Tarchevsky I.A. Plant cell signaling systems

The effect of elicitor drugs is due to the presence of special biologically active substances in their composition. According to modern concepts, signaling substances or elicitors are biologically active compounds of various natures, which in very low dosages, measured in milli-, micro-, and in some cases nanograms, cause cascades of various plant responses at the genetic, biochemical and physiological levels. Their impact on phytopathogenic organisms is carried out by influencing the genetic apparatus of cells and changing the physiology of the plant itself, giving it greater vitality and resistance to various negative environmental factors.

The relationship of plants with the outside world, as highly organized elements of ecological systems, is carried out by perceiving physical and chemical signals coming from the outside and correcting all processes of their life activity through influencing genetic structures, immune and hormonal systems. The study of plant signaling systems is one of the most promising areas in modern cell and molecular biology. In recent decades, scientists have paid a lot of attention to the study of signaling systems responsible for plant resistance to phytopathogens.

The biochemical processes occurring in plant cells are strictly coordinated by the integrity of the organism, which is complemented by their adequate reactions to information flows associated with various impacts of biogenic and technogenic factors. This coordination is carried out through the work of signaling chains (systems), which are intertwined into cell signaling networks. Signaling molecules activate most hormones, usually not by penetrating into the cell, but by interacting with receptor molecules of the outer cell membranes. These molecules are integral membrane proteins, the polypeptide chain of which penetrates the thickness of the membrane. A variety of molecules that initiate transmembrane signaling activate receptors in nano-concentrations (10-9-10-7 M). The activated receptor transmits a signal to intracellular targets - proteins, enzymes. In this case, their catalytic activity or conductivity of ion channels is modulated. In response to this, a certain cellular response is formed, which, as a rule, consists of a cascade of sequential biochemical reactions. In addition to protein intermediaries, relatively small messenger molecules, which functionally act as intermediaries between receptors and the cellular response, can also participate in signal transmission. An example of an intracellular messenger is salicylic acid, which is involved in the induction of stress and immune responses in plants. After the signaling system is turned off, the messengers are quickly broken down or (in the case of Ca cations) pumped out through ion channels. Thus, proteins form a kind of “molecular machine”, which, on the one hand, perceives an external signal, and on the other, has enzymatic or other activity modeled by this signal.

In multicellular plant organisms, signal transmission occurs through the level of cell communication. Cells “speak” in the language of chemical signals, which allows homeostasis of the plant as a whole biological system. The genome and cell signaling systems form a complex self-organizing system or a kind of “biocomputer”. The hard information carrier in it is the genome, and signaling systems play the role of a molecular processor that performs operational control functions. At present, we have only the most general information about the operating principles of this extremely complex biological formation. In many respects, the molecular mechanisms of signaling systems remain unclear. Among the solutions to many issues, it remains to decipher the mechanisms that determine the temporary (transient) nature of the activation of certain signaling systems, and at the same time, long-term memory of their activation, manifested, in particular, in the acquisition of systemic prolonged immunity.

There is a two-way relationship between signaling systems and the genome: on the one hand, enzymes and proteins of signaling systems are encoded in the genome, on the other hand, signaling systems are controlled by the genome, expressing some genes and suppressing other genes. This mechanism includes the reception, transformation, multiplication and transmission of signals to the promoter regions of genes, programming of gene expression, changes in the spectrum of synthesized proteins and the functional response of the cell, for example, the induction of immunity to phytopathogens.

Various organic ligand compounds and their complexes can act as signal molecules or elicitors that exhibit inductive activity: amino acids, oligosaccharides, polyamines, phenols, carboxylic acids and esters of higher fatty acids (arachidonic, eicosapentaenoic, oleic, jasmonic, etc.), heterocyclic and organoelement compounds, including some pesticides, etc.

Secondary elicitors formed in plant cells under the action of biogenic and abiogenic stressors and included in cell signaling networks include phytohormones: ethylene, abscisic, jasmonic, salicylic acids, and

also the systemin polypeptide and some other compounds that cause the expression of protective genes, the synthesis of corresponding proteins, the formation of phytoalexins (specific substances that have an antimicrobial effect and cause the death of pathogenic organisms and affected plant cells) and, ultimately, contribute to the formation of systemic resistance in plants to negative environmental factors.

Currently, seven cell signaling systems are the most studied: cycloadenylate, MAP-kinase (mitogen-activated protein-kinase), phosphatidic acid, calcium, lipoxygenase, NADPH oxidase (superoxide synthase), NO synthase. Scientists continue to discover new signaling systems and their biochemical participants.

Plants, in response to pathogen attack, can use different pathways to form systemic resistance, which are triggered by different signaling molecules. Each of the elicitors, influencing the vital activity of the plant cell along a specific signaling pathway, through the genetic apparatus, causes a wide range of reactions, both protective (immune) and hormonal in nature, leading to changes in the properties of the plants themselves, which allows them to withstand a whole range of stress factors. At the same time, in plants there is an inhibitory or synergistic interaction of various signaling pathways intertwined into signaling networks.

Induced resistance is similar in manifestation to genetically determined horizontal resistance, with the only difference being that its nature is determined by phenotypic changes in the genome. However, it has a certain stability and serves as an example of phenotypic immunocorrection of plant tissue, since as a result of treatment with substances with elicitor action, it is not the plant genome that changes, but only its functioning, associated with the level of activity of protective genes.

In a certain way, the effects that occur when plants are treated with immunoinducers are similar to genetic modification, differing from it in the absence of quantitative and qualitative changes in the gene pool itself. With artificial induction of immune reactions, only phenotypic manifestations are observed, characterized by changes in the activity of expressed genes and the nature of their functioning. However, changes caused by treatment of plants with phytoactivators have a certain degree of persistence, which is manifested in the induction of prolonged systemic immunity, maintained for 2-3 or more months, as well as in the preservation of acquired properties by plants for 1-2 subsequent reproductions.

The nature of the action of a particular elicitor and the effects achieved are closely dependent on the strength of the generated signal or the dosage used. These dependencies, as a rule, are not rectilinear, but sinusoidal in nature, which can serve as evidence of a switching of signaling pathways during their inhibitory or synergistic interactions. It has also been established that under conditions of stress factors, plants respond positively to lower dosages of phytoactivators, which indicates a more high severity of their adaptogenic effect. On the contrary, treatment with these substances in large dosages, as a rule, caused desensitization processes in plants, sharply reducing the immune status of plants and leading to increased plant susceptibility to diseases.

BBK 28.57 T22

Executive Editor Corresponding Member of the Russian Academy of Sciences.I. Grechkin

Reviewers:

Doctor of Biological Sciences, Professor L.Kh. Gordon Doctor of Biological Sciences, Professor L.P. Khokhlova

Tarchevsky I.A.

Signaling systems of plant cells / I.A. Tarchevsky; [Ans. ed. A.N. Grechkin]. -

M.: Nauka, 2002. - 294 p.: ill. ISBN 5-02-006411-4

The links in the information chains of interaction between pathogens and plants are considered, including elicitors, elicitor receptors, G-proteins, protein kinases and protein phosphatases, transcription regulatory factors, reprogramming of gene expression and cell response. The main attention is paid to the analysis of the functioning features of individual plant cell signaling systems - adenylate cyclase, MAP kinase, phosphatidate, calcium, lipoxygenase, NADPH oxidase, NO synthase and proton, their interaction and integration into a single signaling network. A classification of pathogen-induced proteins according to their functional characteristics is proposed. Data are provided on transgenic plants with increased resistance to pathogens.

For specialists in the field of plant physiology, biochemists, biophysicists, geneticists, plant pathologists, ecologists, agrobiologists.

Via AK network

Plant Cell Signaling Systems /1.A. Tarchevsky; . - M.: Nauka, 2002. - 294 p.; il. ISBN 5-02-006411-4

The book discussed the members of signaling chains of interplay of pathogens and plant-host, namely elicitors, receptors, G-proteins, protein kinases and protein phosphatases, transcription factors reprogramming of genes expression, cell response. The main part of the book is devoted to the functioning of separate cell signaling systems: adenylate cyclase, MAP kinase, phosphatidate, calcium, lipoxy-genase, NADPH-oxidase, NO-synthase, protons systems. The concept of interconnections of cell signaling systems and their integration to general cell signaling network is developing. The author has preposed the classification of pathogen-related proteins according to their function properties. The data on transgenic plants with the increased resistance to pathogens are presented.

For physiologists, biochemists, biophysicists, genetics, phytopathologists, ecologists, and agrobiologists

ISBN 5-02-006411-4

(art design), 2002

In recent years, research into the molecular mechanisms of gene expression regulation under the influence of changing living conditions has been rapidly developing. In plant cells, the existence of signaling chains was discovered that, with the help of special receptor proteins, in most cases located in the plasmalemma, perceive signal impulses, convert, amplify and transmit them to the cell genome, causing reprogramming of gene expression and changes in metabolism (including including cardinal ones), associated with the inclusion of previously “silent” genes and the switching off of some active genes. The importance of cell signaling systems was demonstrated by studying the mechanisms of action of phytohormones. The decisive role of signaling systems in the formation of adaptation syndrome (stress) caused by the action of abiotic and biotic stressors on plants was also shown.

The lack of review works that would analyze all the links of various signaling systems, starting with the characteristics of perceived signals and their receptors, the transformation of signal impulses and their transmission to the nucleus, and ending with dramatic changes in cell metabolism and their structure, forced the author to attempt to fill this gap with the help of the book offered to the attention of readers. It must be taken into account that the study of the information field of cells is still very far from completion and many details of its structure and functioning remain insufficiently illuminated. All this attracts new researchers, for whom a summary of publications on plant cell signaling systems will be especially useful. Unfortunately, not all reviews

articles of an experimental nature were included in the bibliography, which to a certain extent depended on the limited volume of the book and the time for its preparation. The author apologizes to colleagues whose research was not reflected in the book.

The author expresses gratitude to his collaborators who took part in the joint study of plant cell signaling systems. The author expresses special gratitude to Professor F.G. Karimova, Candidates of Biological Sciences V.G. Yakovleva and E.V. Asafova, A.R. Mukha-metshin and associate professor T.M. Nikolaeva for assistance in preparing the manuscript for publication.

The work was carried out with financial support from the Foundation of the Leading Scientific School of the Russian Federation (grants 96-15-97940 and 00-15-97904) and the Russian Foundation for Basic Research (grant 01-04-48-785).

INTRODUCTION

One of the most important problems modern biology is to decipher the mechanisms of response of prokaryotic and eukaryotic organisms to changes in the conditions of their existence, especially to the action of extreme factors (stress factors, or stressors) that cause a state of stress in cells.

In the process of evolution, cells have developed adaptations that allow them to perceive, transform and amplify signals of a chemical and physical nature coming from the environment and, with the help of the genetic apparatus, respond to them, not only adapting to changed conditions, rebuilding their metabolism and structure, but also highlighting various volatile and non-volatile compounds into the extracellular space. Some of them act as protective substances against pathogens, while others can be considered as signaling molecules that trigger a response from other cells located at a great distance from the site of action of the primary signal on plants.

We can assume that all these adaptive events occur as a result of changes in the information field of cells. Primary signals through various signaling systems cause a response from the cell genome, manifested in the reprogramming of gene expression. In fact, signaling systems regulate the operation of the main repository of information - DNA molecules. On the other hand, they themselves are under the control of the genome.

For the first time in our country, E.S. began to purposefully study cell signaling systems. Severin [Severin, Kochetkova, 1991] on animal objects and O.N. Kulaeva [Kulaeva et al., 1989; Kulaeva, 1990; Kulaeva et al., 1992; Kulaeva, 1995;

Burkhanova et al., 1999] - on plants.

The monograph presented to the readers contains a summary of the results of studying the influence of biotic stressors on the functioning of plant cell signaling systems. Currently, MAP kinase, adenylate cyclase, phosphatidate, calcium, lipoxygenase, NADPH oxidase, NO synthase and proton signaling systems and their role in the ontogenetic development of plants and in the formation of a response to changing living conditions, especially the effect of various abiotic and biotic stressors. The author decided to focus only on the last aspect of this problem - on the molecular mechanisms of plant response to the action of pathogens, especially since a number of phytohormones are involved in this response and elucidation of the features of the interaction of plant cell signaling systems with them attracts much attention from researchers.

Exposure to biotic stressors results in a plant response that is broadly similar to the response to abiotic stressors. It is characterized by a set of nonspecific reactions, which makes it possible to call it adaptation syndrome, or stress. Naturally, specific features of the response may also be detected, depending on the type of stressor, however, as the degree of its impact increases, nonspecific changes begin to come to the fore to an increasing extent [Meyerson, 1986; Tarchevsky, 1993]. The greatest attention was paid to N.S. Vvedensky (ideas about parabiosis), D.S. Nasonov and V.Ya. Alexandrov (ideas about paranecrosis), G. Selye - in works devoted to stress in animals, V.Ya. Aleksandrov - in research on the molecular basis of stress.

The most significant nonspecific changes during biotic stress include the following:

1. Phasicity in the time course of the response to the action of a pathogen.

2. Increased catabolism of lipids and biopolymers.

3. Increased content of free radicals in tissues.

4. Acidification of the cytosol with subsequent activation of proton pumps, which returns the pH to its original value.

5. An increase in the content of calcium ions in the cytosol with subsequent activation of calcium ATPases.

6. The release of potassium and chlorine ions from cells.

7. A drop in membrane potential (at the plasmalemma).

8. Decrease in the overall intensity of biopolymer synthesis and

9. Stopping the synthesis of certain proteins.

10. Enhanced synthesis or synthesis of missing so-called pathogen-inducible protective proteins (chitinases,(3-1,3-glucanases, proteinase inhibitors, etc.).

11. Intensification of the synthesis of cell wall-strengthening components - lignin, suberin, cutin, callose, hydroxyproline-rich protein.

12. Synthesis of antipathogenic non-volatile compounds -

phytoalexins.

13. Synthesis and isolation of volatile bactericidal and fungicidal compounds (hexenals, nonenals, terpenes and

Dr->- 14. Strengthening synthesis and increasing content (or according to

phenomenon) of stress phytohormones - abscisic, jasmonic, salicylic acids, ethylene, the peptide hormone systemin.

15. Inhibition of photosynthesis.

16. Redistribution of carbon from |4 CO2, assimilated during photosynthesis, among various compounds - a decrease in the inclusion of the label in high-polymer compounds (proteins, starch) and sucrose and an increase (more often relative - as a percentage of the assimilated carbon) - in alanine, malate , aspartate [Tarchevsky, 1964].

17. Increased breathing followed by inhibition. Activation of an alternative oxidase that changes the direction of electron transport in mitochondria.

18. Ultrastructural disorders - changes in the fine granular structure of the nucleus, a decrease in the number of polysomes and dictyosomes, swelling of mitochondria and chloroplasts, a decrease in the number of thylakoids in chloroplasts, restructuring of cyto-

skeleton

19. Apoptosis (programmed death) of cells exposed to pathogens and those neighboring them.

20. The appearance of the so-called systemic nonspecific

high resistance to pathogens in areas remote from the site of pathogen exposure (for example, metameric organs) of the plant.

Many of the changes listed above are a consequence of the “switching on” of a relatively small number of nonspecific signaling systems by stressors.

With increasing study of the mechanisms of plant responses to pathogens, new nonspecific responses of plant cells are being discovered. These include previously unknown signaling pathways.

When elucidating the features of the functioning of signaling systems, it is necessary to keep in mind that these issues are part of a more general problem of regulation of genome functioning. It should be noted that the universality of the structure of the main information carriers of cells various organisms- DNA and genes - predetermines the unification of those mechanisms that serve the implementation of this information [Grechkin, Tarchevsky, 2000]. This concerns DNA replication and transcription, the structure and mechanism of action of ribosomes, as well as the mechanisms of regulation of gene expression by changing conditions of cell existence using a set of largely universal signaling systems. The links of signaling systems are also basically unified (nature, having found at one time the optimal structural and functional solution to a biochemical or information problem, preserves and replicates it in the process of evolution). In most cases, a wide variety of chemical signals coming from the environment are captured by the cell with the help of special “antennas” - receptor protein molecules that penetrate the cell membrane and protrude above its external and internal surfaces.

her sides. Several types of structure of these receptors are unified in plant and animal cells. Non-covalent interaction of the external region of the receptor with one or another signaling molecule coming from the environment surrounding the cell leads to a change in the conformation of the receptor protein, which is transmitted to the internal, cytoplasmic region. In most signaling systems, intermediary G-proteins come into contact with it - another unit of signaling systems that is unified (in its structure and functions). G-proteins perform the functions of a signal transducer, transmitting a signal conformational impulse to the starting enzyme specific for a particular signaling system. The starting enzymes of the same type of signaling system in different objects are also universal and have extended regions with the same amino acid sequence. One of the most important unified links in signaling systems are protein kinases (enzymes that transfer the terminal residue of orthophosphoric acid from ATP to certain proteins), activated by the products of starting signaling reactions or their derivatives. Proteins phosphorylated by protein kinases are the next links in signal chains. Another unified link in cell signaling systems is protein transcription regulatory factors, which are one of the substrates of protein kinase reactions. The structure of these proteins is also largely unified, and modifications of the structure determine the affiliation of transcription regulatory factors to one or another signaling system. Phosphorylation of transcription regulatory factors causes a change in the conformation of these proteins, their activation and subsequent interaction with the promoter region of a certain gene, which leads to a change in the intensity of its expression (induction or repression), and in extreme cases, to the “switching on” or “switching off” of some silent genes. active. Reprogramming the expression of a set of genes in the genome causes a change in the ratio of proteins in the cell, which is the basis of its functional response. In some cases, a chemical signal from the external environment can interact with a receptor located inside the cell - in the cytosol or

Rice. 1. Scheme of interaction of external signals with cell receptors

1, 5, 6 - receptors located in the plasmalemma; 2,4 - receptors located in the cytosol; 3 - starting enzyme of the signaling system, localized in the plasmalemma; 5 - receptor activated under the influence of nonspecific changes in the structure of the lipid component of the plasmalemma; SIB - signal-induced proteins; PTF - protein transcription regulatory factors; i|/ - change in membrane potential

the same core (Fig. 1). In animal cells, such signals are, for example, steroid hormones. This information pathway has a smaller number of intermediates, and therefore has fewer opportunities for regulation by the cell.

Metlitsky et al., 1984; Metlitsky, Ozeretskovskaya, 1985; Kursano-va, 1988; Ilyinskaya et al., 1991; Ozeretskovskaya et al., 1993; Korableva, Platonova, 1995; Chernov et al., 1996; Tarchevsky, Chernov, 2000].

In recent years Special attention focuses on the molecular mechanisms of phytoimmunity. It has been shown that

When plants are infected, various signaling systems are activated that perceive, multiply and transmit signals from pathogens to the genetic apparatus of cells, where the expression of protective genes occurs, allowing plants to organize both structural and chemical protection from pathogens. Advances in this area are associated with cloning genes, deciphering them primary structure(including promoter regions), the structure of the proteins they encode, the use of activators and inhibitors of individual parts of signaling systems, as well as mutants and transgenic plants with introduced genes responsible for the synthesis of participants in the reception, transmission and amplification of signals. In the study of plant cell signaling systems, an important role is played by the construction of transgenic plants with promoters of genes for proteins participating in signaling systems.

Currently, the signaling systems of plant cells under biotic stress are most intensively studied at the Institute of Biochemistry. A.N. Bach RAS, Kazan Institute of Biochemistry and Biophysics RAS, Institute of Plant Physiology RAS, Pushchino branch of the Institute of Bioorganic Chemistry RAS, Bioengineering Center RAS, Moscow and St. Petersburg State Universities, All-Russian Research Institute of Agricultural Biotechnology of the Russian Academy of Agricultural Sciences, All-Russian Research Institute of Phytopathology of the Russian Academy of Agricultural Sciences, etc. .

"Physiological and Molecular Plant Pathology", "Molecular Plant - Microbe Interactions", "Annual Review of Plant Physiology and Pathology"). At the same time, in the domestic literature there is no generalization of works devoted to cell signaling systems, which led the author to the need to write the monograph offered to readers.

PATHOGENS AND ELISITORS

mycoplasma-like microorganisms; mushrooms (lower:

Plasmodiophoromycetes, Chitridomycetes, Oomycetes: higher: Ascomycetes, Basidiomycetes, Deuteromycetes).

synthesis of protective enzymes: phenylalanine ammonia lyase

And anion peroxidase. Wingless forms belonging to this subclass appeared as a result of the loss of these organs during the evolution of winged forms. The subclass includes 20 orders of insects, among which there are polyphages that do not have specificity in relation to the plant, oligophages and monophages, in which the specificity of the interaction between the pathogen and the host plant is clearly expressed. Some insects feed on leaves (the entire leaf blade or skeletalizing the leaf), others feed on stems (including gnawing the stem from the inside), flower ovaries, fruits, and roots. Aphids and cicadas suck sap from vascular vessels using a proboscis or stylet.

Despite the measures taken to combat insects, the pressing problem of reducing the harm they cause continues to be. Currently, over 12% of the agricultural crop yield on the planet is lost as a result of attack by pathogenic microorganisms,

nematodes and insects.

Damage to cells leads to the degradation of their contents, for example, high-polymer compounds, and the appearance of oligomeric signaling molecules. These “shipwrecks” [Tarchevsky, 1993] reach neighboring cells and cause a protective reaction in them, including a change in gene expression and the formation of the protective proteins they encode. Often mechanical damage Infection of plants is accompanied by their infection, since the wound surface opens through which pathogens penetrate the plant. In addition, phytopathogenic microorganisms can live in the mouthparts of insects. It is known, for example, that the carriers of mycoplasma infection are cicadas, in which adult forms and larvae feed on the juice of the sieve vessels of plants, piercing the leaves with their proboscis-stylet and

Rice. 2. Scheme of interaction between a pathogen cell and a host plant / - cutinase; 2 - degradation products of cuticle components (possibly

having signaling properties); 3 - (3-glucanase and other glycosylases excreted by the pathogen; 4 - elicitors - fragments of the host cell wall (CW); 5 - chitinases and other glycosylases that act destructively on the pathogen's CS; 6 - elicitors - fragments of the pathogen's CS; 7 - phytoalexins - inhibitors of proteinases, cutinases, glycosylases and other enzymes of the pathogen; 8 - toxic substances of the pathogen; 9 - strengthening of the host CS due to activation of peroxidases and increased lignin synthesis, deposition of hydroxyproline proteins and lectins; 10 - inducers of hypersensitivity and necrosis of neighboring cells; // - cutin degradation products acting on the pathogen cell

young stems. The roseate leafhopper, unlike other members of the leafhopper, sucks out the contents of the cells. Cicadas cause less damage to plant tissue than leaf-eating insects, however, plants can react to it in the same way as to an associated plant infection.

Upon contact with plants, pathogen cells release various compounds that ensure their penetration into the plant, nutrition and development (Fig. 2). Some of these compounds are toxins that pathogens secrete to weaken the host's resistance. Currently, more than 20 host-specific toxins produced by pathogenic fungi have been described.

Rice. 3. Phytotoxic compound from Cochlio-bolus carbonum

Bacteria and fungi also produce non-selective toxins, in particular fusicoccin, erichosetene, coronatine, phase-olotoxin, syringomycin, tabtoxin.

One of the host-specific toxins secreted

Pyrenophora triticirepentis is a 13.2 kDa protein, others are products of secondary metabolism with a wide variety of structures - these are polyketides, terpenoids, saccharides, cyclic peptides, etc.

As a rule, the latter include peptides whose synthesis occurs outside the ribosomes and which contain D-amino acid residues. For example, the host-specific toxin from Cochliobolus carbonum has a tetrapeptide cyclic structure (D-npo-L-ana-D-ana-L-A3JJ), where the latter abbreviation stands for 2-amino-9,10-epoxy-8-oxo-de -kanoic acid (Fig. 3). The toxin is produced in pathogen cells using toxin synthase. Resistance to this compound in maize depends on the gene encoding NADPH-dependent carbonyl reductase, which reduces the carbonyl group, resulting in

deactivation of the toxin. It turned out that in the host plant the toxin causes inhibition of histone deacetylases and, as a consequence, histone overacetylation. This suppresses the plant's defense response caused by pathogen infection.

Another type of compounds secreted by pathogens is called elicitors (from the English elicit - to identify, to cause). The collective term “elicitor” was first proposed in 1972 to designate chemical signals that arise at sites of plant infection by pathogenic microorganisms, and has become widespread.

Elicitors play the role of primary signals and activate a complex network of processes of induction and regulation of phytoimmunity. This is manifested in the synthesis of protective proteins, non-volatile plant antibiotics - phytoalexins, in the release of antipathogenic volatile compounds, etc. Currently, the structure of many natural elicitors has been characterized. Some of them are produced by microorganisms, others (secondary elicitors) are formed during the enzymatic breakdown of high-polymer compounds of the cuticle and polysaccharides of the cell walls of plants and microorganisms, others are stress phytohormones, the synthesis of which in plants is induced by pathogens and abiogenic stressors. The most important elicitors include protein compounds excreted by pathogenic bacteria and fungi, as well as viral envelope proteins. The most studied protein elicitors can be considered small (10 kDa), conservative, hydrophilic, cysteine-enriched elicitins, secreted by all species studied

Phytophthora and Pythium. These include, for example, cryptogein.

Elisitins cause hypersensitivity and death of infected cells, especially in plants of the genus Nicotiana. The most intensive formation of elicitins by late blight occurs during the growth of micro-

It was found that elicitins are capable of transporting sterols across membranes, since they have a sterol-binding site. Many pathogenic fungi themselves cannot synthesize sterols, which makes clear the role of elicitins not only in the nutrition of microorganisms, but also in inducing a protective response in plants. A 42 kDa glycoprotein elicitor was isolated from late blight. Its activity and binding to the plasma membrane protein receptor, the monomeric form of which is a 100 kDa protein, was ensured by an oligopeptide fragment of 13 amino acid residues. A race-specific elicitor peptide consisting of 28 amino acid residues with three disulfide groups was obtained from the phytopathogenic fungus Cladosporium fulvum, and the peptide was formed from a precursor containing 63 amino acids. This avirulence factor showed structural homology with a number of small peptides, such as carboxypeptidase inhibitors and ion channel blockers, and bound by the plasmalemma receptor protein, apparently causing its modulation, dimerization and transmission of a signal impulse to signaling systems. From the larger pre-protein of Cladosporium fulvum, consisting of 135 amino acids, post-translational processing produces a elicitor protein of 106 amino acids. The elicitor proteins produced by the rust fungus Uromyces vignae are two small polypeptides, 5.6 and 5.8 kDa, with properties unlike other elicitins. Among bacterial protein elicitors, harpins are the most studied

Many phytopathogenic bacteria produce elicitor oligopeptides (created by their synthetic

Chinese analogues), corresponding to the most conserved regions of the protein - flagellin,

which is an important virulence factor for these bacteria. A new elicitor protein has been isolated from Erwinia amylovora, the C-region of which is homologous to the enzyme pectate lyase, which can cause the appearance of elicitor oligomeric fragments - products of pectin degradation. The pathogenic bacterium Erwinia carotovora excretes the elicitor protein harpin and the enzymes pectate lyase, cellulase, polygalacturonase and proteases, which hydrolyze the polymeric components of the cell walls of the host plant (see Fig. 2), resulting in the formation of oligomeric elicitor molecules. Interestingly, pectate lyase, secreted by Erwinia chrysanthemi,

acquired activity as a result of extracellular processing. Some lipids and their derivatives are also classified as

elicitors, in particular 20-carbon polyunsaturated fatty acids of some pathogens - arachidonic acid and eicosapentaenoic acid [Ilyinskaya et al., 1991; Ozerets-kovskaya et al., 1993; Ozeretskovskaya, 1994; Gilyazetdinov et al., 1995; Ilyinskaya et al., 1996a, b; Ilyinskaya, Ozeretskovskaya, 1998], and their oxygenated derivatives. The review work [Ilyinskaya et al., 1991] summarizes data on the elicitor effect of lipids (lipoproteins) produced by pathogenic fungi on plants. It turned out that it is not the protein part of lipoproteins that has the elicitor effect, but their lipid part, which is arachidonic (eicosatetraenoic) and eicosopentaenoic acids, which are not characteristic of higher plants. They caused the formation of phytoalexins, tissue necrosis and systemic plant resistance to various pathogens. Products of lipoxygenase transformation in plant tissues of C20 fatty acids (hydroperoxy-, hydroxy-, oxo-, cyclic derivatives, leukotrienes), formed in the cells of the host plant with the help of the enzyme lipoxygenase complex (the substrates of which can be both C,8 and C20 polyene fatty acids) had a strong effect on the protective response of plants. This is apparently explained by the fact that there is no oxygen in uninfected plants.

nated derivatives of 20-carbon fatty acids, and their appearance as a result of infection leads to dramatic results, such as the formation of necrosis around the infected cells, which creates a barrier to the spread of pathogens throughout the plant.

There is evidence that pathogen induction of lipoxygenase activity led to the formation of a plant response even in the case when the elicitor did not contain C20 fatty acids and the substrate of lipoxygenase activity could only be its own C18 polyenoic fatty acids, and the products were octadecanoids, not eicosanoids. Syringolides [L et al., 1998] and cerebrosides, sphingolipid compounds, also have elicitor properties. Cerebrosides A and C isolated from Magnaporthe grisea were the most active elicitors in rice plants. Cerebroside degradation products (fatty acid methyl esters, sphingoid bases, glycosyl-sphingoid bases) did not exhibit elicitor activity.

Some elicitors are formed as a result of the action of hydrolases secreted by pathogens on plant tissue. The purpose of hydrolases is twofold. On the one hand, they provide nutrition to pathogens necessary for their development and reproduction, on the other hand, they loosen mechanical barriers that stand in the way of pathogens entering their habitats in plants.

One such barrier is the cuticle, which consists primarily of a cutin heteropolymer embedded in wax. More than 20 monomers that make up cutin have been discovered

These are saturated and unsaturated fatty acids and alcohols of various lengths, including hydroxylated and epoxidated, long-chain dicarboxylic acids, etc. In cutin, most of the primary alcohol groups participate in the formation of ester bonds, as well as some of the secondary alcohol groups that provide cross-links between chains and branch points in the polymer. Part of another “barrier” polymer, suberin, is close in composition to cutin. Its main difference is that free fatty acids are the main component of suberic waxes, while there are very few of them in cutin. Moreover, in Suberina

There are mainly C22 and C24 fatty alcohols present, while cutin contains C26 and C28. To overcome the surface mechanical barrier of plants, many pathogenic fungi secrete enzymes that hydrolyze cutin and part of the components of suberin. The products of the cutinase reaction were various oxygenated fatty acids and alcohols, mainly 10,16-dihydroxy-Sk- and 9,10,18-trihydroxy-C|8-acids, which are signal molecules that induce the formation and release of additional amounts of cutinase, “corroding” cutin and facilitating the penetration of the fungus into the plant. It was found that the lag period for the appearance of cutinase mRNA in the fungus after the onset of the formation of the above-mentioned di- and trihydroxy acids is only 15 minutes, and the lag period for the release of additional cutinase is twice as long. Damage to the cutinase gene in Fusarium solani greatly reduced the virulence of this fungus. Inhibiting cutinase using chemicals or antibodies prevented plant infection. The assumption that oxygenated cutin degradation products can act not only as inducers of cutinase formation in pathogens, but also as elicitors of defense reactions in the host plant [Tarchevsky, 1993] was subsequently confirmed.

After the penetration of pathogenic microorganisms through the cuticle, some of them move into the vascular bundles of plants and use the existing there for their development. nutrients, while others are transported inside living host cells. In any case, pathogens encounter another mechanical barrier - cell walls, consisting of various polysaccharides and proteins and in most cases reinforced with a hard polymer - lignin [Tarchevsky, Marchenko, 1987; Tarchevsky, Marchenko, 1991]. As mentioned above, to overcome this barrier and provide their development with carbohydrate and nitrogen nutrition, pathogens secrete enzymes that hydrolyze polysaccharides and cell wall proteins.

Special studies have shown that during the interaction of bacteria and tissues of the host plant, enzymes

degradations do not appear simultaneously. For example, pectylmethylesterase was also present in uninoculated Erwinia carotovora subsp. atroseptia in potato tuber tissues, while polygalacturonase, pectate lyase, cellulase, protease and xylanase activities appeared, respectively, 10, 14, 16, 19 and 22 hours after inoculation.

It turned out that oligosaccharide degradation products of plant cell wall polysaccharides have elicitor properties. But active oligosaccharides can also be formed by polysaccharides that are part of the cell walls of pathogens. It is known that one of the ways to protect plants from pathogenic microorganisms is the formation after infection and the release outside the plasmalemma of enzymes - chitinase and β-1,3-glucanase, which hydrolyze the polysaccharides chitin and β-1,3-polyglucans of the cell walls of pathogens, which leads to suppression of their growth and development. It was found that the oligosaccharide products of such hydrolysis are also active elicitors of plant defense reactions. As a result of the action of oligosaccharides, plant resistance to bacterial, fungal or viral infection increases.

A number of review articles are devoted to oligosaccharide elicitors, their structure, activity, receptors, their “switching on” of cell signaling systems, induction of the expression of protective genes, the synthesis of phytoalexins, hypersensitivity reactions and other plant responses.

In Elbersheim's laboratory, and then in a number of other laboratories, it was shown that oligoglycosides formed as a result of pathogen-induced endoglycosidase degradation of hemicelluloses and pectin substances of plants, chitin and chitosan of fungi, can play the role of biologically active substances. It has even been proposed that they be considered a new class of hormones ("oligosaccharins", as opposed to oligosaccharides, which have no activity). The formation of oligosaccharides as a result of the hydrolysis of polysaccharides, and not during the synthesis from monosaccharides, was shown by the example

AB11 and AB12 play a key role in ABA-induction

bathroom signal path. pH-dependent and Mg2+-dependent activation were observed.

vation ABU.

The main target of MP2C protein phosphatases is MAPKKK, which is activated under the influence of various stressors. This specificity becomes understandable if we consider that some protein phosphatases have binding sites with their corresponding protein kinases

Signaling participants

nal cell systems. This makes it possible to ensure the existence of the protein kinase-protein phosphatase complex and timely and effectively block the transformation and transmission of a signal impulse into the genome. The principle of operation of this mechanism is quite simple: the accumulation of a certain protein kinase - an intermediate of the signal chain - activates phosphoprotein phosphatase and leads to dephosphorylation (inactivation) of the protein kinase. For example, activation of certain protein kinases can lead to phosphorylation and activation of the corresponding protein phosphatases. When studying the functioning of protein phosphatases, specific inhibitors are often used, for example okadaic acid and calyculin.

TRANSCRIPTION REGULATION FACTORS

The synthesis of messenger RNAs is catalyzed by DNA-dependent RNA polymerases, which are one of the largest protein complexes, consisting of two large and 5-13 small subunits, which is determined by the complexity and importance of their functions. These subunits have conservative amino acid sequences, mostly or to a lesser extent common to animals and plants, iRNA polymerase activity and recognition of transcribed genes are regulated by several types of proteins. Transcription regulatory factors have received the most attention." These proteins are able to interact with other proteins, including identical ones, change conformation when phosphorylating several of their constituent amino acids, [recognize regulatory sequences of nucleotides in the promoter regions of genes, which leads to a change in the intensity of their expression.: It is the transcription regulatory factors that direct RNA -polymerase to the transcription initiation point of the corresponding gene (or set of genes), without directly participating in the catalytic act of mRNA synthesis.

In animal organisms, the structural features of more than 1 thousand transcription regulation factors have been determined. Cloning their genes contributed to obtaining information that allowed the classification of these proteins.

All transcription regulatory factors contain three main domains. The most conserved is the DNA-binding domain. The sequence of amino acids in it determines the recognition of certain nucleotide sequences in gene promoters.

Depending on the homology of the primary and secondary structures of the DNA-binding domain, transcription regulatory factors are divided into four superclasses: 1) with domains enriched in basic amino acids; 2) with DNA-binding domains coordinating zinc ions - “zinc fingers”; 3) with domains of the helix-turn-helix type; 4) with domains of the |3-scaffold type, forming contacts with the minor groove of DNA [Patrushev, 2000]. Each superclass is subdivided into classes, families and subfamilies. Notable in superclass 1 are transcription regulatory factors with leucine zipper domains, which are os-helices in which every seventh amino acid is a leucine protruding from one side of the helix. The hydrophobic interaction of leucine residues of one molecule with a similar helix of another molecule provides dimerization (by analogy with a zipper) of transcription regulatory factors necessary for interaction with DNA.

In superclass 2, zinc fingers are amino acid sequences containing four cysteine residues that have a coordinating effect on the zinc ion. Zinc fingers interact with the major groove of DNA. In another class of this superclass, the structure of “zinc fingers” is provided by two cysteine residues and two histidine residues (Fig. 5); in another class, the coordination of two zinc ions in one “finger” is carried out by six cysteine residues. The tips of the zinc fingers contact the major groove of DNA.

The study of the structure of transcription regulatory factors in plants made it possible to establish homology with proteins of this type, characteristic of animal objects. Typical transcription regulatory factors contain the following three main structural elements: DNA binding, oligomerization and regulatory domains. Monomeric forms of transcription factors are inactive, unlike dimeric (oligomeric) forms. The formation of oligomeric forms is preceded by phosphorylation of monomeric forms in the cytosol, then their association occurs and then delivery into the nucleus or using

Rice. 5. Structure of the “zinc finger” transcription regulatory factor

G - histidine residue; C-S - cysteine residue

special transport proteins or due to interaction with receptor proteins in the pores of the nuclear membrane, after which they are transported into the nucleus and interact with promoter regions

corresponding genes. “Transcription regulatory factors are encoded by multigene families, and their synthesis can be induced by pathogens and elicitors, and their activity altered as a result of post-translational modification (mainly phosphorylation or dephosphorylation).

Currently, an ever-expanding database has been created on the structure of various transcription regulatory factors and their genes in plants. It has been shown that the specificity of DNA binding is determined by the amino acid sequences of the stem and loop zones in the already mentioned leucine zippers, which represent one of the most numerous and conserved groups of eukaryotic transcription regulatory factors. Transcription regulatory factors are often classified according to the structure of DNA-binding domains, which may include helical amino acid sequences, “zinc fingers” - regions with two cysteine and two histidine residues or with many cysteine residues, etc. In plants, one to four "zinc fingers" are found in the DNA-binding domains of transcription regulatory factors.

The mechanism of interaction of transcription regulatory factors with DNA-dependent RNA polymerases and gene promoter regions remains one of the key and still insufficiently studied problems in the functioning of the cell genome. Information regarding plant objects is especially scarce.

Mutations in genes encoding transcription regulatory factors in animals can lead to certain diseases.

Members of a family of genes encoding leucine zipper transcription regulatory factors have been described in plants. It has been shown that transcription factors of this type are responsible for the salicylate-induced formation of protective antipathogenic proteins and that mutations in these genes lead to loss of the ability to synthesize these proteins

PROMOTER OF GENES FOR SIGNALING SYSTEMS PROTEINS AND PROTECTIVE PROTEINS

Currently, the structure of the promoter regions of genes responsible for acquiring immunity to various pathogens is being intensively studied. The fact of the almost simultaneous synthesis of a number of pathogen-inducible proteins has long attracted attention: This can be caused either by the divergence of signaling pathways in one signaling system, which causes the activation of several types of transcription regulatory factors, or by the “switching on” of several signaling systems by one or another elicitor, which, functioning in parallel, they activate several types of transcription regulatory factors and, as a result, induce the expression of several types of protective proteins. It is also possible that the gene promoters of several individual proteins have the same structure of regulatory elements, which leads to their simultaneous expression even in the case of signal activation of one representative of transcription regulatory factors.1

The latter option occurs when plants are exposed to the stress phytohormone ethylene, when the transcription regulatory factor interacts with the GCC box of the promoter regions of several ethylene-inducible genes, which ensures more or less simultaneous formation of a whole group of ethylene-inducible proteins. This principle of batch synthesis of protective proteins is implemented when cells respond to various stressors or elicitors (stress phytohormones can also be classified as secondary elicitors). For example, under the influence of elevated temperatures, the transcription of a group of genes containing a common regulation in their promoter regions is induced.

tor element HSE (heat shock element), absent in other genes. This pattern was confirmed using the technique of creating hybrid genes with the heat shock gene promoter coupled with another gene that usually does not change the intensity of expression when exposed to elevated temperatures. In the case of transgenic plants, its expression began. In eukaryotic cells, promoter regions with similar nucleotide sequences are also found in various genes induced by the same intermediate (second messenger) of signaling systems, for example, cyclic AMP. In the latter case, the signal sequence of nucleotides of the promoter region is designated CRE (cyclic AMP response element).

In Arabidopsis, a glucocorticoid system for activating transcription regulatory factors was discovered, the inclusion of which led to the expression of pathogen-induced protective genes [N. Kang et al., 1999]. Common nucleotide sequences in the G-box pro-

motors were CCACGTGG, and in the C-box - TGACGTCA.

Tobacco mosaic virus and salicylic acid caused the induction of two genes of transcription regulatory factors of the WRKY class in tobacco plants, recognizing a certain nucleotide sequence in the promoter regions of protective genes - TTGAC (W-box). Activation of these transcription regulatory factors was accomplished through their phosphorylation by protein kinases. All proteins of the WRKY class, unlike other classes of transcription factors (such as bZIP and myb), have a conserved domain containing a heptameric enzyme.

id WRKYGQK .

(One of the domains of the transcription regulatory factor responsible for the transformation of the jasmonate signal activates the regulatory region of the promoter of several genes encoding jasmonate- and elicitor-inducible proteins, in particular strictosidine synthase. It turned out that the N-terminal acidic domain of the transcription regulatory factor has an activating effect , and the C-terminal domain -I enriched in serine residues is inhibitory.

It has been shown that the promoter of the phenylalanine ammonia lyase gene (the most important starting enzyme of the branched metabolic process of the synthesis of compounds playing a protective role - salicylate, phenolic acids, phenylpropanoid phytoalexins and lignin) contains two copies of regions enriched with AC repeats.

When studying the promoter of the gene for another enzyme synthetizing phytoalexins - chalcone synthase, in cell cultures of beans, tobacco and rice, it was found that the G-box (CACGTG) in the region from -74 to -69 nucleotide pairs and H-boxes (CCTAC) take part in the activation of the promoter ) in the region from -61 to -56 and from -126 to -121 nucleotide pairs.

In other experiments, it was found that under the influence of elicitors, the expression of the chalcone synthase gene in pea plants depends on the promoter region from -242 to -182 nucleotide pairs, in which two regions contain identical AT sequences -TAAAAATAST-, with one of them located in the region from -242 to -226, was necessary for the expression of maximum gene activity.

The promoter of the strictosidine synthase gene, one of the key elicitor-inducible enzymes in the synthesis of terpenoid phytoalexins, has a region activated by transcription regulatory factors from -339 to -145 nucleotide pairs. The G-box located near the -105 nucleotide pair did not affect promoter activity.

When studying the activity of the |3-1,3-glucanase gene in tobacco plants, it was found that it depends on the promoter region from -250 to -217 nucleotide pairs, containing the sequence -GGCGGC-, characteristic of the promoters of genes encoding pathogen-inducible alkali-

ny proteins.

The so-called PR-box of the promoter regions of many pathogen-inducible proteins contains a sequence (5"-AGCCGCC-3"), to which the corresponding transcription regulatory factors bind, which leads to the expression of the genes of these proteins, in particular endochitinases and P-1,3-glucanases in tomato plants.

Many genes of pathogen-inducible proteins contain so-called ocs elements in their promoters, with which transcription regulatory factors that have leucine zippers in their structure interact. In Arabidopsis plants, transcription regulatory factors responsible for transducing the ethylene signal bind to both the GCC box and ocs elements of the promoters, which leads to the expression of a number of protective proteins.

A study of transgenic tobacco plants with an alkaline chitinase promoter and a GUS reporter gene revealed that the promoter region activated by the ethylene signal is located between -503 and -358 nucleotide pairs, where there are two copies of the GCC box (5"- TAAGAGCCGCC-3"), which is characterized -

ren for the promoters of many ethylene-inducible proteins. Further analysis showed that the promoter region responsible for the response to ethylene with two copies of the GCC box is located between -480 and -410 nucleotide pairs.

When studying the response of tobacco plants to treatment with ethylene and infection with mosaic virus, it was found that the activity of the gene promoter (3-1,3-glucanase) depends on the region located between -1452 and -1193 nucleotide pairs, where there are two copies of the heptanucleotide

5-AGCCGCC-3" . Found and additional

tive regions essential for the regulation of promoter activity.

The elicitors, elicitor receptors, G-proteins, protein kinases, protein phosphatases, transcription regulatory factors, and their corresponding gene promoter regions discussed above take part in the functioning of a number of cell signaling systems, on which their response to signals of different nature and intensity depends: adenylate cyclase, MAP- kinase, phosphatidate, calcium, lipoxygenase, NADPH oxidase, NO synthase and proton.

ADENYLATE CYCLASE SIGNALING SYSTEM

This signaling system got its name from the enzyme adenylate cyclase, first characterized by Sutherland, which catalyzes the formation of the main signaling intermediate of this system - cyclic adenosine monophosphate (cAMP). The scheme of the adenylate cyclase system is as follows: an external chemical signal, for example a hormone or elicitor, interacts with the protein receptor of the plasmalemma, which leads to the activation of G-protein (binding of GTP) and the transmission of a signal impulse to the enzyme adenylate cyclase (AC), which catalyzes the synthesis of cAMP from ATP (Fig. .6).

In the adenylate cyclase system, a distinction is made between Gs proteins, which stimulate adenylate cyclase, and (5, proteins, which inhibit the activity of the enzyme. The differences between these two types of proteins are determined mainly by the characteristics of the oc subunits, and not the 3 and y subunits. Molecular masses ocs - subunits of the G protein are 41-46 kDa, ag subunits - 40-41 kDa, (3, - and P2 - subunits - 36-35 kDa, y-subunits - 8-10 kDa. Binding of G-proteins GTP and its hydrolysis to GDP and inorganic orthophosphate ensures the reversibility of the activation processes of adenylate cyclase.

Adenylate cyclase is a monomeric integral protein of the plasma membrane and is therefore difficult to extract and convert into a soluble form. The molecular weight of adenylate cyclase in animal cells is 120-155 kDa; There are also soluble forms of adenylate cyclase 50-70 kDa, insensitive to calmodulin and G-proteins. In plants, the molecular weight of adenylate cyclase is 84 kDa. The curve of the dependence of adenylate cyclase activity on pH had a one-peak character, and the peak of activity for this enzyme

ment was in the pH range of 4.8-5.2.

Data were obtained on the isoform of adenylate cyclase with optimal

mom pH equal to 8.8.

Adenylate cyclase can be modified on the outside of the membrane by glycosylation, and on the inside by phosphorylation by A-kinase [Severin, 1991]. The activity of membrane adenylate cyclase depends on the phospholipid environment - the ratio of phosphatidylcholine, phosphatidyl-ethanolamine, sphingomyelin, phosphatidyls"ery-

on and phosphatidylinositol.

The elicitor-induced increase in cAMP content in cells is transient, which is explained by activation of PDE and, possibly, binding by cAMP-dependent protein kinases. Indeed, an increase in the concentration of cAMP in cells activates various cAMP-dependent protein kinases, which can phosphorylate various proteins, including transcription regulatory factors, which leads to the expression of various genes and the cell’s response to external influences.

The signal multiplication factor achieved during its transmission into the genome and gene expression is many thousands. The signal multiplication scheme for the functioning of the adenylate cyclase signaling system is often used in biochemistry textbooks. This signaling system continues to be intensively studied on various objects, expanding the understanding of the information field of cells and its connection with external information flows.

It should be noted that the question of the functioning of the adenylate cyclase signaling system in plant objects continued to remain controversial for almost a quarter of a century, dividing researchers into its

GENE EXPRESSION

Rice. 6. Scheme of functioning of adenylate cyclase signaling

AC* systems - active form of adenylate cyclase; PKA and PKA* - inactive -

active and active forms of protein kinase A; PLplasmalemma; PDE - phosphodiesterase; PRT* - active form of transcription regulatory factor

supporters [Doman, Fedenko, 1976; Korolev, Vyskrebentseva, 1978; Franco, 1983; Yavorskaya, Kalinin, 1984; Newton and Brown, 1986; Karimova, 1994, Assman, 1995; Trewavas and Malho, 1997; Trewavas, 1999; etc.] and opponents. The first relied on data on an increase in the activity of adenylate cyclase and the content of cAMP under the influence of phytohormones and pathogens, on the imitation of the action of various phytohormones by exogenous cAMP, the second - on facts indicating an insignificant content of cAMP in plants, on the absence in a number of experiments of the influence of phytohormones on the activity of adenylate cyclase and etc.

Advances in the field of molecular genetics and comparison of the gene structure of proteins participating in the adenylate cyclase signaling system in animals and plants have tipped the scales in favor of supporters of its functioning in plants. Result-

The use of exogenous cAMP [Kilev, Chekurov, 1977] or forskolin (an activator of adenylate cyclase) indicated the participation of cAMP in the signal-induced signal transduction chain. The use of theophylline, an inhibitor of cAMP phosphodiesterase, which turned out to be quite active in plants, showed that the incoming part of the cAMP balance is carried out quite intensively [Yavorskaya, 1990; Karimova et al., 1990]. Data were obtained on changes in the content of cAMP in plants under the influence of pathogens, its necessity for the formation of a response to the action of pathogens [Zarubina et al., 1979; Ocheretina et al., 1990].

Noteworthy is the fact of ATP-dependent release into the extracellular environment of a significant part of cAMP formed in the cells of animals, prokaryotes, algae and higher races.

shadows By-

It is significant that in plants, as well as in animals, it was possible to reduce the accumulation of cAMP in cells and its release into the extracellular environment with the help of prostaglandin, which is not found in plants. Possible

but that this role is performed by a prostaglandin-like oxylipin - jasmonate. It is assumed that special ATP-binding proteins are involved in the removal of cAMP from the cell.

ing proteins.

The expediency of secreting cAMP from plant cells into the medium is explained, first of all, by the need to quickly reduce the concentration of this secondary messenger so that overexcitation of the cells does not occur. A relatively rapid decrease in the concentrations of secondary messengers after reaching a maximum level is an indispensable nonspecific feature of the functioning of all signaling systems.

Probably, cAMP released outside the plasmalemma takes part in the regulation of extracellular processes [Shiyan, Lazareva, 1988]. This view may be based on the discovery of ecto-cAMP-dependent protein kinases, which use the secretion of cAMP from cells to activate phosphorylation of proteins outside the plasmalemma. It is also believed that cAMP outside the cell can act as the first messenger [Fedorov et al., 1990], inducing the launch of a cascade of reactions of signaling systems in neighboring cells, as was shown in the example of multicellular slime fungi.

Attracting attention are the data obtained on animal subjects on the inhibition by exogenous adenosine (which can be considered as a product of cAMP degradation) of cell calcium channels [Meyerson, 1986] and activation of potassium channels [Orlov, Maksimova, 1999].

Of great interest is information about the possibility of regulation of the development of pathogenic fungi by secreted cAMP, in particular barley rust, Magnaporthe grisea, infecting plant rice, loose smut Ustilago maydis, Erysiphe graminis, Colletotrichum trifolii, pigmentation Ustilago hordei. Depending on the concentration of cAMP, stimulation or suppression of fungal development occurred. It is believed that heterotrimeric G proteins take part in the transduction of the cAMP signal.

More and more data are accumulating on the influence of various signaling molecules on the secretion of cAMP by plant cells. It has been shown that the role of ABA in plant adaptation to stress may lie in its ability to regulate the content and release of cAMP from cells. It is assumed that the decrease in cAMP content under the influence of ABA is caused by an ABA-induced increase in the Ca2+ content in the cytosol and inhibition of adenylate cyclase. It is known that Ca2+ in high concentration inhibits the activity of adenylate cyclase in eukaryotes. At the same time, Ca2+ can reduce the content of cAMP by inducing an increase in the activity of phosphodiesterase, which hydrolyzes cAMP. Indeed, activation of cAMP phosphodiesterase by the Ca2+-calmodulin complex was discovered in plant objects [Fedenko, 1983].

The dependence of the phosphorylation profile of polypeptides on exogenous cAMP is shown. The number of polypeptides whose phosphorylation was stimulated by cAMP was greatest at micromolar cAMP concentrations. Attention is drawn to the fact of a strong cAMP-induced increase in phosphorylation of the 10 kDa polypeptide at low temperature (Fig. 7) [Karimova, Zhukov, 1991; Yagusheva, 2000]. Interestingly, a polypeptide with such molecular weight is a protein regulator of cAMP phosphodiesterase, which is activated by abscisic acid and Ca2+ and reduces the cAMP content due to its hydrolysis by phosphodiesterase.

Studying the features of activation of cAMP-dependent protein kinases and phosphorylation by them various whites- kov is one of the most important areas of research into the adenylate cyclase signaling system. cAMP-dependent protein kinases (PKAs) are enzymes that are activated by interaction with cAMP and catalyze the transfer of the terminal phosphoric acid residue from ATP to the hydroxyl groups of serine or threonine residues of acceptor proteins. Covalent modification of proteins, carried out during phosphorylation, leads to a change in their conformation and catalytic activity, causing association or dissociation of their subunits, etc.

Molecular mass of proteins, kDa

Rice. 7. Effect of cAMP on protein phosphorylation of three-day-old pea seedlings [Karimova, Zhukov, 1991]

1 - control: cut shoots were transferred by petioles into water for 2 hours, then for another 2 hours - into a solution of 32 P-labeled orthophosphate; 2 - cut plants were transferred for 2 hours to a solution of 1 μM cAMP, then for another 2 hours - to a solution of 32 P-labeled orthophosphate

The substrates in the protein kinase reaction are MgATP and the protein being phosphorylated. Protein substrates can be simultaneously substrates for cGMP- and cAMP-dependent protein kinases at the same serine (threonine) residues, but the rate of cAMP-dependent phosphorylation is 10-15 times higher than that of cGMP-dependent protein kinases. Substrates of cAMP-dependent protein kinases are located in all parts of the cell: cytosol, endoplasmic reticulum (ER), Golgi apparatus, secretory granules, cytoskeleton and nucleus.

Protein kinases activated by exogenous cAMP have been isolated from plant cells, for example, from maize coleoptiles - 36 kDa protein kinase. Kato et al. isolated three types of protein kinases from the duckweed Lemna paucicostata: 165, 85 and 145 kDa, one of which was inhibited by cAMP, the other was activated by cAMP, and the third was cAMP-independent.

The second type of protein kinases phosphorylated polypeptides

59, 19, 16 and 14 kDa.

Exogenous cAMP caused changes (mainly inhibition) in the phosphorylation of a number of chloroplast polypeptides, mediated by the participation of protein kinases

One of the first protein kinase genes cloned in plants was similar in nucleotide sequence to the animal protein kinase A family. There are examples of similarity of amino acid sequences of protein kinases A from plants (their homology) with protein kinases A from animals. Several groups of researchers have reported the cloning of genes homologous to the protein kinase A gene (reviews: ). Protein kinase from petunia phosphorylated a specific synthetic substrate of protein kinase A. The addition of cAMP to plant extracts has been reported to stimulate the phosphorylation of specific proteins. A study of phosphorylation sites in phenylalanine ammonia lyase (PAL), a key enzyme in the biosynthesis of phytoalexins, revealed sites specific for protein kinase A.

The use of a highly specific protein inhibitor (BI) of cAMP-dependent protein kinases made it possible to confirm the assumption that cAMP-dependent protein kinases can be activated by endogenous cAMP during sample preparation: BI suppressed the basal protein kinase activity of leaf extracts in different experiences by 30-50% [Karimova, 1994]. The intermediates of the lipoxygenase signaling system HDK and MeZhK activated protein kinase activity by 33-^8% in the presence of cAMP [Karimova et al., 19996]. Salicylic acid induced an increase in the level of cAMP-dependent phosphorylation of polypeptides of 74, 61 and 22 kDa in pea leaves [Mukhametchina, 2000]. The cAMP-stimulated protein kinase activity of soluble pea leaf proteins depended on the Ca2+ concentration [Karimova et al., 1989; Tarchevskaya, 1990; Karimova, Zhukov, 1991], and enzymatic activity was also detected in isolated cell walls, nuclei, and plasma membranes.

Genes have been found in plants encoding the enzyme protein phosphatase, the target of which is proteins phosphorylated by protein kinase A.

To characterize the adenylate cyclase signaling system, the discovery in plants of genes encoding protein transcription regulatory factors that have extended nucleotide sequences homologous to CREBS, the cAMP-binding transcription factor in animals, is extremely important.

Numerous data on the influence of cAMP on ion channels of plant cells and a relatively weak experimental basis for ideas about the possibility of signal transmission from cAMP through phosphorylation of protein transcription regulatory factors into the genome, on the one hand, strengthen the position of supporters of the existence of an indirect (through activation of ion channels) adenylate cyclase signaling pathway and , on the other hand, force us to intensify attempts to obtain evidence of the functioning of the direct cAMP signaling pathway.

MAR KINASE SIGNALING SYSTEM

Mitogen-activated serine-threonine-type protein kinases (MAPK) and MAP kinase signaling cascade (signal -> receptor -> G-proteins -> MAPKKK - "

-> MAPKK -> MAPK -> PSF -> genome), which have been sufficiently fully studied in animal objects, also function in plant cells (Fig. 8). Review articles are devoted to them

And works of an experimental nature, which provide information about the individual representatives of this signaling system and especially

problems of their regulation.

The MAP kinase cascade is “switched on” during mitosis (which explains the name of these protein kinases), during dehydration

nia, hypoosmosis

tical stress, low temperature, mechanical irritation of plants

Tissue damage, oxidative stress, action of pathogens, elicitors (in

including harpins, cryptogein, oligosaccharides), stress phytohormones jasmonate, sali-

cylate, systemin, ethylene).

The dependence of the functioning of the MAP kinase cascade on various influences is reflected in the names of some MAP kinases, for example WIPK and SIPK (respectively

vein wound-induced protein kinases and salicylate-induced protein

Rice. 8. Scheme of functioning of the MAP kinase signaling system

KKMARK, MAP kinase kinase kinase; KMARK - MAP kinase kinase; MAPK - mitogen-activated protein kinase. Other designations - see fig. 6

Plant resistance to pathogens is determined, as was established by H. Flor in the 50s of the 20th century, by the interaction of a complementary pair of genes of the host plant and the pathogen, respectively, the resistance gene (R) and the avirulence gene (Avr). The specificity of their interaction suggests that the expression products of these genes are involved in the recognition of a pathogen by the plant with subsequent activation of signaling processes to enable defense reactions.

Currently, 7 signaling systems are known: cycloadenylate, MAP kinase (mitogen-activated protein-kinase), phosphatidic acid, calcium, lipoxygenase, NADPH oxidase (superoxide synthase), NO synthase.

In the first five signaling systems, G proteins are the mediator between the cytoplasmic part of the receptor and the first activated enzyme. These proteins are localized on inside plasmalemmas. Their molecules consist of three subunits: a, b and g.

Cycladenylate signaling system. The interaction of a stressor with a receptor on the plasma membrane leads to the activation of adenylate cyclase, which catalyzes the formation of cyclic adenosine monophosphate (cAMP) from ATP. cAMP activates ion channels, including the calcium signaling system, and cAMP-dependent protein kinases. These enzymes activate proteins that regulate the expression of protective genes by phosphorylating them.

MAP kinase signaling system. The activity of protein kinases increases in plants exposed to stress (blue light, cold, drying, mechanical damage, salt stress), as well as treated with ethylene, salicylic acid, or infected with a pathogen.

In plants, the protein kinase cascade functions as a signal transduction pathway. Binding of the elicitor to the plasma membrane receptor activates MAP kinases. It catalyzes the phosphorylation of the cytoplasmic kinase MAP kinase, which activates MAP kinase upon double phosphorylation of threonine and tyrosine residues. It enters the nucleus, where it phosphorylates transcription regulator proteins.

Phosphatidic acid signaling system. In animal cells, G proteins, under the influence of a stressor, activate phospholipases C and D. Phospholipase C hydrolyzes phosphatidylinositol 4,5-bisphosphate to form diacylglycerol and inositol 1,4,5-triphosphate. The latter releases Ca2+ from the bound state. An increased content of calcium ions leads to the activation of Ca2+-dependent protein kinases. Diacylglycerol, after phosphorylation by a specific kinase, is converted into phosphatidic acid, which is a signaling substance in animal cells. Phospholipase D directly catalyzes the formation of phosphatidic acid from membrane lipids (phosphatidylcholine, phosphatidylethanolamine).

In plants, stressors activate G proteins, phospholipases C and D in plants. Therefore, the initial stages of this signaling pathway are the same in animal and plant cells. It can be assumed that the formation of phosphatidic acid also occurs in plants, which can activate protein kinases with subsequent phosphorylation of proteins, including transcription regulatory factors.

Calcium signaling system. Exposure to various factors (red light, salinity, drought, cold, heat shock, osmotic stress, abscisic acid, gibberellin and pathogens) leads to an increase in the content of calcium ions in the cytoplasm due to increased import from the external environment and release from intracellular stores (endoplasmic reticulum and vacuoles)

An increase in the concentration of calcium ions in the cytoplasm leads to the activation of soluble and membrane-bound Ca2+-dependent protein kinases. They participate in the phosphorylation of protein factors regulating the expression of protective genes. However, it has been shown that Ca2+ is able to directly influence the human transcriptional repressor without involving the protein phosphorylation cascade. Calcium ions also activate phosphatases and phosphoinositol-specific phospholipase C. The regulatory effect of calcium depends on its interaction with the intracellular calcium receptor - the protein calmodulin.

Lipoxygenase signaling system. The interaction of the elicitor with the receptor on the plasmalemma leads to the activation of membrane-bound phospholipase A2, which catalyzes the release of unsaturated fatty acids, including linoleic and linolenic acids, from plasmalemma phospholipids. These acids are substrates for lipoxygenase. Substrates for this enzyme can be not only free, but also unsaturated fatty acids contained in triglycerides. The activity of lipoxygenases increases under the action of elicitors and plant infection with viruses and fungi. The increase in lipoxygenase activity is due to stimulation of the expression of genes encoding these enzymes.

Lipoxygenases catalyze the addition of molecular oxygen to one of the carbon atoms (9 or 13) of the cis,cis-pentadiene radical of fatty acids. Intermediate and final products of lipoxygenase metabolism of fatty acids have bactericidal and fungicidal properties and can activate protein kinases. Thus, volatile products (hexenals and nonenals) are toxic to microorganisms and fungi, 12-hydroxy-9Z-dodecenoic acid stimulated the phosphorylation of proteins in pea plants, phytodienic acid, jasmonic acid and methyl jasmonate increase the level of expression of protective genes through activation of protein kinases.

NADPH oxidase signaling system. In many cases, infection by pathogens stimulated the production of reactive oxygen species and cell death. Reactive oxygen species are not only toxic to the pathogen and the infected host plant cell, but are also participants in the signaling system. Thus, hydrogen peroxide activates transcription regulatory factors and the expression of protective genes.

NO synthase signaling system. In animal macrophages that kill bacteria, along with reactive oxygen species, nitric oxide acts, enhancing their antimicrobial effect. In animal tissues, L-arginine is converted to citrulline and NO by the action of NO synthase. The activity of this enzyme was also detected in plants, and the tobacco mosaic virus induced an increase in its activity in resistant plants, but did not affect the activity of NO synthase in sensitive plants. NO, interacting with oxygen superoxide, forms very toxic peroxynitrile. At increased concentrations of nitric oxide, guanylate cyclase is activated, which catalyzes the synthesis of cyclic guanosine monophosphate. It activates protein kinases directly or through the formation of cyclic ADP-ribose, which opens Ca2+ channels and thereby increases the concentration of calcium ions in the cytoplasm, which in turn leads to the activation of Ca2+-dependent protein kinases.

Thus, in plant cells there is a coordinated system of signaling pathways that can act independently of each other or together. A special feature of the signaling system is the amplification of the signal during its transmission. The activation of the signaling system in response to the influence of various stressors (including pathogens) leads to the activation of the expression of protective genes and an increase in plant resistance.

Induced mechanisms: a) increased respiration, b) accumulation of substances that provide stability, c) creation of additional protective mechanical barriers, d) development of a hypersensitivity reaction.

The pathogen, having overcome surface barriers and entering the conducting system and plant cells, causes plant disease. The nature of the disease depends on the resistance of the plant. According to the degree of resistance, plants are divided into four categories: sensitive, tolerant, hypersensitive and extremely resistant (immune). Let us briefly characterize them using the example of the interaction of plants with viruses.

In sensitive plants, the virus is transported from the initially infected cells throughout the plant, multiplies well and causes a variety of disease symptoms. However, even in sensitive plants there are protective mechanisms that limit viral infection. This is evidenced, for example, by the resumption of reproduction of the tobacco mosaic virus in protoplasts isolated from infected leaves of tobacco plants, in which the growth of infectivity has ceased. Dark green zones that form on young leaves of diseased sensitive plants are characterized by a high degree of resistance to viruses. The cells of these zones contain almost no viral particles compared to the neighboring cells of light green tissue. The low level of virus accumulation in the cells of dark green tissue is associated with the synthesis of antiviral substances. In tolerant plants, the virus spreads throughout the plant but reproduces poorly and does not cause symptoms. In hypersensitive plants, the primarily infected and neighboring cells become necrotic, localizing the virus in necrosis. It is believed that in extremely resistant plants the virus reproduces only in initially infected cells, is not transported throughout the plant and does not cause disease symptoms. However, the transport of viral antigen and subgenomic RNAs in these plants was shown, and when infected plants were kept at low temperatures (10-15°C), necrosis formed on the infected leaves.

The mechanisms of resistance of hypersensitive plants are the most well studied. The formation of local necrosis is a typical symptom of a hypersensitive reaction of plants in response to pathogen damage. They arise as a result of the death of a group of cells at the site of pathogen entry. The death of infected cells and the creation of a protective barrier around necrosis block the transport of infectious agents throughout the plant, prevent access to the pathogen of nutrients, cause elimination of the pathogen, lead to the formation of antipathogenic enzymes, metabolites and signaling substances that activate protective processes in neighboring and distant cells, and in ultimately contribute to the healing of the plant. Cell death occurs due to the activation of a genetic death program and the formation of compounds and free radicals that are toxic to both the pathogen and the cell itself.

Necrotization of infected cells of hypersensitive plants, controlled by the genes of the pathogen and the host plant, is a special case of programmed cell death (PCD - programmed cell death). PCD is essential for normal development of the body. Thus, it occurs, for example, during the differentiation of tracheid elements during the formation of xylem vessels and the death of root cap cells. These peripheral cells die even when the roots are growing in water, meaning cell death is part of the plant's development and not caused by soil action. The similarity between PCD and cell death during a hypersensitive reaction is that these are two active processes; in a necrotic cell, the content of calcium ions in the cytoplasm also increases, membrane vesicles are formed, the activity of deoxyribonucleases increases, DNA breaks down into fragments with 3'OH ends, and condensation occurs nucleus and cytoplasm.

In addition to the inclusion of PCD, necrotization of infected cells of hypersensitive plants occurs as a result of the release of phenols from the central vacuole and hydrolytic enzymes from lysosomes due to a violation of the integrity of cell membranes and an increase in their permeability. A decrease in the integrity of cell membranes is caused by lipid peroxidation. It can occur with the participation of enzymes and non-enzymatically as a result of the action of reactive oxygen species and free organic radicals.

One of the characteristic properties of hypersensitive plants is acquired (induced) resistance to repeated infection by the pathogen. The terms systemic acquired resistance (SAR) and localized acquired resistance (LAR) were proposed. LAR is said to occur when cells acquire resistance in the area immediately adjacent to local necrosis (a distance of approximately 2 mm). In this case, secondary necrosis does not form at all. Acquired resistance is considered systemic if it develops in cells of a diseased plant that are distant from the site of initial introduction of the pathogen. SAR manifests itself in a decrease in the level of virus accumulation in cells and a decrease in the size of secondary necrosis, which indicates inhibition of short-range virus transport. It is not clear whether LAR and SAR are different from each other or whether they are the same process occurring in cells located at different distances from the site of initial entry of the virus into the plant.

Acquired resistance is usually nonspecific. Plant resistance to viruses was caused by bacterial and fungal infections and vice versa. Resistance can be induced not only by pathogens, but also by various substances.

The development of SAR is associated with the spread throughout the plant of substances formed in the initially infected leaves. It was assumed that the inducer of SAR is salicylic acid, which is formed during necrosis of initially infected cells.

When plants become diseased, substances accumulate in plants that increase their resistance to pathogens. Antibiotic substances, phytoncides, discovered by B. Tokin in the 20s of the 20th century play an important role in nonspecific plant resistance. These include low molecular weight substances of various structures (aliphatic compounds, quinones, glycosides with phenols, alcohols) that can delay the development or kill microorganisms. Released when onions and garlic are injured, volatile phytoncides protect the plant from pathogens already above the surface of the organs. Non-volatile phytoncides are localized in the integumentary tissues and participate in creating the protective properties of the surface. Inside cells they can accumulate in vacuoles. When damaged, the amount of phytoncides increases sharply, which prevents possible infection of wounded tissues.

Phenols are also classified as antibiotic substances in plants. In case of damage and disease, polyphenol oxidase is activated in cells, which oxidizes phenols to highly toxic quinones. Phenolic compounds kill pathogens and host plant cells, inactivate pathogen exoenzymes and are necessary for lignin synthesis.

Proteins, glycoproteins, polysaccharides, RNA, and phenolic compounds were found among viral inhibitors. There are infection inhibitors that directly affect viral particles, making them non-infectious, or they block viral receptors. For example, inhibitors from beet, parsley and currant juice caused almost complete destruction of tobacco mosaic virus particles, and aloe juice caused linear aggregation of particles, which reduced the possibility of particles penetrating into cells. Reproduction inhibitors change cellular metabolism, thereby increasing cell stability, or inhibit viral reproduction. Ribosome-inactivating proteins (RIPs) are involved in plant resistance to viruses.

In hypersensitive tobacco plants infected with tobacco mosaic virus, proteins initially called b-proteins and now referred to as pathogenesis-associated proteins (PR-proteins) or resistance-associated proteins were found. The common name “PR proteins” suggests that their synthesis is induced only by pathogens. However, these proteins are also formed in healthy plants during flowering and various stress influences.

In 1999, based on the amino acid sequence, serological properties, enzyme and biological activity, a unified nomenclature of PR proteins was created for all plants, consisting of 14 families (PR-1 - PR-14). Some PR proteins have protease, ribonuclease, 1,3-b-glucanase, chitinase activities or are protease inhibitors. Higher plants do not have chitin. It is likely that these proteins are involved in plant defense against fungi, since chitin and b-1,3-glucans are the main components of the cell walls of many fungi and chitinase hydrolyzes the b-1,3-linkages of chitin. Chitinase can also act like lysozyme, hydrolyzing peptidoglucans in bacterial cell walls. However, b-1,3-glucanase may facilitate the transport of virus particles along the leaf. This is explained by the fact that b-1,3-glucanase destroys callose (b-1,3-glucan), which is deposited in the cell wall and plasmodesmata and blocks the transport of the virus.

PR proteins also include low molecular weight (5 kDa) proteins - modifiers of cell membranes of fungi and bacteria: thionins, defensins and lipid transfer proteins. Thionins are toxic in vitro to phytopathogenic fungi and bacteria. Their toxicity is due to their destructive effect on the membranes of pathogens. Defensins have strong antifungal properties, but have no effect on bacteria. Defensins from plants of the Brassicaceae and Saxifragaceae families suppressed the elongation growth of fungal hyphae but promoted their branching. Defensins from plants of the families Asteraceae, Fabaceae, and Hippocastanaceae slowed down the elongation of hyphae, but did not affect their morphology.

When plants are infected with pathogens, the activity of the lytic compartment of cells of sensitive and hypersensitive plants increases. The lytic compartment of plant cells includes small vacuoles - derivatives of the endoplasmic reticulum and Golgi apparatus, functioning as primary lysosomes of animals, that is, hydrolase-containing structures in which there are no substrates for these enzymes. In addition to these vacuoles, the lytic compartment of plant cells includes a central vacuole and other vacuoles, equivalent to secondary lysosomes of animal cells, which contain hydrolases and their substrates, as well as plasmalemma and its derivatives, including paramural bodies, and extracellular hydrolases localized in the cell wall and in the space between the wall and the plasmalemma.

Burachenko D.L. Signal structures. Part 3 (Document)

Modern methods of cell research (manual) (Document)

Signal boards T-4U2, T-6U2, T-8U2, T-10U2. Technical description and operating and repair instructions (Document)

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UDC 58 BBK 28.57 T22

Executive Editor Corresponding Member of the Russian Academy of Sciences A.I. Grechkin

Reviewers:

L.H. Gordon Doctor of Biological Sciences, Professor L.P. Khokhlova

Tarchevsky I.A.

Signaling systems of plant cells / I.A. Tarchevsky; [Ans. ed. A.N. Grechkin]. - M.: Nauka, 2002. - 294 p.: ill. ISBN 5-02-006411-4

For specialists in the field of plant physiology, biochemists, biophysicists, geneticists, plant pathologists, ecologists, agrobiologists.

Via AK network

Tarchevsky I.A.

Plant Cell Signaling Systems /1.A. Tarchevsky; . - M.: Nauka, 2002. - 294 p.; il. ISBN 5-02-006411-4

For physiologists, biochemists, biophysicists, genetics, phytopathologists, ecologists, and agrobiologists

ISBN 5-02-006411-4

(art design), 2002

Articles of an experimental nature were included in the bibliography, which to a certain extent depended on the limited volume of the book and the time for its preparation. The author apologizes to colleagues whose research was not reflected in the book.

INTRODUCTION

One of the most important problems of modern biology is deciphering the mechanisms of response of prokaryotic and eukaryotic organisms to changes in the conditions of their existence, especially to the action of extreme factors (stress factors, or stressors) that cause a state of stress in cells.

The most significant nonspecific changes during biotic stress include the following:

Phasicity in the time course of the response to the action of a pathogen.
Increased catabolism of lipids and biopolymers.
Increased content of free radicals in tissues.
Acidification of the cytosol with subsequent activation of proton pumps, which returns the pH to its original value.
An increase in the content of calcium ions in the cytosol with
subsequent activation of calcium ATPases.
The release of potassium and chlorine ions from cells.
A drop in membrane potential (at the plasmalemma).
Decrease in the overall intensity of synthesis of biopolymers and lipids.
Stopping the synthesis of certain proteins.

Strengthening synthesis or synthesis of missing ones
called pathogen-inducible protective proteins (chi-
tinases (3-1,3-glucanases, proteinase inhibitors, etc.).
Intensification of the synthesis of cell strengthening
wall components - lignin, suberin, cutin, callose,
hydroxyproline-rich protein.
Synthesis of antipathogenic non-volatile compounds - phytoalexins.
Synthesis and isolation of volatile bactericidal and func-
hycidal compounds (hexenals, nonenals, terpenes and

Dr->-

Strengthening synthesis and increasing content (or according to
phenomenon) of stress phytohormones - abscisic, jasmo-
new, salicylic acid, ethylene, peptide hormone
nature of systemin.
Inhibition of photosynthesis.
Redistribution of carbon from |4 CO 2 assimilated in
the process of photosynthesis, among various compounds -
a decrease in the incorporation of the label into high-polymer compounds (proteins, starch) and sucrose and an increase (more often related to
telous - as a percentage of assimilated carbon) - into alanine,
malate, aspartate [Tarchevsky, 1964].

17. Increased breathing followed by inhibition.
Activation of an alternative oxidase that changes the direction of electron transport in mitochondria.

18. Violations of the ultrastructure - changes in the thin
granular structure of the nucleus, a decrease in the number of polysomes and
dictyosomes, swelling of mitochondria and chloroplasts, decrease
decrease in the number of thylakoids in chloroplasts, restructuring of cyto-
skeleton

Apoptosis (programmed death) of cells subjected to
exposed to pathogens and those adjacent to them.
The appearance of the so-called systemic nonspecific
high resistance to pathogens in remote locations
exposure to pathogens in areas (for example, metameric
organs) of plants.

Many of the changes listed above are a consequence of the “switching on” of a relatively small number of nonspecific signaling systems by stressors.

With increasing study of the mechanisms of plant responses to pathogens, new nonspecific responses of plant cells are being discovered. These include previously unknown signaling pathways.

When elucidating the features of the functioning of signaling systems, it is necessary to keep in mind that these issues are part of a more general problem of regulation of genome functioning. It should be noted that the universality of the structure of the main information carriers of the cells of various organisms - DNA and genes - predetermines the unification of those mechanisms that serve the implementation of this information [Grechkin, Tarchevsky, 2000]. This concerns DNA replication and transcription, the structure and mechanism of action of ribosomes, as well as the mechanisms of regulation of gene expression by changing conditions of cell existence using a set of largely universal signaling systems. The links of signaling systems are also basically unified (nature, having found at one time the optimal structural and functional solution to a biochemical or information problem, preserves and replicates it in the process of evolution). In most cases, a wide variety of chemical signals coming from the environment are captured by the cell with the help of special “antennas” - receptor protein molecules that penetrate the cell membrane and protrude above its external and internal surfaces.

No side. Several types of structure of these receptors are unified in plant and animal cells. Non-covalent interaction of the external region of the receptor with one or another signaling molecule coming from the environment surrounding the cell leads to a change in the conformation of the receptor protein, which is transmitted to the internal, cytoplasmic region. In most signaling systems, intermediary G-proteins come into contact with it - another unit of signaling systems that is unified (in its structure and functions). G-proteins perform the functions of a signal transducer, transmitting a signal conformational impulse to the starting enzyme specific for a particular signaling system. The starting enzymes of the same type of signaling system in different objects are also universal and have extended regions with the same amino acid sequence. One of the most important unified links in signaling systems are protein kinases (enzymes that transfer the terminal residue of orthophosphoric acid from ATP to certain proteins), activated by the products of starting signaling reactions or their derivatives. Proteins phosphorylated by protein kinases are the next links in signal chains. Another unified link in cell signaling systems is protein transcription regulatory factors, which are one of the substrates of protein kinase reactions. The structure of these proteins is also largely unified, and modifications of the structure determine the affiliation of transcription regulatory factors to one or another signaling system. Phosphorylation of transcription regulatory factors causes a change in the conformation of these proteins, their activation and subsequent interaction with the promoter region of a certain gene, which leads to a change in the intensity of its expression (induction or repression), and in extreme cases, to the “switching on” or “switching off” of some silent genes. active. Reprogramming the expression of a set of genes in the genome causes a change in the ratio of proteins in the cell, which is the basis of its functional response. In some cases, a chemical signal from the external environment can interact with a receptor located inside the cell - in the cytosol or

SIGNALS

NIB

Rice. 1. Scheme of interaction of external signals with cell receptors

1,5,6- receptors located in the plasmalemma; 2,4 - receptors located in the cytosol; 3 - the starting enzyme of the signaling system, localized in the plasmalemma; 5 - a receptor activated under the influence of a nonspecific change in the structure of the lipid component of the plasmalemma; SIB - signal-induced proteins; PTF - protein transcription regulatory factors; i|/ - change in membrane potential

Same core (Fig. 1). In animal cells, such signals are, for example, steroid hormones. This information pathway has a smaller number of intermediates, and therefore has fewer opportunities for regulation by the cell.

Our country has always paid great attention to the problems of phytoimmunity. A number of monographs and reviews by domestic scientists are devoted to this problem [Sukhorukov, 1952; Verderevsky, 1959; Vavilov, 1964; Gorlenko, 1968; Rubin et al., 1975; Metlitsky, 1976; Tokin, 1980; Metlitsky et al., 1984; Metlitsky, Ozeretskovskaya, 1985; Kursano-va, 1988; Ilyinskaya et al., 1991; Ozeretskovskaya et al., 1993; Korableva, Platonova, 1995; Chernov et al., 1996; Tarchevsky, Chernov, 2000].

In recent years, special attention has been paid to the molecular mechanisms of phytoimmunity. It has been shown that

When plants are infected, various signaling systems are activated that perceive, multiply and transmit signals from pathogens to the genetic apparatus of cells, where the expression of protective genes occurs, allowing plants to organize both structural and chemical protection from pathogens. Advances in this area are associated with the cloning of genes, deciphering their primary structure (including promoter regions), the structure of the proteins they encode, the use of activators and inhibitors of individual parts of signaling systems, as well as mutants and transgenic plants with introduced genes responsible for the synthesis of receptor participants , transmission and amplification of signals. In the study of plant cell signaling systems, an important role is played by the construction of transgenic plants with promoters of genes for proteins participating in signaling systems.

Currently, the signaling systems of plant cells under biotic stress are most intensively studied at the Institute of Biochemistry. A.N. Bach RAS, Kazan Institute of Biochemistry and Biophysics RAS, Institute of Plant Physiology RAS, Pushchino branch of the Institute of Bioorganic Chemistry RAS, Bioengineering Center RAS, Moscow and St. Petersburg State Universities, All-Russian Research Institute of Agricultural Biotechnology of the Russian Academy of Agricultural Sciences, All-Russian Research Institute phytopathology of the Russian Academy of Agricultural Sciences, etc.

The problem of deciphering the molecular mechanisms of biotic stress, including the role of signaling systems in its development, has united plant physiologists and biochemists, microbiologists, geneticists, molecular biologists, and phytopathologists over the past ten years. A large number of experimental and review articles are published on various aspects of this problem (including in special journals: "Physiological and Molecular Plant Pathology", "Molecular Plant - Microbe Interactions", "Annual Review of Plant Physiology and Pathology"). At the same time, in the domestic literature there is no generalization of works devoted to cell signaling systems, which led the author to the need to write the monograph offered to readers.

PATHOGENS AND ELISITORS

Plant diseases are caused by thousands of species of microorganisms, which can be divided into three groups: viruses (more than 40 families) and viroids; bacteria (Agrobacterium, Corynebacterium, Erwinia, Pseudomonas, Xanthomonas, Streptomyces) and mycoplasma-like microorganisms; fungi (lower: Plasmodiophoromycetes, Chitridomycetes, Oomycetes; higher: Ascomycetes, Basidiomycetes, Deuteromycetes).

Thesis of protective enzymes: phenylalanine ammonia lyase and anion peroxidase. Wingless forms belonging to this subclass appeared as a result of the loss of these organs during the evolution of winged forms. The subclass includes 20 orders of insects, among which there are polyphages that do not have specificity in relation to the plant, oligophages and monophages, in which the specificity of the interaction between the pathogen and the host plant is clearly expressed. Some insects feed on leaves (the entire leaf blade or skeletalizing the leaf), others feed on stems (including gnawing the stem from the inside), flower ovaries, fruits, and roots. Aphids and cicadas suck sap from vascular vessels using a proboscis or stylet.

Despite the measures taken to combat insects, the pressing problem of reducing the harm they cause continues to be. Currently, over 12% of the crop yield of agricultural plants on the planet is lost as a result of attack by pathogenic microorganisms, nematodes and insects.

Damage to cells leads to the degradation of their contents, for example, high-polymer compounds, and the appearance of oligomeric signaling molecules. These “shipwrecks” [Tarchevsky, 1993] reach neighboring cells and cause a protective reaction in them, including a change in gene expression and the formation of the protective proteins they encode. Often, mechanical damage to plants is accompanied by infection, since the wound surface opens through which pathogens penetrate the plant. In addition, phytopathogenic microorganisms can live in the mouthparts of insects. It is known, for example, that the carriers of mycoplasma infection are cicadas, in which adult forms and larvae feed on the juice of the sieve vessels of plants, piercing the leaves with their proboscis-stylet and

Rice. 2. Scheme of interaction between a pathogen cell and a host plant

/ - cutinase; 2 - degradation products of cuticle components (possibly having signaling properties); 3 - (3-glucanase and other glycosylases excreted by the pathogen; 4 - elicitors - fragments of the host cell wall (CW); 5 - chitinases and other glycosylases that act destructively on the pathogen's CS; 6 - elicitors - fragments of pathogen CS; 7 - phytoalexins - inhibitors of proteinases, cutinases, glycosylases and other pathogen enzymes; 8 - toxic substances of the pathogen; 9 - strengthening of the host's CS due to activation of peroxidases and increased lignin synthesis, deposition of hydroxyproline proteins and lectins; 10 - inducers of hypersensitivity and necrosis of neighboring cells; // - cutin degradation products acting on the pathogen cell

Young stems. The roseate leafhopper, unlike other members of the leafhopper, sucks out the contents of the cells. Cicadas cause less damage to plant tissue than leaf-eating insects, however, plants can react to it in the same way as to an associated plant infection.

Rice. 3. Phytotoxic compound from Cochlio-bolus carbonum

Bacteria and fungi also produce non-selective toxins, in particular fusicoccin, erichosetene, coronatine, phase-olotoxin, syringomycin, tabtoxin.

One of the host-specific toxins secreted by Pyrenophora triticirepentis is a 13.2 kDa protein, others are products of secondary metabolism with a wide variety of structures - these are polyketides, terpenoids, saccharides, cyclic peptides, etc.

As a rule, the latter include peptides whose synthesis occurs outside the ribosomes and which contain D-amino acid residues. For example, the host-specific toxin from Cochliobolus carbonum has a tetrapeptide cyclic structure (D- npo- L- ana- D- ana- L- A3 JJ), where the last abbreviation means 2-amino-9,10-epoxy-8-oxo-de-canoic acid (Fig. 3). The toxin is produced in pathogen cells using toxin synthase. Resistance to this compound in maize depends on the gene encoding NADPH-dependent carbonyl reductase, which reduces the carbonyl group, resulting in

Deactivation of the toxin. It turned out that in the host plant the toxin causes inhibition of histone deacetylases and, as a consequence, histone overacetylation. This suppresses the plant's defense response caused by pathogen infection.

Elisitins cause hypersensitivity and death of infected cells, especially in plants of the genus Nicotiana. The most intensive formation of elicitins by late blight occurs during the growth of micro-

Sky analogs), corresponding to the most conserved regions of the protein - flagellin, which is an important virulence factor of these bacteria. A new elicitor protein has been isolated from Erwinia amylovora, the C-region of which is homologous to the enzyme pectate lyase, which can cause the appearance of elicitor oligomeric fragments - products of pectin degradation. The pathogenic bacterium Erwinia carotovora excretes the elicitor protein harpin and the enzymes pectate lyase, cellulase, polygalacturonase and proteases, which hydrolyze the polymeric components of the cell walls of the host plant (see Fig. 2), resulting in the formation of oligomeric elicitor molecules. Interestingly, pectate lyase secreted by Erwinia chrysanthemi acquired activity as a result of extracellular processing.

Some lipids and their derivatives also belong to elicitors, in particular 20-carbon polyunsaturated fatty acids of some pathogens - arachidonic acid and eicosapentaenoic acid [Ilyinskaya et al., 1991; Ozerets-kovskaya et al., 1993; Ozeretskovskaya, 1994; Gilyazetdinov et al., 1995; Ilyinskaya et al., 1996a, b; Ilyinskaya, Ozeretskovskaya, 1998], and their oxygenated derivatives. The review work [Ilyinskaya et al., 1991] summarizes data on the elicitor effect of lipids (lipoproteins) produced by pathogenic fungi on plants. It turned out that it is not the protein part of lipoproteins that has the elicitor effect, but their lipid part, which is arachidonic (eicosatetraenoic) and eicosopentaenoic acids, which are not characteristic of higher plants. They caused the formation of phytoalexins, tissue necrosis and systemic resistance of plants to various pathogens. Products of lipoxygenase transformation in plant tissues C 20 fatty acids (hydroperoxy-, hydroxy-, oxo-, cyclic derivatives, leukotrienes), formed in the cells of the host plant with the help of the enzyme lipoxygenase complex (the substrates of which can be either C, 8 or and C 20 polyene fatty acids) had a strong effect on the protective response of plants. This is apparently explained by the fact that there is no oxygen in uninfected plants.
nated derivatives of 20-carbon fatty acids, and their appearance as a result of infection leads to dramatic results, such as the formation of necrosis around the infected cells, which creates a barrier to the spread of pathogens throughout the plant.

There is evidence that the induction of lipoxygenase activity by a pathogen led to the formation of a plant response even in the case when the elicitor did not contain C20 fatty acids and the substrate of lipoxygenase activity could only be its own C18 polyene fatty acids, and the products were octadecanoids, not eicosanoids. Syringolides [L et al., 1998] and cerebrosides, sphingolipid compounds, also have elicitor properties. Cerebrosides A and C isolated from Magnaporthe grisea were the most active elicitors in rice plants. Cerebroside degradation products (fatty acid methyl esters, sphingoid bases, glycosyl-sphingoid bases) did not exhibit elicitor activity.

One such barrier is the cuticle, which consists primarily of a cutin heteropolymer embedded in wax. More than 20 monomers have been discovered that make up cutin. These are saturated and unsaturated fatty acids and alcohols of various lengths, including hydroxylated and epoxidated, long-chain dicarboxylic acids, etc. In cutin, most of the primary alcohol groups participate in the formation of ester bonds, as well as some of the secondary alcohol groups that provide cross-links between chains and branch points in the polymer. Part of another “barrier” polymer, suberin, is close in composition to cutin. Its main difference is that free fatty acids are the main component of suberic waxes, while there are very few of them in cutin. Moreover, in Suberina

Mainly C22 and C24 fatty alcohols are present, while cutin contains C26 and C28. To overcome the surface mechanical barrier of plants, many pathogenic fungi secrete enzymes that hydrolyze cutin and part of the components of suberin. The products of the cutinase reaction were various oxygenated fatty acids and alcohols, mainly 10,16-dihydroxy-Sk- and 9,10,18-trihydroxy-C|8-acids, which are signal molecules that induce the formation and release of additional amounts of cutinase, “corroding” cutin and facilitating the penetration of the fungus into the plant. It was found that the lag period for the appearance of cutinase mRNA in the fungus after the onset of the formation of the above-mentioned di- and trihydroxy acids is only 15 minutes, and the lag period for the release of additional cutinase is twice as long. Damage to the cutinase gene in Fusarium solani greatly reduced the virulence of this fungus. Inhibiting cutinase using chemicals or antibodies prevented plant infection. The assumption that oxygenated cutin degradation products can act not only as inducers of cutinase formation in pathogens, but also as elicitors of defense reactions in the host plant [Tarchevsky, 1993] was subsequently confirmed.

After the penetration of pathogenic microorganisms through the cuticle, some of them move into the vascular bundles of plants and use the nutrients available there for their development, while others are transported inside the living cells of the host. In any case, pathogens encounter another mechanical barrier - cell walls, consisting of various polysaccharides and proteins and in most cases reinforced with a hard polymer - lignin [Tarchevsky, Marchenko, 1987; Tarchevsky, Marchenko, 1991]. As mentioned above, to overcome this barrier and provide their development with carbohydrate and nitrogen nutrition, pathogens secrete enzymes that hydrolyze polysaccharides and cell wall proteins.

Special studies have shown that during the interaction of bacteria and tissues of the host plant, enzymes

Degradations do not appear simultaneously. For example, pectylmethylesterase was also present in non-inoculated Erwinia carotovora subsp. atroseptia in potato tuber tissues, while polygalacturanase, pectate lyase, cellulase, protease and xylanase activities appeared, respectively, 10, 14, 16, 19 and 22 hours after inoculation.

Xyloglucan oligosaccharide with antiauxin action.

The structure of a number of physiologically active oligosaccharides was deciphered: a branched heptaglucoside obtained from the cell walls of a pathogenic fungus [Elbersheim, Darvill, 1985]; penta- and hexamers of N-acetyl-glucosamine obtained from the hydrolysis of chitin, as well as glucosamine formed from the hydrolysis of chitosan; 9-13-mer linear oligogalacturonides formed during the hydrolysis of pectin substances; decagalacturonide with 4-5 unsaturated terminal galacturonosyl residue; oligogalacturonosides with a degree of polymerization of 2-6, exhibiting certain activity. Data have been published on physiologically active xyloglucans obtained from hemicelluloses with a degree of polymerization of 8-9, chitobiose, chito-triose and chitotetrose, branched xyloglucan fragments with the formula Glu(4)-Xi(3)-Gal(1 or 2)-Fuc and their natural O-acetylated derivatives. It was found that branched p-glucoside has the highest phytoalexin-inducing activity. Chemical modification of this oligosaccharin or a change in the branching pattern led to a decrease in elicitor properties.

The study of the mechanism of action of oligosaccharides on plants made it possible to establish that the spectrum of responses depends on the concentration and structure of the substances under study. Various oligosaccharide elicitors exhibit the highest activity at different concentrations. For example, the induction of the synthesis of protective compounds (chitinases) in rice cell culture was maximum at a concentration of N-acetylchitohexaose of 1 μg/ml, while to achieve the same effect in the case of laminarine hexaose (fragment (3-1,3-glucan) it was required 10 times higher concentration.

It was found that the degree of plant resistance to the pathogen is determined (along with other factors) by the ratio of various polysaccharides of plant cell walls. This can be judged based on a comparison of Colletotrichum linde-resistant and susceptible to the pathogen.
muthianum bean lines that were exposed to the pathogen's endopolygalacturonase. Oligomeric fragments of pectin were isolated; It turned out that in the resistant variety, residues of neutral sugars predominate, while in the unstable variety, galacturonate residues predominate.

Recently, results have been obtained indicating that oligogalacturonate fragments are formed in plants not only under the influence of pectin-degrading enzymes of pathogens, but also as a result of the expression of polygalacturonase genes in host cells in response to systemin and oligosaccharide elicitors.

The multidirectional regulation of the protective response of cells by degradation products of cell wall polysaccharides attracts attention. It turned out that small oligogalacturonides with a degree of polymerization of 2-3 are active elicitors, and fragments of rhamnogalacturonic pectins with a high degree of polymerization are suppressors of the formation of hydroxyproline proteins of cell walls. In other words, degradation processes in cell walls caused by pathogens can regulate (as a result of a complex sequence of reactions of cell signaling systems) biosynthetic processes that increase the stability of cell walls due to the accumulation of hydroxyproline proteins and the formation of covalent bonds between them.

Fucose-containing fragments of xyloglucan (tri- and pentasaccharides) had immunosuppressive properties, but when replacing xylose with another monosaccharide, they changed the suppressor activity to elicitor activity [Ilyinskaya et al., 1997]. Deprivation of the oligosaccharide fucose deprived it of both suppressor and elicitor properties. Low active doses and high selectivity of specific suppressors indicate the receptor nature of their action [Ozeretskovskaya, 2001].

There are other examples of pathogens producing not only elicitors, but also suppressors of plant defense reactions. Thus, the pycnosgyurs Mycosphaerella pinodes secreted both types of such compounds.

It should be noted that oligosaccharide fragments of polysaccharides of the cell walls of plants and fungi are from

They are used as non-race specific elicitors that cause non-specific protective responses on the part of infected plants. This is understandable, since during the degradation of polysaccharides a wide range of oligosaccharides are formed, in which the species specificity of the pathogen or host is very weakly expressed. At the same time, protein (or peptide) bacterial virulence factors, which are recognized by “their” plant cell receptors, are race-specific. The latter type of interaction is called genetic ping-pong, or gene-on-gene interaction, since the specificity of the elicitor or receptor is determined by the genes encoding them, and the resistance or susceptibility of plants to the pathogen is determined by the ability of the receptor to recognize the elicitor.

To study the mechanisms of response of plant cells to the action of elicitors, not individual oligosaccharides are often used, but a mixture of oligosaccharides formed during the hydrolysis of polysaccharides of the cell walls of pathogenic fungi. This approach is justified if we take into account that even in the first moments of infection by pathogens, plant cells can be affected by not one, but several elicitors. By the way, there are relatively few works devoted to the study of the characteristics of the action of several elicitors simultaneously. For example, it has been shown that the elicitins parasiticein and cryptogein, as well as oligosaccharide elicitors from cell walls, cause rapid activation of the 48 kDa SIP-type protein kinase and phenylalanine ammonium lyase in tobacco. At the same time, it was the elicitins, and not the oligosaccharides, that activated the 40 kDa protein kinase. Glucan and Ca 2+ enhanced the effect of arachidonate and eicosapen-taenoate. The fact that EGTA (a specific Ca 2+ ligand) inhibited the synthesis of phytoalexins suggests that calcium ions play an important role in the regulation of protective function plants. It is possible that signaling substances are also products of degradation of cell wall proteins rich in hydroxyproline residues and containing oligoglycosyl branches.

ELICITOR RECEPTORS

It was already mentioned in the introduction that receptors for elicitor signals can be located in the cell membrane, in the cytosol, and in the nucleus, but we are especially interested in the first, most common case, when the elicitor itself does not penetrate the cell, but interacts with the extracellular part of the plasma membrane protein receptor , which is the first link in a complex chain of signaling events that culminate in the cell’s response to changed living conditions. The number of molecular antennas of one type of cell plasma membrane receptor can apparently reach several thousand. The number of types of molecular antennas remains unknown, but it can be argued that they have unified basic structural properties. They have three main domains: an external variable N-terminal domain (acceptor in relation to elicitors), a transmembrane domain with an increased content of the hydrophobic amino acid leucine, and a cytoplasmic variable C-terminal domain, the structure of which determines the transmission of a signal impulse to a particular signaling system. A receptor may be specific for only one type of elicitor or for a group of related (eg, oligomeric) elicitors. Several types of receptor proteins of cell membranes in animals have been described: in some receptors, the transmembrane chain of the protein crosses the membrane only once, in others (serpentine) - seven times, in others, interaction with the elicitor ligand leads to the formation of a homo- or heterodimer (oligomer), which and is the primary converter of an external signal. The structure of receptor proteins of the plant plasmalemma has been studied to a lesser extent, but the principles of their construction are the same

ATP

ATP

Rice. 4. Scheme of the structure of a two-component receptor signaling system

A - simple receptor; b - multi-link receptor. 1 - “input” domain; 2 - autokinase histidine domain; 3 - response regulator receptive domain; 4 - "output" domain of the response regulator; 5 - histidine-containing phosphate transfer domain; A - aspartic acid residue; G - histidine residue; P is an orthophosphate residue transferred during kinase reactions. The external signal is indicated by a lightning symbol

The same as in animal cells. The two-component receptor structure, which has the properties of a protein kinase, attracts special attention (Fig. 4). It was first discovered in prokaryotic organisms, and then, in a modified form, in eukaryotic organisms, including plants, such as Arabidopsis. If in the first case two components - the receptor itself and the executive - are independent, although interacting, protein molecules, then in the second they are two domains of the same protein.

Confirmation of the role of elicitor-receptor interactions in the transmission and transduction of signals from pathogens into the genome was the establishment of a positive correlation between the ability of elicitors to non-covalently bind to receptors and cause a protective cell response, such as the accumulation of phytoalexins. Binding to the outer portion of plasma membrane protein receptors was characteristic of oligosaccharide elicitors of plant cell walls, oligochitin fragments of fungal cell walls, elicitor proteins and peptides, syringolides, stress phytohormones systemin, ethylene, abscisic acid, methyl jasmonate, and brassinosteroids. In the latter case, there is a fundamental difference from animal cells, in which steroid hormone receptors are located in the nucleus.

A number of membrane protein elicitor receptors have been isolated. To do this, after the receptors bind labeled elicitors, the membranes are released from the cells, destroyed, and the protein with the retained elicitor is identified by its radioactivity. It has been discovered, for example, that the receptor for systemin is a 160 kDa protein, the bacterial elicitor flagellin is a 115 kDa membrane protein, and a glycoprotein from the cell wall of late blight, which has a signal oligopeptide fragment of 13 amino acid residues -91 kDa or 100 kDa.

The concept of molecular gene-to-gene interaction between pathogens and plants often involves indirect (mediated by signaling systems) recognition of the pathogen's avirulence gene (avr gene) by its corresponding resistance gene (R gene) of the plant cell.

The molecular basis of the “gene-to-gene” interaction between a pathogen and a plant was the elicitor-receptor model. Receptor proteins have been isolated and purified, and the genes encoding these proteins have been cloned. There are a number of review works devoted to the structure of receptor proteins

It turned out that many of them have similar conserved leucine-rich repeats (from 12 to 21), necessary for protein-protein interaction. These repeats mediate the binding of the receptor R protein to elicitors. Studies of mutants with impaired resistance to pathogenic bacteria caused by the replacement of glutamate with lysine in one of the leucine repeats confirm that protein-protein interaction is an important link in the transformation and transmission of elicitor signals into the cell genome.

Currently, several models of receptor structure and methods of transmitting an elicitor signal from outside to inside a plant cell are accepted. A family of 35 serpentine receptors has been found in Arabidopsis. The receptor perceives the signal molecule at the N-terminal site on the outer side of the membrane, and transmits the signal impulse into the cytoplasm through the internal C-site. Binding of a signal molecule leads to a change in the conformation of the entire receptor molecule, which causes the activation of protein molecules associated with it in the cytoplasm that transduce the signal.

One of the fundamentally important mechanisms used in cell signaling systems is dimerization (oligomerization) of some protein intermediates of these systems. Examples include the dimerization of receptors after binding of ligands to them, the dimerization of some intermediates of signaling systems, and the dimerization of transcription regulatory factors. Both homo- and heterodimerization (oligomerization) are observed. In animals, the mechanism of dimerization of tyrosine kinase receptors of the cell membrane is characteristic, for example, for the transduction of polypeptide hormones (placental growth factor, etc.). Serine/threonine kinase receptors function in a similar way. Little is known about which forms of receptors - monomeric, homodimeric or heterodimeric - are involved in the transformation of elicitor signals in plant cells. A scheme of heterodimer re-
receptor, which is activated by a ligand, which leads to phosphorylation of the cytosolic kinase domain and activation of proteins associated with it, some of which transmit a signal impulse to the following intermediates of signaling systems. One of the associated proteins is protein phosphatase, which inactivates the kinase domain.

In animal cells, the tyrosine kinase receptor consists of three domains - extracellular, transmembrane and cytosolic. The specific structure of the first and third domains (consisting, for example, in the fact that they are not capable of phosphorylation) determines, on the one hand, which hormone the receptor interacts with and, on the other hand, which signaling systems are “turned on” by this hormone. The interaction of the external domain with a signaling ligand leads to autophosphorylation of the tyrosine residue of this domain, which increases its kinase activity. Typically, protein kinases contain multiple phosphorylation sites. This also applies to receptor protein kinases. The cytoplasmic domain of the monomeric form of the growth factor receptor in animal cells contains at least nine autophosphorylatable tyrosine residues. One of them, Tyr 857, is important for the manifestation of kinase activity, and eight others determine the specificity of the connection with molecules that convert the signal. There is reason to believe that the same principles of receptor functioning are also used in plant cells, however, mainly serine-threonine receptor protein kinases involved in pathogen-induced plant defense reactions are found in them.

Currently, 18 Arabidopsis receptor-like serine-threonine protein kinases are divided into four groups depending on the structure of their extracellular domain:

1. Protein kinases with domains enriched with leucine repeats, usually characteristic of fragments involved in protein-protein interactions. In animals, such receptors bind polypeptide (or peptide) signaling molecules. It is assumed that this group includes brassinolide receptors with enriched

Myleucine repeats in the N-terminal supramembrane region. In tomato, a gene for a similar protein was isolated, but without the cytosolic kinase domain.

2. Protein kinases with S-domains, which contain
many cysteine residues.

Protein kinases with leucine-rich domains
repetitions, but, unlike the first group, is associated
with lectins. This creates the possibility of reception by these
protein kinases of oligosaccharide elicitors.
Cell wall-associated protein kinases.

These groups did not include some protein kinases, in particular a protein kinase that has an extracellular domain that binds to a protein that accumulates in the intercellular space when plants are infected with various pathogens. As already noted, many receptor kinases can interact with other proteins, and this provides both a greater variety of associated chemical signals and the regulation of these processes. Perhaps the mentioned protein kinase is one of the receptor proteins responsible for plant defense reactions.

One of the ancient, conservative and widespread types of membrane receptors are transmembrane autophosphorylating histidine kinases, which can be activated by a wide range of elicitor signaling molecules. Binding of the elicitor by the external N-terminal region of the receptor, protruding above the lipid layer of the plasmalemma, causes a change in its conformation and autophosphorylation of the histidine residue (see Fig. 4). Then the phosphoric acid residue is transferred to the aspartate residue of the internal (cytoplasmic) region of the protein, which also causes a change in its conformation and, as a result, activation of the enzyme associated with the receptor (directly or through intermediaries - most often G-proteins). Enzyme activation is the most important link in the signaling system, the purpose of which is the transmission and multiplication of the elicitor signal, culminating in the expression of protective genes and the appearance of proteins that

The response of cells and the plant as a whole to infection and the effects of elicitors is determined. The specificity of receptors for elicitors is determined by the variable outer N-terminus of the protein, and the specificity for the enzyme is determined by its internal C-terminus. This type of receptor has been shown to interact with the stress phytohormone ethylene IBleecker et al., 1998; Hua and Meyerowitz, 1998; Theologis, 1998; Woeste and Kieber, 1998; Alonso et al., 1999; Chang, Shockey, 1999; A.E. Hall et al., 1999; Hirayama et al., 1999; Cosgrove et al., 2000; Savaldi-Goldstein, Fluhr, 2000; etc.], which elicits protective reactions of plant cells. Cloning and determination of the primary structure of the histidine receptor gene in Arabidopsis revealed that its N-terminal membrane domain is similar to metal ion transporters.

Currently, a transmembrane receptor protein has been described, the N-terminus of which interacts with the cell wall, and the C-terminus is located in the cytoplasm and has the properties of serine-threonine protein kinases. According to the authors, this receptor protein performs signaling functions, providing signaling contact between the cell wall and the internal contents of the cell.

Since the interaction of the signal molecule and the receptor occurs without the formation of covalent bonds between them, the possibility of their decoupling cannot be ruled out. On the other hand, the association of these two types of molecules can be quite strong, and a change in the conformation of the receptor protein creates the prerequisites for facilitating the attack on it by proteases that recognize proteins with a disrupted structure and destroy these molecules. In this regard, the ability of cells to quickly restore the number of receptors is of great importance. various types. Noteworthy are the experiments devoted to the study of the effect of protein synthesis inhibitors on the intensity of binding of elicitors by receptor proteins of the plasmalemma. It turned out that treatment of cells with cycloheximide, an inhibitor of protein synthesis with the participation of cytoplasmic ribosomes, caused a fairly rapid decrease in the level of systemin binding to cells, which indicates that

The high rate of turnover of the receptor protein is 160 kDa. There is data on the elicitor-induced synthesis of receptors located in the plasmalemma, but, as far as is known, there is currently still no information on the degree of specificity of the synthesis of a particular receptor protein depending on the type of elicitor.

Tarchevsky I.A. Plant cell signaling systems - file n1.doc

n1.doc