Plant Adaptations to Stressors & Herbivory: Toxin Production & Induced Responses, Manuais, Projetos, Pesquisas de Química

Baixe Plant Adaptations to Stressors & Herbivory: Toxin Production & Induced Responses e outras Manuais, Projetos, Pesquisas em PDF para Química, somente na Docsity! 14 Biochemical Plant Ecology Jeffrey B. Harborne 14.1 Introduction 503 14.2 Plant responses to the environment 503 14.3 Plant response to herbivory: toxin production 506 14.4 Constitutive defense mechanisms 508 14.5 Induced phytochemical response to herbivory 512 14.6 Conclusion 515 References 515 14.1 INTRODUCTION Much plant biochemistry is carried out on plants that are growing under optimal conditions in a glasshouse. Even where field-grown plants are studied, such organisms are unusually well pro- tected from stress. They are protected from nutritional inadequacies (by fertilization), from drought (by irrigation), from disease and pests (by spraying), from other plants (by herbicide treatment) and from mammalian herbivores (by fences). Few wild plants are so fortunate. Indeed, the natural vegetation that proliferates in uncultivated parts of the land surface has had to struggle to survive adversity. The evolutionary history of the plant kingdom is a story of constant adaptation to environmental stresses and to the threat of uncontrolled herbivory by grazing animals. Yet in spite of such environmental pressures, there is scarcely any habitat in the world where plants are not to be found. That plants have survived so well is due to their enormous flexibility to adapt to the diversity of growth conditions and environments that are present on this planet. Plants have adapted by modifying morphological and anatomical features (e.g. spines instead of leaves in desert habitats), through physiological adaptation (e.g. by reducing transpiration under a hot midday sun) or by biochemical means. In recent years, increasing attention has been paid to the ways that plants are biochemically adapted to their differing environ- ments. Biochemical adaptation may involve both primary and secondary metabolism. It may be focused on environmental variables (drought, frost, salinity, heavy metal toxicity) or on the PLANT BIOCHEMISTRY ISBN 0-12-214674-3 response of plants to other forms of life, whether they be bacteria, fungi, insects, molluscs or grazing mammals. The study of biochemical adaptation of plants to ecological factors is the subject matter of ecological biochemistry, or chemical ecology as it is often termed (Harborne, 1993). The purpose of this chapter is to provide a general introduction to such biochemistry. The emphasis will naturally be on the plant and its responses, although much of the literature of plant-animal interactions has been concerned with animals (especially insects) rather than with plants. Plant-microbial inter- actions will not be considered here since they are the subject matter of Chapter 13. The first section of this chapter will deal with plant adaptation to climatic and edaphic factors in the natural environment. The second section will consider the various toxins produced constitu- tively by plants in response to pressures of herbivory. The third section will discuss how far the distribution patterns of these toxins which accumulate in plants are correlated with a defensive role. The fourth and final section will be devoted to the ways that plants can respond dynamically to the damaging effects of grazing animals by producing new chemicals, becoming unpalatable in the process. 14.2 PLANT RESPONSES TO THE ENVIRONMENT The biochemical responses of plants to various forms of environmental stress are listed in Table 14.1. Each response involves one or more changes Copyright © 1997 Academic Press Ltd All rights of reproduction in any form reserved 504 JEFFREY B. HARBORNE Table 14.1 Biochemical responses of plants to differing environmental stresses Stresses Biochemical responses High temperatures (moist subtropics) Drought (deserts) Long term: C4 (Hatch-Slack) photosynthesis Short term: Heat-shock proteins Crassulacean Acid Metabolisms Increase in abscisic acid production Increase in proline or pinitol levels Low temperatures (arctic or alpine areas) Salt (sea coast, salt marsh) Accumulation of sugars and/or polyols Synthesis of anti-freeze proteins Increases in the proportion of unsaturated fatty acids in the chloroplast membrane lipids Accumulation of cytoplasmic osmotica (proline, glycinebetaine) Heavy metals Modification of the enzymes at the root surface (lead, zinc or nickel containing soils) Binding of metal to sites in the root cell wall Accumulation/detoxification as peptide chelates Accumulation/detoxification as organic acid chelates (e.g. nickel citrate) in the biochemistry of the plant cell. Thus it may require the development of a new metabolic pathway. This happens in the case of photosyn- thetic adaptation to subtropical and tropical climates, when the Hatch-Slack C4 cycle comes into operation (Chapter 2). It may result in the accumulation of a low-molecular-weight meta- bolite, from the carbohydrate or amino acid pool, which is otherwise only a trace component. An example is the imino acid proline, which may accumulate in both drought- and salt-stressed plants. Such a compound may have a special protective, e.g. osmotic, effect within the plant cell. This is true of proline and glycinebetaine, zwitterionic substances which are both formed under saline stress. Another response to stress may be a change in plant hormone levels. For example, plants subject to drought commonly produce higher levels of abscisic acid in the guard cells of the leaf, in order to close the stomata and reduce the loss of water by transpiration. Related analogs may increase, instead of abscisic acid, in a few plants; thus phaseic acid increases in concentration and pro- duces stomatal closure in drought-stressed vine plants, Vitis vinifera, A further change in biochemistry may be the synthesis of special proteins (e.g. heat-shock or anti-freeze proteins) which are not otherwise detectable in the unstressed plant. Such proteins may have a special role in limiting the damage within the cell or within the cell membranes, caused by that particular stress. In the case of adaptation to heavy metal (zinc, lead or copper) contamination in the soil, plants may respond by the synthesis of proteins or peptides, which have the ability through the presence of cysteine residues, to chelate those metals. Chelation of heavy metals with organic acids such as citric or oxalic is also possible in such plants. The biochemical responses to stresses in plants (Table 14.1) are probably more complex than is suggested here. There may be more subtle changes in cellular biochemistry that are not so readily detected as those listed in Table 14.1. Biochemical responses are often correlated with physiologi- cal or anatomical adaptations, which have often been studied in much greater detail by botanists (e.g. Fitter & Hay, 1987; Crawford, 1989). The biochemical responses to stress in plants are often paralleled by similar responses in animals. The ability to respond to mild temperature shock by the synthesis of heat-shock (HS) proteins within two hours of the shock is a general one and has been observed in microbes and animals as well as in plants. Typically in a plant such as the soybean, a two-hour preincubation at 40°C will protect against a two-hour treatment at 45°C, which otherwise would be lethal (Schoeffl et al,, 1984). Two groups of HS protein are produced of low and high molecular ranges and they persist as long as the increases in external temperature are maintained. These HS proteins are channeled within the cell to particular sites (e.g. the nucleus) to provide the necessary protection from the temperature increase. The ecological significance BIOCHEMICAL PLANT ECOLOGY 507 Table 14.2 The major classes of plant toxins Class of compund^ Example Toxicity Alkaloids (10000) Cardenolides (200) Cyanogenic glycosides (60) Furanocoumarins (400) Glucosinolates (150) Iridoids (250) Isoflavonoids (1000) Non-protein amino acids (400) Peptides (50) Polyacetylenes (650) Proteins (100) Quinones (800) Saponins (600) Sesquiterpene lactones (3000) Senecionine in ragwort, Senecio jacobaea Ouabain in Acokanthera ouabaio Amygdalin in Prunus amygdalus Xanthotoxin in Pastinaca sativa Sinigrin in Brassica oleracea Aucubin in Aucuba japonica Rotenone in Denis elliptica /^-Cyanoalanine in Vicia sativa seed Viscotoxin in Viscum album berry Oenanthetoxin in Oenanthe crocata roots Abrin in Abrus precatorius seed Hypericin in Hypericum perforatum leaf Lemmatoxin in Phytolacca dodecandra Hymenoxin in Hymenoxys odorata Cumulative poison in cattle (hepatotoxin) Heart poison; LD50 in rats 14mgkg"^ Universal toxin; fatal dose in humans ca. 50 mg Molluscicide; toxic to toads; allergenic in humans Damages thyroid, liver and kidney in cattle Toxic to mammals, birds and insects Insecticide and fish poison; LD50 in mice 2.8mgkg-i Neurotoxin; LD50 in rats 13.4 mg kg ^ Toxic to heart muscle in mammals Toxic to sheep and cattle Fatal dose 0.5 mg in humans Causes facial eczema in sheep Toxic to snails, LD50 1.5 mg 1~̂ Livestock poison ^Approximate numbers of known structures are given in parentheses. These toxins can be broadly divided on biosynthetic grounds into nitrogen-based and non- nitrogenous toxins. The best-known nitrogen- based compounds are the alkaloids, v^hich are biosynthetically derived from amino acid precur- sors. These substances have been used since time immemorial for poisoning purposes. Although the general toxicity of plant alkaloids in mammals is w^idely recognized, their teratogenic effects have only been recorded in more recent times. Adult female cattle and sheep may imbibe alkaloids in their diet in insufficient amounts to cause their death, but as a result of feeding on alkaloid- containing plants such as species of Lupinus, congenital defects may occur in the offspring. The malformed offspring usually suffer skeletal damage and defects of the digits or of the palate and have a low^ survival rate. Other nitrogen-based toxins are the non-protein amino acids, cyano- genic glycosides, glucosinolates, certain peptides and proteins. The biosynthetic origins and biolo- gical properties of these various metabolites are discussed in more detail in Chapter 12. The non-nitrogenous toxins in plants are mainly of terpenoid origin. Thus iridoids, sesquiterpene lactones, cardenolides and saponins are biosynthe- sized from mevalonolactone and isopentenyl pyrophosphate (Chapter 11). Other classes of terpenoid not listed here also have toxic members (e.g. the monoterpene, thujone and the diterpene, acetylandromedol of Rhododendron leaves). Iso- flavonoids, quinones and furanocoumarins listed in Table 14.2 are of shikimic acid origin and their biosynthesis and properties are discussed in more detail in Chapter 10. 14.3.3 The cost of chemical defense The synthesis of secondary metabolites is costly to the plant, requiring as it does a steady flow^ of precursors from primary metabolism, together w îth enzymes and energy-rich cofactors (ATP, NADPH, etc.) to drive the biosynthetic pathways. Photosynthesis normally ensures a more than adequate supply of precursors for carbon com- pounds (e.g. terpenoids). By contrast, nitrogen uptake by the plant is limited and the synthesis of nitrogen compounds (e.g. alkaloids) can compete for precursor with protein synthesis. Indeed, the cost of synthesis of alkaloids has been estimated at 5g of photosynthetic CO2 per gram of toxin compared to a value of 2.6 g for a phenolic (Gulmon & Mooney, 1986). This cost has to be balanced against the cost of new plant growth. Thus, all plants face a dilemma when attacked by herbivores expressed in the words of Herms & Mattson (1992): to grow or to defend. The competition for resources may be met in different ways and various theories have been put forward in recent times to explain the phenotypic variation that occurs in secondary metabolism and hence in chemical defense. The growth-differentiation balance (GDB) hypothesis 508 JEFFREY B. HARBORNE (Tuomi et ai, 1990) has as its fundamental premise the existence of a physiological trade-off between growth and differentiation, the latter term includ- ing secondary metabolism synthesis. This hypoth- esis suggests that perennial plants might be divided into two groups: first, growth-dominated plants, with rapid growth, poor chemical defense but with a highly inducible resistance system (see section 14.5) and second differentiation-dominated plants with a slow growth rate, well defended with high levels of toxin but with poorly developed inducible resistance. There is evidence supporting such a dichotomy in growth characteristics (Herms & Mattson, 1992). These hypotheses help to explain the many dif- ferences that are apparent in the chemical armory of flowering plants. They also indicate why the concentrations of secondary metabolites can increase in plants as a result of environmental stress. Growth on low-nitrogen soils or under drought stress can cause plants to stop producing new leaves, with the precursors and energy thus released flowing into secondary synthesis. Experi- ments with the composite plant Heterotheca subaxillaris, which is defended by monoterpe- noids, illustrate the changes that can occur in growth and metabolism. The younger, softer leaves have higher terpene levels than the older, tougher leaves. Feeding leaves taken from plants growing in nutrient-rich soils to the generalist insect Fseudoplusia includens results in a larval survival rate of 78 and 98% according to leaf age (Mihalaik &: Lincoln, 1989). Plants growing in soils with low nitrate levels immediately become more resistant to insect feeding and larval survival rates are reduced to 14% on young leaves and 38% on old leaves. In this case, when nutrient levels in the soil are adequate for growth, the plant accepts a degree of insect feeding on the leaves and maintains mono- terpene production at a moderate level, principally to protect the very young leaves. However, nitrate stress induces increases in terpene production so that insects can no longer feed successfully. This change can be seen as a dynamic response to herbivory when the plant cannot grow new leaves and is then more vulnerable to leaf loss. 14.4 CONSTITUTIVE DEFENSE MECHANISMS 14.4.1 Localization of toxins at the plant surface If secondary compounds do have a protective function against herbivory, then they are most likely to be located where they are most readily perceived by animals, namely at the plant surface. There is increasing evidence that this is so for many different kinds of plant; toxins are indeed often concentrated at or near the plant surface. Secondary compounds have been detected vari- ously in glandular hairs (or trichomes), in leaf waxes, in leaf resins and in bud exudates. Even when they are present in the leaf vacuole, they are often found specifically in the epidermal cells of the upper surfaces. This is true, for example, of the localization of quinolizidine alkaloids in the leaves of lupin plants (Wink, 1983). Again, in the case of lactiferous plants, toxins are found in the latex and are detected by animals as the latex exudes from the leaf following damage. Localization at the surface particularly affects insect feeders and many of the adverse effects of surface toxins are on insect pests. One of the most bizarre types of trichome defense has been observed in the wild potato species Solatium berthaultii (Table 14.3). The defense is two- pronged, with different agents secreted in mor- phologically distinct trichomes A and B, and is aimed primarily at aphids. Type B trichomes contain a volatile signal, jE-/?-farnesene, which occurs with other sesquiterpenes. Farnesene is actually a well-known aphid alarm pheromone and its release by the plant causes a similar Table 14.3 Trichome toxins of Solatium leaves^ Type of trichome Toxins present Effect on insect A B B Polyphenol/phenolase system —> Aphid arrested, stops feeding and starves sticky exudate on disruption Sucrose acid esters (e.g. 3,4-di-isobutyl-6-caproyl Aphid arrested, stops feeding and starves sucrose) /3-Caryophyllene and £-/3-farnesene Aphid alarm pheromone disrupts feeding ^Detected mainly in Solanum berthaultii and/or S. tuberosum leaf trichomes; sucrose esters also occur widely in many solanaceous plants in their trichome secretions. BIOCHEMICAL PLANT ECOLOGY 509 disturbance to aphid feeding. The second defense in the A-type trichome consists of a phenoHc- containing exudate, Hnked to a phenolase/ peroxidase enzyme system. When an aphid lands on the leaf, it disrupts the droplet, the enzyme then reacts with the phenolic substrate and a brown, sticky residue is formed. This immobilizes the aphid, prevents it from feeding and causes its death from starvation (Gregory et al., 1986). The sticky exudate is reinforced by a mixture of non- volatile sucrose esters, which are secreted along with the alarm pheromone in type B trichomes. This defense must be effective since S. berthaultii is virtually free of insect attack; indeed, potato breeders have considered hybridizing this species with S. tuberosum in order to improve the aphid resistance of the domestic potato. The range of substances found in trichomes is considerable and includes some water-soluble compounds, as in the case of the tomato. Here, two classes of toxin are present: the simple hydrocarbon 2-tridecanone and the phenolic compounds, rutin and chlorogenic acid. These substances have adverse effects on tomato pest insects, such as Heliothis zea. The concentrations of these trichome constituents are much higher in the wild tomato species from which the cultivated tomato is derived. Such toxins secreted in the leaf glands do not necessarily have to kill insect herbivores. All they need to do is to generally weaken them, retard their growth or delay their pupation. As a consequence, the insects will be more vulnerable to disease, to predation and to the environment (Stipanovic, 1983) and the plant will benefit from the reduced herbivory. One final point needs to be mentioned about trichome constituents: they are almost inevitably multifunctional in their effects. Many of the same substances which are toxic to insects are anti- microbial (see Chapter 13) and also have deleter- ious effects on mammalian herbivores. The allergenic effects in man of the trichome constitu- ents of Farthenium hysterophorus and Primula obconica are well known. Many other allergenic plants have similarly yielded, on chemical exami- nation, related toxins in the trichomes (Rodriguez et al., 1984). Leaf waxes provide a second line of defense in some plants, since they may be a barrier to certain insect feeding. More importantly, at least 50% of angiosperm species contain 'extra' secondary constituents (e.g. sterols, methylated flavonoids, etc.) at the leaf surface mixed in with the wax. These substances almost certainly have a protec- tive role, although less work has been done with them than with the trichome compounds. There is also evidence that the actual constituents of the leaf wax may, on occasion, be repellent to insects. Certain varieties of Sorghum are distasteful to Locusta migratoria, because of the presence of leaf alkanes of chain length C19, C21 and C23 and of fatty acid esters of chain length C12-C18. These and other effects of surface wax constituents on insects are reviewed by Woodhead &c Chapman (1986). A third line of defense in plants from insect predation is latex production. Latex has been reported in over 12 000 plant species and one of its main functions would appear to be to protect plants from herbivory. The effectiveness of latex, a white viscous liquid consisting of a suspension of rubber particles, as a feeding deterrent is often reinforced by the presence of terpenoid toxins. One particularly unpleasant series of latex con- stituents are the phorbol esters of Euphorbiaceae. These were first isolated from croton oil, Croton tiglium, but they occur widely in the family. Although most notorious as skin irritants and co- carcinogens in mammals, these diterpene esters have recently been shown to be toxic to insects as well. The effectiveness of sticky latex as an insect feeding deterrent is nicely illustrated by the case of the larvae of the Monarch butterfly, Danaus plexippus, when feeding on its food plant, the milkweed Asclepias syriaca. Here the larvae care- fully cut the leaf veins before feeding. This releases the latex as a series of white blobs along the leaf. The larvae then feed safely on the outer parts of the leaf, avoiding the latex in the process. The value of a sticky latex as an insect feeding deterrent may often be enhanced by the presence of toxic secondary substances within the flow of the latex. For example, the latex of chicory is rich in bitter-tasting sesquiterpene lactones (lactupicrin and 8-deoxylactucin, Fig. 14.2) and feeding experiments against the locust Schistocerca gre- garia show that these are antifeedants at a concentration of 0.2% dry wt. Analyses of the concentrations of these lactones in roots, stems OCOCH2/ \ o H Lactupicrin 8-Deoxylactucin Figure 14.2 Feeding deterrents of chicory root, stem and leaf. 512 JEFFREY B. HARBORNE of the plant are more expendable than others. Floral tissues, for example, may not be obviously protected from predation because they are gen- erally so short-lived compared to leaves. Finally, there is the possibility of variation within the same tissues. This may be apparent especially in leaves of long-lived plants, i.e. trees, which are the most vulnerable of all plant tissues to herbivore attack. Deciduous trees produce leaves year after year, sometimes for centuries so that insect populations can 'home in' on a readily available and pre- dictable food resource. Since insect predation has the potential to remove such a tree, there must be some other factor which provides resistance in the plant. The most recent suggestions of ecologists are that one protective factor is variable defense. The experimental evidence in favor of chemical variation within leaf tissues is of two sorts: the patterns of insect grazing on trees and chemical analyses within tree canopies. A study of insect grazing on some trees, such as birch and hazel, shows that there is an overdispersion of grazing initiations and that a proportion of leaves receive a low level of grazing. Such a pattern would be consistent with between-leaf variations in palat- ability. This idea has been supported by chemical analyses of leaves within the canopy in Betula lutea and Acer saccharum, which indicate sig- nificant variations in chemical parameters from leaf to leaf on the same branch (Schultz, 1983). Palatability is principally defined by tannin levels, total phenolic content, toughness (or degree of lignification), water content and nutrient content and all these measurements can change from leaf to leaf. Variation of this type may be due in part to genetic factors, the light regime, nutrient status of the tree, position of leaf on the tree and so on. There may also be considerable variations in leaf chemistry in different individuals within a tree population. This occurs, for example, in the neotropical tree Cecropia peltata (Moraceae), where leaves of some individuals are rich in tannin and are low in herbivore damage and the leaves of others are low in tannin and suffer considerable insect feeding. The tannin levels in the leaves vary from 13 to 58 mgg~^ dry wt (Coley, 1986). Tests with the army worm, Spodoptera latifascia, showed that there was reduced feeding on leaves of the high-tannin individual. There was, however, a significant cost to the tree in terms of tannin production and the high-tannin trees produced fewer leaves than the low-tannin trees, growing under the same conditions. For the insect seeking food on trees, there are considerable risks, since having to spend more time moving around within the canopy must increase the chance of it being the victim of one of its predators. Certainly, within-leaf variation, where it occurs, must place a constraint on the insect grazer. As Schultz (1983) has put it: The situation can be said to resemble a 'shell game', in which a valuable resource (suitable leaves) is hidden among many other similar- appearing but unsuitable resources. The insect must sample many tissues to identify a good one. The location of good tissues may be spatially unpredictable, and may even change with time. For a choosy or discriminating insect, finding suitable food in an apparently uniform canopy could be highly complex. 14.5 INDUCED PHYTOCHEMICAL RESPONSE TO HERBIVORY 14.5.1 Phytochemical induction in plants One way that a plant might reduce the metabolic costs of synthesizing and storing toxins for chemical defense is only to produce the defensive chemicals when they are actually needed, i.e. in direct response to herbivore attack. Such a mechanism was first described from experiments in plant pathology, when it was discovered that plants can respond to fungal infection by the de novo synthesis of antifungal agents at the site of attack. These so-called 'phytoalexins' were pro- duced within a few hours of infection and reached a maximum within 48 h of the invasion (see Chapter 13). It is now apparent that insect feeding on plant tissues can produce a related dynamic response to damage. With insect feeding, however, the effect is systemic and the whole plant is alerted to the fact of invasion. Insect feeding can be mimicked by mechanical damage, i.e. punching a hole in a leaf, and the response is quite separate from the 'wound response', which is a localized repair mechanism of plants. Three distinctive types of herbivore-induced defense have been recognized so far. The first, the so-called 'PIIF induction', produces completely new defense agents, the synthesis of proteinase inhibitors, within 48 h. These cause the leaf to become unpalatable and insect feeding is arrested. The second type of induced defense is the increase in the synthesis of a single class of toxin, which is already being produced in lower amounts before attack. Again, the effect is the production of unpalatable leaves, the effect increasing over several days after the induction. The third and most remarkable type of induced defense is the BIOCHEMICAL PLANT ECOLOGY 513 production of volatile chemicals at the feeding site on the leaf, which has the apparent effect of attracting insect parasitoids. These parasitoids then visit the plant and destroy the herbivore. Each of these three types of herbivore-inducing defense will now be considered. 14.5.2 De novo synthesis of proteinase inhibitors 1990). As PIIF-like activities have been detected in extracts of 37 plant species representing 20 families, this mechanism may well be a general one. Ecological experiments have also shown that PIIF induction in the tomato reduces the grazing by larvae of the armyworm Spodoptera littoralis within 48 h, with avoidance being most pro- nounced on the young leaves (Edwards et al., 1992). It has been shown that a Colorado beetle feeding on potato or tomato leaves can cause the rapid accumulation of proteinase inhibitors, even in parts of the plant distant from the site of attack. The process is mediated by a proteinase inhibitor inducing factor, known as PIIF, which is released into the vascular system. Within 48 h of leaf damage, the leaves may contain up to 2% of the soluble protein as a mixture of two proteinase inhibitors. Subsequently, the presence of the proteinase inhibitors in the leaf is detected by the beetle, which avoids further feeding and moves onto another plant (Fig. 14.3). In theory, the inhibitors, if taken in the diet, will have an adverse effect on the insects' ability to digest and utilize the plant protein, since they inhibit the protein-hydrolyzing enzymes, trypsin and chymotrypsin. In fact, proteinase inhibitors are well known as constitutive constituents of many plant seeds, where they have a similar protective role in deterring insect feeding. PIIF induction is also brought about by mechanical wounding of plant tissue so that it is not yet entirely clear how specific this effect is to herbivore grazing. The nature of the chemical signal PIIF has been explored in tomato plants and it is a small protein, called systemin. This protein is 10000 times more active than oligosaccharides, which also have the ability to trigger this defense system (Ryan, 1992). A volatile chemical, the fatty acid metabolite methyl jasmonate, may also be involved in the signaling process (Farmer & Ryan, 14.5.3 Increased synthesis of toxins A related form of induced defense, apparently quite distinct from the PIIF system, has been observed in a variety of plants. The effect is relatively rapid and the leaves become unpalatable to animals within a matter of hours or a few days. It may be short term, disappearing after the insect has stopped feeding, or long term, extending over to the following season in trees. The chemical changes involve an increase in the concentration of existing toxins (Fig. 14.4), sufficient to lead to herbivore avoidance. Such increases in toxin synthesis have been observed in two alkaloid-containing plants. One is the wild tobacco species Nicotiana sylvestris, which contains nicotine and nornicotine as the major alkaloids. Larval feeding induced a 220% increase in alkaloid content throughout the plant CH2OH Nicotine {Nicotiana sylvestris) Atropine {Atropa belladonna) Colorado beetle feeding Potato or tomato leaf 1-2 h Inhibition of Beetle's digestion of leaf protein Proteinase inhibitor inducing factor (systemin) ± methyl jasmonate (volatile signal) [ Proteinase inhibitor I (MW 39000) [ Proteinase inhibitor II (MW 21000) Figure 14.3 De novo synthesis of proteinase inhibitors in potato and tomato leaves. Xanthotoxin {Pastinaca sativa) Figure 14.4 Chemicals which increase in concentration in plant leaves in response to insect herbivory. 514 JEFFREY B. HARBORNE over a period of 5-10 days. Mechanical damage which avoids cutting the secondary veins produced a smaller response (170%). In fact, the tobacco hornworm Manduca sexta w^hen feeding on the tobacco leaf avoids cutting through the secondary veins. It thus avoids triggering off the fullest response in the leaf, which can be as much as 400% of the control if the simulated damage includes damaging the vein. The nicotine alkaloids are synthesized in the roots and transported up into the leaf and this was apparent in experiments in which pot-bound plants with confined roots failed to show any significant alkaloid increase after mechanical damage (Baldwin, 1988). Similar experiments with the tropane alkaloids in leaves of Atropa acuminata showed a maximum increase of 153-164% over the control 8 days after mechani- cal damage or slug feeding. Repeated mechanical damage at 11-day intervals initially increased the response to 186% of the control but this effect fell off with time. Further experiments showed that only 9% of the leaf area needed to be removed mechanically or by animal feeding to produce the maximum response (Khan & Harborne, 1991). Another well-investigated example of induced chemical defense is the wild parsnip, Pastinaca sativa, which produces five furanocoumarins in the leaves. Artificial damage increased furanocoumarin synthesis to 162% of the control, whereas feeding by the generalist insect Trichoplusia ni increased it to 215%. Furthermore, larvae of T. ni grew very slowly on induced leaves, and larvae on an artificial diet supplemented with furanocoumarins were similarly affected (Zangerl, 1990). The response of oil seed rape, Brassica napus, to insect infestation or leaf damage is quite distinctive and involves the massive accumulation of indole glucosinolates, which are barely detectable in the control. There is a corresponding reduction in the amounts of the aliphatic glucosinolates of the plant, but the total titer of glucosinolate does appear to increase under these treatments (Koritsas et ai, 1989). Other examples where induced changes in protective chemistry have been recorded are given in Tallamy & Raupp (1991). For every plant that shows a positive response, there is another plant where no detectable change in palatability occurs. Rapid inducible resistance appears to be weak in or absent from the leaves of slow-growing plants (section 14.3.3). Environ- mental factors also determine the magnitude of the response. Finally, the response may disappear as the plant grows older and concomitantly more resistant to grazing. For example, two-year-old trees of Pinus contorta respond to defoliation by increasing the concentration of both terpenes and tannins in the needles, whereas ten-year-old trees fail to show any increases. 14.5.4 Release of predator- attracting volatiles An even more interesting and remarkable plant- animal interaction involving induced chemical changes has been observed by Dicke et al. (1990). In response to herbivory, some plants have developed the means to release volatile chemicals (Fig. 14.5), which are particularly attractive to parasitoids of their herbivores, which then visit the plant and destroy the herbivores. As Dicke puts it, plants may 'cry for help' when attacked by spider mites and predatory mites come to the rescue. Much research has been conducted on the spider mite Tetranychus urticae, the predatory mite Phytoseiulus persimilis, and the host plants. The chemicals released seem to be plant species- specific. Cucumber plants infested by the spider mite release /?-ocimene and 4,8-dimethyl-l,3,7- nonatriene (see Fig. 14.5) and are only moderately attractive to the predatory mites, whereas lima beans release a cocktail of linalool, /3-ocimene, the nonatriene and methyl salicylate which is highly attractive. A further advantage to the plant world is that the volatile released may alert uninfested neighboring plants so that they become better protected from spider mite attack. Thus cotton seedlings, when infected by these mites, release volatile cues which both attract predatory mites and also alert neighboring plants to withstand herbivore attack. The systematic release of volatile chemicals which mediate in plant-herbivore-predator inter- actions has been observed in other plant systems. Thus, it has been recorded that corn {Zea mays) seedlings respond to beet armyworm [Spodoptera exigua) attack by releasing volatiles, which attract parasitic wasps, Cotesia marginiventris, to attack CH3 HO^ CH3 Linalool (Lima bean) E-p-Ocimene (Cucurbita) CH3 CHs H3C ^^ ^ CH2 E-4,8-Dimethyl-1,3,7-nonatriene Figure 14.5 Predator-attracting volatiles released from plant leaves during herbivory.