What self-defense mechanisms do plants use

Defense against predators: Self-defense in the plant kingdom

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1 quarterly publication of the Natural Research Society in Zurich (2002) 147/4: Defense against predators: Self-defense in the plant kingdom Andreas Schaller, Zurich Summary The face of the earth is shaped by plants, regardless of the herbivorous diet of half of all insect species. The resistance of plants to herbivorous insects is due to an abundance of factors that have evolved over millions of years of mutual adaptation. Anatomical features of plants represent structural barriers that make access difficult for insects. Toxic ingredients contribute to resistance just like the natural enemies of the pests. Many resistance factors are only induced by insect infestation, such as the wound defense reaction in Solanacea, which is the system that has been best investigated experimentally. After being wounded by insect caused damage, systemin is formed, a messenger substance that triggers systemic resistance. The defense reaction manifests itself in a dramatic change in gene expression. As a result, defense proteins accumulate in the leaves of the plant. With the consumption of the leaves, these proteins are absorbed and then impair the growth and development of the insect. This thesis tries to give an overview of the diversity and the fascinating complexity of the resistance-determining factors in plants. Herbivore resistance: Self-defense in the plant kingdom Plants are the most prevalent organisms on this planet, despite the abundance of insect species, half of which feed on living plant tissue. Plant resistance is due to a multitude of characteristics that evolved in millions of years of reciprocal adaptation. Plant anatomical features constitute structural barriers that prevent access to the predator. Toxic secondary compounds contribute to resistance as do the natural enemies of insect herbivores. Many determinants of resistance are induced only after insect attack. The wound response in Solanaceae is the one system that has been investigated in more detail. Wounding by insect herbivores results in the release of systemin, a signal molecule that induces systemic resistance of the plant. Hallmark of the wound response is a dramatic change in gene expression resulting in the accumulation of defense proteins in the plant leaves. Upon consumption of the leaves these proteins are taken up by the insect and, as a consequence, insect growth and development is retarded. In this article, a synopsis is attempted of the diversity and fascinating complexity of the factors contributing to plant resistance against herbivores. Key words: herbivorous insects coevolution microarray analysis resistance signal transduction system in wounding 1 INTRODUCTION The harmful effects of the Colorado potato beetle (Leptinotarsa ​​decemlineata) are well known. Both the larva and the adult insect are herbivores. Herbivores are animals that feed on living plant material, in this case the leaves of the potato plant. From the farmer's point of view, the Colorado beetle is a predator of the plant, and it will appear again and again as a representative of all herbivorous insects in the course of this article. The plant on the other hand is by no means helpless in its predation

2 Andreas Schaller delivers. It has a number of different ways to ward off predators and these will be the subject of this article. The defense strategies of plants against herbivorous insects are presented and the decisive factors that contribute to the resistance of the plant are shown. In the following, a selected example is used to explain which biochemical and molecular processes underlie the plant defense reactions and how such processes can be experimentally investigated. Plants and insects are by no means always opponents in the struggle for existence. On the contrary, they are interdependent in a variety of ways. If we want to understand plant resistance mechanisms, then we cannot ignore these interrelationships. It is therefore necessary to take a brief look at the history of the development of plants and insects before discussing the defense strategies of plants. 2 ALTERNATIVE ADAPTATIONS OF PLANTS AND INSECT COEVOLUTION 135 million years ago, at the beginning of the Cretaceous period, the era of the tree ferns, horsetail and moss, which previously covered the planet in huge forests, was long gone. At this time, naked-seed plants (gymnosperms), such as conifers and gingko, dominated. But now the first flowering plants (angiosperms) appeared, and they had a tremendous success on the world stage: 70 million years later, towards the end of the Cretaceous, they were already absolutely predominant (and still are). Today we know about plant species, of which angiosperms are. The flowering plants are extremely diverse and colonize all imaginable habitats. In turn, they provide the insects with a habitat and food. With the developing diversity of flowering plants, a myriad of new niches opened up for insects, and the colonization of these ecological niches accelerated the evolution of insects. It is obvious that the resulting diversity of insects could not remain without influence on the plant world. Most flowering plants are pollinated by insects; they are dependent on the insects for the targeted transmission of the pollen. In adaptation to the different forms and diets of the insects, the most varied of flower shapes have developed, which has led to strong mutual dependence of the respective plant and the pollinating insect species. These mutual adaptations are most evident in the orchid family, the youngest, but species-richest family of higher plants. Many orchids, especially those of the Ophrys genus, are pollinated by just one insect, a single species of bee or wasp. Outwardly, the flower resembles the female of the respective species. The corresponding male is not only attracted by the appearance of the flower. In many cases, the plant also produces fragrances that are similar to the sex attractant of female bees. Attracted by this optical and chemical mimicry 1, the males pollinate the flower in a process known as pseudo-population. As a result of such mutual adaptations, a process that is also known as coevolution, today's species diversity of insects and flowering plants came about. Within insects, about half of all species are herbivorous, i.e. they feed on plants. It is easy to imagine that the relationships between herbivorous insects and their host plants have led to mutual adaptations similar to those just described for flower development (a comprehensive discussion of mutual adaptations between plants and insects can be found in BENZ, 1998). 3 PLANT RESISTANCE MECHANISMS The Colorado potato beetle, as the name suggests, is a pest on potato plants; but he also accepts tomatoes, aubergines and some other representatives of the nightshade family, the Solanaceae. The Colorado beetle eats a few selected plant species and is therefore referred to as oligophagous 2, which corresponds to the majority of the 1 As mimicry, in the narrower sense, is the imitation of body shape, color, smell or behavior of an animal that is due to its ability to fight or its bad taste is protected from enemies. Mimicry is used to deter predators. Many hover flies (Syrphidae) imitate wasps or bees in this way. The term is used here in a broader sense; the optical and chemical mimicry of the orchids is not a deterrent, but rather attractive to the pollinating insects. In contrast to this, an imitation costume that serves as camouflage is called a mimetic. The bodies of many insects resemble twigs, leaves or bird droppings and are therefore difficult to spot by their predators. 2 Insects that, as outspoken specialists, feed on only one plant species are called monophag, those that eat a few related species are called oligophagous. Generalists who accept a wide range of host plants from different plant families are called polyphagous. 142

3 Defense against predators: Self-defense in the plant kingdom of herbivorous insects applies. This suggests that it is specially adapted to Solanaceae. Other plants, on the other hand, seem to be resistant to it. But there are also differences within the Solanaceae: Why are some species more sensitive than others? On the other hand, why are potato plants resistant to most other feeding pests? The aim here is to ask about the resistance mechanisms that have developed in plants when they adapt to herbivorous insects, and it is these resistance mechanisms that we should now be concerned with. Understanding such resistance mechanisms is not only an esoteric, but also an economic and social interest. Every year, regardless of the use of chemical pesticides and weedkillers, large parts of the harvest are lost worldwide (around 35%), in almost equal proportions due to plant diseases (12%), weeds (10%), but also insect caused damage (13th century) %; PIMENTEL, 1991). By understanding the natural resistance and defense mechanisms, we hope to be able to avoid the losses caused by predators and at the same time dispense with the use of pesticides. Before going into more detail about the resistance mechanisms of plants, the options available to agriculture in combating pests should be considered. First of all, there are purely mechanical means. For example, as a preventive measure, you can put up a net to keep the birds away from the cherries. Or as a reaction to the infestation, the Colorado beetle and its larvae can be collected. This may be tedious and inefficient, but it was the best method before the development of chemicals, i.e. pesticides. Both mechanical and chemical means are mostly directed against the pest. In addition, indirect, biological pest control is also conceivable, and this means control by the natural enemies of the pests, such as the use of ladybirds against the aphids on the roses in our gardens. The resistance mechanisms of the plant fall into exactly the same categories (Fig. 1). They can be mechanical, chemical or biological in nature; as a preventive measure, they can be constantly pronounced, i.e. constitutive, Fig. 1. The factors that contribute to resistance to herbivorous insects can be classified as mechanical (structural barriers), chemical (secondary plant constituents) or biological (including other organisms). They can always be (constitutive) pronounced in a preventive manner or they can be induced in response to pest infestation. You can turn against the pest directly or indirectly involve its enemies. Fig. 1. Mechanical (structural barriers), chemical (secondary plant compounds) or biological (involving other organisms) factors contribute to herbivore resistance. These factors can be either constitutively present, or induced after pest attack. They either aim directly at the predator, or in an indirect fashion, involve the natural enemies of the predator. or they can be triggered, ie induced, by the infestation with the pest. They can target the pest directly, or they can include the pests' natural enemies and then act indirectly. This will be illustrated in the following with a few examples. 3.1 Mechanical resistance factors Mechanical resistance factors are barriers that make it more difficult for the pest to access and thus increase the resistance of the plant. Lignified or silicified cell walls represent such barriers. Another form of mechanical defense should be well known: Who has not been injured on the thorns of a cactus or the thorns of a rose? Thorns and prickles are very effective in repelling predators. However, the mechanical barriers are not always so conspicuous: if they are directed against small insects, then much smaller structures are also effective. Most of the time, hairs (trichomes) 3 are found on the leaf surface, which among other things serve to repel insects. In hair, mechanical and chemical mechanisms are often combined, for example in 3 hair or trichomes are appendages of the epidermis. A distinction is made between single-cell and multi-cell trichomes, which can have a protective or supportive function. Glandular hairs secrete secondary phytonutrients, which often serve to protect against predators. Hair can also act as protection against dehydration. In this case, the cells have died and the hair appears white. 143

4 Andreas Schaller stinging hair of the nettle (Urtica dioica). When touched, the tip of the hair breaks off and the remaining shaft is a real injection cannula, through which substances irritating to the pest, including histamine, acetylcholine and formic acid, are injected. Amazingly, stinging hairs are not only a constitutive, but also an inducible resistance factor: If stinging nettles are wounded, for example on a grazed meadow, the density of the stinging hairs on all newly developing leaves will be greater than on the leaves that developed before the wounding (PULLIN and GILBERT, 1989). 3.2 Chemical resistance factors The behavior of insects is regulated in a variety of ways by chemical substances in the plant. The chemistry of the plant has a decisive influence on the selection of the host plant, the laying of eggs and the feeding behavior of the insects. What kind are these substances? Every living cell, be it a plant cell or an animal cell, has a metabolism, the primary metabolism, which is essential for the cell to function. In addition, there is the so-called secondary metabolism. The substances of the secondary metabolism are not necessary for the life of the individual cell. Their function only becomes clear in the interaction of the cells or in the interaction of the organism with its environment. The function of secondary plant constituents can be illustrated well using the example of flower pigments. The dyes are not necessary for the survival of the cells that produce them. But they attract the flower-pollinating insects and in this context their function becomes clear: They are important for the reproduction of the organism. To date, more than secondary phytonutrients have been identified, and many of them contribute to resistance to feeding pests. Secondary plant constituents can have a deterrent effect, then the insect will avoid the plant from the outset. It can be bitter substances, taste spoilers so to speak, or the substances can have a downright toxic effect. Many of these substances are well known because humans also make use of their toxic effects. Above all, the alkaloids should be mentioned here, such as the nicotine of tobacco, the morphine of the opium poppy, strychnine or quinine. The terpenoids represent another large class of phytochemicals. These include, for example, menthol in mint, gossipol in cotton and glucosides that have an effect on the heart, such as digitoxigenin in thimble. Others are derived from phenols, such as tannins or coumarin. With the production of poisons, the plant poses a problem for itself, because these substances are often not only toxic to the insects, but also harmful to the plant itself. The plant must therefore have developed strategies to protect itself from its own poison 4. Such a strategy will be explained using the example of coumarin. The coumarin serves as protection against eating in the sweet clover (Melilotus albus) and the woodruff (Galium odoratum). In order not to expose oneself to the toxic effect, not the coumarin itself, but a non-toxic precursor, namely the coumaric acid glucoside, which is stored in the vacuole (Fig. 2). For the formation of the Cu Fig. 2. Coumarin as a chemical resistance factor. The formation of coumarin from its inactive precursor, the coumaric acid glucoside, requires the enzymatic activity of a ß-glucosidase. The precursor (in the vacuole) and the ß-glucosidase (in the cell wall) are constitutively formed, but stored spatially separated from each other. Only when wounded do the precursor and enzyme come together and the coumarin is released (according to MATILE, 1984). Figure 2. Coumarin as chemical factor of resistance. The formation of coumarin from glucosylcoumaric acid requires the activity of a ß-glucosidase. Both the precursor and the ß-glucosidase are formed constitutively, but stored in different compartments (the vacuole and the cell wall, respectively). Upon wounding, the enzyme acts upon the precursor resulting in the formation of coumarin (redrawn from MATILE, 1984). 4 Such a teleological description of resistance factors is chosen here, as in the further course of this work, only for the sake of better clarity. Of course, the plant is unaware of the problem of toxic substances, and the adaptations that allow such substances to accumulate are not the result of a conscious choice. 144

5 Defense against predators: Self-defense in the marine vegetable kingdom requires an enzyme, a glucosidase. However, this enzyme is formed in a spatially separate manner from the precursor. If the plant is now wounded, the vacuole breaks open and the precursor comes into contact with the glucosidase: the glycosidic bond is hydrolyzed, the coumaric acid is released and spontaneously continues to react to form coumarin. In a very similar way, the dhurrin serves as protection against feeding on millet (sorghum bicolor). Dhurrin is a cyanogenic glucoside and is not itself toxic. After being wounded, however, hydrocyanic acid is released, which serves as protection not only against insects, but also against snails and mammals (MATILE, 1984). 3.3 Biological resistance factors Defensive strategies that include the natural enemies of the pests are referred to as biological resistance factors. A well-known example of this is the symbiosis (Greek: symbiosis = living together) between tree acacias in Central America or Africa and ants (JANZEN, 1966). These tree acacias (e.g. Acacia cornigera in Central America) have very large hollow thorns, so-called domatia, which offer the ants a habitat and protection. The ant species of such symbioses (in this case the species Pseudomyrmex ferruginea) are unusually aggressive. They attack any herbivorous insect that might attack the tree. So the tree benefits from the presence of the ants. The ants, for their part, benefit from a sugar-rich nectar that the plant makes available to them in extra-floral nectaries. This arrangement seems to be of great mutual benefit for both partners, because such dependencies have developed several times independently of one another in the course of evolution (KLÖTZLI, 1993). In our latitudes, too, there are examples of such complex interactions, such as the defense reactions of cabbage plants (Brassica oleracea) against the cabbage whitefly (Pieris brassicae). The larva of the cabbage whitefly is an outspoken specialist. It feeds on cabbage and is specially adapted to this food source. She has learned, so to speak, to deal with the chemical defense substances in cabbage (so-called glucosinolates), which is why this resistance factor no longer works. So the plant needs help, which it finds in a wasp (Cotesia glomerata). This wasp is a parasitoid; it lays its eggs in the cabbage white caterpillar, which then serves as a host for the wasp larvae. The cabbage recruits Cotesia glomerata by synthesizing and emitting volatile substances in response to being wounded by the pests, fragrances that can be interpreted as a chemical cry for help. The scents are recognized by the wasp and signal the presence of the herbivorous larva. The wasp is attracted, parasitizes the larva and thus combats the cabbage pest (MATTIACCI et al., 1994). 4 INDUCED RESISTANCE THE WOUND REACTION IN SOLANACES The arsenal of possibilities thus includes mechanical, chemical and biological resistance factors. These can be constitutively expressed or induced by infestation, they can turn directly against the pest or also include their enemies (Fig. 1). In the following, the biochemical and molecular processes on which these resistance mechanisms are based will be discussed in more detail. It is of particular interest how it is actually possible for the plant to react, ie. H. So only after infestation by a feeding pest develop new resistance factors. Only one system will be presented as an example: namely the wound defense reaction in Solanaceae, especially in tomato plants. This system has been studied intensively over many years and is therefore a model for similar resistance mechanisms (RYAN, 2000). This reaction is a chemical defense mechanism that is induced by infestation and that is directed directly against the pest. Thirty years ago Green and Ryan (GREEN and RYAN, 1972) observed that tomato plants synthesize a proteinase inhibitor (PI) and accumulate in the leaves after being infected with adult Colorado beetles or their larvae, or after mechanical wounding. This proteinase inhibitor is a small protein that is able to inactivate the digestive enzymes trypsin and chymotrypsin. When the leaves are eaten, the proteinase inhibitor enters the insect's digestive tract, the digestive enzymes are inhibited, and as a result, deficiency symptoms occur in the insect, which delay growth and development. The authors suspected that this reaction is an inducible defense mechanism that increases resistance to insects. Further work has shown that in addition to this proteinase inhibitor, a whole series of other defense proteins (so-called "systemic wound response proteins", SWRPs) are formed, which together contribute to the induced resistance (BERGEY et al., 1996; SCHALLER et al., 1995). 145

6 Andreas Schaller 4.1 Signal transduction in the wound reaction How does the accumulation of defense proteins (SWRPs) occur? It is true that the plant is still the same after being wounded; it is genetically unchanged. Their physiological state, however, characterized by the accumulation of SWRPs, is different. The synthesis of the defense proteins is an expression of a change in gene expression: the genes of the SWRPs have been activated. There is an increased transcription of the genes; this leads to an accumulation of the corresponding messenger RNAs (mrnas), and these are translated into the SWRPs in the process of translation 5, which then accumulate. Interestingly, all of these processes are not limited to wounded plant parts. The expression of the SWRPs is induced to the same extent in wounded as in unwounded leaves. Green and Ryan therefore postulated the existence of a mobile factor which is formed at the site of the wounding, which is then distributed in the shoot of the plant and which is able to trigger the expression of the defense genes even in unwounded parts of the plant (GREEN and RYAN, 1972; Fig. 3). This initially hypothetical factor has been searched intensively for many years. The work culminated in the discovery of Systemin in 1991 (PEARCE et al., 1991). Systemin is an oligopeptide 6, consisting of 18 amino acids, making it the first peptide with hormone-like effects that was ever found in plants. In the following year, the gene for systemin was also characterized in the same working group. It turned out that this gene codes for a larger precursor protein called Prosystemin. Prosystemin consists of a chain of 200 amino acids, and the 18 amino acids of the systemin sequence are found embedded near the carboxy-terminal end of the precursor (Fig. 4). Fig. 4. Schematic representation of systemin and prosystemin. The prosystemin polypeptide of 200 amino acids in length is a precursor of systemin. The 18 amino acids of the systemin sequence are found embedded near the carboxy terminus of the precursor. Fig. 4. Schematic representation of Systemin and Prosystemin. The 200 amino acid polypeptide prosystemin is a precursor of systemin. The 18 amino acids of the system in sequence are located close to the carboxy terminus of the precursor. Fig. 3. Schematic representation of the wound reaction in Solanaceae. As a result of insect damage or mechanical wounds, a messenger substance is released which is distributed in the plant and triggers the defense reaction in local and systemic tissues. Figure 3. Schematic representation of the wound response in Solanaceae. As a result of insect attack or mechanical wounding, a signal molecule is released at the wound site and translocated throughout the plant. It induced the defense response locally as well as in systemic, undamaged tissues. 4.2 Systemin is sufficient and necessary for triggering the wound response Today it is undisputed that systemin or prosystemin play a central role in mediating the wound response. This knowledge is based on experiments which show that (pro) systemin is both sufficient and necessary to develop the wound response. From a large number of experiments that prove this, only two should be selected as examples. So if systemin suffices 5 The relationship between DNA, RNA and protein is described by the so-called “central dogma of molecular biology”. According to this, the genetic information is contained in the base sequence of the DNA sequence and encoded in a code according to which three successive bases each encode an amino acid. This information, conveyed by the mrna, serves as a template for the synthesis of the corresponding protein. The flow of information therefore always runs from nucleic acid (DNA, mrna) to the protein, never the other way around. 6 Like proteins, peptides consist of amino acids linked by peptide bonds. Depending on the number of amino acids (length of the chain), a distinction is made between di-, tri-, tetra-, oligo- or polypeptides. 146

7 Defense against predators: self-defense in the plant kingdom should trigger the wound response, then one should expect that the synthetically produced peptide induces the same gene expression changes as the wound itself. To test this hypothesis, it requires an experimental approach that allows the Compare gene expression in unwounded tomato plants with that in plants treated with systemin. The so-called microarray analysis is suitable for this, with which the relative concentration of certain mrnas in two RNA extracts can be determined (Fig. 5). In this experiment, the mrna of the two tomato plants (untreated control plant, systemin-treated plant) is first isolated. Then the entire mrna population is rewritten into complementary DNAs, in cdnas. This process is known as reverse transcription, and in this process all cdna molecules are marked with a fluorescent dye: red for the treated plants, green for the control. The two samples are then mixed. What does that mean for the mixed color? If a particular gene is wounded, then the corresponding mrna will accumulate and the mixture will be dominated by the red color. Otherwise, should the expression of a gene be suppressed, the concentration of the corresponding transcript will be correspondingly reduced, and consequently the mixture will be predominantly green. If the expression of a gene does not change, then the mrnas are present in a ratio of 1: 1. The color-marked mixture of cdnas is then placed on a microarray which comprises any number of DNA fragments in a grid arrangement. Each point on the grid contains DNA from a specific, known to us tomato gene. The marked cdnas will now bind to the DNA of the corresponding gene in a process known as hybridization. Then the red and green color component is determined for each gene (each point in the grid) and the expression ratio of wounded compared to healthy plants can easily be determined. The result of such an experiment is shown for a small selection of genes in Fig. 6, which shows systemin-induced changes in gene expression 1, 3 and 6 hours after treatment of the plants. After 6 hours a strong accumulation of the mrnas of the SWRPs can be observed (PI-I, PI-II, CPI, CDI, LAP, PPO, TD). In contrast, the transcripts of proteins that contribute to resistance to pathogenic microorganisms are not induced (PR-1a PR-3b). In addition, there is a rapid but temporary accumulation of a number of other Fig. 5. Sequence of a microarray experiment for the analysis of gene expression. The entire mrna is isolated from treated as well as from control plants and transcribed into cdna by reverse transcription. The two cdna samples are marked with different fluorescent dyes. The cdnas are mixed and hybridized against a grid of DNA fragments of known genes. For each point of the grid, the relative proportion of the two dyes is determined in order to derive the expression ratio of the corresponding gene (further details in the text). Fig. 5. Design of a microarray experiment for the analysis of gene expression. In a first step, mrna is isolated from both treated and control plants. The mrna is then converted into cdna by reverse transcription and, in that process, the two samples are each labeled with a different fluorescent dye. The cdna samples are mixed and hybridized against an array of DNA fragments. For each point of the array, the relative intensity of the two dyes is determined, and the expression ratio of the respective genes is deduced (further detail is given in the text). mrnas (LOXD, AOS1, AOC, OPR3), which code for enzymes of jasmonic acid biosynthesis (the relevance of this finding is discussed in more detail in the following chapter). These gene expression changes coincide with 147

8 Andreas Schaller Fig. 6. Systemin-induced gene expression changes. Changes in gene expression in tomato plants 1, 3 and 6 hours after treatment with Systemin were examined with the aid of microarray analysis. The lighter a field, the greater the accumulation of the corresponding transcript in treated versus control plants. The upper block shows genes of wound defense (SWRPs: proteinase inhibitor I and II (PI-I, PI-II), carboxypeptidase inhibitor (CPI), cathepsin D inhibitor (CDI), leucine aminopeptidase (LAP), polyphenol oxidase (PPO) and threonine deaminase (TD )), as well as genes of the pathogen defense ("pathogenesis-related" (PR) proteins 1a, 2a, 3a, 1b, 2b, 3b). The lower block shows some genes for which a role in signal transmission is being discussed (Prosystemin (ProSYS), lipoxygenase (LOX) C and D, allene oxide synthase (AOS) 1 and 2, allene oxide cyclase (AOC), oxophytodienoic acid reductase ( OPR) 1, 2 and 3, hydroperoxide lyase (HPL), divinyl ether synthase (DES) and a transcription factor (LeJA2). Fig. 6. Systemin-induced changes in gene expression. Systemin-induced changes in gene expression were analyzed in tomato plants 1, 3, and 6 hours after treatment with systemin using cdna microarray analysis. The brighter a field, the more abundant was the respective mrna in treated versus control plants. The upper panel shows wound response genes (SWRPs: proteinase inhibitors I and II (PI-I, PI-II), carboxypeptidase inhibitor (CPI), cathepsin D inhibitor (CDI), leucine aminopeptidase (LAP), polyphenol oxidase (PPO) and threonine deaminase (TD)), as well as pathogen defense genes (pathogenesis-related (PR) -proteins 1a, 2a, 3a, 1b, 2b, 3b). The lower panel shows genes with putative roles in signal transduction (prosystemin (Pro- SYS), lipoxygenase (LOX) C and D, alleneoxid synthase (AOS) 1 and 2, alleneoxid cyclase (AOC), oxo-phytodienoic acid reductase (OPR) 1, 2 and 3, hydroperoxide lyase (HPL), divinylether synthase (DES) and a transcription factor (LeJA2). men largely agree with the changes observed after wounding. It turns out that wounding is not necessary: ​​Systemin is enough to trigger the wound reaction. In order to show that systemin is not only sufficient but also necessary for the wound reaction, transgenic plants (i.e. plants in whose genome additional DNA segments were introduced using genetic engineering methods) were used. The expression of prosystemin was literally switched off in transgenic tomato plants (MCGURL et al., 1992). To do this, they made an artificial gene called an antisense gene. This gene codes for a mrna that is complementary to the normal prosystemin mrna. This antisense gene suppresses the expression of prosystemin in transgenic plants. The resistance of the transgenic plants to the herbivorous larva of the tobacco hawk (Manduca sexta) was then compared with the resistance of normal tomato plants. It turned out that the transgenic plants are considerably more susceptible. The larvae of the tobacco hawk consumed significantly more leaf material from the transgenic plants and showed a significantly faster weight gain on these plants compared to the wild type (OROZCO-CARDENAS et al., 1993). Apparently, Prosystemin is indispensable for developing the wound reaction and the associated resistance to herbivorous insects. On the one hand, the experiment shows how effective this inducible resistance factor is, and it also makes it clear that Prosystemin is necessary for the wound reaction. 4.3 A model of wound signal transduction We have seen that (Pro) Systemin is sufficient and necessary for the development of the wound reaction. The question now arises as to how the (pro) systemin triggers the expression of the SWRPs. Which molecular processes are involved in this? A model developed by Farmer and Ryan (1992) summarizes our current ideas of how systemin and prosystemin work (Fig. 7). As a result of the wounding, systemin is released from its precursor protein, prosystemin. In target tissues, it interacts with a receptor protein on the cell surface. This interaction activates a lipase (a hydrolytic enzyme) that releases a fatty acid, linolenic acid, from membrane lipids. Through a series of enzymatic transformations, the linolenic acid is converted into another signaling molecule, jasmonic acid, which in turn induces the expression of the SWRPs

9 Defense against predators: Self-defense adorns the plant kingdom (FARMER and RYAN, 1992). In the previously described microarray experiment, in addition to the accumulation of mrnas of the SWRPs, a more rapid and transient accumulation of a second group of transcripts was observed. This mrnas (LOXD, AOS1, AOC, OPR3, see Fig. 7. A model of the wound signal transduction pathway. As a result of the wounding, systemin is released from its precursor. It interacts with a receptor that is permanent in the plasma membrane, which leads to the activation of a lipase.Linolenic acid is released and converted to jasmonic acid in a series of enzymatic steps. Jasmonic acid induces the defense genes (according to FARMER, 1992). Figure 7. A model of the wound signal transduction pathway. Systemin is released from its precursor as a consequence of wounding. It interacts with a plasma membrane receptor resulting in the activation of a lipase. Linolenic acid is released from membrane lipids. A series of enzymatic reactions results in the formation of jasmonic acid which ultimately induces defense gene expression (acc. To FARMER, 1992). Fig. 6 and Fig. 7) code for the enzymes that are responsible for jasmonic acid biosynthesis. The chronological sequence of gene activation observed here, namely the rapid induction of the genes for jasmonic acid biosynthesis, followed by the induction of the defense genes, is consistent with the wound signal transduction model shown in Fig. 7. Such a model must always be viewed as provisional, as hypothetical. It is only valid as long as it takes into account all experimental observations. A large number of experiments have been carried out over the past few years to test the durability of this model and in fact it seems to describe the realities quite well 7. Even so, many questions remain unanswered. For example, the lipase, the existence of which must be postulated for the release of linolenic acid, has not yet been identified. Furthermore, it is still unknown how signals are transmitted from the cell surface to the inside of the cell. In addition, research over the next few years will have to deal with even more complex problems: Do the results obtained in the greenhouse and laboratory really reflect the conditions in nature? There, wounding is always associated with potential microbial infections. The induced resistance to herbivory cannot be viewed in isolation from pathogen resistance. How do both relate to each other? In addition, the development of resistance factors is not in vain: the plant invests resources that could otherwise benefit the yield. So does induced resistance also have disadvantages? Which are they? All of these questions need to be clarified in the interest of environmentally friendly, sustainable agriculture that can largely dispense with the use of pesticides. 5 THANK YOU I would like to thank Christina Sie, who carried out the microarray experiment to analyze systemin-induced gene expression changes as part of her diploma thesis at ETH Zurich. I would also like to thank the ETH Zurich, the Swiss National Fund for the Promotion of Scientific Research and, above all, Prof. Dr. Nikolaus Amrhein for the support given. 7 For a more detailed discussion of signal transduction and the supporting experimental data, reference is made to the review articles in RYAN, 2000, and SCHALLER and RYAN, 1995. 149

10 Andreas Schaller 6 LITERATURE BENZ, G Mutual relationships between insects and plants as examples of coevolution. New year's paper of the Natural Research Society in Zurich 201. BERGEY, D.R., HOWE, G.A. & RYAN, C.A Polypeptide signaling for plant defensive genes exhibits analogies to defense signaling in animals. Proc. Natl. Acad. Sci. USA 93, FARMER, E.E. & RYAN, C.A Octadecanoid precursors of jasmonic acid activate the synthesis of wound-inducible proteinase inhibitors. Plant Cell 4, GREEN, T.R. & RYAN, C.A Wound-induced proteinase inhibitor in plant leaves: A possible defense mechanism against insects. Science 175, JANZEN, D Coevolution of mutualism between ants and acacias in Central America. Evolution 20, KLÖTZLI, F.A Ecosystems: structure, functions, disorders. UTB 1479, Fischer, Stuttgart, 447 pp. MATILE, P The toxic compartment of the plant cell. Naturwissenschaften 71, MATTIACCI, L., DICKE, M. & POSTHUMUS, M.A Induction of parasitoid attracting synomone in Brussels sprouts plants by feeding of Pieris brassivcae larvae: role of mechanical damage and herbivore elicitor. J. Chem. Ecol. 20, MCGURL, B., PEARCE, G., OROZCO-CARDENAS, M. & RYAN, C.A Structure, expression and antisense inhibition of the systemin precursor gene. Science 255, OROZCO-CARDENAS, M., MCGURL, B. & RYAN, C.A Expression of an antisense prosystemin gene in tomato plants reduces the resistance toward Manduca sexta larvae. Proc. Natl. Acad. Sci. USA 90, PEARCE, G., STRYDOM, D., JOHNSON, S. & RYAN, C.A Apolypeptide from tomato leaves induces wound-inducible proteinase inhibitor proteins. Science 253, PIMENTEL, D Diversification of biological control strategies in agriculture. Crop Protection 10, PULLIN, A.S. & GILBERT, J.E The stinging nettle, Urtica dioica, increases trichome density after herbivore and mechanical damage. Oikos 54, RYAN, C. A. The systemin signaling pathway: differential activation of plant defensive genes. Biochim. Biophys. Acta 1477, SCHALLER, A. & RYAN, C.A Systemin a polypeptide defense signal in plants. BioEssays 18, SCHALLER, A., BERGEY, D.R. & RYAN, C. A. Induction of wound response genes in tomato leaves by bestatin, an inhibitor of aminopeptidases. Plant Cell 7, Dr. Andreas Schaller, ETH Zurich, Institute for Plant Sciences, Universitätsstrasse 2, LFW E51, 8092 Zurich Current address: University of Hohenheim, Institute for Plant Physiology and Biotechnology (260), D Stuttgart, Germany 150