Tarchevsky, Igor Anatolyevich - Plant cell signaling systems = Plant cell signaling systems. Signaling systems of plant defense reactions to pathogens Tarchevsky and a signaling systems of plant cells
AB11 and AB12 play a key role in ABA-induced
bathroom signal path. pH-dependent and Mg2+-dependent activity was observed.
ation ABU .
In protein phosphatases MP2C, the main target is MAPKKK, which is activated under the influence of various stressors. This specificity becomes understandable, given that some protein phosphatases have found binding sites with their corresponding protein kinases.
Participants of the signal
ny systems of cells. This makes it possible to ensure the existence of the protein kinase-protein phosphatase complex and to block the transformation and transmission of the signal impulse into the genome in a timely and effective manner. 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. In the study of the functioning of protein phosphatases, specific inhibitors are often used, such as okadaic acid and caliculin.
TRANSCRIPTION REGULATION FACTORS
The synthesis of messenger RNAs is catalyzed by DNA-dependent RNA polymerases, 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, "RNA polymerase activity and recognition of transcribed genes are regulated by several types of proteins. Transcriptional regulation factors have attracted the most attention." These proteins are able to interact with other proteins, including identical ones, change conformation upon phosphorylation of several of their amino acids, [recognize regulatory nucleotide sequences in the promoter regions of genes, which leads to a change in the intensity of their expression.: It is transcription regulation factors that direct RNA -polymerase to the point of initiation of transcription of the corresponding gene (or set of genes), without directly participating in the catalytic act of mRNA synthesis.
In animal organisms, structural features of more than 1,000 transcription regulation factors have been determined. The cloning of their genes contributed to obtaining information that made it possible to classify these proteins.
All transcription regulation factors contain three main domains. The DNA-binding domain is the most conservative. The amino acid sequence 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 regulation 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 helix-turn-helix domains; 4) with domains of the |3 scaffold type, which form contacts with the minor groove of DNA [Patrushev, 2000]. Each superclass is subdivided into classes, families, and subfamilies. In superclass 1, transcription regulatory factors with leucine zipper domains, which are oc-helices, in which every seventh amino acid is a leucine protruding from one side of the helix, attract attention. The hydrophobic interaction of leucine residues of one molecule with a similar helix of another molecule provides dimerization (similar to a zipper) of transcription regulation 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 DNA major groove. 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" are in contact with the DNA major groove.
The study of the structure of transcription regulation factors in plants made it possible to establish homology with proteins of this type, which are characteristic of animal objects. Typical transcription regulation 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 they are associated and then delivered to the nucleus or via
Rice. 5. Structure of the "zinc finger" transcription regulation factor
G - histidine residue; C-S - cysteine residue
special transport proteins or through interaction with receptor proteins in the pores of the nuclear membrane, after which they are transferred to the nucleus and interact with promoter sites
the corresponding genes. "Transcriptional regulatory factors are encoded by multigene families, and their synthesis can be induced by pathogens and elicitors, and their activity can be changed as a result of post-translational modification (mainly phosphorylation or dephosphorylation).
An ever expanding database has now been created on the structure of various transcription regulation 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 core and loop zones in the already mentioned leucine zippers, which are one of the most numerous and conservative groups of eukaryotic transcription regulation factors. Often, transcription regulation factors are classified precisely according to the structure of DNA-binding domains, which may include helical sequences of amino acids, "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 regulation factors.
The mechanism of interaction of transcription regulation factors with DNA-dependent RNA polymerases and promoter regions of genes remains one of the key and still insufficiently studied problems of the functioning of the cell genome. Information concerning plant objects is especially scarce.
Mutations in genes encoding transcription regulation factors in animals can lead to certain diseases.
Representatives of a family of genes encoding transcription regulation factors with leucine zippers have been described in plants. It has been shown that transcription factors of this type are responsible for the salicylate-induced formation of protective anti-pathogenic proteins and that mutations in these genes lead to a loss of the ability to synthesize these proteins.
PROMOTORS OF GENES OF PROTEINS OF SIGNALING SYSTEMS AND PROTECTIVE PROTEINS
Currently, the structure of the promoter regions of the genes responsible for the acquisition of immunity to various pathogens is being intensively studied. The fact of the almost simultaneous synthesis of a number of pathogen-induced proteins has long attracted attention: This can be caused both by divergence of signaling pathways in one signaling system, which causes the activation of several types of transcription regulation factors, and by the “switching on” of several signaling systems by one or another elicitor, which, functioning in parallel, they activate several types of transcription regulation factors and, as a result, cause the expression of several types of protective proteins. It is also possible that the promoters of the genes 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 regulation factors.1
The latter variant occurs under the action of the stress phytohormone ethylene on plants, when the transcription regulation factor interacts with the GCC box of the promoter regions of several ethylene-inducible genes, which provides 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 action of elevated temperatures, transcription of a group of genes containing in the promoter regions a common regulation is induced.
the torus element HSE (heat shock element), which is absent in other genes. This pattern was confirmed by creating hybrid genes with a heat shock gene promoter docked with another gene, which usually does not change the intensity of expression under the action of elevated temperatures. In the case of transgenic plants, its expression began. In eukaryotic cells, promoter regions with similar nucleotide sequences have also been found in different genes induced by the same intermediate (second messenger) of signaling systems, for example, cyclic AMP. In the latter case, the nucleotide signal sequence of the promoter region is designated CRE (cyclic AMP response element).
In Arabidopsis, a glucocorticoid system for activating transcription regulation factors was found, 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 are pro-
motors were CCACGTGG, and in the C-box - TGACGTCA.
Tobacco mosaic virus and salicylic acid caused in tobacco plants the induction of two genes of transcription regulation factors of the WRKY class, which recognize a certain nucleotide sequence, TTGAC (W-box), in the promoter regions of protective genes. The activation of these transcription regulation factors was carried out by their phosphorylation by protein kinases. All proteins of the WRKY class, in contrast to other classes of transcription factors (such as bZIP and myb), have a conserved domain containing a heptameric pep-
type WRKYGQK .
(One of the domains of the transcription regulation factor responsible for the conversion 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 regulation factor has an activating effect , and the C-terminal domain -I enriched with serine residues is inhibitory.
It was shown that the promoter of the phenylalanine-ammonia-lyase gene (the most important starting enzyme of the branched metabolic process for the synthesis of compounds that play a protective role - salicylate, phenolic acids, phenylpropanoid phytoalexins, and lignin) contains two copies of regions enriched in AC repeats.
When studying the promoter of the gene of another enzyme syntheia of phytoalexins - chalcone synthase, in a cell culture of beans, tobacco and rice, it was found that the G-box (CACGTG) in the region from -74 to -69 base pairs and H-boxes (CSTACC) take part in the activation of the promoter. ) in the region from -61 to -56 and from -126 to -121 base pairs.
In other experiments, it was found that under the action of elicitors, the expression of the chalcone synthase gene in pea plants depends on the promoter region from -242 to -182 base pairs, in which two regions contain identical AT sequences -TAAAATAST-, and one of them, located in the region from -242 to -226, was necessary for the manifestation of the maximum activity of the gene.
The promoter of the strictosidine synthase gene, one of the key elicitor-inducible enzymes for the synthesis of terpenoid phytoalexins, has a region activated by transcription regulation factors from -339 to -145 bp. The G-box, located near -105 bp, did not affect the activity of the promoter.
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 base pairs, containing the -GGCGGC- sequence, which is characteristic of the promoters of genes encoding pathogen-induced alkaline
ny proteins.
The so-called PR-box of the promoter regions of many pathogen-induced proteins contains the sequence (5'-AGCCGCC-3'), which binds the corresponding transcription regulation factors, 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 regulation factors that have leucine zippers in their structure interact. In Arabidopsis plants, transcription regulation factors responsible for ethylene signal transduction bind to both the GCC box and the ocs promoter elements, resulting in the expression of a range of defense proteins.
The study of transgenic tobacco plants with the alkaline chitinase promoter and the GUS reporter gene revealed that the promoter region activated by the ethylene signal is located between -503 and -358 base pairs, where there are two copies of the GCC box (5"-TAAGAGCCGCC-3"), which is characterized -
ren for promoters of many ethylene-inducible proteins. Further analysis showed that the site of the promoter with two copies of the GCC box responsible for the reaction to ethylene is located between -480 and -410 bp.
When studying the response of tobacco plants to ethylene treatment and mosaic virus infection, it was found that the activity of the gene promoter (3-1,3-glucanase) depends on the region located between -1452 and -1193 base pairs, where there are two copies of the heptanucleotide
5-AGCCGCC-3 ". Found and added
filamentous regions essential for the regulation of promoter activity.
The elicitors discussed above, elicitor receptors, G-proteins, protein kinases, protein phosphatases, transcription regulation factors, their corresponding promoter regions of genes are involved in the functioning of a number of cell signaling systems, on which their response to signals of various 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, such as a hormone or an elicitor, interacts with the plasma membrane protein receptor, which leads to the activation of the G-protein (binding GTP by it) 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, there are Gs proteins that stimulate adenylate cyclase and (5,) proteins that 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 (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 GTP and its hydrolysis to GDP and inorganic orthophosphate ensure the reversibility of adenylate cyclase activation processes.
Adenylate cyclase is a monomeric integral protein of the plasma membrane and therefore is difficult to extract and convert to 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, which are not sensitive to calmodulin and G-proteins. In plants, the molecular weight of adenylate cyclase is 84 kDa. The curve of the dependence of the activity of adenylate cyclase on pH had a unimodal character, and the peak of activity for this enzyme
menta was in the pH range of 4.8-5.2.
Data on the isoform of adenylate cyclase with optimal
Imo pH equal to 8.8.
Adenylate cyclase can be modified from the outside of the membrane by glycosylation, and from the inside by phosphorylation by A-kinase [Severin, 1991]. The activity of membrane adenylate cyclase depends on the phospholipid environment - the ratio of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phosphatidyls "eri-
on and phosphatidylinositol.
The elicitor-induced increase in the content of cAMP in cells is transient, which is explained by PDE activation 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 regulation 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 scheme of signal multiplication in the functioning of the adenylyl cyclase signaling system is often used in biochemistry textbooks. This signaling system continues to be intensively studied on various objects, replenishing ideas about 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 be debatable for almost a quarter of a century, dividing researchers into its
GENE EXPRESSION
Rice. 6. Scheme of the functioning of adenylate cyclase signaling
AC* systems - active form of adenylate cyclase; PCA and PCA*- inactive-
naya and active forms of protein kinase A; PLplasmalemma; PDE - phosphodiesterase; PGF* - active form of transcription regulation factor
supporters [Doman, Fedenko, 1976; Korolev and Vyskrebentseva, 1978; Franco, 1983; Yavorskaya and Kalinin, 1984; Newton and Brown 1986; Karimova, 1994; Assman, 1995; Trewavas, Malho, 1997; Trevavas, 1999; etc.] and opponents. The former 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 latter on facts indicating a low content of cAMP in plants, on the absence in a number of experiments of the effect of phytohormones on the activity of adenylate cyclase and etc.
Advances in the field of molecular genetics, a comparison of the structure of the genes of proteins participating in the adenylate cyclase signaling system in animals and plants, tipped the scales in favor of supporters of its functioning in plants. Result-
The use of exogenous cAMP [Kilev and Chekurov, 1977] or forskolin (an adenylate cyclase activator) indicated the involvement 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 input 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].
Attention is drawn to the fact of the 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 oxylipin, similar to prostaglandin, jasmonate. The possibility of participation in the removal of cAMP from the cell of special ATP-binding
ing proteins.
The expediency of cAMP secretion from plant cells into the medium is explained, first of all, by the need for a sufficiently rapid decrease in the concentration of this second messenger so that cell overexcitation does not occur. A relatively rapid decrease in the concentrations of second messengers after reaching the maximum level is an indispensable non-specific feature of the functioning of all signaling systems.
It is likely that cAMP, which is excreted 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 that use cAMP secretion from cells to activate protein phosphorylation outside the plasmalemma. It is also believed that cAMP outside the cell can act as the first messenger [Fedorov et al., 1990], inducing the triggering of a cascade of signaling system reactions in neighboring cells, which was shown in the example of multicellular slime fungi.
Attention is drawn to the data obtained in animals on inhibition by exogenous adenosine (which can be considered as a product of cAMP degradation) of calcium channels in cells [Meyerson, 1986] and activation of potassium channels [Orlov, Maksimova, 1999].
Of great interest is information on the possibility of regulation of the development of pathogenic fungi by secreted cAMP, in particular, barley rust, Magnaporthe grisea, which affects rice plants, loose smut Ustilago maydis, Erysiphe graminis, Colletotrichum trifolii, pigmentation of Ustilago hordei. Depending on the concentration of cAMP, the development of fungi was stimulated or suppressed. It is believed that they have heterotrimeric G proteins involved in cAMP signal transduction.
More and more data are accumulating on the effect of various signaling molecules on cAMP secretion by plant cells. It was 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 the content of cAMP under the action of ABA is caused by an ABA-induced increase in the content of Ca2+ in the cytosol and inhibition of adenylate cyclase. It is known that high concentrations of Ca2+ inhibit the activity of adenylate cyclase in eukaryotes. At the same time, Ca2+ can reduce the content of cAMP, inducing an increase in the activity of phosphodiesterase, which hydrolyzes cAMP. Indeed, the activation of cAMP phosphodiesterase by the Ca2+-calmodulin complex was found in plant objects [Fedenko, 1983].
The dependence of the polypeptide phosphorylation profile on exogenous cAMP was shown. The number of polypeptides whose phosphorylation was stimulated by cAMP was greatest at the micromolar concentration of cAMP. Attention is drawn to the fact of a strong cAMP-induced increase in the phosphorylation of the 10 kDa polypeptide at low temperatures (Fig. 7) [Karimova, Zhukov, 1991; Yagusheva, 2000]. Interestingly, a polypeptide with this molecular weight is a protein regulator of cAMP phosphodiesterase, which is activated by abscisic acid and Ca2+ and reduces the content of cAMP due to its hydrolysis by phosphodiesterase.
The study of the features of activation of cAMP-dependent protein kinases and their phosphorylation of various proteins is one of the most important areas of research on the adenylate cyclase signaling system. cAMP-dependent protein kinases (PKA) are enzymes that are activated upon interaction with cAMP and catalyze the transfer of a 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 the association or dissociation of their subunits, etc.
Molecular weight of proteins, kDa
Rice. Fig. 7. Influence of cAMP on protein phosphorylation in three-day-old pea seedlings [Karimova and Zhukov, 1991]
1 - control: cut shoots were transferred for 2 hours with petioles into water, then for another 2 hours - into a solution of orthophosphate labeled with 32 R; 2 - cut plants were transferred for 2 h into a solution of 1 μM cAMP, then for another 2 h into a solution of 32 P labeled orthophosphate
The substrates in the protein kinase reaction are MgATP and the phosphorylated protein. Protein substrates can simultaneously be substrates for cGMP- and cAMP-dependent protein kinases for 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 (EPR), Golgi apparatus, secretory granules, cytoskeleton, and nucleus.
Protein kinases activated by exogenous cAMP have been isolated from plant cells, for example, from maize coleoptiles, a 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 to the animal protein kinase A family in nucleotide sequences. There are examples of amino acid sequence similarities between plant protein kinases A (their homology) and animal protein kinases A. Several research groups have reported the cloning of genes homologous to the protein kinase A gene (reviews: ). A protein kinase from petunia phosphorylated a specific synthetic substrate for protein kinase A. The addition of cAMP to plant extracts has been reported to stimulate the phosphorylation of specific proteins. The study of phosphorylation sites in phenylalanine ammonia lyase (PAL), a key enzyme in the biosynthesis of phytoalexins, revealed sites specific to 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 even during sample preparation: BI suppressed the basal protein kinase activity of leaf extracts in different experiments by 30-50% [Karimova, 1994]. Intermediates of the lipoxygenase signaling system HDA and MeFA 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 74, 61, and 22 kDa polypeptides in pea leaves [Mukhametchina, 2000]. cAMP-stimulated protein kinase activity of soluble pea leaf proteins depended on Ca2+ concentration [Karimova et al., 1989; Tarchevskaya, 1990; Karimova, Zhukov, 1991], and enzymatic activity was also found in isolated cell walls, nuclei, and plasma membranes.
In plants, genes have been found that encode the enzyme protein phosphatase, the target of which are proteins phosphorylated by protein kinase A.
To characterize the adenylyl cyclase signaling system, it is extremely important to find genes in plants that encode protein transcription regulation factors that have long nucleotide sequences homologous to CREBS, the cAMP-binding transcription factor in animals.
Numerous data on the effect of cAMP on ion channels of plant cells and a relatively weak experimental base of ideas about the possibility of signaling from cAMP through phosphorylation of protein factors regulating transcription into the genome, on the one hand, strengthen the positions of supporters of the existence of an indirect (through activation of ion channels) signaling adenylate cyclase pathway and , on the other hand, force us to intensify attempts to obtain evidence of the functioning of the direct cAMP signaling pathway.
MAP-KINASE SIGNALING SYSTEM
Mitogen-activated serine-threonine-type protein kinases (MAPK) and the MAP-kinase signaling cascade (signal -> receptor -> G-proteins -> MAPKKK -»
-> MARCK -> MAPK -> PGF -> genome), which have been sufficiently 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
features of their regulation.
The MAP kinase cascade is “turned on” during mitosis (which explains the name of these protein kinases), during dehydration
nii, hypoosmo-
tic stress, low temperature, mechanical irritation of plants
Tissue damage, oxidative stress, the action of pathogens, elicitors (in
including harpins, cryptogaine, 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,
venous 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 - MAPkinase kinase; MAPK is a mitogen-activated protein kinase. Other designations - see fig. 6
The action of elicitor preparations 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 nature, 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 viability, resistance to various negative environmental factors.
The relationship of plants with the outside world, as highly organized elements of ecological systems, is carried out through the perception of physical and chemical signals coming from outside and correcting all the processes of their life by influencing genetic structures, the 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 much attention to the study of signaling systems responsible for plant resistance to phytopathogens.
Biochemical processes occurring in plant cells are strictly coordinated by the integrity of the organism, which is complemented by their adequate responses to information flows associated with various effects of biogenic and technogenic factors. This coordination is carried out due to the work of signal chains (systems), which are woven into signal networks of cells. Signaling molecules turn on most hormones, as a rule, without penetrating inside the cell, but interacting with receptor molecules of 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 at 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 the conductivity of ion channels is modulated. In response to this, a certain cellular response is formed, which, as a rule, consists in a cascade of successive biochemical reactions. In addition to protein messengers, signal transduction can also involve relatively small messenger molecules that are functionally mediators between receptors and the cellular response. An example of an intracellular messenger is salicylic acid, which is involved in the induction of stress and immune responses in plants. After switching off the signaling system, the messengers are rapidly split or (in the case of Ca cations) are pumped out through the ion channels. Thus, proteins form a kind of “molecular machine”, which, on the one hand, perceives an external signal, and on the other hand, has enzymatic or other activity modeled by this signal.
In multicellular plant organisms, signal transmission is carried out through the level of cell communication. Cells "speak" the language of chemical signals, which allows the homeostasis of a plant as an integral 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 the signaling systems play the role of a molecular processor that performs the functions of operational control. At present, we have only the most general information about the principles of operation of this extremely complex biological entity. In many ways, the molecular mechanisms of signaling systems still remain unclear. Among the solution of many issues, it is necessary to decipher the mechanisms that determine the temporary (transient) nature of the inclusion of certain signaling systems, and at the same time, the long-term memory of their inclusion, which manifests itself, 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 others. This mechanism includes reception, transformation, multiplication, and signal transmission to the promoter regions of genes, programming of gene expression, changes in the spectrum of synthesized proteins, and a functional response of the cell, for example, induction of immunity to phytopathogens.
Various organic compounds-ligands 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. .
The secondary elicitors formed in plant cells under the action of biogenic and abiogenic stressors and included in the 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 the 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.
At present, seven signaling systems of cells 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.
In response to the attack of pathogens, plants can use various pathways for the formation of systemic resistance, which are triggered by different signaling molecules. Each of the elicitors, acting on the vital activity of a plant cell through a certain signaling pathway, through the genetic apparatus, causes a wide range of reactions, both protective (immune) and hormonal, leading to a change in the properties of the plants themselves, which allows them to withstand a whole range of stress factors. At the same time, inhibitory or synergistic interaction of various signaling pathways intertwined into signaling networks occurs in plants.
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. Nevertheless, it has a certain stability and serves as an example of phenotypic immunocorrection of plant tissue, since as a result of treatment with eliciting substances, 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 arising from the treatment of plants with immunoinductors are related to gene modification, differing from it in the absence of quantitative and qualitative changes in the gene pool itself. With the artificial induction of immune responses, only phenotypic manifestations are observed, characterized by changes in the activity of the expressed genes and the nature of their functioning. Nevertheless, the changes caused by the treatment of plants with phytoactivators have a certain degree of stability, which manifests itself in the induction of prolonged systemic immunity, which is maintained for 2-3 months or more, as well as in the preservation of the acquired properties by plants during 1-2 subsequent reproductions.
The nature of the action of a particular elicitor and the effects achieved are most closely dependent on the strength of the generated signal or the dosage used. These dependences, as a rule, are not linear, but sinusoidal in nature, which can serve as evidence of switching signaling pathways during their inhibitory or synergistic interactions. high severity of their adaptogenic action. On the contrary, treatment with these substances in high doses, as a rule, caused desensitization processes in plants, sharply reducing the immune status of plants and leading to an increase in plant susceptibility to diseases.
Plant resistance to pathogens is determined, as established by H. Flor in the 1950s, by the interaction of a complementary pair of host plant and pathogen genes, 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 plant recognition of a pathogen with subsequent activation of signaling processes to trigger defense responses.
Currently, 7 signaling systems are known: cycloadenylate, MAP-kinase (mitogen-activated protein-kinase), phosphatidic acid, calcium, lipoxygenase, NADP H-oxidase (superoxide synthase), NO-synthase.
In the first five signaling systems, G proteins mediate between the cytoplasmic part of the receptor and the first activated enzyme. These proteins are localized on the inner side of the plasmalemma. Their molecules consist of three subunits: a, b and g.
Cycloadenylate 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 phosphorylation.
MAP kinase signaling system. The activity of protein kinases is increased 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 passes into the nucleus, where it phosphorylates transcriptional regulatory proteins.
Phosphatido acid signaling system. In animal cells, G proteins activate phospholipases C and D under the influence of a stressor. 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 phosphatidic acid is also formed in plants, which can activate protein kinases with subsequent phosphorylation of proteins, including transcription regulation factors.
calcium signaling system. The impact of 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 an increase in import from the external environment and out of intracellular storage (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 are involved in the phosphorylation of protein factors regulating the expression of protective genes. However, Ca2+ has been shown to be able to directly affect the human transcriptional repressor without triggering the protein phosphorylation cascade. Calcium ions also activate phosphatases and phosphoinosit-specific phospholipase C. The regulatory effect of calcium depends on its interaction with the intracellular calcium receptor, the calmodulin protein.
Lipoxygenase signaling system. The interaction of the elicitor with the receptor on the plasma membrane leads to the activation of membrane-bound phospholipase A2, which catalyzes the release of unsaturated fatty acids, including linoleic and linolenic acids, from plasma membrane phospholipids. These acids are substrates for lipoxygenase. Substrates for this enzyme can be not only free, but also unsaturated fatty acids that are part of triglycerides. The activity of lipoxygenases increases under the action of elicitors, infection of plants with viruses and fungi. The increase in the activity of lipoxygenases is due to the 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 end products of lipoxygenase metabolism of fatty acids have bactericidal, 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 protein phosphorylation in pea plants, phytodienoic, jasmonic acids and methyl jasmonate increase the expression level of protective genes through activation of protein kinases.
NADP·N-oxidase signaling system. In many cases, infection with pathogens stimulated the production of reactive oxygen species and cell death. Reactive oxygen species are not only toxic to the pathogen and infected host plant cells, but are also participants in the signaling system. Thus, hydrogen peroxide activates transcription regulation factors and the expression of protective genes.
NO synthase signaling system. In macrophages of animals that kill bacteria, along with reactive oxygen species, nitric oxide acts, enhancing their antimicrobial effect. In animal tissues, L-arginine is converted by NO synthase into citrulline and NO. The activity of this enzyme was also found 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 a very toxic peroxynitrile. With an increased concentration 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 feature of the signaling system is the amplification of the signal in the process of its transmission. Activation of the signaling system in response to the impact of various stressors (including pathogens) leads to 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 the surface barriers and getting into the conducting system and plant cells, causes the disease of the plant. The nature of the disease depends on the resistance of the plant. According to the degree of resistance, four categories of plants are distinguished: sensitive, tolerant, hypersensitive and extremely resistant (immune). Let us briefly characterize them using the example of the interaction of plants with viruses.
In susceptible plants, the virus is transported from the initially infected cells throughout the plant, multiplies well and causes a variety of disease symptoms. However, in susceptible 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 ended. The dark green zones that form on young leaves of diseased susceptible plants are characterized by a high degree of resistance to viruses. The cells of these zones contain almost no viral particles compared to neighboring cells of light green tissue. The low level of virus accumulation in dark green tissue cells is associated with the synthesis of antiviral substances. In tolerant plants, the virus spreads throughout the plant but does not reproduce well and cause no symptoms. In hypersensitive plants, initially 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, does not transport through 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 a low temperature (10–15°C), necrosis formed on infected leaves.
The resistance mechanisms 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 a pathogen attack. They arise as a result of the death of a group of cells at the site of the introduction of the pathogen. The death of infected cells and the creation of a protective barrier around necrosis block the transport of the infectious principle throughout the plant, prevent access to nutrients to the pathogen, 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 recovery of the plant. Cell death occurs due to the inclusion of the genetic death program and the formation of compounds and free radicals that are toxic both to the pathogen and to 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). PCD is essential for the 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 grow in water, which means that cell death is part of the development of the plant and not caused by the action of the soil. The similarity between PCD and cell death in a hypersensitive reaction is that these are two active processes, in a necrotizing cell the content of calcium ions in the cytoplasm also increases, membrane vesicles are formed, the activity of deoxyribonucleases increases, DNA decomposes into fragments with 3'OH ends, 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 disruption of the integrity of cell membranes and an increase in their permeability. The decrease in the integrity of cell membranes is due to lipid peroxidation. It can occur with the participation of enzymes and in a non-enzymatic way 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 re-infection with a pathogen. The terms systemic acquired resistance (SAR) and localized acquired resistance (LAR) have been proposed. LAR is said to be in cases where resistance is acquired by cells in the area immediately adjacent to the local necrosis (distance of approximately 2 mm). In this case, secondary necrosis does not form at all. Acquired resistance is considered systemic if it develops in diseased plant cells remote from the site of initial pathogen introduction. SAR is manifested in a decrease in the level of accumulation of viruses in cells, a decrease in the size of secondary necrosis, which indicates inhibition of the short-range transport of the virus. It is not clear whether LAR and SAR differ from each other, or whether this is the same process occurring in cells located at different distances from the site of the initial entry of the virus into the plant.
Acquired resistance is usually non-specific. 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 has been suggested that the inducer of SAR is salicylic acid, which is formed during necrosis of initially infected cells.
When a disease occurs, substances accumulate in plants that increase their resistance to pathogens. An important role in the nonspecific resistance of plants is played by antibiotic substances - volatile, discovered by B. Tokin in the 20s of the 20th century. These include low-molecular substances of various structures (aliphatic compounds, quinones, glycosides with phenols, alcohols) that can retard the development or kill microorganisms. Being 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 integumentary tissues and are involved in creating the protective properties of the surface. Inside cells, they can accumulate in vacuoles. In case of damage, the amount of phytoncides increases sharply, which prevents possible infection of wounded tissues.
Phenols are also classified as antibiotic compounds in plants. In case of damage and diseases, 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 required for lignin synthesis.
Proteins, glycoproteins, polysaccharides, RNA, phenolic compounds were found among viral inhibitors. There are inhibitors of infection that directly affect viral particles, making them non-infectious, or they block the receptors of viruses. For example, inhibitors from beetroot, parsley, and currant juice caused almost complete destruction of tobacco mosaic virus particles, while aloe juice caused linear particle aggregation, which reduced the possibility of particle penetration into cells. Reproduction inhibitors alter cellular metabolism, thereby increasing cell resistance, 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 were found, originally called b-proteins, and now they are referred to as pathogenesis-related proteins (PR-proteins) or resistance-associated proteins. 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 stresses.
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 bonds of chitin. Chitinase can also act as lysozyme by hydrolyzing the peptidoglucans of bacterial cell walls. However, b-1,3-glucanase can facilitate the transport of viral particles across the leaf. This is due to 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.
The composition of PR proteins also includes low molecular weight (5 kDa) proteins - modifiers of cell membranes of fungi and bacteria: thionins, defensins, and lipid transfer proteins. Thionins are toxic under in vitro conditions for phytopathogenic fungi and bacteria. Their toxicity is due to the destructive action on the membranes of pathogens. Defensins have strong antifungal properties, but do not act on bacteria. Defensins from plants of the Brassicaceae and Saxifragaceae families suppressed the growth of fungal hyphae by stretching, but promoted their branching. Defensins from plants of the Asteraceae, Fabaceae, and Hippocastanaceae families slowed down hyphal elongation 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 the Golgi apparatus, functioning as primary animal lysosomes, that is, structures containing hydrolases that do not have substrates for these enzymes. In addition to these vacuoles, the lytic compartment of plant cells includes the central vacuole and other vacuoles equivalent to secondary lysosomes of animal cells that 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.
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; [Resp. ed. A.N. Grechkin]. -
M.: Nauka, 2002. - 294 p., ill. ISBN 5-02-006411-4
The links of information chains of interaction between pathogens and plants are considered, including elicitors, elicitor receptors, G-proteins, protein kinases and protein phosphatases, transcription regulation factors, gene expression reprogramming, and cell response. The main attention is paid to the analysis of the features of the functioning of individual signaling systems of plant cells - 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 features is proposed. Data on transgenic plants with increased resistance to pathogens are presented.
For specialists in the field of plant physiology, biochemists, biophysicists, geneticists, phytopathologists, ecologists, agrobiologists.
On the 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 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
© Russian Academy of Sciences, 2002 © Nauka Publishing House
(art design), 2002
In recent years, studies of the molecular mechanisms of regulation of gene expression under the influence of changing living conditions have been rapidly developing. In plant cells, the existence of signal chains was discovered, which, 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) associated with the inclusion of previously "silent" and the exclusion of some active genes. The significance of cell signaling systems was demonstrated in the study of the mechanisms of action of phytohormones. The decisive role of signaling systems in the formation of an adaptation syndrome (stress) caused by the action of abiotic and biotic stressors on plants was also shown.
The lack of review papers 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 being completed, and many details of its structure and functioning remain insufficiently illuminated. All this attracts new researchers, for whom the generalization of publications on the signaling systems of plant cells will be especially useful. Unfortunately, not all reviews
articles of an experimental nature were included in the list of references, 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 his gratitude to his collaborators who took part in the joint study of the signaling systems of plant cells. The author is especially grateful to Professor F.G. Karimova, candidates of biological sciences V.G. Yakovleva and E.V. Asafova, A.R. Mucha-metshin and associate professor T.M. Nikolaeva for help in preparing the manuscript for publication.
This work was supported financially by 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 of modern biology is the deciphering of 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 the 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 changing conditions, rebuilding their metabolism and structure, but also highlighting various volatile and non-volatile compounds into the extracellular space. Some of them play the role of protective substances against pathogens, while others can be considered as signaling molecules that cause a response of 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 with the help of various signaling systems cause a reaction on the part of the cell genome, which manifests itself in the reprogramming of gene expression. In fact, signaling systems regulate the operation of the main receptacle 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. 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 attention of readers contains a generalization of the results of studying the effect of biotic stressors on the functioning of the signaling systems of plant cells. MAP kinase, adenylyl cyclase, phosphatidate, calcium, lipoxygenase, NADPH oxidase, NO synthase, and proton signaling systems and their role in the ontogenetic development of plants and in shaping the response to changing living conditions, especially to the action 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 this response involves a number of phytohormones and elucidation of the features of the interaction of plant cell signaling systems with them attracts much attention of researchers.
The impact of biotic stressors leads to a plant response that is basically similar to the response to abiotic stressors. It is characterized by a set of non-specific reactions, which made it possible to call it an adaptation syndrome, or stress. Naturally, specific features of the response depending on the type of stressor can also be detected, however, as the measure of its impact increases, nonspecific changes come to the fore more and more [Meyerson, 1986; Tarchevsky, 1993]. The greatest attention was paid to them by 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 studies of the molecular basis of stress.
The most significant non-specific changes in biotic stress include the following:
1. Phase in the deployment in time of the response to the action of the pathogen.
2. Increased catabolism of lipids and biopolymers.
3. An increase in the content of free radicals in tissues.
4. Acidification of the cytosol followed by activation of proton pumps, which returns the pH to its original value.
5. An increase in the content of calcium ions in the cytosol, followed by activation of calcium ATPases.
6. Exit from cells of potassium and chlorine ions.
7. Drop in membrane potential (on the plasmalemma).
8. Decreased overall intensity of biopolymer synthesis and
9. Stopping the synthesis of some proteins.
10. Increased synthesis or synthesis of absent so-called pathogen-induced protective proteins (chitinases,(3-1,3-glucanases, proteinase inhibitors, etc.).
11. Intensification of the synthesis of components that strengthen cell walls - lignin, suberin, cutin, callose, a protein rich in hydroxyproline.
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 the synthesis and increasing the content (or according to
phenomenon) of stress phytohormones - abscisic, jasmonic, salicylic acids, ethylene, the hormone of the peptide nature of systemin.
15. Inhibition of photosynthesis.
16. Redistribution of carbon from |4CO2, assimilated in the process of photosynthesis, among various compounds - a decrease in the inclusion of the label in high-polymer compounds (proteins, starch) and sucrose and an increase (often relative - as a percentage of the assimilated carbon) - in alanine, malate , aspartate (Tarchevsky, 1964).
17. Increased breathing followed by its inhibition. Activation of an alternative oxidase that changes the direction of electron transport in mitochondria.
18. Violations of the ultrastructure - a change 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, rearrangement of cyto-
skeleton.
19. Apoptosis (programmed death) of cells exposed to pathogens and neighboring cells.
20. The appearance of the so-called systemic nonspecific
resistance to pathogens in plant sites (for example, metameric organs) remote from the place of pathogen impact.
Many of the changes listed above are a consequence of the “switching on” of a relatively small number of nonspecific signaling systems by stressors.
As the mechanisms of plant responses to the action of pathogens are being studied more and more, 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 should be borne in mind that these issues are part of a more general problem of regulating the functioning of the genome. It should be noted that the universality of the structure of the main information carriers of cells of various organisms - DNA and genes - predetermines the unification of the 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 the optimal structural and functional solution of a biochemical or informational problem in its time, 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 surfaces from the outside and inside.
her side. Several types of structure of these receptors are unified in plant and animal cells. The non-covalent interaction of the outer region of the receptor with one or another signal molecule coming from the environment surrounding the cell leads to a change in the conformation of the receptor protein, which is transmitted to the inner, cytoplasmic region. In most signaling systems, intermediary G-proteins are in contact with it - another unified (in terms of its structure and functions) link of signaling systems. G-proteins perform the functions of a signal transducer, transmitting a signal conformational impulse to the starting enzyme specific for a particular signal system. 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 of 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 signal reactions or their derivatives. Phosphorylated proteins by protein kinases are the next links in the signal chains. Another unified link in cell signaling systems is protein transcription regulation factors, which are one of the substrates of protein kinase reactions. The structure of these proteins is also largely unified, and structural modifications determine whether transcription regulation factors belong to one or another signaling system. Phosphorylation of transcription regulation 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 "turning on" of some silent genes or "turning off" active. Reprogramming of the expression of the totality of genome genes 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 yes -
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 non-specific changes in the structure of the lipid component of the plasmalemma; SIB - signal-induced proteins; PGF - protein transcription regulation factors; i|/ - change in membrane potential
same nucleus (Fig. 1). In animal cells, such signals are, for example, steroid hormones. This information pathway has a smaller number of intermediates, and therefore it has fewer opportunities for regulation by the cell.
In our country, great attention has always been paid 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 and Ozeretskovskaya, 1985; Kursanov, 1988; Ilinskaya et al., 1991; Ozeretskovskaya et al., 1993; Korableva, Platonova, 1995; Chernov et al., 1996; Tarchevsky and Chernov, 2000].
In recent years, special attention has been paid to the molecular mechanisms of phytoimmunity. It was 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 protective genes are expressed, which allows plants to organize both structural and chemical protection against pathogens. Advances in this area are associated with gene cloning, deciphering their primary structure (including promoter regions), the structure of the proteins encoded by them, 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 the genes of proteins involved 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 RAAS, All-Russian Research Institute of Phytopathology RAAS, 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 on various aspects of this problem are published (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 the signaling systems of cells, which led the author to the need to write a monograph offered to readers.
PATHOGENS AND ELICITERS
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; mushrooms (lower:
Plasmodiophoromycetes, Chitridomycetes, Oomycetes: higher: Ascomycetes, Basidi-omycetes, Deuteromycetes).
theses for protective enzymes: phenylalanine-ammonia-lyase
And anionic peroxidase. The 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 plant specificity, oligophages and monophages, in which the specificity of the interaction between the pathogen and the host plant is pronounced. Some insects feed on leaves (the entire leaf blade or skeletonizing the leaf), others feed on stems (including gnawing the stem from the inside), flower ovaries, fruits, and roots. Aphids and cicadas suck the juice from conducting vessels with the help of a proboscis or stylet.
Despite the measures taken to combat insects, the problem of reducing the harm they cause continues to be a topical issue. Currently, over 12% of the world's agricultural crops are lost as a result of attack by pathogenic microorganisms,
nematodes and insects.
Damage to cells leads to the degradation of their contents, such as high-polymer compounds, and the appearance of oligomeric signaling molecules. These "wreckage fragments" [Tarchevsky, 1993] reach neighboring cells and induce a protective reaction in them, including changes in gene expression and the formation of protective proteins encoded by them. Often, mechanical damage to plants is accompanied by their infection, since a wound surface opens through which pathogens penetrate into the plant. In addition, phytopathogenic microorganisms can live in the oral organs 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 leaf covers with a stylet proboscis and
Rice. 2. Scheme of interaction of a pathogen cell with 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 (CS); 5 - chitinases and other glycosylases that act destructively on the pathogen CS; 6 - elicitors - fragments of the pathogen 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 the activation of peroxidases and increased synthesis of lignin, 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 rose leafhopper, unlike other representatives of the leafhopper, sucks out the contents of the cells. Cicadas cause less damage to plant tissues than leaf-eating insects, however, plants can react to it in the same way as to the infection of plants associated with it.
Upon contact with plants, pathogen cells secrete 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. More than 20 host-specific toxins produced by pathogenic fungi have been described so far.
Rice. 3. Phytotoxic compound from Cochlio-bolus carbonum
Bacteria and fungi also form non-selective toxins, in particular fusicoccin, erihoseten, coronatin, phase-olotoxin, syringomycin, tabtoxin.
One of the host-specific toxins released
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, the synthesis of which occurs outside the ribosomes and which contain residues of D-amino acids. For example, the host-specific toxin from Cochliobolus carbonum has a tetrapeptide ring structure (D-npo-L-ana-D-ana-L-A3JJ), where the last abbreviation is 2-amino-9,10-epoxy-8-oxo-de -canoic acid (Fig. 3). The toxin is produced in pathogen cells by 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 body of the host plant, the toxin causes inhibition of histone deacetylases and, as a consequence, histone overacetylation. This suppresses the plant's defense response to pathogen infection.
Another type of compounds secreted by pathogens is called elicitors (from the English elicit - to identify, cause). The collective term "elicitor" was proposed for the first time in 1972 to designate chemical signals arising at the sites of infection of plants by pathogenic microorganisms and has become widespread.
Elicitors play the role of primary signals and set in motion a complex network of processes of induction and regulation of phytoimmunity. This is manifested in the synthesis of protective proteins, nonvolatile plant antibiotics - phytoalexins, in the isolation of antipathogenic volatile compounds, etc. At present, the structure of many natural elicitors has been characterized. Some of them are produced by microorganisms, others (secondary elicitors) are formed during the enzymatic cleavage of high-polymer compounds of the cuticle and polysaccharides of the cell walls of plants and microorganisms, and others are stress phytohormones, the synthesis of which in plants is induced by pathogens and abiogenic stressors. Among the most important elicitors are protein compounds excreted by pathogenic bacteria and fungi, as well as viral envelope proteins. Small (10 kDa), conservative, hydrophilic, cysteine-enriched elicitins secreted by all studied species can be considered the most studied protein elicitors.
Phytophthora and Pythium. These include, for example, cryptogein.
Elicitins cause hypersensitivity and death of infected cells, especially in plants of the genus Nicotiana. The most intensive formation of elicitins by phytophthora occurs during the growth of mi-
It was found that elicitins are capable of transporting sterols across membranes, since they have a sterol-binding site. Many pathogenic fungi are unable to synthesize sterols themselves, which explains the role of elicitins not only in the nutrition of microorganisms, but also in inducing the defense response of plants. A 42 kDa glycoprotein elicitor was isolated from Phytophthora. Its activity and binding to the plasma membrane protein receptor, the monomeric form of which is a 100 kDa protein, was provided 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 to a number of small peptides, such as carboxypeptidase inhibitors and ion channel blockers, and bound to the plasma membrane receptor protein, apparently causing its modulation, dimerization, and transmission of a signal impulse to signaling systems. The larger Cladosporium fulvum pre-protein of 135 amino acids is post-translationally processed into an elicitor protein of 106 amino acids. The elicitor proteins produced by the rust fungus Uromyces vignae are two small polypeptides of 5.6 and 5.8 kDa, unlike other elicitins in properties. Among bacterial protein elicitors, harpins are the most studied.
Many phytopathogenic bacteria produce elicitor oligopeptides (their synthetic
sian analogues), corresponding to the most conservative regions of the protein - flagellin,
which is an important factor in the virulence of these bacteria. A new elicitor protein has been isolated from Erwinia amylovora, the C-region of which is homologous to the pectate lyase enzyme, which can cause the appearance of elicitor oligomeric fragments - pectin degradation products. The pathogenic bacterium Erwinia carotovora excretes the elicitor protein harpin and the enzymes pectate lyase, cellulase, polygalacturonase, and proteases that hydrolyze the polymeric components of the host plant cell walls (see Fig. 2), resulting in the formation of oligomeric elicitor molecules. Interestingly, the pectate lyase secreted by Erwinia chrysanthemi,
acquired activity as a result of extracellular processing. Some lipids and their derivatives are also
elicitors, in particular 20-carbon polyunsaturated fatty acids of some pathogens - arachidonic and eicosapentaenoic [Ilyinskaya et al., 1991; Ozeretskovskaya et al., 1993; Ozeretskovskaya, 1994; Gilyazetdinov et al., 1995; Ilyinskaya et al., 1996a, b; Ilyinskaya, Ozeretskovskaya, 1998], and their oxygenated derivatives. The review paper [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 eliciting effect, but their lipid part, which is arachidonic (eicosatetraenoic) and eicosapentaenoic 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 conversion in plant tissues of C20 fatty acids (hydroperoxy-, hydroxy-, oxo-, cyclic derivatives, leukotrienes) formed in host plant cells with the help of an enzymatic lipoxygenase complex (substrates of which can be both C,8 and C20 polyene fatty acids) had a strong influence on the defense response of plants. This is apparently due to the fact that there is no oxygen in uninfected plants.
derivatives of 20-carbon fatty acids, and their appearance as a result of infection leads to dramatic results, for example, the formation of necrosis around 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 could be octadecanoids rather than eicosanoids. Syringolides also have eliciting properties [L et al., 1998] and cerebrosides - sphingolipid compounds. Cerebrosides A and C isolated from Magnaporthe grisea were the most active elicitors for rice plants. Cerebroside degradation products (fatty acid methyl esters, sphingoid bases, glycosyl-sphingoid bases) showed no elicitor activity.
Some elicitors are formed as a result of the action on plant tissues of hydrolases released by pathogens. The purpose of hydrolases is twofold. On the one hand, they provide nutrition for pathogens necessary for their development and reproduction, on the other hand, they loosen the mechanical barriers that prevent pathogens from penetrating into their habitats in plants.
One such barrier is the cuticle, which consists mainly 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 epoxidized, long-chain dicarboxylic acids, etc. In cutin, most of the primary alcohol groups participate in the formation of ether bonds, as well as some of the secondary alcohol groups that provide crosslinks 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. In addition, in the sub
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 some of the components of suberin. The products of the cutinase reaction were various oxygenated fatty acids and alcohols, mainly 10,16-dihydroxy-CK- and 9,10,18-trihydroxy-C|8-acids, which are signal molecules that induce the formation and release of additional amounts of cutinase, which "corrode" cutin and facilitate 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 di- and trihydroxy acids was only 15 min, while the release of additional cutinase was twice as long. Damage to the cutinase gene in Fusarium solani greatly reduced the virulence of this fungus. Inhibition of cutinase with 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 into the living cells of the host. In any case, pathogens encounter yet another mechanical barrier - cell walls, consisting of various polysaccharides and proteins and in most cases reinforced with a rigid polymer - lignin [Tarchevsky, Marchenko, 1987; Tarchevsky and Marchenko, 1991]. As mentioned above, in order to overcome this barrier and ensure 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
degradation does not appear simultaneously. For example, pectylmethylesterase was also present in non-inoculated Erwinia carotovora subsp. atroseptia in the tissues of potato tubers, while polygalacturonase, pectate lyase, cellulase, protease, and xylanase activities appeared 10, 14, 16, 19, and 22 h after inoculation, respectively.
It turned out that oligosaccharide degradation products of plant cell wall polysaccharides have elicitor properties. However, 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 release outside the plasma membrane of enzymes - chitinase and β-1,3-glucanase, which hydrolyze the polysaccharides chitin and β-1,3-polyglucans of pathogen cell walls, which leads to inhibition 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.
Oligosaccharide elicitors, their structure, activity, receptors, their “switching on” of cell signaling systems, induction of expression of protective genes, synthesis of phytoalexins, hypersensitivity reactions, and other plant responses are the subject of a number of review articles.
In the laboratory of Elbersheim, 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 suggested that they be considered a new class of hormones ("oligosaccharins", as opposed to oligosaccharides that have no activity). The formation of oligosaccharides as a result of the hydrolysis of polysaccharides, and not in the course of synthesis from monosaccharides, was shown by the example
To narrow the search results, you can refine the query by specifying the fields to search on. The list of fields is presented above. For example:
You can search across multiple fields at the same time:
logical operators
The default operator is AND.
Operator AND means that the document must match all the elements in the group:
research development
Operator OR means that the document must match one of the values in the group:
study OR development
Operator NOT excludes documents containing this element:
study NOT development
Search type
When writing a query, you can specify the way in which the phrase will be searched. Four methods are supported: search based on morphology, without morphology, search for a prefix, search for a phrase.
By default, the search is based on morphology.
To search without morphology, it is enough to put the "dollar" sign before the words in the phrase:
$ study $ development
To search for a prefix, you need to put an asterisk after the query:
study *
To search for a phrase, you need to enclose the query in double quotes:
" research and development "
Search by synonyms
To include synonyms of a word in the search results, put a hash mark " #
" before a word or before an expression in brackets.
When applied to one word, up to three synonyms will be found for it.
When applied to a parenthesized expression, a synonym will be added to each word if one was found.
Not compatible with no-morphology, prefix, or phrase searches.
# study
grouping
Parentheses are used to group search phrases. This allows you to control the boolean logic of the request.
For example, you need to make a request: find documents whose author is Ivanov or Petrov, and the title contains the words research or development:
Approximate word search
For an approximate search, you need to put a tilde " ~ " at the end of a word in a phrase. For example:
bromine ~
The search will find words such as "bromine", "rum", "prom", etc.
You can optionally specify the maximum number of possible edits: 0, 1, or 2. For example:
bromine ~1
The default is 2 edits.
Proximity criterion
To search by proximity, you need to put a tilde " ~ " at the end of a phrase. For example, to find documents with the words research and development within 2 words, use the following query:
" research development "~2
Expression relevance
To change the relevance of individual expressions in the search, use the sign " ^
" at the end of an expression, and then indicate the level of relevance of this expression in relation to the others.
The higher the level, the more relevant the given expression.
For example, in this expression, the word "research" is four times more relevant than the word "development":
study ^4 development
By default, the level is 1. Valid values are a positive real number.
Search within an interval
To specify the interval in which the value of some field should be, you should specify the boundary values in brackets, separated by the operator TO.
A lexicographic sort will be performed.
Such a query will return results with the author starting from Ivanov and ending with Petrov, but Ivanov and Petrov will not be included in the result.
To include a value in an interval, use square brackets. Use curly braces to escape a value.
