Stress physiology: study guide. 9786010410985

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AL-FARABI KAZAKH NATIONAL UNIVERSITY

S. Atabayeva S. Kenzhebayeva I. Blavachinskaya

STRESS PHYSIOLOGY Study guide

Almaty «Qazaq university» 2015

UDC 159.9 (075.8) LBС 88.3 я 73 A 90

Recommended for publication by the Science Committee of Faculty of Biology and Biotechnology and Publishing Council of al-Farabi Kazakh National University

Reviewers: Doctor of Biological Science, Professor A.A. Nurzhanova Candidate of Biological Science S.K. Turasheva

A 90

Atabayeva S. Stress physiology: study guide / S. Atabayeva, S. Kenzhebayeva, I. Blavachinskaya. – Almaty: Qazaq university, 2015. – 84 p. ISBN 978-601-04-1098-5 In the study guide the current state of knowledge about the physiology of plant resistance to adverse environmental conditions, stress physiology, plant adaptation mechanisms, the system of regulation of plants under stress 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. These types of regulation together define response of the whole organism to plant stress The study guide is intended for university students studying in the field of «Biology», «Biotechnology, «Ecology» as well as for teachers, graduate students and researchers.

UDC 159.9 (075.8) LBС 88.3 я 73 © Atabayeva S., Kenzhebayeva S., Blavachinskaya I., 2015 ISBN 978-601-04-1098-5 © Al-Farabi КazNU, 2015

Content

List of acronyms................................................................................... 4 Introduction.......................................................................................... 5 1. Plants under stress...................................................................... 7 2. Mechanisms of stress................................................................. 9 3. Strategies of plant adaptation to the effects of stress............... 15 4. Systems of regulation under stress conditions......................... 18 4.1. Perception and transduction of signal...................................... 20 4.2. Genetic regulation.................................................................... 31 4.3. Metabolic regulation................................................................ 40 4.4. Membrane regulation............................................................... 46 4.5. Hormonal system of regulation................................................ 52 4.6. Trophic regulation system........................................................ 60 4.7. Electro-physiological regulation.............................................. 62 Conclusion........................................................................................... 67 Glossary............................................................................................... 69 Test tasks............................................................................................. 72 Self study work tasks for students.................................................... 80 Recommended literature................................................................... 82

List of acronyms

АA – АBA – АDC – АDH – АGC – АFO – АОS – AP – АPО – АTP – CAT – DNA – EP – EPR – HSP – HB – GDP – Glu – GS – GTP – FA – IAA – IP3 – LDG – LP – MDA – MDG – ODC – PА – PO – RP – PPP – Putr – RNA – ROS – Spd – Spm –

аminoacid abscisic acid argynine decraboxylase аlcohol dehydrogenase аscorbic gluthathione cycle active forms of oxygen аntioxidant system action potential аscorbate peroxidase adenosine triphosphate catalase deoxyribonucleic acid excitation potentials endoplasmic reticulum heat shock proteins gibberellin guanosine diphosphate glutathione glutathione synthetase guanosine diphosphate fatty acids indolacetic acid inositol triphosphate lactate dehydrogenase lipid peroxidation malonic dialdehyde malate dehydrogenase ornitine decarboxylase polyamines peroxidase resting potential penthose phosphate pathway putrescin ribonucleic acid reactive oxygen substances spermidine spermine

Introduction

I

n 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 like 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

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Stress physiology

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.).

1. Plants under stress

In the second half of 30-iesth 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. Ac-cording 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: a) physical: insufficient or excess lighting or temperature humidity, radioactive radiation, mechanical impact;

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Stress physiology

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 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 the 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 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.

2. 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 moved beyond 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 agrophytocenoses 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 included in 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. 2. The entry of Ca2+ ions in the cytoplasm (from the cell walls and intracellular compartments: vacuoles, endoplasmic reticulum, mitochondria).

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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 light-scattering 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, reduction of 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 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

2. Mechanisms of stress

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 (Field, 1986; Alehinа et al., 2007). 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

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Stress physiology

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. In addition to the above mentioned soluble carbohydrates and their derivatives protein osmotin has osmophilic properties and is synthesized under water deficit. 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 penthoses required for synthesis (in particular, nucleic acids). At the level of the whole organism adaptation mechanisms inherented in the cell are complemented by new reactions. They are based on the competitive relationship between the organs for the physiologically active substances and nutrients and are built on the principle of at-tracting 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 res-

2. Mechanisms of stress

ponses 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 this mechanism of adaptation is intrapopulation variability of resistance level to the different factors or their complexes. Under 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 toxic substances 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 supply the Selye triad with 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 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 maintaining of biological systems balance with the environment, ensuring their reliability in a rapidly changing environment.

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Stress physiology

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, 986; 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.

3. Strategies of plants adaptation to the effects of 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 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 acclimation plants acquire resistance to the stressor. Acclimation occurs during the life of the organism and 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 ne-

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Stress physiology

gative 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 suсculents, 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 (glycoalkaloids) 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.

3. Strategy of plant adaptation to the effects of stress

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. The entire set of isozymes in general 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 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 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?

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4. Systems of regulation under stress conditions

The integrity of 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 parameters of the internal environment. Intracellular regulation systems must have appeared during evolution. This includes the regulation on the enzymes level, genetic, membrane regulation. When multicellular organisms appear the intercellular regulation systems develop. This includes trophic, hormonal, electrophysiological systems (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 short-lived and unable to protect the organisms from death (Figure 1). 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 repairation of changes is possible after returning to normal conditions the length of the adaptation phase

4. Systems of regulation under stress conditions

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). Plants Sensitive

Tolerant

Short term defense reactions

The time of adaptation phase is longer

Resource depletion reliability, irreversible processes

Maintain of homeostasis, reparative processes

Death of plants

Survival

Figure. 1. The degree of resistance of the tolerant and sensitive plants to the action of the stressor SYSTEMS OF REGULATION Intercellular

At the level of the organism

Effect of factors

Electrophysiological regulation Hormonal

Trophical Intracellular

At the cell level

Membrane regulation Enzymatic regulation Genetic regulation Figure. 2. Systems of plant regulation (T.V. Chirkova, 2002)

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4. Systems of adjusting to stress conditions

4.1. Perception and transduction of signal 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). 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). Work prerequisite of regulation operation systems is the perception (reception or perception), transmission and transformation (transduction) 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). 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:

4.1. Perception and transduction of signal

– 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).

A

B Figure. 3. G-protein-linked receptors (http://philschatz.com/biology-book/contents/ m44451.html, https://flochalmers.wordpress.com)

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4. Systems of regulation under stress conditions

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. 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 localized at the outer side of the membrane and the portion contacting the G-protein at its cytoplasmic side. 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 adenylate cyclase, 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 4A, 4B).

4.1. Perception and transduction of signal

Cations

Cytoplasm

А B С1 – an external signal, Р – a receptor protein: an asterisk denotes the components of the signaling system in the «on» state Figure. 4. Ion-channel-linked receptors (http://bioserv.fiu.edu)

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 of receptors associated with the enzyme is on the side which faces the extracellular space 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 (Fig. 5A, 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. 5A, right; 5B). 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 the receptor 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.

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4. Systems of regulation under stress conditions

Receptor-enzyme

Receptor-«anchor»

Cytoplasm Catalytic center

The protein kinase

A С1 – an external signal, Р – a receptor protein: an asterisk denotes the components of the signaling system in the «on» state

B Figure. 5. Enzyme-linked receptors (https://flochalmers.wordpress.com)

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 are involved in the transmission of signals and relatively small molecules which serve as secondary signals. These are called the secondary intermediaries 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

4.1. Perception and transduction of signal

small molecules (e.g. calcium ions) actually serve as messengers (messengers) between different proteins, multienzyme complexes or cell structures (Chirkova 2002). 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. 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 (Fig. 6). 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 is transmited 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 form. Eukaryotes have also small G-proteins (Rasproteins), 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. 6. GTP-dependent activation of Gα-phospholipase

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 high-

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speed 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, this 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. 7), which contributes to the accumulation of calcium in the vacuoles or transport across the plasma membrane in the cell wall. Inosytol -1,4,5-triphosphate Cytosole Vacuole Calmodulin Calmodulin

ATP

ADP + P

Figure. 7. Effect of ATP – translocator of Ca2+ ions (T.V. Chirkova, 2002)

The phosphoinositol way. Calcium channels are controlled by the phosphoinozytol 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 1,4, 5-triphosphate (IP3) and diacylglycerol (DAG). These compounds are also involved in signal transduction; IP3 opens calcium channels (Fig. 8), 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

4.1. Perception and transduction of signal

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. Phospholipase С, stimulated by G-protein ↓ Degradation of phosphatidylinositol ↓ ↓ 1,4,5-triphosphat (IP3) diacylglycerol (DAG). ↓ ↓ The opening of Ca-channels, Activation of Са2+-calmodulin release of Са2+ into cytosole dependent protein kinase (СDРК) ↓ Biosynthesis of stress proteins A

B Figure. 8. The phosphoinozitol way (http://www.picscience.net)

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Calmodulin. Calcium acts as a mediator only after interaction with calmodulin (Figure 9). 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 9). 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 – Ca 2+ – phospholipid dependent pyruvate kinase, activated by DAG, Ca2+ and phospholipids, and included in the phosphoinozytol cascade of protein kinases, G – cGMP-dependent protein kinases and Ca2+-calmodulin-dependent protein kinases.

Inactive protein kinase

Calmodulin

Active protein kinase

Calmodulin + protein kinase

Figure. 9. Role of calmodulin in activation of protein kinases (T.V.Chirkova, 2002)

4.1. Perception and transduction of signal

MAP (mitogen activated proteins) is a kinases cascade. It consists of three protein kinases. Protein kinases are enzymes capable to catalyze 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. Kinases of MAP cascade work as follows (Fig. 10). MAPKK-dependent piruvate kinase (МАР­ККК) ↓ Activation МАРК-dependent protein kinase (МАР­КК) ↓ Activation Mitogen-activated protein kinase (МАРК) A

B Figure. 10. The kinases of MAP cascade

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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. For this process in the cell there is another protein kinase – a kinase of kinase of MAP kinase (Fig. 11). 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. 11. The MAP cascade (http://cc.scu.edu.cn)

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

4.2. Genetic regulation

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. Their behavior may be regulated by cell-cell interactions that are mediated by by integrating into the outer cell membrane receptors. In 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 scheme of response to a stressor? 3. What is studying the field of biochemistry, called «cell signaling»? 4. What types of membrane receptors are known? 5. What are the components of the signal transduction. 6. What are the functions of G-proteins? 7. What refers to the second messenger?

4.2. Genetic regulation 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 and transmission of genetic information. Information is recorded in the chromosomal DNA using the nucleotide triplet code. 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

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destination. 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. 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 the transition from cytoplasm to membrane leading to disruption of the properties of proteins, which are essential for the signal function. The most common mechanism of transcriptional regulation is a specific interaction of protein transcription factors of cytoplasm to regulatory regions of DNA. There are identified three main options for this interaction (Fig. 12, 13). In the first type of these interactions (Figure 13A) cytosolic protein 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. 13B) a signal transmits to the nucleus protein phosphorylated phosphorylated by it. Prior to this process it was a

4.2. Genetic regulation

latent transcriptional factor, and after the phosphorylation it becomes active, enters the nucleus and binds specifically to DNA. In the third variant (Fig. 13C) 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.

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 trunsduction. Figure. 12. The main signal transduction pathway from the cytosol to the nucleus (V.I. Kulinskiy, 1997) Protein kinases of cytosole /МАР-kinases, proteinkinase A/

NUCLEUS Phosphorilation of transcriptional factors

NUCLEUS Changes in affinity of the transcription factor to DNA and in level of activity Figure. 13A. The first type of interaction of transcription factors with DNA

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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. Latent transcriptional factor

Protein phosphorilated by proteinkinase

Phosphorilation Proteinkinase

NUCLEUS Specific binding to DNA

Figure. 13B. The second type of interaction of transcription factors with DNA

Protein complex Phosphorilation and release of inhibitor or «anchor» subunit субъединицы

The active transcription factor

Phosphorilation

Proteinkinase

NUCLEUS Specific binding to DNA

Figure. 13C. The third type of interaction of transcription factors with DNA

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

4.2. Genetic regulation

phosphatases either phosphorylate transcription factors in the nucleus or their phosphorylated proteins enter into the nucleus and interact with transcription factors. This results in activation of stress-inducible gene and as a consequence, in the synthesis of mRNA and stress proteins such as chaperones, ubiquitin, aquaporins, that enhance the plant tolerance (Figure 14). 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. Perception of signal

Plasmalemma

Stress

Intermediates of signal trunsduction

Nucleus Promotor

Tolerance

Transcription factor Gene, induced by stress

Protein

Figure. 14. Role of perception and transduction of stress signal in the genome activation (T.V. Chirkova, 2002)

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 cytosole 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

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bind to amino acids of the polypeptide chain, as it will appear on the other side of the membrane and perform the folding. 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 induction of biosynthesis of inhibitors of proteases that prevent proteolytic degradation of proteins and cells retain their structure and functional properties (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. 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. 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. 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). The overall function of osmolytes is the participation in osmoregulation. Many of the inorganic ions, such as Na+ and Cl- in the 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

4.2. Genetic regulation

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. 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 of soil salinity effect. Along with osmoregulation the compatible substances perform another very important function in dehydration – protective function 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). 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 (Fig. 15, 16).

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Figure. 15. Aquaporins (http://cstl-csm.semo.edu)

Figure. 16. Water movement across membrane http://plantphys.info)

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 (Fig. 17). Changes in the activity of existing water channels of the membrane play an important role in the regulating of water conductivity in

4.2. Genetic regulation

membrane under stress. One of the mechanisms of such regulation is the phosphorylation and dephosphorylation of aquaporins.

Figure. 17. 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.

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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 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?

4.3. Metabolic regulation 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. 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

4.3. Metabolic regulation

effect). The intensity of the enzyme is defined as the presence of cofactors, coenzymes for two-component enzymes (nicotinamide adenine dinucleotide 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 (Fig. 1). 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) occurs, which leads respectively to activation or inhibition of the functional activity of the catalytic center. An example 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 phosphoenolpyruvate, 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. 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

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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 electrochemical 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. 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. There is another mechanism of active transport of substances, which is called the secondary active transport. Specific proteins function as transporters of ions and the energy of ATP is released by using ATPase and spent on their movement through the membrane. 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

4.3. Metabolic regulation

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 biochemical pH-stat to maintain proton homeostasis (Chirkova 2002). Classic pH-stat consists of a complex carboxylates (phospoenolpiruvate (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) (Fig. 18). PEP- carboxylase /рН 8/ ↓ Oxaloacetate (ОА) ↓ Malatdehydrogenase (МDH) ↓ Malat ↓ NAD-malic enzyme (МE), /рН 6/, ↓ Decarboxylation of malate ↓ Shift of рН to alkaline side Figure. 18. The metabolic regulation, PH-state

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 19). 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. Another unique feature of glycolysis in plants is the way of its control. If in non-plant systems the glycolytic flux is controlled by

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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 8).

GK – gexokinase, PFK – phosphofructokinase, GAPD – glyce-raldehyde phosphate dehydrogenase, CG – carboanhydrasa, PK – piruvat kinase, АА – acetaldehyde, АО – alternative oxydase, CО – cytochrome oxydase, LDG – lactate dehydrohenase, PDK – piruvat decarboxylase, ADH – alcohol dehydrogenase, PEP – phosphoenolpi-ruvate, МE – malik enzyme, Н+ – formation of protons, – release of protons. Figure. 19. The structure and function of biochemical рН-state (по K.Sakano, 1998)

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 19). 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.

4.3. Metabolic regulation

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. 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 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).

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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?

4.4. Membrane regulation 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. And of particular importance in all of these mechanisms is a system of membrane chemo-, photoand 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. Membrane regulation is carried out due to changes in membrane transport, binding or release of enzymes and regulatory proteins and by altering the activity of membrane enzymes. Membrane as a natural barrier is first exposed to stress factors. As dynamic structures, the membranes are able to respond rapidly to variations in the conditions of existence of the changes occurring in them, involving cascade changes in metabolism of the whole cell. Receptor-regulatory function is determined by the presence in the membranes of chemo-, photo-, mechanical and other receptors of protein nature, perceiving signals from the environment and contribute

4.4. Membrane regulation

to responses to changes in the conditions of existence (Chirkova, 2002). 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. Possible changes chain length fatty acids, a positional arrangement of double bonds, the amount of polar groups. Due to the close 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. 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 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 (10-5-10-8 mol/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

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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 exoosmos 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 include 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

4.4. Membrane regulation

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 supplies necessary for the working of pumps in a stressful environment. 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) (Fig. 20, 21).

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Figure. 20. The polyamines structure (http://www.mdpi.com)

A

B Figure. 21. The polyamine biosynthetic pathway (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

4.4. Membrane regulation

phospholipid membranes, thereby enhancing the stability and 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. So, among the causes of greater stability of cell membranes of resistant plants we 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). Test questions 1. What are the main functions of biological membranes? 2. Why does the membrane react quickly to changing environmental conditions? 3. What determines the receptor-regulatory function of membranes? 4. How does the structure of the membrane react to the action of stressors? 5. How do the stress factors influence the function of membrane proteins? 6. What is the role of calcium ions in the regulation of membrane? 7. What are the properties of membranes modified by the action of stressors? 8. What characterizes plasmalemma membrane and cell organelles in stable and unstable plants? 9. How do polyamines stabilize membranes? 10. What are the reasons for greater stability of cell membranes in resistant plants.

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4.5. Hormonal system of regulation Hormonal system is a key factor in the regulation and management of plants. Phytohormones auxin (indole-3-acetic acid), cytokinins (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 (Fig. 22).

Figure. 22. 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

4.5. Hormonal system of regulation

produced in the root tips. 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 (Fig. 23).

Figure. 23. 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. 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 in-

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teraction 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 or other molecular complexes are involved in hormonal signal transduction,. Hormonal system is one of the most important regulatory systems 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. Typically under the influence of stressors the growth of plants is inhibited,the content of indolyl-acetic acid (IAA), gibberellins and cytokinins reduces, but the number of inhibitors, abscisic acid (ABA), ethylene, jasmonic acid, increases. ABA and ethylene are even called stress hormones. Reduction of the level of hormones-stimulants and accumulation of growth inhibitors during stress play an important adaptive role because they lead to decrease in the intensity of metabolic processes, stop 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). 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»)

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, Mg-dependent protein phosphatase, MAP kinase cascade. As a result of cytoplasmic processes phosphorylation and dephosphorylation are enhanced. Thus, regulation of the activity

4.5. Hormonal system of regulation

of both, the key enzymes of different metabolic pathways and transcription factors, is performed. 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. Thus, the following occurs: 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. 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 plants during their loss of water during the water, salinity and low temperature stress. 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 leads

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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 drought-resistant 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 1 min. Nevertheless, it manages to contact with receptor located in the plasma membrane. The system of signal transduction involves GTP

4.5. Hormonal system of regulation

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 (Fig. 24).

Figure. 24. The role of phytohormones under stress conditions (https://secure.jbs.elsevierhealth.com)

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.

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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 proccesses 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 enhancement 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

4.5. Hormonal system of regulation

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 too. However, the signal transduction of Ca2+ in the nucleus is not clear yet. Cytosolic second messengers and protein kinases may 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?

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4.6. 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). 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 (Figure 25). 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 25). 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

4.6. Trophic regulation system

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.

Figure. 25. Regulation of genomes by stressors and sugars as signal molecules (Chirkova,2002)

Carbohydrate signal transduction cascade comprises a protein kinase phosphatase. In transduction involving MAP kinases, calcium ions, as intermediates, they can probably be included in the trophic system of education and the regulation of carbohydrate consumption. 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

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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. Under stress the competition of various organs for nutrients intensifies, but usually plant development continues, it may even 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. 4.7. 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 MP 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 plasmodesmata in cells of the cell at a speed of 0.1-1.0 cm/s. Action potential is induced only when 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. Bioelectrogenesis of plants is important for the coordination of their functional activity and morphogenesis (Chirkova, 2002).

4.7. Electro-physiological regulation

As a result of bioelectrogenesis the bioelectric potentials occur. There are a resting potential (RP), the potential difference 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 RP) and signal (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 compression). 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).

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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 AP of animals Na+ and K+ and Cl- and K+ are not involved. 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 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 AP. 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, AP 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, AD may cause them a response. Thus the occurrence of AP in response to an external stimulus is nonspecific (Fig. 26).

4.7. Electro-physiological regulation

Distributing AP 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 some specific processes specific to the organ (e.g., the leaves change photosynthesis, roots increased absorption of substances and etc.). 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 potassium chanells ↓ Repolarization of membrane ↓ Restore the original values of the potential Figure. 26. The electrophysiological regulation

The signal (information) role of AP 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 AP in plants occurs at moderate changes in the environment, even when the temperature difference is 1-20 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 AP is reduced to a temporary increase in the stability of organs and tissues of plants to adverse effects, which is nonspecific. Thus, at weak effects associated with the function of AP 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.

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4. Systems of regulation under stress conditions

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 form necessity for the survival ability in a gradual and sequential interaction of different systems of regulation. 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. 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?

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 for the growth of the identification of adaptation mechanisms of various kinds of stressors is the most important challenge faced by scientists around the world. The adverse environmental factors are called stressors, and the body’s response to abnormal stress. Plants are characterized by three phases of stress: 1) primary stress response, 2) the 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 non-specific, independent of the type of stressor and specific, peculiar only to certain types of stress. Under conditions of stress, plants use different strategies for survival, how to avoid stress and to use resistance mechanisms. Under the effect of the stressor during acclimation plants acquire resistance to the stressor, but this stability is not inherited. At low doses, repeated stress leads to hardening of the body, and hardening to one stressor enhances stability of the organism and other damaging factors. In the process of evolution in plants also develop adaptive to the action of certain stressors features that are transmitted genetically. At the organismal level retains all the cellular mechanisms of adaptation and supplemented with new, reflects the interaction of the whole plant. First of all, it is a competitive relationship for the physiologically active substance 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, accelerates the aging process and abscission of the lower leaves, and the products of hydrolysis

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of organic compounds are used to power the young leaves and the formation of generative organs. Plants are able to replace lost or damaged organs through regeneration and growth of axillary buds. In all these processes are involved correlative growth intercellular regulation system (hormonal, trophic and electrophysiological). Under conditions of prolonged and severe stress first of all are dying sensitive to stress plants. They are eliminated from the population, and the seed progeny form more resistant plants. As a result, the overall level of resistance in the population increases. Thus, at the population level is included selection, leading to the emergence of a more adapted organisms and new species. Therefore, a basic knowledge of the physiology of stress, 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 in the evolution of the processes is necessary to create new tolerant species of economically valuable crops.

Glossary

Adaptation – is genetically fixed constitutive feature present in the plant regardless of whether it is or not under stress. 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 – is, for example, the biosynthesis of substances that can help reduce the negative impact of stressors. Acclimation – responses that allow plants to adapt to the new stress conditions; they are affected by changes in gene expression, metabolism and physiological functions called homeostasis. During acclimation plants acquire resistance to the stressor. Acclimation occurs during the life of the plant and is not inherited (hardening). 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. Includes several enzymes and low molecular weight substances that are present in plants (superoxide dismutase (SOD), catalase (CAT), ascorbate-glutathione 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. 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.

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Diffusion – type movement substances driving force is the concentration gradient or a chemical potential gradient. Catalase (CAT) – this enzyme catalyzes the oxidation of hydrogen peroxide to form molecular oxygen H2O2 + O2 → 2H2O. Prosthetic groups of CAT software serves heme, which includes the iron atom. 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 (endurance) – are mechanisms that allow plants to survive stressful conditions, not avoiding action stressor. Such mechanisms include, biosynthesis of several isozymes, perform catalysis of the same reaction. In addition, each isoform possesses the necessary catalytic properties in a relatively narrow range of environmental parameters. Reaction norm – hereditarily determined amplitude of possible changes in the genotype. 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. Lipid peroxidation (LP) – is the formation of lipid radicals due to hydrogen abstraction from unsaturated fatty acids (FA) by ROS, which leads to a cascade of of cyclic reactions, to recurrent formation of short chain alkanes and fatty acid aldehydes, completely destroy the structure of lipids. The consequence of this is the dimerization and polymerization of proteins that damaging to the membrane. Increased lipid peroxidation involves a high lipolytic activity on membranes and membrane oxidation. 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.

Glossary

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. 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. Development – a qualitative change in the structure and activity functional activity of plant and its parts (organs, tissues and cells) during ontogenesis. Fenton reaction – oxidation of the hydroxyl group α-hydroxy acids and α-glycol 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, associated with malignancy elements of their structures. This concept reflects the quantitative changes that accompany the development of an organism or its parts. 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. Resistance – the ability of plants to maintain a constant internal environment (maintain homeostasis) and to implement life cycle under the effect of stressors.

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Test tasks

1. What is a paranecrosis? A. The set of specific reactions. B. Synthesis of metallothioneins. C. The phase exhaustion. D. The complex of nonspecific reactions. E. The change of pH. 2. To biotic stressors belongs: A. Salinity B. The change in temperature. C. Diseases of animals. D. Oxydative stresss E. Pathogens 3. The 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 studies… 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. ... is not inherited. B. .. occurs after exhaustion. C. ... is inherited.

Test tasks

D ... comes in the first phase of the Selye triad. E. …occurs after restitution phase 6. Phase exhaustion ... A. .. occurs immediately after stress. B. ... occurs in plants adapted to stress. C. … is a first phase of Selye triad 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 the physiologically active and 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. Phase restitution: 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. Occur on the second level of Selye triade G. Manifested in the third phase of the triad. D. Occur after restitution phase. 11. Responses… A. Allow plants to adapt to new stress conditions B. Only aimed to preservation of homeostasis.

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C. Present only in sensitive to stress plants. D. Arise in a phase of exhaustion. E. Occur only on the phase of restitution. 12. Acclimation ... A. Is inherited. B. There is a third phase of the triad. C. Synonym of adaptation. D. Is not inherited. E. Depletion of the resource of reliability. 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 not 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. Are not inherited. E. Synthesis of polyamines 16. Mechanisms of resistance… A. There are only for acclimation. B. Observed only during adaptation C. Allow the plants to survive 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. Are not inherited.

Test tasks

D. Were observed in the first phase of the Selye triad. E. None of the answer is correct. 18. The mechanisms of resistance are: A. Only the specific reaction. B. The reactions 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. 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 repair phase D. They can not survive the adaptation phase 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 RNA precursor: A. The regulation at the translational level. B. The regulation at the level of transcription

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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 C. Responsibile for early maturation D. Responsible for the synthesis of all stress proteins. E. They are responsible for the response during 15 hours. 26. Which compound is called «service stations»? A. LEA-proteins. B. Hormones. C. Chaperones. D. Proteases E. Ubiquitines 27.The 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 crate 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

Test tasks

C. Zwitteriones D. Osmoprotectors E. Proteins 30. At drought conditions the functional genes are responsible: 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 compunds. 31. Regulatory genes… 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. 32. The genes of transcription factors belong 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

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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 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

Test tasks

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 leads to increase of : A. Malat B. PEP B. Oxaloacetate G. Piruvic acid E. Glutamic acid 44. Which are responsible for the metabolic regulation in plants : A. Biophysical and biochemical components B. Physiological and biochemical components C. Electrophysiological component D. The electrochemical component E. Metabolic component 45. To biochemical component belongs to the processes: A. Oxidation and reduction B. Synthesis and hydrolysis B. Carboxylation and decarboxylation G. Dehydration E. Desamidization

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Tasks for Self Study works of students

1. Subject: Physiology of stress. Objective: To summarize and systematize the knowledge about plant responses to stress.Tasks: 1. Create a diagram or table that describes the different types of stress (biotic, abiotic, examples) 2. Create a diagram or table description of specific and nonspecific reactions of plants to stress. 3. Describe in a table or diagram the stages of Selye triad (primary stress response, adaptation, resource depletion reliability.). 4. Describe in a table or diagram adaptation strategy of plants to stress (damage responses, adaptation, acclimation). 2. Subject: Systems of regulation under stress in plants. Objective: To summarize and systematize the knowledge of various systems of regulation under stress. Tasks: 1. Describe in a diagram or table the performance of the signal system of the plant organism (perception and signal transduction types of membrane receptors, components of signal transduction, second messengers). 2. Describe in a diagram or table the hormonal system regulation. 3. Describe in a diagram or table the characteristics of stress proteins (chaperones, heat shock proteins, proteases, ubiquitin, LEA-proteins, aquaporins). 4. Describe in a diagram or table the metabolic regulation (pH-stat). 5. Describe a circuit of the membrane regulation. 6. Describe in a diagram or table the trophic system of regulation. List the reports (optional) and presentations of self study work of students 1. Resistance to freezing plants. 2. Plant resistance to high temperatures.

Tasks for Self study work tasks for students

3. 4. 5. 6. 7. 8.

Work signaling systems under the action of cold. Work signaling systems under the action of heat. Mechanisms of thermoregulation in plants. Effect of oxygen deficiency on plants. Effect of ambient ozone on plants. Effect of pesticides on physiological and biochemical processes in plants. 9. Phytoremediation of soils contaminated with pesticides. 10. Resistance of plants to salinity. 11. Resistance to drought. 12. Mechanisms of resistance of plants in conditions of dehydration. 13. Genetically engineered approach to the study of plant resistance to cold. 14. Genetically engineered approach to the study of plant resistance to heat. 15. Genetically engineered approach to the study of plant resistance to salinity. 16. Study of the resistance of plants to heavy metals using molecular biology techniques. 17. The study of plant resistance to drought effects using methods using molecular biology.

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Recommended literature

The basic 1. Lisar S.Y.S., R. Motafakkerazad, Hossain M.M., Rahman I.M.M. Water Stress in Plants: Causes, Effects and Responses. A Review. In «Water stress», Ed. by Prof. Ismail Md. M. Rahman – 2012. – 300 p. (www. intechopen.com). 2. Alekhina N.A., Balnokin J.V., V.F. Gavrylenko «Plant Physiology». Ed. I.P.Ermakov. – M., 2007. – 640 p. 3. Bitrián M., Zarza X., Altabella T., Tiburcio A. F. , Alcázar R. Polyamines under Abiotic Stress: Metabolic crossroads and hormonal crosstalks in plants. Review // Metabolites. – 2012. V. 2. – Pp. 516-528. 3. Chirkova T.V. «Physiological basis of tolerance» (Russian). Manual. – SPb .: Publishing House of St. Petersburg. University Press, 2002. – 244 p. 4. Alkazar R., Altabella T., Marco F., Bortolotti K., Reymond M., Konez C., Carrasco P., Tiburcio A.F. Polyamines: molecules withregulatory functions in plant abiotic stress tolerance. Review // Planta, 2010. – V. 231. – Pp. 1231-1249. 5. Wasternack C., Hause B. Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. Review // Annals of Botany – 2013. – V. 111 Pp. 1021-1058. 6. Depuydt S., Hardtke C. S. Hormone signalling crosstalk in plant growth regulation. Review // Current opinion in Plant Biology. – 2011. – V. 21. – Pp. 365-373. 7. Gupta B. Mechanism of Salinity Tolerance in Plants: Physiological, Biochemical, and Molecular Characterization . Review // International Journal of Genomics. – 2014. – http://dx.doi.org/10.1155/2014/701596. – 18 p. Additional 1. Gill S. S., Tuteja N. Polyamines and abiotic stress tolerance in plants // Plant Signaling & Behavior. – 2010. – V. 5(1). – Pp. 26-33. 2. P Rahdari, Hoseini S. M. Salinity Stress. A Review // TJEAS – 2011. – V.1(30) –Pp. 63-66.

Recommended literature

3. Peleg Z., Blumwald E.Hormone balance and abiotic stress tolerance in crop plants // Current Opinion in Plant Biology. – 2011. – V. 14. – Pp. 290–295. 4. Reactive oxygen species in plants: their generation, signal transduction, and scavenging // AJCS. – 2011. – V. 5(6). – Pp. 709-725. 5. Savitri E. S., Nurul N. B., Estri A., Arumingtyas L. Identification and characterization drought tolerance of gene LEA-D11 soybean (Glycine max L.) based on PCR-sequencing // American Journal of Molecular Biology – 2013. – V. 3. – Pp. 32-37. 6. Galston A.W. Polyamine and plant response to stress // The physiology of polyamines (Eds. U.Bachrach, U.M.Heimer). – CRC. Press. Inc. Boca Raton,. Fla., 2001 – Pp. 99-106. 7. Prasad MNV, Stralka K. Physiology and biochemistry of metal toxicity and tolerance in plants. – Kluewer Academic Publishers. Dordirecht, 2010. – 432 p.

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Еducational issue

Atabayeva Saule Kenzhebayeva Saule Blavachinskaya Irina STRESS PHYSIOLOGY Study guide Managing Editor E. Suleimenova Typesetting and cover design G. Kaliyeva Cover design used photos from sites www.zelle.com

IB No. 8250

Signed for publishing 15.05.2015. Format 60x84 1/16. Offset paper. Digital printing. Volume 5,25 printer’s sheet. 120 copies. Order No 1082. Publishing house «Qazaq University» Al-Farabi Kazakh National University KazNU, 71 Al-Farabi, 050040, Almaty Printed in the printing office of the «Qazaq university» publishing house