsci.missouri.edu/faculty/mendoza-cozatl.cfm)

The relatively high concentration in the cell walls of Pb and Cd detected in a number of species. The study of compartmentalization of metals in plant cellsfor plant tolerance it is important metal accumulation in vacuoles of root cells. Cell walls of monocotyledonous and dicotyledonous plants differed in content of pectin and hemicelluloses, thereby it determine the differences in their ability to bind cations. The bond strength of certain metal ions with the components of the cell wall varies. It correlates with different values of stability 100

constants (Log K) metal complexes with functional groups of carbohydrates. For Pb, it is equal to 6.4, for Cd – 4,9. Large amounts of Pb are detected in cortical parenchyma in comparison with central cylinder. This fact was explained by Pb lower mobility as compared to other metals. Therefore, Pb binds more strongly with cellular membranes than Cd, and slowly moves along the apoplast. The affinity of other metals to polygalacturonic acid decreases in the following order: Pb> Cr> Cu>Ca> Zn. It was not studied the binding of Pb and Cd by apoplast proteins. In barley leaf apoplast cadmium increased the content of these proteins. However, the role of these proteins is uncertain. Under the influence of heavy metals can be enhanced suberin and callose deposition, reducing the absorption of metals (Figure 57).

Figure 57. Heavy metal effect on cell wall compounds (http://www.mdpi.com/22237747/4/1/112http://www.mdpi.com/2223-7747/4/1/112)

Heavy metals were also detected in the intercellular space, dictyosome, endoplasmic reticulum, nuclear membrane. In the cytoplasm ions can bind to biomolecules. In this case, the chelate is de101

rived from a cell or accumulated therein (mostly in the vacuoles). Accumulation of toxic ions into vacuoles in the form of inactive compounds are more typical for plant tolerance to heavy metals. Fraction remaining in the cytosol as free ions or soluble complexes is moving simplistically from the root to the stem and then – to the leaves of plants by charged sites of xylem or carried away by the transpiration stream of water. Cadmium is accumulated in cell wall in less amounts than inside the cell. It can bind to intracellular lysosomеs like granules and largest amount of the metal is in the cytoplasm. It is known that the usual response to the Cd is the induction of synthesis of low molecular weight, cysteine-rich proteins – metallothioneins, phytochelatins. The chemical bond of cadmium with organic ligands is much stronger than other metals. At high concentrations of Cd-phytochelatins complexes are localized in the vacuoles. At low concentrations 86-100% of Cd found in the cytoplasm in Datura inoxia (Rauser, 1987). Under these conditions, there is no need to isolate the cells of Cd in the vacuoles. At high concentrations of Cd binds with organic acids, and low – with glutathione in the cytosol. Cadmium is found in the cytoplasm and nucleus vacuoles in bentgrass and roots of corn, and also it was indicates the presence of Cd in the cell wall of roots of maize. Other studies have detected a high concentration of cadmium, associated with phosphorus and calcium in the sea fern Azolla filiculoides L. Most of them have been identified in the cell wall of the xylem vascular bundles. It is shown that the Cd concentration decreases with increasing content of Se, Al, K, Ca, P. The aerial parts and roots of wheat subjected to the effect of Cu, the metal was found in the cell wall and vacuoles. In the presence of Cu in the growth medium, the ratio of the metal content in the cell walls and vacuoles increased in favor of the latter. Copper was found in the matrix of the cell walls of Enteromorpha compressa. The study of Pb effect on Lemna minor L. has shown that Pb is present in the vacuoles, vesicles and in cell wall. At high concentrations lead was found in symplast. Changing the ratios of content of Pb in vacuoles and cell walls after a 6- and 12-hour of exposure in favor of the latter indicates a redistribution of the metal cell walls (Samardakiewicz et al., 2000). 102

It was found that in Thlaspi caerulescens L., subjected to the effect of Zn, the metal is concentrated in the epidermal cells and vacuoles. At low concentrations the greatest amounts of Zn was found in the vacuole. Perhaps tonoplast of epidermal cells of leaves of T. caerulescens has a higher ability to transport Zn in the vacuole than mesophilic cells. The ability to isolate in T. caerulescens Zn in epidermal vacuole is an important aspect in this type of hypertolerance to heavy metals (Figure 58). The preferential localization of Zn in the epidermis, apparently contributes to the protection of mesophyll cells from damage and maintains the functions of mesophyll cells at a high concentration of Zn in the leaves. Arabidopsis halleri L. is pseudometallophyte, i.e.it is growing in contaminated and uncontaminated soils. It is hyperaccumulators of zinc, as well as cadmium. Recent studies using electron microscopy demonstrated the cellular distribution of Zn in the tissues A. halleri, grown in a hydroponic environment. Zinc in the plant leaves mainly found in the base of trichomes – hairs, present on the surface of plant leaves (Dahmani-Muller et al., 2000). Thus, the character distribution of heavy metals in cell organelles plays an important role in protective mechanism in plants. The above data show that not all ions uptake by plants actively influence at its metabolism, so as a definite part of metals can bind with organic acids and low-molecular proteins and concentrate in an metabolically low-active compartments. Part of toxic ions turns out of firmly bound with reactive-capable portions on the surface cell wall and in the apoplast, and are penetrating across the plasmalemma – with intracellular biomolecules. What proportion of given metal ions would be in the free form, and what – bound with organic molecules, it depends from pH of the environment and chemical properties of element. The stability of metals complexes decreases in the case of deviation pH of environment from neutral: at low pH there is a competition of protons with metal ions for binding sites in molecules, at high pH – by the reason of the competition of hydroxyl groups with ligand. It is also important to take into account, that the multiply charged ions form a more stable complexes, than the singly charged, possessing lesser charge density. 103

Figure 58. localization of Zn in vacuole http://journal.frontiersin.org/article/10.3389/fpls.2013.00144/full

Thus, the heavy metals are mainly concentrated in the roots of plants, which limits their movement in the generative organs. A common feature of interstitial and intracellular distribution is the concentration of a large amounts of metals in the surface structures and protecting cells from the toxic effect of metals. Concentration of heavy metals occurs by binding them into soluble compounds having a different nature. Despite the significant accumulation of heavy metals in a metabolically inactive cell compartments (vacuoles and cell walls), some of the metals enters the cytoplasm and exerts multiple toxic effects, and this may be due to both direct effect of metals and reducing the activity of some of the processes as a result of violation of others. Test questions 1. What plant organs of plants is contained the greatest quantity of heavy metals? 2. From what the localization of metals in plant parts depend? 3. Which heavy metal is considered as a less movable in comparison with others? 4. In what part of plant roots can be accumulated heavy metals? 5. What does play the main role in the tolerance to heavy metals?

104

6. Why in the response to the effect of heavy metals is amplified the deposition of of callose and suberin? 7. In what cellular organelles is accumulated cadmium? 8. In what cellular organelles is accumulated lead? 9. In what cellular organelles is accumulated zinc? 10. From what the distribution of metals to organs and cellular organelles depends? 11. How does the character of the distribution of heavy metals in cellular organelles play an important role in a protective the mechanism of plant organism? 12. What is a common feature of interstitial and intracellular distribution of heavy metals?

2.4. Toxic effects of heavy metals for plants According to the classification D.Wood (1974), a very toxic heavy metals include the following elements: Co, Ni, Cu, Zn, Sn, As, Se, Fe, Rb, Ag, Cd, Hg, Pb. It is shown that the toxicity of certain metals is reduced with increasing degree of oxidation. The solubility of the metal compounds is often dependent on the anion included in their composition. 2.4.1. The effect of heavy metals on the properties of cell membranes Cell membranes are the primary target of action of heavy metals that promote the deformation of the membrane, leading to its fragmentation. Zinc changes the amount of phospholipids in the needles of Scots pine. The effect of the amount of copper changed slightly, Manganese has no effect on the number of membrane lipids. Other authors suggest that the target for heavy metals are also plasma membrane proteins. The violating in the integrity of the root cell membrane leads to an increase in the electrolytes leakage. More resistant wheat varieties possess the greater acidic secretion activity of roots under the effect of Zn, Cu, Ni and Mn. This suggests that the acidic root secretions can partly bind excess metal in the radicular solution, preventing the movement of them in the aerial organs. The treatment with Cd (10 μM) leads to a change in the composition of the lipid membranes of the roots of rice plants, increasing 105

the degree of unsaturated of the phospholipid-bound fatty acids and an increase in the content of stigmasterol (30-42%). These changes were accompanied by a decrease in the content of sitosterol (from 26.8 to 17.4 mg/g). Under the influence of heavy metals there is a decrease in the content of minerals in plants. Divalent cations may compete with other cations (Ca2+, Mg2+, Fe2+) in their transport through the membrane. The direct effect on the properties of the membrane expressed in the decreasing of cell entering of the required elements. The effect of cadmium on cell membrane expressed in a change in membrane permeability and transmembrane electrical potential (Em). The damage of the membrane is expressed in increasing of the output of K+ ions or electrolytes leakage of Cd-treated plants. Heavy metals can damage the membranes of root cells of susceptible species. An increase in membranes the process of lipid peroxidation (LPO), indicating the damage of the membrane, depolarize the cell membrane by divalent metal cations, including cadmium in sensitive plants. 2.4.2. The impact of heavy metals on the activity of enzymes. Oxidative stress Oxidative stress occurs in plants under conditions promoting to the formation of reactive oxygen species (ROS) – H202 hydrogen peroxide, hydroxyl radical HO*, singlet oxygen 1O2 and ozone O3. High concentrations of ROS in the cells leads to damage of biomolecules. Oxidative stress in plants is the result of effect of all environmental factors, including drought, soil salinity, air pollution with toxic compounds, such as for example, ozone, nitrogen and sulfur oxides, heavy metals, high and low temperature, high light intensities, UV radiation, lack of mineral nutrients, some herbicides such as paraquat (methyl viologen), pathogens of various nature, and others. Oxidative stress is included as a component in most of the other stresses. This universality in the induction of oxidative stress under the action a variety of environmental factors indicates the crucial role of antioxidant systems, which reduce the intracellular concentration of ROS, as well as systems, eliminates toxic products of the reactions 106

of ROS with biopolymers and increases plant resistance to adverse environmental factors in general (Figure 59). Antioxidant defense system of plants involves several enzymes and low molecular weight substances which are present in plants (Figure 60). Superoxide radicals are generated in plants are converted into hydrogen peroxide by the enzyme superoxide dismutase (SOD). The accumulation of hydrogen peroxide, a strong oxidant is prevented by catalase (CAT) and ascorbate-glutathione cycle (AGC) where ascorbate peroxidase (APO) restores it to the water. Ascorbate and glutathione, the other components of the antioxidant protective system, also increase under stress conditions.

Figure 59. Heavy metals induced oxidative stress (https://www.researchgate.net/figure/236785078_fig3_Figure-1-Heavy-metalinduced-oxidative-stress-tolerance-and-detoxification-mechanisms)

107

Specifically, glutathione (Glu) plays an important role in plants exposed to Cd. In fact, glutathione a monomer molecule of phytochelatins (PC) – the metal-binding peptides, which form complexes with Cd and isolate it in the vacuoles assisted protein with properties similar to glutathione transferase. Glutathione in a reduced form of thiol groups protected plant cells from the oxidation. For this purpose it is controlled by the concentration of the complex homeostatic mechanisms. Superoxide dismutase is a key enzyme against oxidative stress. This enzyme catalyzes the conversion of superoxide radical (O.-) into H2O2 and O2. In the analysis of SOD in the presence of cadmium decreased activity of all isoenzymes present in the leaves. Isoform of CuZn-SOD was the most sensitive to cadmium, especially a cytosolic isoform, as well as the chloroplast isoform. Fe-SOD was also sensitive, while Mn-SOD localized in mitochondria and peroxisomes, it has been the most stable isoform (Figure 61). Immuno-blot analysis with antibodies against CuZn-SOD showed a linear decrease in the protein content in the CuZn-SOD in response to the increase of Cd in the environment. Reduced absorption Cu, Zn, Fe and Mn in the leaves in the presence of cadmium decreased synthesis isozymes containing these metals.

Figure 60. ROS scavenging by antioxidative enzymes (http://www.intechopen.com/books/abiotic-stress-in-plants-mechanisms-andadaptations/towards-understanding-plant-response-to-heavy-metal-stress) 108

Heavy metal ions disrupt the activity of enzymes in carbohydrate and energetic metabolism. For example, Ni and Cd increased activity of the enzymes glucose 6-phosphate dehydrogenase, malate dehydrogenase, glutamate dehydrogenase. In plants Silene italic L. сadmium facilitated accumulation of proline and increased peroxidase activity in roots of rice seedlings. Under the influence of heavy metals in rice SOD activity increased and it deactivated superoxide anion radicals. Heavy metals also increases the activity of glutathione reductase and ascorbate in cyanobacterium Anacystis nidulans L. Сadmium alters the activity of the enzymes malate dehydrogenase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase involved in energy metabolism and NADPH regeneration and guaiacol peroxidase, ascorbate, glutamate, involved in removing oxidative stress. In most cases, under the effect of Cd and Pb inactivation of enzymes due to the interaction of metals with the SH-groups of the enzyme, but also with the groups responsible for the stabilization of the tertiary structure, thereby altering the conformational structure of the enzyme (Table 2).

Figure 61. Types of superoxide dismutase (http://www.slideshare.net/azfarali2/seminar-on-sods) 109

With an excess of Zn in the environment of growing plants increased oxidative transformation of exogenous phenolic compounds (pyrocatechols and hydroquinone) to quinones. Phenolic compounds are relatively resistant to heavy metals in intact tissues. A significant part of the phenolic compounds concentrated in the vacuoles, and diphenol oxidase activity is detected in the cytoplasm. The oxidation of phenolics is not possible without the transition of them to the vacuoles. Manifestation of inhibitory properties of native enzyme under extreme environmental conditions is possible only when the disturbances of membrane permeability and release of phenolics from vacuoles in the cytoplasm followed by oxidative conversion to quinones. High concentrations of Ni and Cr significantly increase the activity of polyphenol oxidase (PPO), oxidizing phenols in leaves and roots of a sunflower. There are changes in the metabolism of nucleic acids. Caffeic acid and scopoletin inhibit RNA and DNA synthesis. Polyphenol oxidase is a membrane-bound enzyme, so in violation of membrane permeability caused by an excess of heavy metals, the transition from latent membrane-bound form in free, active form is occurred, which increases the content of quinones. Table 2 Effect of Cd and Pb on enzymes activities (I.V. Seregin and V.B. Ivanov, 2001) Enzymes

Process

1 Aminolavulenic acid dehydrates

2 Chlorophyll synthesis

Protochlorophyllide reductase Ezymes system of photolysis Ribulosa-1,5-bis phosphate carboxylase (oxygenase)

Chlorophyll synthesis

Cd

↓

Photooxydation of H2O

Cd

↓

Cd Pb

↓ Сd2+>Cu2+ > Pb2+>Zn2+

CO2 fixation

Metal Enzyme activity 3 4 Pb ↓

110

↓

Effect

Plant species

5 6 1) Binding to Pennisetum SH-groups Typhoideum 2) Pb-induced Zn-deficiency Binding to Hordeumvulgare SH-groups Binding to SH-groups Binding to SH-groups Цис 173 и Цис 458

Lycopercsicum Eskulentum Hordeumvulgare Cajanuscajan Avena sativa

1 Phosphoenolpyruvate – carboxylase Glyceraldehyde-3phosphodehydrogenase Ribulosa-5phosphokinase Nitrogenase

2 Chlorophyll synthesis

3 Cd Pb

↓

Calvin cycle

Cd

↓

Calvin cycle

Cd

↓

Reduction of N2

Cd Pb

↓ Cd2+>Pb2+

Nitranereductase Reduction of NO3-

Cd Pb

↓

Н+-АТPase (roots)

Cd

↓ Zn2+>Cd2+ > Ca2+>Mn2

Ions transport

+

Hexokinase Glycosi-6phosphatr dehydrogenase Carboanhydrase

Cu-Znsuperoxydedismutase Mn-superoxyde dismutase Peroxydase

Protease Catalase

4

5

6 Zeamays Cajanuscajan

Binding to SH-groups

Valerianella locusta Cajanuscajan Binding to Valerianella SH-groups Locusta Glycinemax Azolla Filiculoides 1) Binding to Pisumsativum SH-groups Phaseolus 2) Decrease of vulgaris NO3- uptake Lycopercsicum Eskulentum Change of Zea mays conformation Helianthus annuus Triticumaestivum Pisumsativum Pisumsativum

Glycolysis Penthose phosphate pathway Reversible dehydration CO2 Neutralisation of О2*-

Cd Cd

↓ ↓

Cd Pb

↓

Cd

↓

Neutralisation of О2*Oxidationofpol yphenolsusing H2O2 Proteins hydrolysis Thedestruction ofН2О2

Cd Pb Cd

↑ ↓ ↑

-

Cd

↑

-

Lemnasp.

Pb

↑

-

Oryzasativa Zeamays

Zn deficiency Modification of molecules Zn deficiency

Footnote: «↑» – activation, «↓» – inhibition

111

-

Glycinemax Melicanutans Phaseolusvulgaris Lupinusluteus Phaseolusvulgaris Phaseolusvulgaris

Transition metals like copper catalyze the formation of hydroxyl radicals (OH.) from the non-enzymatic chemical reaction between superoxide (O2.-) and H2O2 (Haber-Weiss reaction). Hence, the presence of excess of copper can cause oxidative stress in plants and subsequently increase the antioxidant responses due to increased production of highly toxic oxygen free radicals. An excess of copper in plants lead to oxidative stress inducing changes in the activity and content of some components of the antioxidative enzyme-ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbatereductase (DHAR), glutathione reductase (GR), superoxide dismutases (SODs), guiacol peroxidase. The antioxidant responses were observed in leaves and roots being both Cuconcentration dependent and time-dependent. The ascorbateglutathione cycle has been reported to be involved in response to excess of copper (Drazkiewicz et al., 2003). Determination of heavy metals such as copper, may be toxic due to their participation in redox cycles that produce hydroxyl radicals (*OH), which are extremely toxic to living cells. Copper (Cu2+) ions are bound with cell wall polymers (histidine-rich glycoprotein) can be reduced to Cu+ by apoplastic ascorbate as an electron donor and superoxide ions. Copper ions (I) (Cu+) may then be subjected to Fenton reaction to the apoplastic hydrogen peroxide to generate hydroxyl radicals (OH*)(Fry et al., 2002). Fenton reaction – an oxidation of a hydroxyl group of α-hydroxy acids and glycols to a carbonyl group and the formation of hydroxyl radicals in the presence of hydrogen peroxide. This process can cause non-enzymatic separation of cell wall polysaccharides. In the cell wall of tomato fruits *OH-radicals cause the dissolution of pectins. It was demonstrated that acidic pectins isolated Cu2+ not strong enough to prevent the attack of OH* radicals. Cadmium is not a redox metal and does not participate in Fenton-type reactions, but it can also indirectly produce oxidative stress, causing damage in the chloroplasts. Catalase activity decreased with increasing of cadmium in the medium in Phaseolus vulgaris L. Heavy metals may reduce content of the antioxidant glutathione, as it is the substrate for the synthesis of phytochelatins, that are involved in detoxification of heavy metals. 112

Heavy metals cause an increase in reaction products with thiobarbituric acid (TBA), which is an index of lipid peroxidation, thus oxidative stress. Lipid peroxidation (LPO) of membranes damage their function and integrity and may produce irreversible damage to cell function. Lipid peroxidation, induced by copper, is the cause of the degeneration of the membrane. Another possible target proteins are oxidized radicals. On the functions of proteins reactive oxidize substances (ROS) can act as oxidants of amino acid side chains, react with aldehyde products of lipid peroxidation. Lipid peroxidation causes an oxidative modification of proteins and generates reactive aldehydes as the products of lipid peroxidation. Feedback of lipid peroxidation is a direct oxidation of the amino acid residues or reaction with the secondary products of lipid peroxidation, or glycosylation, thereby increasing a formation of carbonyl groups (C = O) per molecule of protein. The presence of these reactive groups as well as lipid peroxidation is an index of oxidative stress. Modified proteins alter their hydrophobicity, produce the formation of the aggregates or fragments of peptides. Some authors have demonstrated that the modified proteins are oxidized selectively and used as a target for the protease (Hall, 2002). 2.4.3. Heavy metals effect on photosynthesis and respiration of plants As described above, the heavy metals are toxic to membranes and their properties. Due to the violations of the membrane structure of mitochondria and chloroplasts, heavy metals have an adverse effect on the processes of photosynthesis and respiration (Figure 62). It was revealed that an excessive amount of heavy metals suppress respiration and photosynthesis in plants. Cadmium at concentration of 1 mM decreased absorption of O2by roots cell suspension culture of tobacco. The authors suggest that cadmium inhibited the transport of electrons and protons in the mitochondria, resulting in a function of the electron transport chain. It was also shown that cadmium is not substantially affect the glycolysis and pentose phosphate pathway, but significantly inhibits succinate oxidation in the Krebs cycle. 113

Hence, the authors concluded that succinate dehydrogenase complex – one of the primary target affected cadmium in mitochondria. However, other authors have demonstrated the inhibitory effect of cadmium (in vitro and in vivo) on the key enzymes of glycolysis and the pentose phosphate pathway. Cadmium inhibited the fixation reaction of carbon dioxide in chloroplasts. Lead is a potent inhibitor of cell metabolism and can increase the toxicity of other metals. M. Trice (1985) indicated that the formation of a complex of Pb with ATP. Lead promoted the reduction of the rate of cell division in the roots at lowest concentrations. The study of the effect of lead salts on the at the mitochondrial electron transport inhibition is detected, particularly in the absence of phosphorus. Salts like PbCl2, Pb (NO3) 2at a concentration of 0,1-2,4 mMol capable of inhibiting the activity of Photosystem II (PS) in the oxidation step, and the addition of Mn salts removed the inhibitory effect of lead. Lead concentration of 20 mMol Pb (NO3)2decreased the fluorescence, inhibited photosynthesis. There were abnormal electron transport reactions in thylakoid membranes of chloroplasts in plants grown in the medium with an excess of zinc and copper. Apparently, this is associated with changes in the content and function of metalcontaining components in the electron transport chain. Changes on the donor side of Photosystem II are associated with disorders of the Mn-containing proteins. Copper, lead and zinc strongly inhibited the transport of electrons. There have been structural changes in proteins as well as an increased concentration of lipids. The mechanism of copper toxicity on photosynthetic electron transport has been extensively studied in vitro and it was found that PSII (Figure 62?63) is a more sensitive site to copper toxicity than Photosystem I (PSI) (Gonzalez-Mendoza et al., 2013). The most apparent effect of copper toxicity on Photosystem II (PSII) is the inhibition of oxygen evolution accompanied by quenching of variable fluorescence. It was suggested that both the acceptor and the donor sides of PSII as the main targets of copper toxic action. On the reducing side of PSII, the QB binding site and the Phe-Fe-QA domain as the most sensitive sites for copper toxicity. The electron flow from Tyrz to P680+ is blocked at toxic copper concentrations. It is proposed that copper interacts not only with Tyrz, but also with TyrD on D2 protein. A 114

possible direct interaction between copper and calcium at the oxidizing side of PSII was also shown in vitro and in vivo (Yruela, 2005).

A (http://journal.frontiersin.org/article/10.3389/fpls.2013.00280/full)

B (http://www.hindawi.com/journals/jb/2012/872875/fig1/) Figure 62. Effect of heavy metals on plant metabolism 115

Nickel and cadmium ions in a concentration of 5-10 mMol inhibited the rate of photosynthesis. Under heavy metals stress in the isolated tissues of chloroplasts chlorophyll molecules had a substitution central Mg2+ atom to the corresponding atom of heavy metal (Cu, Zn, Cd, Pb). This substitution resulted in damage to the lightharvesting pigment complexes. The effect of Pb decreased the rate of photosynthesis, inhibits the Hill reaction in isolated chloroplasts. Copper reduces the concentration of chlorophyll. Reducing the concentration of chlorophyll caused by iron deficiency, causes a disturbance in the PS II. The decrease in chlorophyll content and sensitivity to photoinhibition were overcome by the addition of iron. It is assumed that an excess of copper inhibits the donor and acceptor sites of PS II. The reducing of chlorophyll concentration was accompanied by a change in the ultrastructure of the thylakoid membranes.

Figure 63. Toxic effect of copper on photosystem II (http://www.scielo.br/scielo.php?script=sci_arttext&pid=S167704202005000100012&lng=en&nrm=iso&tlng=en) 116

Excess of copper affects the biosynthesis of photosynthetic apparatus and modifies pigment protein complexes of photosynthetic membranes. 2.4.4. Heavy metals effect on cell division and nuclear apparatus Inorganic metal salts influence cell division and a nuclear device. The ability to inhibit the mitosis and cause chronic abnormalities increases with increasing molecular weight of heavy metals. Under cadmium stress it was observed a disturbance in the cells fission of the root meristem of maize during mitosis. It was shown that these violations are irreversible and lead to the cell death. Heavy metals induce in plants and animals chronic abnormalities include changes in chromosome structure – fragmentation, inversions, translocations, polyploidy. Prolonged exposure to metal salts leads to a sharp change in cellular activity, chromosomal aberration and reduced mitotic index, increase in the frequency of sister chromatid volume (Figures 64, 65). Total DNA and RNA content decreases with increasing dose and timing of treatment. This may be as consequence of significant conformational changes in the nucleosome. Under the influence of heavy metals, many interphase cells are returned to the state of prophase. There is a presence of the destroyed nuclei. Some cells completely lose the nucleus as a result of the complete disintegration of the nuclear material. In some cells is indicated an uneven distribution of chromosomes. There are anomalies such as K-anaphase, indicating that complete suppression of the formation of the spindle. It was observed the K-metaphase and 2-nucleated cells due to the delay in cell cycle in which DNA chromosome is replicated, but not distributed normally. In 48 hours after the start of treatment on equator plate chromosome fragments and daughter chromosomes were detected and were found 3- and 4-polar cells. Zinc induces changes in the mitotic index. Duration of cell cycle increases linearly with increasing concentration of zinc in the environment. 117

Figure 64. Effects of different concentrations of Pb on nucleolar organizing regions (NORs) in root tip meristematic cells of Allium cepa during mitosis (http://www.mdpi.com/1422-0067/15/8/13406/htm) (a–h) Normal mitotic process. (a) The interphase cell; (b,c) Showing decondensed chromatin fibers were around the nucleoli; (d) Showing that the nucleoli disappeared in their characteristic structures; (e) Showing NORs localized on metaphase chromosomes; (f) Showing NORs migrated with the chromosomes to the poles at anaphase; (g) Showing nucleoli rebuilt at early telophase and the size increased; (h) Showing the two daughter nuclei entered interphase, and mitosis was completed; (i–l) Mitotic process under Pb stress. (i,j) Showing persistent nucleoli during metaphase ((i) 10 μMPb, 24 h; (j) 1 μMPb, 48 h);(k) Showing sticky chromosomes with Ag-stained NOR particles at metaphase (100 μMPb, 48 h);and (l) Showing sticky chromosomes with Ag-stained NOR particles at anaphase (100 μMPb, 48 h). Scale bars = 10 μM. Nucleoli and NORs: dark brown; nuclei and chromosomes: green; cytoplasm: yellow. Arrowhead shows NORs

Under cadmium stress it was observed a stimulation of cell divisions of quiescent center and the braking of divisions of initial cells adjacent to the quiescent center. This leads to the «opening of meristem», which consists in changing the orientation of cell division of quiescent center so that their derivatives form an initial group of cells common for primary cortex and root cap. In the future root growth accelerated after the initial braking. 118

Figure 65. Effect of lead on plant cell (http://link.springer.com/chapter/10.1007/978-3-642-38469-1_7)

Having the affinity to the cell surface, ions of Cd, Pb, Zn absorbed on the surface of the cells and reduced the surface charge of the cells. After penetration into the cell metals are bound to DNA and produce a genotoxic effect. Genotoxic metals form covalent bonds, unlike physiologically essential, which form typical ionic bonds. Reducing the speed of cell division and elongation tension under the influence of heavy metals cells occurs due to different mechanisms: direct binding to DNA, metal-induced aberrations, the elongation of the mitotic cycle, impaired formation of microtubules, reduce the plasticity of cell membranes (Kozhevnikova et al. 2007). 2.4.5. The impact of heavy metals on mineral nutrition of plants Toxic effects of heavy metals may be due to a violation of the relations between the different metals in plants. Excessive intake of one of the metals may lead to a shortage of other essential mineral 119

elements as a result of limitations of its receipt, or binding. Mechanisms leading to mineral deficiency, can be a competition of heavy metals and essential mineral elements for binding sites on specific carriers when they enter the cell and transport of this element by the metabolic chain. Antagonism of ions can occur between heavy metals and macroelements like Ca, Mg, or K, and phosphorus (Figure 65).

Figure 65. Mineral interaction(http://www.balancingbrainchemistry.co.uk /petersmith/28/Heavy-Metal-Toxicity-Depression-&-Anxiety.html)

It is believed that heavy metals primarily lead (Pb), can form insoluble phosphates, thereby causing phosphorus deficiency in plants. It is important not only the impact of heavy metals on particular cell process, but also the combination of a number of elements, i.e., cooperative action. For example, Fe is a specific catalyst for the basic processes of respiration and photosynthesis in plants, being a part of a number of carriers of electron transport chains. At a deficiency of Fe it is disrupted the formation of reducing equivalents 120

necessary to implement enzymatic reactions of nitrate reduction. Thus, Fe-deficiency may cause the shortage of nitrate accumulation in plants, i.e. even in the absence of an excess of nitrogen in the culture medium. Inharmonious, unbalanced ratio of minerals may be affected indirectly, and give an excess of nitrate in the plant, which is now one of the most urgent vital problems. Iron deficiency causes the changes in the ratio of Fe-Mn during their accumulation in plants. Iron-containing enzymes are inactivated at the excess of manganese, resulting in retarded Mn delivery at the cellular level. Mn-deficiency cause the decrease the oxygen release process in photosynthesis, since Mn included in the O2-releasing component and it is responsible for the decomposition of water and recovering O2. Mercury bichloride is neutralized by the addition of mercury compounds containing SH-groups (glutamine, cysteine), which react by forming mercaptides. Toxicity of Zn in Lactobacillus arabimosis is removed by addition of salts of Mn, Mg, Ca, and the inhibitory effect of Ni, Co, Zn for E. coli is reduced by adding of Mg. Toxicity of copper decreased in the presence of organic matter (protein, sugar), with which it forms a complex compounds. Increasing of Cu concentration causes an increase in nitrate reductase activity. Mn, Mo, Cu at moderately high doses reduced activity of cytochrome oxidase. The synergistic effect is manifested in the fact that Pb increased toxicity of other metals, copper oxide increased the effect of oxides of other metals (Pb, Zn, Fe). With an excess of calcium intake decreased Mn content. Copper facilitated the uptake of Mn, Zn, B. All of the above changes in the metabolism of cells by the action of heavy metals lead to the substantial suppression of plant growth and development. 2.4.6. Heavy metals effects on growth and development of plants The most common manifestations of the toxic effect of heavy metals are the inhibition of growth and biomass accumulation, chlorosis, disruption of water exchange and, as a result, the decline in yields (Figure 66). 121

Inhibition of growth is widely used to test the presence of heavy metals in the environment. Inhibition of plant growth by the action of heavy metals occurs, on the one hand, due to metabolic disorders, on the other hand – as a result of direct metal effect on growth, for example, due to the reaction of a metal with polysaccharides and decrease the plasticity of cell walls (Ivanov and Seregin 2001). The most resistant to heavy metals process is a germination. This is due to low permeability for heavy metals the seed peel of most species that has been shown by many authors via histochemical methods. Root growth is more sensitive to heavy metals than the growth of shoots, which correlates with a predominant accumulation of heavy metals in the roots. Basically, root growth is suppressed, reducing the number of root hairs, root biomass. Under the influence of heavy metals primarily is disturbed the meristem zone in the roots, then – the violation of the stretching zone of cells, which are usually in the process of formation of root hairs. This reduces the total volume and total absorption surface of the roots.

Figure 66. Effect of heavy metals on plants growth (http://phys.org/news/2013-06-south-american-purifying-soils-contaminated.html)

The decrease of the root ability to absorb nutrients leads to suppression of growth, development and plant death. Heavy metals decrease the growth of above-ground parts of plants too. This disturbed the development of assimilation organs, weakened an accumulation of dry biomass, which leads to a decrease in the viability of the plant.

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It is shown that the ions of Cd and Pb cause changes in the morphology of the roots. If the metal concentration is not too high, number of lateral roots is reduced to less than the length of the main root, and the root system have more compact form. The denser root system is due to the reduction of distance between the lateral root primordia. The investigation of the effect of different concentrations of Cd2+, Co2+, Cu2+, Fe2+, Fe2+, Hg2+, Mn2+ on pollen germination and pollen tube growth of Lillium longiflorum L. by light microscopy revealed that the concentration of heavy metals 3 and 100 μM cause a toxic effect. The most toxic effect was observed with Cd, Cu, Hg, while Mn has the least influence on the germination and pollen tube growth rate. The tips of damaged tubes swall, it was observed the abnormal organization of cell walls to form circular fibrillar aggregates with changing shape and size of the bubbling of the Golgi apparatus. Cadmium induced effects on the intracellular level: there is a disruption of the spatial distribution of organelles in the tips of the tubes. The study of the effects of lead on the growth of barley and oats showed that the inhibition of growth is one of the early symptoms of adverse effect of the metal. Plant height and weight gain of the dry plants varied to a greater extent than the dimensions of the leaf blade. Ascending the inhibition parameters studied were located as follows: width and length of leaf, plant height, growth of above-ground mass of plants. Action of lead in both cereal was more pronounced in the phase of seedlings (10-13 days) than in subsequent phases of development. Morphological and physiological analysis showed that lead in the early phases of ontogenesis even in the absence of apparent differences between the control and experimental plants variants significantly slowed the pace of organogenesis. With increasing concentrations of lead is occurred the reduction in the length of the growth cone. The increasing of concentrations of heavy metals in a growth medium inhibited the growth processes: are reduced the size and area of the leaf, which reduces the assimilation surface, obviously a total 123

amount of assimilates that in turn can cause the reduction in plant productivity Thus, the heavy metals have severe toxic effects on physiological and biochemical processes in plants, such as photosynthesis and respiration, change the structure and properties of the cell membrane, induce oxidative stress, have a denaturing effect on important metabolic proteins, resulting in suppressed growth and development of plants. Test questions: 1. What is the primary target of the action of heavy metals in the plant? 2. What is the effect of heavy metals on the cell membrane? 3. What is the effect of heavy metals on lipids and membrane proteins? 4. What is the effect of heavy metals on the uptake of mineral elements in plants? 5. What is the oxidative stress caused by heavy metals? 6. Describe the antioxidant system of plants. 7. What is the role of glutathione in the reduction of oxidative stress? 8. What are the reactions catalyzed by the enzyme superoxide dismutase (SOD)? 9. What enzymes activity influence heavy metals on? 10. What is the mechanism of the effect of heavy metals on the activity of the enzymes? 11. What is the effect of heavy metals on the peroxidase, catalase and polyphenoloxidase activities? 12. What are the metals involved in redox cycles and how they affect the development of oxidative stress in plants? 13. What is the Fenton reaction? 14. How cadmium can produce oxidative stress in plants? 15. How does the lipid peroxidation of cell membrane effect on the integrity on the function of cell membranes? 16. How reactive oxygen species effect on proteins? 17. What is the effect of heavy metals on plant respiration? 18. What is the effect of heavy metals on the photosynthesis of plants? 19. How do heavy metals effect on the cell division? 20. What is the effect of heavy metals on mineral nutrition of plants? 21. Which metals have an antagonistic effect? 22. What metals have a synergistic effect? 24. What is the most common manifestations of the toxic effect of heavy metals? 25. Why root growth is more sensitive to the action of metals? 26. What is the area of the roots more susceptible to the action of heavy metals? 27. What has been the change in the morphology of roots under the influence of heavy metal?

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2.5. Mechanisms of resistance of plants to heavy metals The specificity of reactions of different objects to the action of heavy metals depends not only on the degree of violation of physiological and biochemical processes of plants, but also the ability to synthesize different metal-binding compounds and excretion of absorbed heavy metals from the active metabolism. The resulting response of the biological system to the excessive concentration of metal ions depends on the efficiency of the detoxification them in plants. In plants, there are a number of mechanisms of resistance to heavy metals. These mechanisms can be divided into two groups: 1) limiting the admission of metals in the plant and the cytosol; 2) changes in cell metabolism, to reduce the toxic effect of metals and their removal from the plants organism. The processes under consideration in the first group can not completely eliminate heavy metal ions flow into the cell and have a maximum efficiency at low concentrations of heavy metals in the environment, and in the case of a short exposure to heavy metals. Multiple intracellular detoxification mechanisms and the formation of plant resistance to heavy metals, along with an evolutionarily conserved mechanisms of maintenance of genome integrity, provide a stable functioning of the plants under stress caused by the presence in the environment of high concentrations of heavy metals. 2.5.1. Heat shock proteins Many intracellular detoxification mechanisms, as well as processes limiting the entering of heavy metals in plants are nonspecific. Thus, associated with heat shock proteins, cell response is not highly specialized is the generalized system, which activates the transcription of several genes that ensure cell survival under extreme conditions. All organisms respond to stress at the cellular level, the socalled rapid synthesis of stress proteins and the simultaneous inhibition of normal protein synthesis. Heat shock proteins (HSPs) are in125

volved in a number of important of folded layers of proteins, their transport across membranes, modulation of receptor activity and an a assembling of oligomeric proteins (Figure 67).

Figure 67. Heat shock proteins (http://www.bio.miami.edu/tom/courses/bil255/bil255goods/03_proteins.html)

It is expected that under stressful conditions, these proteins help repair denatured proteins and protect others from damage. This allows to recover and survive the cell under stress. Some of the main conservative heat shock proteins shows the high degree of homology among prokaryotes and eukaryotes. Many heat shock proteins act as chaperones. They restore the structure of other proteins denatured during thermal exposure. Some heat shock proteins are synthesized in the cells during development of plants and not induced by thermal effects, but they are referred to heat shock proteins by homology with the amino acid sequences of heat shock proteins induced by heat. Heat shock proteins are subdivided into five families according to their molecular weights. 126

HSP-100 act as chaperones. They carry out the disintegration of protein aggregates formed due to protein denaturation during thermal exposure, and to prevent errors in the folding of the polypeptide chain during the formation of the tertiary structure of the protein molecules. HSP-90 is found in the cytosol, nucleus and endoplasmic reticulum (ER). It is believed that they, too, can function as molecular chaperones, but unlike typical chaperones are highly specific to certain proteins and interact with them for a long time. HSP-70 – ATP-dependent molecular chaperones. Some members of this family are expressed constitutively, while others are induced by heat or cold shock. HSP-70 is found in the cytosol, endoplasmic reticulum, mitochondria, plastids and other organelles. HSP70 participate in folding and unfolding of the polypeptide chains, the assembly and disassembly of the quaternary structure of the protein. During heat shock HSP-70 are in the nucleolus and migrate into the cytoplasm after the heat exposure. N-terminal region of HSPI-70 containing ATP-binding domain are highly conserved, whereas their C-terminal sequence of various organisms is variable and determines the substrate specificity. HSP-60, apparently also are molecular chaperones. They are found in the mitochondrial matrix and the stroma of the chloroplast. HSP-60 not only induced by heat shock, but also present in plants and at normal temperatures. Their principal function consists in the assembly of protein molecules composed of subunits. One of the HSP-60 nuclear encoded chloroplast genome is a protein that is involved in assembly of ribose bisphosphate carboxylase – Rubisco. In virto HSP-60 prevent proteins from aggregation. Low molecular weight heat shock proteins with molecular weight from 15 up to 30 kDa are detected in cells of plants in large quantities. They are distributed to the different compartments of the cell. Low molecular heat shock proteins form complexes with molecular weights from 200 to 800 kDa. Although their role is unclear in many respects, it is assumed that they contribute significantly to the heat tolerance of plants. There is no evidence that they are re127

quired for normal cellular function, i.e. in the absence of heat shock. Low molecular weight heat shock proteins do not require ATP. It demonstrated that HSP-18, in pea (Pisum sativa) protein prevents aggregation at high temperatures. The most studied in plants HSP70 – a protein with molecular mass of 70 kDa, having in amino acid sequence the conserved regions. The mechanism of the protective effect of HSP-70 is the disaggregation of abnormal protein-protein interactions. It communicates with the dissociated or denatured proteins, which are produced during the thermal shock, exposure to toxic substances or radiation. HSP-70 protein facilitates the formation of de novo the folded layers of different proteins or rebuild these layers during stress. This protein is found in the cytoplasm, plasma membrane and nucleus of plant cells in response to Cd, and when stress caused by Cu, usually synthesized HSP-17. 2.5.2. Polyamines In response to heavy metals in plant to maintain a homeostasis the activation of various protection systems is occurred. One of these responses is the change in nitrogen-containing components, including polyamines (PA). Polyamines are widely distributed in plants. As described above, polyamines are low molecular polycations and are present in all living organisms. Diamine putrescine (Putr) and polyamines spermidine (Spd) and spermine (Spm) are low molecular aliphatic amines (Figure 68). Putrescine is the precursor of spermidine and spermine. In higher plants putrescine is synthesized from ornithine by the enzyme ornithine decarboxylase (ODC). An alternative biosynthesis pathway of putrescine in higher plants include an arginine decarboxylase. The product of this reaction–agmantin, then turns into putrescine. In animal cells putrescine is synthesized only by the ODC. Spermidine and spermine are synthesized by sequential addition of aminopropyl group to putrescine (Figure 69).

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Figure 68. Structure of polyamines (https://www.google.kz/search?q=polyamines&source=lnms&tbm=isch&sa=X&ved =0ahUKEwj7gtLDusPWAhXsDpoKHb72A18Q_AUICigB&biw=1920&bih=901#i mgrc=xYuJGlFVB8cbAM:)

They stimulate the reactions involved in the synthesis of DNA, RNA and proteins. As polycations polyamines have high affinities for biomolecules heaving negative charges, in particular DNA, RNA, phospholipids, and acidic proteins, and to anionic groups of components of cell walls and membranes. In plants polyamines are involved in many physiological processes, including cell division, cytoskeleton, initiation of root growth, embryogenesis and ripening. Polyamines prevent damage of biomolecules caused by drought, salinity, low temperatures and ozone. The structures of DNA, RNA, ribosomes, and membrane are in combination with polyamines are stabilized. By reducing the activity of RNA-ase and protease increased in stressful conditions, the polyamines improve the accuracy of reading the information in the synthesis of proteins and inhibit lysis of cell structures. The mechanism of the stabilizing effect of polyamines can be considered an example of DNA, a strong polybasic acid. It is fully 129

ionized at a pH above 4. At cytoplasmic pH the negatively charged phosphate groups on the periphery of the double helix of DNA form ionic bonds with a positively charged polyamines Spd and Spm, which protects DNA from damage. Polyamines plays an important role in the homeostatic regulation of cell pH and stabilize the cell membranes. Recently, much attention is paid to the study of changes in the content of polyamines in plants exposed to different kinds of stress (Figures 70).

Figure 69. Biosynthesis pathway of polyamines http://journal.frontiersin.org/article/10.3389/fgene.2015.00022/full

It observed that spermidine and spermine inhibit the hydrolysis of RNA and DNA. It is believed that this effect is due to inhibition of RNA-ase activity, through the binding with enzyme’s substrate. Polyamines binds to phosphate groups, form cyclic molecular compounds, called nuclear nuclear aggregates of polyamines. These molecules interact with the genomic DNA and thus protect them from the action of nucleases. The protective effect of these aggregates are more effective than individual effect of polyamines (putrescine, spermidine, spermine). The importance of polyamines in cell function is reflected in the direct regulatory control of their intracellular 130

level. An adequate level of polyaminesis achieved bycareful balance between biosynthesis, breakdown and absorption of amines (Galston, 2001). It is known that heavy metals affect the content of polyamines – putrescine, spermine, spermidine. The presence of copper in the growth medium induced an increase of polyamines content (putrescine, spermidine and spermine) in the cotyledons and hypocotyl 4-day-old sprouts of Brassica rapa L (turnips). There was an increase of content of putrescine in the leaves of rice under copper stress. In cotyledon leaves of celery with an excess of Cr (III) in the growing medium it was observed in a dose-dependent increase in the content of free and conjugated forms of putrescine.

Figure 70. Polyamines production in cells under stress conditions (http://journal.frontiersin.org/article/10.3389/fenvs.2015.00021/full) AOX, Alternative oxidase; ADC, Arginine decarboxylase; ASC, Ascorbate; APX, Ascorbate peroxidase; CAT, Catalase; DAO, Diamine oxidases; ETS, Electron transport chain; GSH, Glutathione; ODC, Ornithine decarboxylase; PA, Polyamine; PAO, Polyamine oxidases; SPDS, Spermidine synthase; SPMS, Spermine synthase; SOD, Superoxide dismutase. 131

Changing the content of the polyamines in the time range may indicate a change in the ratio of polyamines. In tobacco plants, it was found that the content of certain polyamines, their free and conjugated forms is changed at a certain time during the light and dark periods. It is known that heavy metals cause potassium deficiency in plants. In tissues, treated by heavy metals, increased electrolytes leakage, including potassium. It is believed that the inorganic cation is replaced by an organic polyamines to maintain the pH within the cell. The main difference between the polyamines and inorganic cations is that the content of potassium in cells depends on their entry into the cell from the outside, whereas polyamines synthesis occurs inside cells and their intracellular level can be controlled by the balance between their synthesis and degradation. In the cells of wheat high levels of potassium reduces the synthesis of putrescine and stimulated the conversion of the diamine into spermidine. An increase in the polyamines, in response to stress is often attributed to increased cell membrane peroxidation and minimize lipid membranes, as it is also known that at high concentration (10-50 mM) amines reduce accumulation of enzymatically generated reactive hydroxyl and superoxide radicals. The study of tobacco leaf, damaged by ozone showed that the oxidative effect of ozone is prevented by the use of putrescine through the roots. The level of reactions of polyamines with hydroxyl (*OH), sulfite- (SO3*) and superoxide/peroxide radicals (O2*, HO2*) has been relatively slow, but the reaction of natural conjugates of putrescine with hydroxyl decinnamate, coumarate, kofeate and ferulate were more active (100-1000 times) in quenching of radicals. According to the literature, under heavy metals polluted environment, polyamines have also signaling function and act as antioxidants (Figures 71, 72, Table 3). In plants, the polyamines can be converted to higher polyamines (conjugated forms) and accumulate the latest amines for greater resistance to environmental stresses. For example, in wheat was found covalently conjugated (CC) and non-covalently conjugated (NCC) 132

polyamines with DNA. In the resistant variety it was observed the highest elevation of the NCC spermidine. The increase of the polyamines like as diamine putrescine and the polyamines spermidine and spermine, under the influence of heavy metals indicates the great significance of polyamines in response protective reaction of plants under stress conditions as stabilizers of cell membranes, is a buffer mechanism for cells to maintain homeostasis, as well as participating in the protection of plant cell from oxidative stress.

Figure 71. The role of endogenous and exogenous polyamines in maintaining redox homeostasis under salinity stress (http://journal.frontiersin.org/article/10.3389/fenvs.2015.00021/full)

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ANN, Annexin-formed channel; CAX, Cation/H+ exchangers; CNGCs, Cyclic nucleotide-gated channels; cGMP, cyclic guanylcyclase; DA-NSCCs, Depolarizationactivated NSCCs; DAO, Diamine oxidases; FV, Fast vacuolar channel; HACC, Hyperpolarization-activated Ca2+ influx channel; KIRC, K+ inward-rectifying; KORC, K+ outward rectifying; VK, K+-selective channels; ROSIC, Non-selective voltage-independent conductance; NHX, Na+/H+ antiporters; PA, Polyamine; PAO, Polyamine oxidases; ROS-NSCC, ROS activated Non-selective cation channel; SOS1, SOS2, SOS3, respectively, Salt overly sensitive 1,2,3; SV, Slow vacuolar channel; VI-NSCCs, Voltage-independent nonselective cation channels. Figure 72. Relationship between polyamines and ROS during salinity in the context of ion transport regulation at plasma membrane and vacuole (http://journal.frontiersin.org/article/10.3389/fenvs.2015.00021/full)

Plant polyamines are thought to contribute to cellular responses during salt stress through modulation of ROS homeostasis via two distinct mechanisms (Takahashi and Kakehi, 2010). First, polyam134

ines promote ROS degradation by scavenging free radicals and activating antioxidant enzymes during stress conditions (Gupta et al., 2013). Free polyamines detoxify of superoxide anions and hydrogen peroxide, and the conjugated polyamines are responsible for scavenging other ROS (Kubis, 2005). Conjugated polyamines showed more antioxidant ability than free polyamines. Polyamines promote ROS production through polyamine catabolism in the apoplast (Yoda et al., 2006; Marina et al., 2008; Mohapatra et al., 2009; Campestre et al., 2011). In transgenic Nicotiana tabacum overexpressing an ornithine decarboxylase (ODC) gene, free polyamine content increased by 2-4 fold and germination increased by 33–45% on high salt medium (Kumria and Rajam, 2002). When S-adenosyl methionine decarboxylase (SAMDC) gene was introduced in Nicotiana tabacum increased content of soluble polyamines, seed weight, photosynthetic rate and expression of antioxidant enzymes (APX, Mn-SOD, and glutathione S-transferase) in comparison with untransformed lines (Wi et al., 2006). The result of the overexpression of SAMDC cDNA from Tritordeum in Oryza sativa was the higher production of free polyamine content (Put, Spd, Spm), and a reduction in salt-induced shoot growth repression compared to non-transgenic rice plants (Roy and Wu, 2002). It is considered that exogenous application of polyamines has a significant effect on the plant, and this is a potential strategy to increase plant survival under salt stress. Application of spermidine promoted the tolerance to osmotic and salt stress in Arabidopsis and rice due to enhanced polyphenol accumulation, enchanced activities of catalase and superoxide dismutase (Sreenivasulu et al., 2000; Cheruiyot et al., 2007; Roychoudhury et al., 2011; Radhakrishnan and Lee, 2013). Application of putrescine increased the activity of antioxidant enzymes and carotenoids content in leaf tissues under salt stress in Brassica juncea and enhanced seedling growth as compare to the untreated controls (Verma and Mishra, 2005). These studies show that alteration of polyamine accumulation through manipulation of polyamines biosynthesis genes or direct application could have an effect on physiological responses to salt stress. 135

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Table 3 Effect of polyamines in the regulation of various enzymatic and non-enzymatic antioxidant components in salt stressed plants. (http://journal.frontiersin.org/article/10.3389/fenvs.2015.00021/full)

Under salt stress, exogenous application of spermidine blocks VI-NSCC reducing the inward flow of Ca2+ and Na+ and efflux of K+ in barley (Zhao et al., 2007). Polyamine accumulation under salt stress leads to higher vacuolar Na+ sequestration and an improved cytosolic K+/Na+ homeostasis (Zepeda-Jazo et al., 2008). It was assumed that polyamines are involved in the process of modulation of the activity of certain Ca2+- channels, prevents Na+ and K+ entry into the cytosol, sequestration Na+ into the vacuole (Yamaguchi et al., 2006). Polyamines have a great effect on ion homeostasis under salinity stress (Figure 73).

Figure 73. Effect of polyamine in ion homeostasis in plant cells

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2.5.3. Effect of heavy metals on organic acids and thiols content in plant cell The most effective detoxification mechanism is the binding heavy metals ions to organic acids and thiols in the cytoplasm and then transport these complexes into vacuole (Figure 74). The accumulation in vacuoles – the most effective mechanism for detoxification of heavy metals tolerant organisms. And resistant species mostly accumulate toxic ions in vacuoles of roots and shoots. The metals in the reached leaf sheets are accumulated in the vacuoles of the epidermis and its derivatives – trichomes and glands that serve to remove metals from the plants organism. Citrate, and other low molecular weight dicarboxylic anions and organic cations in xylem stream are involved in the translocation of heavy metals to the shoots. Copper acropetally moves through the xylem. In xylem exudation copper ions are present in one or more negatively charged complexes, possibly, including the amino acids. Over 99% of copper is in a complex form in xylem sap of sunflower. Amino acids that are usually present in higher amounts than copper ions in the xylem sap could form stable complexes with copper. There are asparagine, histidine in bean, asparagine and glutamine in tomato exudate. Nicotinamine (NA) is a major transporter of Cu in the xylem sap of tomato. Subtoxic concentrations of Cd in stems of tomato, not preexposed and exposed for 24 hours in 2,5 x10-4 M citrate solution increased the Cd translocation from the roots to the aerial organs. Increasing the concentration of cadmium up to 5x10-6 M increased translocation of cadmium to aerial parts in 6-8 times as compared to control variant without pre-incubation in citrate and variant with simultaneous addition of cadmium and citrate. Pre-incubation with citrate doubles the amount of citrate, collected from the xylem exudate. In other studies citrate showed a decrease the lateral extraction of cadmium in xylem vessels and decreased the adsorption by xylem cell walls of cadmium. The study of the bean plants demonstrated that there is a competition between cadmium and other cations of xylem sap for binding sites in the xylem wall, mainly between Cd2+ 138

and Mg2+. This process is simplified by mechanism that can be compared with the ion exchange chromatography.

Figure 74. Schematic representation of major functions, interrelationships among thiol and non-thiol compounds, and their coordination with other defense system components in metal(loid)-exposed plants(http://journal.frontiersin.org/article/10.3389/fpls.2015.00192/full) (A) metal ion binding to the cell wall and root exudates; (B) reduction of metal influx across the plasma membrane; (C) membrane efflux pumping into the apoplast (ATP-binding-cassette (ABC) and P1B-ATPase transporter); (D) heavy metal (HM) chelation in the cytosol by ligands such as phytochelatins (PC), metallothioneins (MT), organic acids, and amino acids; (E) ROS defense mechanism [Antioxidant enzymes (SOD: superoxide dismutase, CAT: catalase, APX: ascorbate peroxidase, GPX: glutathione peroxidase, GSH: glutathione reduce and GSSG: glutathione oxidase)]; (F) hormone signaling pathway. (G) Transport and compartmentalization in the vacuole (ABC and P1B-ATPase transporter, NRAMP: natural resistance associated macrophage protein, CAX: cation/proton exchanger). Metal ions are shown as black dots.

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Citric acid and other low molecular weight dicarboxylic acids are common components of the xylem sap. Cadmium entering the xylem, forms stable complexes. The stability of these complexes is enhanced when citric acid was added to the culture medium. Organic complex 109Cd-citrate was detected by electrophoresis in xylem sap of rice. It is believed that 50% of xylem cadmium is in inorganic form, possibly, in Cd2+or Cd (OH)2 form. Phytochelatin (PC) in Cd-exposed plants may facilitate or reduce the translocation of cadmium. Nevertheless, most of the works with high and low levels of cadmium exposure indicates that the citrate and optionally other dicarboxylic acids, and Ca2+ and Mg2+ions as a result of competing for binding sites in the xylem wall may facilitate the translocation of cadmium. It was found the relationship between Ni content and citric acid in mature leaves of 15 species of plants- hyperaccumulators. The study of complexes of organic acids with Ni, using ion exchange and liquid chromatography (HPLC) have shown that in Psychotria douarrei Ni is present mainly in the form of negatively charged malate complex balanced by cationic hydrated complex of nickel. In the plant Ni-hyperaccumulator Phyllanthus serpentinus L. it was found 42% of citric acid and 40% of malic acid bound to nickel. «Nickel» plants are ideal objects for studying the biochemistry of organic components, as Ni content is so high in them that are isolated milligrams of these complexes as in conventional plants – micrograms. L. Pancaro et al. (1978) in the study of Alyssum bertolonii L. found that in plants growing on serpentine soils, Ni was bound to malic acid and malonic acid in the ratio 1: 1. Stockley (1980) found malic acid in the crude extract of the plant and is suggested that heavy metals binding to malic acid inhibits the citric acid cycle in the step of conversion of succinate to fumarate. The conversion of oxaloacetate into citrate is controlled by malate produced by the condensation of acetate units and small residues of the conversion of succinate to fumarate. It was analyzed the organic acids and amino acids composition of xylem sap of hyperaccumulator Thalspi caerulescens L. and non-hyperaccumultor Thlaspi arvense L. The authors suggested that a significant amounts of Zn, entering the aerial organs, make complexes or associated with one or 140

many low-molecular ligands. The prevalent organic acid in xylem sap of Thlaspi were oxalic and malic acids. The oxalic acid content was increased in response to a Zn-exposure. Zinc-resistant plants usually accumulate a large amount of organic acids and form Zncytrate complexes. Zinc is also associated with malonic acid. Zinc accumulation in vacuoles in resistant plants may be determined by generation of a large amount of malic acid and citric acid as compared to sensitive to Zn plant species. Organic acids are involved in the transport of Zn from the cytoplasm to the vacuole to form chelate complexes with metal cations. It was defined the chemical forms of Zn in tolerant hyperaccumulator Arabidopsis halleri L. and non tolerant Arabidopsis lyrata L. In the above-ground parts of plants Zn was octahedrally coordinated and complexed with malate. Secondary organic substances were identified on the base of trichomes, which contained a significant amount of Zn, which was tetrahedrally coordinated and complexed with carboxyl or hydroxyl groups. In the roots of Arabidopsis halleri, grown in hydroponic medium, it was found phosphates of Zn. The roots of Arabidopsis halleri, grown on the contaminated soil, Zn was detected as malate, citrate and phosphate complexes. Theoretically, it is assumed that most of Fe (II) and Zn (II) in the xylem sap exist in the form of citrate chelate, whereas Cu (II) is chelated by different aminoacids, including histidine and asparagine. Other chelating components can also play significant role in the transport of metal ions in plants. Nicotinamine has the ability to form complexes with various divalent metal ions including Cu, Ni, Co, Zn, Fe, Mn. Studies on tomato mutants (Lycopercicon esculentum Mill.), chloronerva, which lacks the ability to synthesize nicotinamine (NA), clearly demonstrated that nicotinamine included in the distribution of Fe (II) and optionally, Zn (II) and Mn in young growing tissues through phloem in dicots. In addition, the metals can be transported in floem chelated with other low molecular weight metabolites or proteins. Thus, organic acids play an important role in detoxification of heavy metals in the cell by chelating metals and participating in the transport of metabolically inactive compartments. 141

2.5.4. Metallothioneins To identify mechanisms of hypertolerance and hyperaccumulation of heavy metals in plants it is a necessary step in the development of phytoremediation. The researchers suggest that the increase in concentration of metal-binding proteins or peptides in plant cells can increase the ability to bind metals and plant tolerance. Detoxification processes can be specific or nonspecific. It depends on whether the synthesis of binding heavy metals compounds is induced or they are formed in a cell constitutively. Specificity for binding metal ions are considered cysteine-rich proteins – metallothioneins synthesized in animal cells and plant organisms in response to heavy metals. Metallothioneins, metal-binding proteins, got their name due to the high metal content, which can reach 20% of the molecular weight (Salt and Rauser 1995). Metal-binding proteins are commonly synthesized in small quantities. Their content in the cell increases rapidly if they are affected by heavy metals and decreases in the case of reducing their concentration in the nutrient substrate (Gobbett 2000). Sulfur is present in metallothioneins usually in the form of thiolate and it’s content is generally equal to 10-13% (Oven et al. 2001). Moreover, increased concentrations of heavy metals in the environment not only stimulate the synthesis of metallothioneins, but assist to bind these proteins to metals. Nevertheless, both the process of synthesis of metal-binding proteins and the system heat shock is an integral response of a cell to the effect of many stressful agents (Figure 75). According to recommendations of the Committee on Nomenclature of Metallothioneins the 2-nd International Congress on Metallothioneins and other low molecular weight metal-binding proteins (Zurich,1985) any polypeptide which is similar in structure and function to the mammalian metallothioneins may be considered to be in the group of these compounds. Metallothioneins are divided into three classes based on the chemical structures of their molecules (Rauser, 1995). First class (MT1) – metal-binding proteins invertebrates. In the MT1groupthe metal-binding domain molecule contains 20 cysteine residues, the location of which is always constant for this class. 142

Figure 75. The role of metallothioneins in plant cell (http://web2.mendelu.cz/af_239_nanotech/J_Met_Nano/0214/02-use-of-massspectrometry-technique.html)

The second class (MT2) – polypeptides that are similar in structure to the MT1, but do not have such a conservative position of cysteine residues. They are common for invertebrates, plants, fungi, cyanobacteria and other prokaryotes, algae and yeast. The third class (MT3) – phytochelatins (PC), kadastins, glutamylpeptides, i.e. polypeptides of some algae, higher plants and fungi containing-γ-glutamylcystein residues, and differ from other metallothioneins by enzymatic method of their synthesis. All MT I (mammals) and MT II are composed of one polypeptide chain containing 60 amino acid residues (sometimes 25 and 70). Number of Cys(cysteine) there in up to 30%. Often there is such sequence as Cys-X-Cys, where X is anyone different from the amino acid cystein (Cys). Among the basic amino acids the metallothioneins more often contain Lys (lysine), rarely – Arg (arginine). Metallothioneins of higher plants and algae (III) are composed of two or more amino acids and polypeptides, including primarily cysteine, γ-glutamic acid and glycine. The most common sequence is Cys-γ-Glu-Cys. In most metallothioneins all Cys residues are deprotonated and are able to bind heavy metalsin the ratio 3 ligands to one metal ion. Glutamine (Glu) performs a definite role in the mechanism of re143

sistance. This compound restores the metallothionein molecule oxidized bysuperoxide anion radicals (Ruttkay-Nedecky et al., 2013). Glutamine is involved in the synthesis of phytochelatins in the cells of higher plants. The specificity of this group of plant metallothioneins is a large gap the length of which is about 40 amino acid residues including the aromatic acid residues, separating it into two metal binding domains. The length of the gap of other groups of metallothioneins is less than 10 amino acid residues, and contains no aromatic amino acids (Van Hoof, 2001). Stimulation of metallothionein synthesis of the corresponding class is dependent on many factors. The main ones are the properties of heavy metal and its concentration,the ionic environment, specific features of the plant. Various metal ions stimulate the synthesis of metallothioneins not to the same extent. Metals such as Ca, Al, Na, Mg, Udo not induce metallothioneins synthesis (Grennan 2011; Emamverdian et al. 2015). Elevated levels of metal binding peptides in the cell under the effect of Fe and Cs are obviously observed in certain cases, which depend on the type of a plant and the concentration of heavy metals (Dahmani-Muller et al. 2000). Predominantly Cd, Zn, Cu, Hg, Au, Ag, Co, Ni, Pb induce metallothionein synthesis. However, the effectiveness of the activation of the MT synthesis is different. For example, according to some data the synthesis of MT1 and MT2 is already stimulated by Cd at concentration of about 10-7 M, whereas to obtain the same effect concentration of Zn exceeds 3x10-4 M (Schat et al. 2002). The same metal also affects the formation of metal binding proteins differently. For example, Cd induces MT2 synthesis some time after the beginning of MT3 synthesis (Van Hoof et al. 2001). The formation of metallothionein shows the dependence on the ionic environment. It was established (Okuyama et al. 1999) that he synthesis of CUP1 in Saccharomyces cerevisiae, inducible by Cu, while culturing yeast in a medium which contains ions of other heavy metals is largely reduced. At the same time to inhibit the synthesis of CUP1 the content of Co2+, Ni2+, Zn2+ should be at least an order of magnitude higher than of Cd2+and Mn2+ . 144

The plants can form metallothioneins of several classes and the synthesis of metallothioneins is carried out in different organs. For example in Arabidopsis were found metal binding proteins of all known types and in Silene vulgaris L. – MT2 only (Van Hoof et al. 2001; Jack et al. 2007). Under the effect of Cu in Arabidopsis MT2 is synthesized in trichomes and MT1 – in leaves, roots and flowers . Differences in the expression level of metal binding proteins in Silene vulgaris populations were associated with their different tolerance to copper (Jack 2007). The most widespread plants metallothioneins are metallothioneins of the third class (MT3) – phytochelatins (PC) found in almost all species of plants, as well as some fungi and invertebrates (Schmаger et al. 2000; Cobbett 2000; Ruttkay-Nedecky et al. 2013) (Fig. 72). Therefore the investigation of genetic and molecular basis of metal detoxification in plants by this group of metal binding peptides is of greatest interest. First PCs were found by researchers in the Schizosaccharomyces pombe, in cell culture of Rauvolfia serpentine. Nowadays there is considerable progress in understanding the molecular mechanisms of synthesis and functioning of MT3. Structure of phytochelatins (PC). Phytochelatins are compounds of general formula (γ-Glu-Cys)n-Gly, where n is equal maximum to 11, but usually varies from two to five (Clemens et al. 2002). On this basis MT3 are divided into two groups: with low and high molecular weights (Schmаger 2000). The amino acid in the amino terminal MT3 structure can vary: in addition to glycine(PC) it can be serine (hydroxymethyl-PC), β-alanine (homo-PC), and also glutamine (Glu) and cysteine(Cys) (Clemens et al. 2002) (Figures 76, 77). The ratio of PC and their derivatives is dependent on the plant species as well as on the ratio of metals in the soil or nutrient solution (Cobbett 2000). For example, resistance to Cd of Oryza sativa L. is provided by hydroxymethyl-PC (Batista et al. 2014). Thiol-peptide level and proteomic changes in response to cadmium toxicity in Oryza sativa L. roots (Wu, 2013), in Vigna 145

angularis are only provided by homo-phytochelatins (Oven et al., 2001). Biosynthesis of PC. There are many mechanisms that regulate the synthesis of phytochelatins, which includes several stages (Cobbett, 2000; Tsuji et al., 2003).

Figure 76. Phytochelatins (http://www.scielo.br/scielo.php?script= sci_arttext&pid=S1677-04202005000100006)

Figure 77. Biosynthesis of phytochelatins in higher plants (http://www.scielo.br/scielo.php?script=sci_arttext&pid=S167704202005000100006)

For example, in Brassica juncea L. the synthesis of phytochelatins is preceded by a series of stages, the initial one is the reaction between cysteine and glutathione. This process is regulated by genes involved in the transport and sulfur assimilation and the biosynthesis of glutathione. In Arabidopsis thaliana L. biosynthesis of PC begins with metal activating transcription of genes encoding glutathione 146

reductase (GR), and enzymes involved in biosynthesis of glutathione: γ-glutamyl cysteine synthetase (γ-Glu-Cys-synthetase) and glutathione sinthetase (GS) (Rauser 1995). Glutathione is the main substrate for PC formation and the key enzyme with crucial activity in the process of biosynthesis is PC-synthase. In Triticum aestivum L. the synthesis of PC from glutathione can be carried out without intermediate steps. This process is catalyzed by PC-synthase (Ahner et al., 2001; Clemens et al., 2002). The specific activator of this enzyme is mainly Cd, but some other heavy metals can provide this role as well. In descending order of their specificity they can be arranged as the following: Ag, Bi, Pb, Zn, Co, Hg, Au (Schat et al. 2002). Previously it was thought that only free metal ions are able to activate PC-synthase and subsequent PC synthesis. Now it is known that anions (AsO4)3-, AsO2-(Schmаger et al., 2000), phosphate anions (Kvesitadze et al. 2002) and jasmonic acid can participate in this process. It is also shown that heavy metal-thiolates, glutathionates and heavy metal complexes with low molecular weight PC, are active substrates for the synthesis of MT3, being either a catalyst or a substrate. Thus, the formation of MT3 is activated when exposed to a large number of heavy metals. However, only the Cd detoxification mechanism can be considered universal: more than 90% of the Cd2+ ions penetrating into the cells of studied 200 species of three taxa (Bryophyta, Pteridophyta, Spermatophyta) are associated with phytochelatins synthesis (Oven et al. 2001; Jack et al., 2007). The role of PC in the detoxification of metal ions. A number of research clearly demonstrate that MT3 are involved in the detoxification of heavy metals in plants, although there are some other hypotheses about the role of PC (Oven et al. 2001). Metal detoxification mechanism by PC includes a number of steps: 1) PC-synthase activation by metals; 2) the complex formation of MT3 with metals; 3) the complex transfer to the vacuole. Moreover it is considered that low molecular weight MT3 transport Cd to the vacuole where it accumulates in the form of a complex with macromolecular MT3 or organic acids (Inouhe et al. 2000). 147

Any violation of at least one stage of detoxification leads to lessening the plant tolerance to heavy metals (Oven 2001). For example, either damage of PS synthase gene or glutathione synthase gene lead to hypersensitivity of organisms to Cd (Oven et al. 2001). Conversely, overexpression of these genes increases tolerance of plants, as it was demonstrated in cell cultures Lycopersicon esculentum and Saccharomyces cerevisiae. The violation of enzymes involved in the metabolism of sulfur (Schmаger et al. 2000) and reduced amount of necessary for the synthesis of glutathione sulfurcontaining amino acids – Cys (Dominguez et al. 2001), also affects the resistance of plants to Cd. With the growth of polymerization degree the affinity of MT3 to metal ion (Delhaize et al. 1989) increases and hence does the efficiency of detoxification. It is proved that the tolerance to heavy metals increases with the growth of degree of polymerization MT3 and with saturation of the coordination valence with sulfide anions, i.e. with the formation of additional bonds to S2- (Oven et al. 2001). In its turn, the increase in the proportion of molecules with high degree of polymerization occurs with the concentration growth of Cd and time of the exposure (Inouhe 2000). Formation of complexes is largely dependent on the pH of the solution. In the acidic medium metal is replaced by hydrogen and hence the efficiency of detoxification of heavy metals decreases. It is important to mention that the activation of MT3 synthesis is already observed within a few minutes after the treatment of plants with heavy metals (Gachot et al. 1994), but the highest concentration of peptides in the tissues is revealed only after a certain time after the start of exposure. Some authors suggest that phytochelatins play no significant role in the hypertolerance of plants to heavy metals. Although in cell culture the expression of metallothioneins (Grill et al. 1989) or phytochelatins (Rauser, 1995) increased plant tolerance to cadmium, the transfer of genes responsible for the synthesis of metallothioneins in higher plants had no effect on the accumulation of metal ions. While testing plants-hyperaccumulators there were no changes inthe concentration of phytochelatins that suggests that hypertolerance to Cd and Zn is provided not by phytochelatin synthesis. The 148

evidence for the certain role of phytochelatins is that there was found correlation between their presence and normal rate of tolerance to the metals. The mutation leading to inability to produce phytochelatins resulted Arabidopsis thaliana L. in being hypersensitive to Cd. It was found that high phytochelatins content correlates with the ability of plants to transport Cd to aerial organs. The alternative point of view is that phytochelatins supply plants with normal resistance to metal excess in the environment. For plants with normal resistance (A. thaliana) the synthesis of phytochelatins is definitely necessary if there is an excess of metals in the environment while for hypertolerance of plants – hyperaccumulators PCs unlikely play any role (Lombi et al. 2001). To determine the limiting factors of heavy metals accumulation and to obtain tolerant transgenic plants with enhanced ability to accumulate heavy metals, gene of Escherichia coli gshii, encoding the synthesis of γ-glutathione synthetase(GS) was activated in the cytosol of Indian mustard. Transgenic plants significantly more than wild species accumulated metal: the concentration of Cd in the aboveground organs was higher by 25%. However, these plants showed enhanced tolerance to Cd in the phase of seedlings and mature stage. The accumulation of Cd and tolerance was correlated with the level of gshii gene expression. Cadmium treated plants contained a larger amount of glutathione, phytochelatins, thiols, S and Ca as compared with the wild type. It was assumed that the enzyme (GS) at Cd presence is a limiting factor for the biosynthesis of glutathione and phytochelatins. Gamma-glutathione synthetase (GS) overexpression is a promising strategy for the production of plants with necessary for phytoremediation superabilities. Reduced glutathione GSH plays an important role in the protection of plants against various stresses. Glutathione is not only a substrate for glutathione-S-transferase, neutralizing the potentially toxic xenobiotics but also a reducing agent of dehydroascorbate. Moreover, GSH is a precursor of phytochelatins. Phytochelatins contain high percentage of Cys-sulfhydryl residues that bind and isolate ions 149

into stable complexes and are induced by metals such as Cd in all tested plants . Glutathione is synthesized from its constituent aminoacids in 2 consequences. ATP-dependent reaction is catalyzed by γ-glutamylCys-synthetase (GCS) and by γ-glutathione synthetase (GS), respectively. Phytochelatine synthase sequentially catalyzes elongation of (γ-Glu-Cys)n by the transfer of γ-Glu-Cys-group to glutathione or phytochelatins (Chen et al. 1997). Manipulation by the expression of enzymes involved in the synthesis of glutathione and phytochelatins can be an excellent approach for the improvement of plant resistance to heavy metals. Phytochelatine synthase enzyme can be not a limiting factor for the synthesis of phytochelatins due to their constitutive expression in plants (Steffens, 1990) and the activation at the presence of metals. Genes encoding enzymes involved in the synthesis of glutathione are more promising in this regard. Limiting step for the synthesis of glutathione in the absence of metal is a reaction catalyzed by γ-glutamyl-Cys-synthetase since the activity of this enzyme is regulated by glutathione feedback and depends on the accessibility of Cys. Overexpression of the gene gshi of E.coli encoding γ-glutamylCys-synthetase increased glutathione levels in poplar. Furthermore, the expression in tomato of γ-GCS can restore the tolerance of glutathione-deficient mutant of Arabidopsis cad2. However, the overexpression of this gene did not increase tolerance to Cd in wild-type of Arabidopsis. Normally the GS is not a limiting factor as glutathione content does not change much due to the low concentration of phytochelatins. Overexpression of the gene gshii of E.coli, coding GS did not increase the level of glutathione in poplars (Foyer et al. 1995). Nevertheless, the presence of heavy metals affects the regulation of the biosynthesis of glutathione which is substantially altered. Heavy metals activate phytochelatins synthase and thereby induce biosynthesis of phytochelatins resulting in reduced glutathione levels. Successively as a feedback, glutathione removes the inhibition of γ-glutamyl-cys-glutathione synthetase. 150

Moreover, expression of γ-glutamyl-Cys-synthetase can be increased by heavy metals. It was demonstrated that Cd increases the gene transcription of γ-glutamyl-Cys-synthetase and deactivates the GS. There is a decrease of glutathione and its accumulation is inhibited by γ-Glutamyl-Cys while reducing the activity of GS. The corn roots exposure in the presence of Cd caused decrease of glutathione and γ-glutamyl accumulation of cysteine by reducing the activity of GS. Therefore, GS can become a limiting factor for the biosynthesis of glutathione and phytochelatins. Overexpression of gshii gene can increase the content of glutathione and phytochelatins synthesis (Figures 78, 79).

Figure 78. Regulation of phytochelatin synthesis in plants (Atabayeva, 2016).

High levels of glutathione in the roots of transgenic plants produces greater resistance to cadmium. Cd-PC form complexes with sulfide groups in vacuoles. It is believed that tolerance to metal scan be limited by the availability of sulfur to the synthesis of Ca sand sulfides. The level of total sulfur was higher in aboveground organs. 151

Cadmium significantly reduced the concentration of calciumin wild and transgenic plants, but overexpression of the GS gene decreased the rate of decline in Ca in the aerial parts. The roots with the calcium content are not much different in the transgenic plants and the wild type. Cd is a calcium channel blocker, Ca interferes with binding to calmodulin, the protein which regulates the activity of many enzymes and cellular processes (Marchiol et al. 1996). Increased levels of Cd-bound peptides in transgenic plants may reduce the effect of cadmium on interaction with calcium. Transgenic plants store more Cd in the aboveground organs. Cd translocation from the roots to the aerial parts of the xylem transpiration stream was provided by transpiration flow (Petit, 1978). The more Cd binds phytochelatins stored in vacuoles in transgenic plants, the smaller are destroyed vital biochemical and physiological processes. This leads to the leaf surface increase thus to greater accumulation of Cd (as a result of increasing the transpiration).

Figure 79. Phytochelatins synthesis (http://rydberg.biology.colostate.edu/ epsmitslab/PCproject.htm)

Transgenic plants absorb more cadmium due to less damage to the root surface. Water absorption is the primary mechanism for in152

creasing the movement of Cd in the plant (Petit et al. 1978). High level of phytochelatins in the roots of transgenic plants reduces the negative effect of Cd on water absorption. Thus, regulation of synthesis of glutathione promotes accumulation of heavy metals and increases the tolerance of transgenic plants. Transgenic plants allow to increase the efficiency of the manipulation of gene expression of glutathione synthesis, may be one of promising approach to increase phytoextraction of heavy metals and resistance of plants. Test questions: 1. What groups can be divided into mechanisms of plant resistance to heavy metals? 2. What function is performed by heat shock proteins (HSP)? 3. What groups are divided into HSP? 4. What function is performed by HSP-100? 5. What function is performed by HSP-90? 6. What function is performed by HSP-70?7. What is the function performed HSP-60? 8. What function is performed by the low molecular weight HSP? 9. The role played by organic acids in the detoxification of heavy metals in a plant cell? 10. What organic acids involved in the binding of heavy metals? 11. What is the metallothioneins? 12. What groups are divided metallothioneins? 13. What is the chemical structure of metallothionein? 14. What stimulates the synthesis of metallothionein metals? 15. What is the structure phytochelatin? 16. What is the function performed phytochelatins? 17. As the synthesis phytochelatin? 18. What is the role phytochelatin in the detoxification of heavy metals in a plant cell?

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CONCLUSION In ever-changing climate and increasing anthropogenic pollution solution of problems of plant resistance to adverse environmental conditions acquires global concern. The need to increase crop yields in unfavorable conditions, identification of adaptation mechanisms to various kinds of stressors is the most important challenge faced by scientists around the world. The adverse environmental factors are called stressors. Plants are characterized by three phases of responses to stress: 1) primary stress response, 2) adaptation, 3) depletion. The stress depends on the magnitude of the damaging factor, duration of exposure and the resilience of its plants. Plant responses to stress are divided into 2 types: non-specific, independent of the type of stressor and specific, peculiar only to certain types of stress. Under stress conditions, plants use different strategies for survival to avoid stress and use resistance mechanisms. Under the effect of stressors during acclimation plants acquire resistance to the stressor, but this tolerance is not inherited. At low doses repeated stress leads to hardening of the plant organism, and tolerance to one stressor enhances resistance of the plant organism to other damaging factors. During evolution process in plants develop adaptive reactions to the certain stressors also that are transmitted genetically. At the organism level retain all cellular mechanisms of adaptation and supplemented with new, reflect the interaction of the whole plant. First of all, it is a competitive relationship for the physiologically active substances and foods. This allows the plants under extreme conditions to form only a minimum of the generative organs, which they are able to provide the necessary nutrients for maturing. Under adverse conditions, the aging process and abscission of the lower leaves accelerate, and the products of hydrolysis of organic compounds are used to power the young leaves and the formation of 154

generative organs. Plants are able to replace lost or damaged organs through regeneration and growth of axillary buds. In all these processes correlative growth and intercellular regulation system (hormonal, trophic and electrophysiological) are involved. Under conditions of prolonged and severe stress first of all are dying plants sensitive to stress. They are eliminated from the population, and the seed progeny form more resistant plants. As a result, the level of resistance in the population increases. Thus, at the population level nature selection process is included, leading to the emergence of a more adapted organisms and new species. Therefore, a basic knowledge of the physiology of stress, the study of the mechanisms of action of different stressors, specific and nonspecific reactions of plants, as well as the mechanisms of resistance, developed in plants during the evolution process are necessary to create new tolerant species of economically valuable crops.

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GLOSSARY Adaptation – is genetically fixed constitutive feature present in the plant regardless of whether stress there is not or it is. Adaptations do populations of organisms adapted to the respective environmental conditions (morphological changes depending on the growing conditions). Adaptations are also manifested at the biochemical level, for example, the biosynthesis of substances that can help reduce the negative impact of stressors. Acclimation – responses of plants that allow plants to adapt to the new stress conditions. During the process of acclimation plants acquire resistance to the stressor. Acclimation occurs during the life of the plant and is not inherited. However, it is based on the opportunities inherent in the genotype, i.e. within normal reaction. Active transport – transport of ions against an electrochemical gradient. Implement transport ATPase using ATP energy. Antioxidant system – plant protection system aimed at reducing oxidative stress. It includes several enzymes and low molecular weight substances that are present in plants (superoxide dismutase (SOD), catalase (CAT), ascorbateglutathione cycle (AGC), ascorbate peroxidase (APO), glutathione). Antiport of ions – transfer of some ions through the membrane into the cell due to the removal of other ions. Apoplast – a unified system of cell walls. Resistance – the ability of plants to maintain a constant internal environment (maintain homeostasis) and to implement life cycle under the effect of stressors. ATPase – specific phosphatase which catalyzes the cleavage reaction of a phosphate group from a molecule of ATP. The biological role of ATPases is releasing energy pyrophosphate bond in the hydrolysis of ATP Heat shock proteins (HSPs) – a protein synthesized under stress conditions, they contribute to the repair of denatured proteins and other protected from damage. ATPase V-type – vacuolar type ATPase, acting at the level of the tonoplast. ATPase P-type – ATPase, acting at the level of the plasma membrane (plasmalemma). ATPase F- type – ATPase, acting at the level of the thylakoid membrane and the inner mitochondrial membrane. Casparian strip – the endoderm cell walls impregnated with suberin, a waxy substance that create a water impermeable barrier. Passage of ions through the endoderm is a limiting factor in the translocation of metals to the aerial organs. Catalase (CAT) – this enzyme catalyzes the oxidation of hydrogen peroxide to form molecular oxygen H2O2 + O2 → 2H2O. Prosthetic groups of CAT is heme, which includes the iron atom.

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Development – a qualitative change in the structure and functional activity of plant and its parts (organs, tissues and cells) during ontogenesis. Diffusion – type movement substances driving force is the concentration gradient or a chemical potential gradient. Heavy metals – metals with an atomic weight greater than 50 and a density > 5 g/cm3. In the definition of the term «heavy metal» takes into account the following parameters: their high toxicity to living organisms at relatively low concentrations, as well as the potential to bioaccumulate. As applied studies to heavy metals often are included Pt, Ag, W, Fe, Au, Mn. Fenton reaction – oxidation of the hydroxyl group α-hydroxy acids and αglycol to a carbonyl group and the formation of hydroxyl radicals in the presence of hydrogen peroxide) to the apoplastic hydrogen peroxide to generate hydroxyl radicals. In such reactions can be involved iron (Fe), and other redox metals of variable valency (Cu). Growth – an irreversible increase in the size and mass of the cell, organ or whole organism. It reflects the quantitative changes that accompany the development of an organism or its parts. Lipid peroxidation (LP) – is the formation of lipid radicals due to hydrogen peroxidation from unsaturated fatty acids (FA) by reactive oxidized substances (ROS), which leads to a cascade of cyclic reactions, to recurrent formation of short chain alkanes and fatty acid aldehydes, completely destroy the structure of lipids. The consequence of this process is the dimerization and polymerization of proteins that damaging the membrane. Increased lipid peroxidation involves a high lipolytic activity on membranes and membrane oxidation. Mechanisms to avoid stress – enable plants to avoid the action of stressors. This category also includes the mechanisms of ion homeostasis in the cytoplasm of plants resistant to soil salinity or excess chemical toxicants. Mechanisms of resistance – the mechanisms that allow plants to survive in stressful conditions, not avoiding the action of stressor. Such mechanisms include biosynthesis of several isozymes, that perform catalysis of the same reactionas their izoforms. In addition, each isoform possesses the necessary catalytic properties in a relatively narrow range of environmental parameters. Metallothioneins (MT) – metal-binding proteins, got their name due to the high metal content, which can reach 20% of the molecular weight. Metal-binding proteins are synthesized normally in small quantities. Their content in the cell increases rapidly by the action of heavy metals and is reduced in the case of decreasing their concentration. Sulfur is present in them, usually, in the form of the thiolate, and consists of 10-13%. Metallothioneins 1 (MTI), first class – metal-binding proteins in vertebrates. The metal-binding domain of MT1 molecule contains 20 cysteine residues, the location of which is always constant for this class. Metallothioneins II (MTII), the second class- polypeptides similar in structure to the MT1, but do not have such a conservative position of cysteine residues. They are widespread in invertebrates, plants, fungi, cyanobacteria and other prokaryotes, algae and yeast. 157

Metallothioneins III (MTIII), third class – phytochelatins (PC), kadastins, glutamyl peptides, i.e. polypeptides of some algae, higher plants and fungi containing γ-glutamyl cystein residues uncharacteristic for the first two classes, which differ from other MT by enzymatic synthesis. They have about 40 amino acid residues including the aromatic amino acid residues, separating two metal binding domain. Oxidative stress – a condition that occurs in plants under stress conditions promoting to the formation of reactive oxygen species (ROS). ROS refers to the anion radical O-2, hydrogen peroxide H202, hydroperoxide radical HO2*, hydroxyl radical HO* and singlet oxygen02* and ozone O3. High concentrations of ROS in the cells leads to damage of the biomolecules. Oxidative stress in plants is the result of the actions of virtually all environmental factors. Passive transport – transport by chemical and electrical gradients. Implemented through the phospholipid phase, if the substance is soluble in lipids by lipoprotein carriers, as well as ion channels. Peroxidase (PO) – is a multifunctional enzyme protective-adaptive system of plants to stress factors. PO is an enzyme that uses hydrogen peroxide as an oxidant and operates as follows: AH2 + H2O2 → A + 2H2O. This enzyme catalyzes the oxidation of various polyphenols, which are in plants in free or bound, and aromatic amines. Huge value is in the normal course of the oxidative processes in various types of adverse effects on plants, in particular, with the defeat of tissues by various pathogenic agents, influence of salts of heavy metals, under the effect of atmospheric toxicants. It is known that it is localized on the surface of cell membranes, which explains its high sensitivity to external influences. Plants-hyperaccumulators of heavy metals – plant species that accumu-late 10-100 times more metals than conventional plants, primarily in aerial organs. Polyamines – a low molecular weight polycations present in all living organisms. Representatives are polyamines putrescine, spermidine and spermine. Putrescine (NH2 (CH2) 3NH2) is a precursor of spermidine (Spd) (NH2 (CH2)3 NH (CH2)4 NH2) and spermine (NH2(CH2)3 NH(CH2)4 NH(CH2)3NH2). In higher plants, putrescine is synthesized from ornithine by the enzyme ornithine decarboxylase (ODC). An alternative way of putrescine biosynthesis in higher plants include arginine decarboxylase. Resistance – the ability of plants to maintain a constant internal environment (maintain homeostasis) and to implement life cycle under the effect of stressors. Symport of ions – joint transport of ions through the membrane. Symplast – unified system of the cytoplasm of cells, tissues and organs. Stress – is the collection of all non-specific changes occurring in the body of an animal under the influence of any strong influences (stressors), including the rearrangement of the body's defenses. Superoxide dismutase (SOD) – is an enzyme that catalyzes the conversion of superoxide anion (O- *) to H2O2 and O2. Transpiration – a physiological process of water evaporation plants. The transpiration rate is usually expressed in grams of water evaporated for 1h per unit area or per 1 g of dry weight. 158

TEST TASKS 1. What is a paranecrosis? A. The set of specific reactions. B. Synthesis of metallothioneins. C. Phase of exhaustion. D. The complex of non specific reactions. E. The change of pH. 2. Tobioticstressors belongs: A. Salinity B. The change of temperature. C. Diseases of animals. D. Oxidative stress E. Pathogens effect 3. Specific plant response to stress: A. The lowering of acidity of the cytoplasm. B. Calcium release into the cytoplasm. С.Reducing the rate of photosynthesis. D. Synthesis of metal binding proteins. E. Synthesis of heat shock proteins. 4. Phytostressology is studying… A. Natural phenomenon. B. Plant diseases. C. Specific plant response to stress D. Processes reactivity of plants under stress. E. Non-specific plant responses to stress. 5. Adaptation... A....not inherited. B...occurs after exhaustion. C....inherited. D. ...comes in the first phase of the Selye triad. E.…occurs after restitution phase 6. Exhaustion phase... A...occurs immediately after stress. B....occurs in plants adapted to stress. C. … is a first phase of Selye triad 159

D ..is the third phase of Selye triad E. …. is the second phase of Selye triad 7. Adaptation at the organismal level: A. Decrease in membrane permeability B. Sequestration in vacuoles of toxic substances. C. Compartmentalization. D. Based on the competitive relationship between species for the nutrients E. Activation of ATPase 8. Adaptation at the population level: A. Natural selection B. Synthesis of osmolytes C. Increase of calcium in the cytoplasm. D. The decrease of the intensity of respiration. E. Increase in membrane permeability. 9. Restitution phase: A. The second phase of the triad B. Comes after a phase of anxiety. C. Occurs in sensitive to stress organisms. D. Comes after the removal of the stressor. E. Occurs in the oxidative stress. 10.Damages… A. Appear only on the cell level. B. Appear on the different levels of structural and functional organization of the plant B. Occurs on the second level of Selye triade G. Manifested in the third phase of the triad. D. Occurs after restitution phase. 11. Responses… A. Allows plants to adapt to new stress conditions B. Only aimed to preservation of homeostasis. C. Presents only in sensitive to stress plants. D. Arise in a phase of exhaustion. E. Occurs only on the phase of restitution. 12.Acclimation is... A. Is inherited. B. There is at third phase of the triad. C. Synonym of adaptation. D. Is non- inherited. E. Depletion of the resource of reliability. 160

13. Adaptation …. A. Occurs only after the removal of the stressor. B. The third phase of the Selye triad. C. Reactions unique to plants. D. The second phase of the triad E. Is non-inherited. 14. Reaction norm... A. Typical only for resistant plants. B. Occurs in unadapted plants C. Occurs during the first phase of the Selye triad. D. Manifested only by the effect of biotic stressors. E. Hereditarily determined amplitude of possible changes in the process of realization of genotype 15.Mechanisms to avoidance of stress… A. Common reactions for all plants. B. Characteristic of paranecrosis C. Elongation of roots under drought conditions. D.Is non-inherited. E. Synthesis of polyamines 16.Mechanismsof resistance… A. There are only for acclimation. B. Observed only during adaptation C. Allow the plants to survive under stress D. Arise in a phase of exhaustion. E. Allow to avoid stress. 17.Constitutive features… A. Hereditarily fixed signs. B. Arise during acclimation. C.Is non-inherited. D. Was observed in the first phase of the Selye triad. E. None of the answer is not correct. 18. To mechanisms of resistance belong: A. Only the specific reaction. B. There actions manifested only at the cell level. C. Changes in the expression of genes synthesis of stress proteins. D. Reactions only by the action of biotic stressors. E. Only the mechanisms of homeostasis. 19.Mechanisms of the stress avoidance include: A. All of the protective reaction of the organism. 161

B. The increase of the permeability of membranes. B. Synthesis of osmolytes. G. Deep root system in drought conditions. D. The appearance of small vacuoles. 20. For resistant plants the following future is true... A. Phase adaptation is slow B. Phase adaptation passes quickly C. After the alarm phase occurs immediately the repair phase D. They cannot survive E. Increase of ATPase activity 21. To intracellular regulatory system belongs: A. Hormonal regulation B. Electrophysiological regulation B. Membrane regulation G. Trophic regulation D. Physiological regulation 22. How many levels of cellular response is known: A. Two B. Three C. Four D. Five E. Six 23. What type of regulation include the fact that the regulation is subject the protein synthesis, its subsequent processing or degradation of mRNA precursor: A. The regulation at the translational level. B. The regulation at the level of transcription. C. The regulation at the level of mature proteins D. Metabolical regulation E. Hormonal regulation 24. How many versions of a specific protein interactions of transcription factors with cytoplasmic regulatory regions of DNA: A. Two B. Three C. Four D. Five E. Six 25. The «early»genes… A. They are responsible for the quick response of cells. B. Responsible for phosphorilation of proteins 162

C. Responsible for early maturation D. Responsible for the synthesis of all stress proteins. E. They are responsible for the response during 15 hours. 26. Whichcompoundis called "service stations"? A. LEA-proteins. B. Hormones. C. Chaperones. D. Proteases E. Ubiquitines 27. Osmolytes are.... A. Organic acids B. Proteins C. The low molecular weight organic compounds D. Amines E. Amino acids 28. For «late» genes is true…. A. For exclusion of the work of "early genes" B. Provides response rate for 25 minutes. C. Stimulate protein synthesis. D. Their activity is realized in a few hours E. Their activity is realized during 15 min 29. The aquaporins are..: A. Inhibitors of proteases B. Amphiphilic compounds C. Zwitteriones D. Osmoprotectors E. Proteins 30. At drought conditions the functional genes are responsible for: A. For the synthesis of aquaporins B. For the synthesis of osmolytes C. For the synthesis of LEA-proteins D. For the synthesis of proline E. For synthesis of all mentioned above compounds. 31.Regulatorygenes… A. Participate in the synthesis of osmolytes B. Participants in the synthesis of ubiquitins C. Participate in signal transduction by expression of other genes D. Participate in the synthesis of proteases E. Responsible for the synthesis of chaperones. 163

32. The genes of transcription factors belongs to: A. To the regulatory genes. B. Functional genes C. To "early» genes D. To protein kinases E. To "late» genes 33. The functional genes are: A. Genes that are directly responsible for the formation mechanisms of plant tolerance B. The genes of transcription factors. C. Genes of protein kinases D. Genes of phospholipases E. The regulatory genes 34.The isosteric regulation: A. Regulation only by substrate B. Regulation only by cofactors C. Regulation only by the reaction product D. It is realized only on the level of their catalytic centers E. Regulation by substrate, cofactors, final product of reaction and realized on the level of their catalytic centers 35. For allosteric enzymes is true: A. Include only catalytical subunit B. Include only the regulatory subunit C. Have only negative effectors D. Have only positive effectors E. Include catalytical, regulatory subunits and negative effectors . 36. Zimogen is: A. The form of active enzyme B. Any protein-enzyme C. The latent form of enzymes D. Isozymes E. Zeatin 37. ATP-dependent proton pump participates in: A. pH regulation... B. Hormonal regulation. C. Genetic regulation E. Phosphorylation D. Electrophysiological regulation

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38. Classic pH-stat consists of a complex: A. …of hydrolyzing enzymes B. ..of oxidative enzymes C....dehydrating enzymes D....carboxylating enzymes E....carboxilating and decarboxylating enzymes. 39. For secondary active transport is true: A. It is associated only with the hydrolysis of ATP. B. It is associated only with redox reactions in chloroplasts C. The transporters are special proteins, and the energy of ATP is spent on their movement. D. It is associated with the redox reaction in mitohondria E. Without ATP 40. Driving force of secondary active transport: A. The electrogenic H-pump. B. The gradient of sodium ions C. The gradient of potassium ions. D. Gradient of transported substances E. Gradient of chlorine ions 41. The optimum of the function of malate enzyme at.. A. pH 9 B. pH 8 C. pH 7 D. pH 6 E. pH 5 42. The pH optimum of the function of PEP-carboxylase: A. pH 9 B. pH 8 C. pH 7 D. pH 6 E. pH 5 43. The activation of PEP-carboxylase lead to increase of : A. Malate B. PEP B. Oxalic acetate G. Piruvic acid E. Glutamic acid 44. Which are responsible for the metabolic regulation in plants: A. Biophysical and biochemical components 165

B. Physiological and biochemical components C. Electric physiological component D. The electric chemical component E. Metabolic. 45. To biochemical component belongs the processes: A. Oxidation and reduction B. Synthesis and hydrolysis B. Carboxylation and decarboxylation G. Dehydration E. Deamidization 46. What is a man-made source of heavy metals contamination of the environment? 1. Chemical industry. 2. Mining. 3. Transport 4. Metallurgical industry. 5. All the answers are correct. 47. What characteristics are used as the criteria for heavy metals? 1. Odour, density, color. 2. The atomic mass, density, toxicity. 3. The roughness, the density, the smell. 4. Color, atomic weight, shape. 5. Transparency, weight, stiffness. 48. Heavy metals include metals with mass ... 1. > 30 at. Units 2. > 50 at. Units 3. < 30 at. Units 4.< 50 at. Units 5. All answers are incorrect 49. Heavy metals are metals with a density ... 1. > 3 g/cm3 2. < 3 g/cm3 3. > 5 g/cm3 4. < 5 g/cm3 5. 5 g/cm3 50. Heavy metals ... 1. Al 2. K 3. Cu 166

4. Na 5. Mg 51. … includes copper(Cu) 1. Ferrodoxin 2. Plastoquinone. 3. Plastocyanin. 4. Chlorophyll. 5. Protein Riske. 52.Which of the trace elements belong to heavy metals? 1. Zn 2. Cu 3. Mn 4. Li 5.Allanswers are correct 53. In what process is involved zinc (Zn)? 1. The synthesis of chlorophyll 2. Activation of enzymes in carbohydrate metabolism. 3.Activation of enzymes of energy metabolism. 4. It protects chlorophyll from disintegration. 5. All the answers are correct. 54. Cadmium enters the soil from... 1. Fertilizers. 2. Pesticides 3. Settles from the atmosphere. 4. From the wastewater. 5. All answers are correct. 55. Maximum allowable concentration of cadmium for crops. 1.0.01mg/kg 2.0.1mg/kg 3.0.001mg/kg 4. 1 mg/kg 5. No answer is correct 56. The main source of lead (Pb) in the environment. 1. Motor vehicles. 2. Pesticides 3. Mineral fertilizers. 4. From the wastewater. 5. All the answers are correct

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57. Lead (Pb) is a part of ... 1. Fertilizer 2. Liquid fuel. 3. Pesticides. 4. Herbicides 5. Mineral fertilizers. 58. Like as heavy metals in plants? 1. Through the roots 2. Leaves 3. Cuticle 4. Stomata 5. All the answers are correct 59. Transport of cadmium through cell membranes is a passive process .. 1. At low concentrations of cadmium. 2. At high concentrations of cadmium. 3. At pH > 7 4. At pH < 7 5. No answer is correct. 60. Transport of cadmium through cell membranes is an active process. 1. At low concentrations of cadmium. 2. At high concentrations of cadmium. 3. In the presence of other metals. 4. In a neutral environment. 5. All the answers are correct. 61. The cations are transported through the membrane ... 1. Due to the negative charge on its surface. 2. Due to the positive charge on the surface. 3. Due to the high ionic strength. 4. Due to the high concentration. 5. All the answers are correct. 62. Receipt of ions into the cell depends on .. 1. The gradient trains. 2. The concentrations. 3. The presence of other ions. 4. pH 63. Ion mobility 1. The accumulation of metal ions in the headspace of the cell wall is determined by... 2. The value of the coefficient of ion exchange. 168

2. The amount of concentration. 3. The magnitude of the charge. 4. pH value. 5. permeability of the membrane 64. The magnitude of the ion exchange ratio depends on the number ... 1. Hydroxyl groups. 2. Histidine groups. 3. Carbonyl group. 4. Amine groups. 5. Aldehyde group 65. The linear portion of the dependence of the absorption of metals on the time correspond to .. 1. Binding to components of the apoplast. 2. Transport across the cytoplasmic membrane. 3. Saturation ion cell cytoplasm. 4. Low concentration of metal in the cell. 5. No answer is correct. 66. Oxidative stress cause the increase of …in proteins 1. Hydroxyl groups. 2. Hystidine groups. 3. Carbonyl group. 4. Amine groups. 5. Aldehyde group 66. Transport ions is carried out by the xylem, if the ions pass through ... 1. Rizoderm 2. Endoderm 3 .Protoderm 4. Cortex 5. All answers are correct. 67. Caspari strips… 1. Endoderm cell walls impregnated with suberin and wax compounds. 2. The cell walls of the cortex, impregnated suberin and wax compounds. 3. Xylem cell walls impregnated with suberin and wax compounds. 4. Cell wall rizoderm impregnated with suberin and wax compounds. 5. Phloem cell walls impregnated with suberin and wax compounds. 68. For the transport of cations in the xylem against the electrochemical gradient used ... 1. Ionic transporters. 2. Hydrophobic substances. 169

3. Ionophores. 4. The cationic-proton antiporter. 5. Lipophilicsubstances. 69.The absorption of cadmium cell walls reduce... 1. Proteins. 2. Carbohydrates. 3. Citrate. 4. Fat. 5. The ionic environment. 70.The lowest concentration of heavy metals is found... 1. In the leaves. 2.In the roots. 3.Cortex. 4. Stems. 5. Trichomes. 71. Iron(Fe)moves through xylem 1. In a related form. 2. In the cationic form. 3. Theionic form. 4. The toxic form. 5. No answer is correct. 72. With an excess of heavy metals in the cell, they are mainly associated with ... 1. Chloroplasts 2. Cell wall 3. Mitochondria 4. Membranes 5. Golgi apparatus. 73. Most of the heavy metals in the cell is stored in the ... 1. Dictyosome 2.Vesicles 3.Vacuoles 4. Liposomes 5. Glioxysoma 74. What absorbed ions affect the metabolism of the cells? 1. All absorbed ions. 2. Only the free ions. 3. Only ions bound with organic acids. 4. Only ions associated with proteins. 170

5. Ions associated with cell walls. 75. Which ions form more stable complexes? 1. Cations. 2. Anions. 3. Multiply charged ions. 4. Singly charged ions. 5. No answer is correct. 76. Affinity of metals to polygalacturonic acid cell wall decreases in the following order: 1. Zn > Ca > Cu > Cr > Pb 2. Ca > Zn>Pb> Cr> Cu 3. Cu > Ca > Zn>Pb> Cr 4. Cr > Pb > Ca > Cu > Zn 5. Pb > Cr > Cu > Ca > Zn 77. Plants……accumulate of heavy metals in vacuoles 1. …non tolerant to heavy metals… 2. …tolerant to heavy metals … 3. …which have ability to accumulate heavy metals 4. Any plants... 5. No answer is correct. 78.The stability of metals complexes decreases when... 1.pH>7 2.pH9 5. All answers are correct. 79. The first target for heavy metals is... 1. Mitochondria. 2. Chloroplasts. 3. The cell membranes. 4. Vacuoles. 5. All answers are correct. 80. Effect of heavy metals on the membrane expressed in ... 1. ..increase the pH 2 .. changes in membrane permeability. 3. … lower the pH. 4. …increase the protein content. 5. …increasing the carbohydrate content.

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81. Antioxydative system of plants consists of enzymes ... 1. Superoxide dismutase 2. Peroxidase. 3. Catalase. 4. Ascorbate peroxidase. 5. All answers are correct. 82. Glutathione ... 1. Reduces pH. 2. Protects thiol group oxidation. 3. Increases heavy metals 4. Increases in pH. 5. …is the enzyme 83. The activity of which enzyme changes under the influence of heavy metals? 1. Glucose-6-phosphate dehydrogenase. 2. Peroxidase. 3. Nitrate reductase. 4. Glutathionedehydrogenase. 5. All answers are correct. 84. Enzymeactivity against oxidative stress. 1. Polyphenol oxidase. 2. Peroxidase. 3. Catalase. 4. Superoxide dismutase 5. All answers are correct. 85.The carbonyl group is... 1-COOH 2-C=O 3-CHOH 4-CH2OH 5-SNNN2 86. Heavy metals effect on proteins.. 1. Cause the breakdown of the tertiary structure of proteins. 2. Hydrolyze proteins. 3. Form a carbonyl group. 4. Only reduce the activity of the enzyme protein. 5. All answers are correct. 87. What is the index of oxidative stress? 1. Lipid peroxidation. 2. The formation of hydroxyl groups. 172

3. The increase in pH. 3. Reduction of pH. 5. Change the properties of proteins. 88. Heavy metals effect on? 1. Glycolysis. 2. Krebs cycle. 3. Pentoza phosphate pathway. 4. The electron transport in the mitochondria. 5. All answers are correct. 89. Which process of photosynthesis influence heavy metals? 1. Inhibits photosystem II 2. Reduce the fluorescence. 3. Violate electron transport reactions. 4. Disable the light-collecting complex. 5. All answers are correct. 90. Heavy metals affect the nuclear apparatus of the cell? 1. Violate nuclear fission. 2. Change the structure of chromosomes. 3. Reduce the mitotic index. 4. Reduce the total content of DNA and RNA. 5. All the answers are correct. 91. Heavy metals cause ... 1. …the reduce the length of the main root. 2. …the reduce the length of lateral roots. 3. …the increase the length of the main root. 4. ...stimulation the growth of roots. 5. No response is correct. 92. Heavy metals cause ... 1. Reduction of morphogenesis processes. 2. Reduce the size of the leaf blade. 3. Reduce the growth of biomass. 4. Reduce the height of the plants. 5. All the answers are correct. 93. At what pH is increased content of free cadmium? 1. pH = 9 2. pH = 7 3. pH> 7 4. pH