Duration of Priming of Two Indirect Plant Defenses

When plants are infested by herbivores, they emit herbivore-induced plant volatiles (HIPVs) that attract carnivorous natural enemies of herbivores. Furthermore, there are increasing evidences that defenses of intact plants against herbivores are primed when exposed to HIPVs. We previously reported that lima bean leaf volatiles induced by the herbivorous mites Tetranychus urticae primed two T. urtiae-induced indirect defenses in neighboring conspecific plants: HIPV emission and extrafloral nectar (EFN) secretion. An intriguing unanswered question is whether the durations of these two defenses are the same. Here, we show that the durations of the two defenses were the same for up to two days after the initiation of T. urticae damage. The two induced primed defense would act as a battery of defense in exposed plants.Key Words: herbivore-induced plant volatiles, indirect, defense, induced response, plant-plant interaction, primingWhen infested by herbivores, plants defend themselves indirectly by emitting herbivore-induced plant volatiles (HIPVs). One of the ecological functions of HIPVs is to attract carnivorous natural enemies of the herbivores.^1,2 Recently, it was reported that the emission of HIPVs primed defenses against herbivores in neighboring intact plants.^3–7 Thus, HIPVs also mediate interactions between infested and intact plants.⁸ The enhanced defense in response to HIPVs in intact plants is called ‘priming’, which has been studied intensively in plant-pathogen interactions,⁹ but not so in plant-insect interactions.We previously reported that exposure to HIPVs emitted from lima bean leaves infested by Tetranychus urticae primed HIPV production in detached intact conspecific leaves.³ We also reported that exposure to HIPVs, produced in response to T. urticae damage,⁴ primed the induced production of extrafloral nectar (EFN; an alternative food source for predators^10,11 in lima bean plants. An intriguing question is whether the two primed defenses work as a battery against T. urticae. To answer this, we examined the duration of primed HIPV production by lima bean plants using the same experimental set-up as our previous study of EFN priming by conspecific plants.⁴For exposure of plants to HIPVs, we used a 60 × 60 × 60 cm cage with two 30 × 30 cm windows on opposite sides of the cage.¹² As odor sources, we used eight plants that had been infested with 60 adult T. urticae females per plant for 1 day. Eight uninfested plants were used as control odor sources. Two uninfested plants were placed in a cage with the odor source plants and exposed to either HIPVs or uninfested plant volatiles (UPVs) for 10 days in a climate-controlled room (25 ± 2°C, 60–70% RH, 16:8; L:D).A Y-tube olfactometer¹³ was used to examine the response of the predators to HIPVs. Adult female P. persimilis were randomly selected from a colony and individually positioned at the beginning of the iron wire. When test mites reached the end of one arm of the olfactometer, their choice was recorded. We tested the olfactory responses of the predator toward (1) plants infested by T. urticae for two days after exposure to UPVs vs. plants infested by T. urticae for two days after exposure to HIPVs, and (2) plants infested by T. urticae for four days after exposure to UPVs vs. plants infested by T. urticae for four days after exposure to HIPVs.HIPV-exposed plants attracted more predators than UPV-exposed plants in a Y-tube olfactometer when infested by T. urticae for two days (Fig. 1A). By contrast, the predators did not distinguish between HIPV- and UPV-exposed plants when infested by T. urticae for four days (Fig. 1B). Our previous study showed that HIPV-exposed plants secreted significantly larger amounts of EFN secretion than UPV-exposed plants infested by T. urticae for two days under the same experimental condition as in this study.⁴ However, the difference was not significant when they were infested for four days.⁴Open in a separate windowFigure 1The olfactory response of P. persimilis females to volatiles from the odor-exposed plants, as determined in a Y-tube olfactometer: (A) plants infested by T. urticae for two days after exposure to UPVs (UPV-exposed—T. urticae 2d) vs. plants infested by T. urticae for two days after exposure to HIPVs (HIPV-exposed—T. urticae 2d), and (B) plants infested by T. urticae for four days after exposure to UPVs (UPV-exposed—T. urticae 4d) vs. plants infested by T. urticae for four days after the exposure to HIPVs (HIPV-exposed—T. urticae 4d). Asterisks beside each bar indicate a significant difference between the first trifoliate leaves and the primary leaves. Asterisks beside a bar indicate a significant difference (binomial test: p < 0.001).Lima bean plants increase the amount of endogenous jasmonic acid after exposure to HIPVs.¹⁴ Jasmonic acid, an important plant hormone regulating a defense signaling pathway against herbivores and pathogens,^15,16 is reported to be involved in the induction of both volatile emission^17,18 and EFN secretion¹⁹ in response to T. urticae damage in lima bean plants. The increase of endogenous jasmonic acid in HIPV-exposed plants may partly explain the simultaneous priming of the two defenses.In this study, we showed that the durations of priming of two indirect defenses were roughly the same for up to two days. Priming of these two indirect defenses would thus be a battery of defense at the outset of T. urticae damage. Further study is necessarily to test whether the primed battery of induced defense increases the fitness of the exposed plants.     

Induced defenses of Veronica spicata: Variability in herbivore-induced volatile organic compounds

Plants release volatile organic compounds (VOCs) that have many eco-physiological functions. Induction of plant VOCs is known to occur upon herbivory. Herbivore-induced VOCs are involved in the attraction of predators and parasitoids, a phenomenon known as an indirect defense of plants. We measured the VOC profiles of the wild species Veronica spicata with and without larval feeding and oviposition by the specialist butterfly Melitaea cinxia. V. spicata showed great plasticity when deploying indirect defences. The induction of several ubiquitous terpenoids and green leaf volatiles (GLVs) was associated with larval feeding, whereas the increase of two ketones, 6-methyl-5-hepten-2-one and t-geranylacetone and the suppression of GLVs were associated with oviposition by the butterfly.     

Qualitative variability of lima bean's VOC bouquets and its putative ecological consequences

Studies on direct and indirect defenses of lima bean (Phaseolus lunatus L.) revealed a quantitative trade-off between cyanogenesis and the total quantitative release of herbivore-induced volatile organic compounds (VOCs). In this addendum we focus on the qualitative variability in the VOC bouquets. We found intraspecific and ontogenetic variation. Five out of eleven lima bean accessions lacked particular VOCs in the bouquets released from secondary and/or primary leaves. These compounds (cis-3-hexenyl acetate, methyl salicylate and methyl jasmonate) can induce and prime indirect defenses in neighboring plants. Thus, the variability in VOC quality as described here might have substantial effects on plant-plant communication.Key words: indirect defense, herbivore-induced plant volatiles, Phaseolus lunatus, trade-off, tritrophic interactions, plant-plant communicationPlants can be attacked by multiple enemies and accordingly have evolved multiple defense strategies comprising direct and indirect mechanisms. Lima bean (Fabaceae: Phaseolus lunatus L.) represents a well established experimental plant for the analysis of three defenses: herbivore-induced volatile organic compounds (VOCs), extrafloral nectar (EFN) and plant cyanogenesis. Herbivore-induced VOCs have manifold functions associated to indirect plant defenses. For example, VOCs attract arthropod predators or parasitoids and thus can act as an indirect defense.^1–5 They also may be perceived by neighboring, yet-undamaged plant individuals (plant-plant signaling) or plant parts (within-plant signaling) and they prime or induce defensive responses.⁶ VOC-exposed plants may upregulate the secretion of EFN^7–9 or the release of VOCs.¹⁰ In addition to these indirect defenses, lima bean shows cyanogenesis as a direct defense. Cyanogenesis means the release of toxic hydrogen cyanide (HCN) from preformed precursors in response to cell damage¹¹ and is considered a constitutive direct defense against herbivores¹² (but see ref. ¹³).Recently we demonstrated that cyanogenesis and total release of VOC in lima bean are negatively correlated to each other.¹⁴ Accessions characterized by strong cyanogenesis in secondary leaves released little amounts of VOCs in response to jasmonic acid (JA) treatment, whereas total VOC production in accessions with low cyanide concentrations was high. Interestingly, these findings also held true on the ontogenetic level, since primary leaves generally showed low concentrations of cyanide and released high amounts of VOCs. However, the question remained unanswered whether these differences are merely of quantitative or also of qualitative nature. We therefore selected eleven accessions from the larger set of lima beans that had been used in our previous study and searched for qualitative differences in their VOC bouquets.Eight out of the eleven volatiles that were quantified in our study were consistently released from both primary and secondary leaves of all accessions. However, five accessions indeed lacked individual compounds in secondary or primary leaves. For example, we did not detect cis-3-hexenyl acetate in the headspace of secondary leaves of accessions CV_2357 and CV_8078. The latter accession also lacked methyl salicylate in all individual plants analyzed (N = 10 plants). In contrast to secondary leaves, primary leaves of these accessions showed the complete blend of eleven VOCs. While CV_2357 and CV_8078 showed qualitative variability in VOC blends depending on leaf developmental stage, the accessions WT_2233 and WT_PYU consistently lacked methyl jasmonate in both, secondary and primary leaves. Strikingly, accession CV_8073, which was characterized by high quantitative release of cis-3-hexenyl acetate and methyl salicylate from secondary and primary leaves, showed a very low release of methyl jasmonate from secondary leaves—and lacked this compound completely in the headspace of primary leaves (Fig. 1).Open in a separate windowFigure 1Qualitative variation in VOC mixtures of lima bean. Secondary (A) and primary leaves (B) of cultivated (CV) and wildtype (WT) lima beans characterized by high (HC) or low (LC) concentration of cyanogenic precursors in secondary leaves were analysed for release of three selected VOC compounds. Volatiles were collected in a closed-loop stripping over an experimental period of exactly 24 hrs. Values shown represent the mean (±SE) of five plants per accession. Different letters on top of the columns indicate means that differ significantly (according LSD post-hoc analysis after one-way ANOVA. Statistical analyses were conducted separately for each compound and each leaf stage).In addition to qualitative differences, the quantitative release of volatiles was significantly different among the accessions. This holds true for the three compounds we have focused on in this addendum (Fig. 1) as well as for all eleven volatiles that we have quantified in our previous study (data not shown). However, while quantitative release of total VOCs from secondary leaves was negatively correlated to their cyanide concentration,¹⁴ the qualitative differences in VOC composition were not that strictly correlated to cyanogenesis. Accessions CV_2357, CV_8078, WT_2233 and WT_PYU were characterized by high cyanogenic secondary leaves—and lacked at least one compound. However, also the generally low cyanogenic primary leaves did not always show the complete VOC blend (Fig. 1B). These findings demonstrate a high qualitative variability of VOC composition that did depend neither on cyanogenic leaf features nor on leaf ontogeny.So far it remains elusive whether the observed intraspecific and ontogenetic variability is of relevance in natural systems, but ecological effects are highly likely. For some tritrophic systems an intriguing degree of sophistication in the communication between plants and the third trophic level has been demonstrated: bouquet compositions can provide specific information on the identity of the attacking herbivore and, hence, on its suitability for the prey-seeking carnivores.¹⁵ Variability in the composition of VOC compositions as observed for lima bean (and as also known for corn, cotton or cabbage^16–20) may compromise the reliability of herbivore-specific signals across a range of plant genotypes,²¹ although the ability of parasitoids to learn and associate successful foraging and egg-laying experience with the encountered odor pattern may help them in part overcome this problem.^22,23In contrast to tritrophic interactions, the efficiency of volatiles in plant-plant signaling appears more restricted to specific compounds. Recent studies on lima bean demonstrated that cis-3-hexenyl acetate causes priming or induction of extrafloral nectar.^7,24 In other plant species, the release of gaseous methyl jasmonate in response to herbivore attack has been demonstrated to induce the synthesis of proteinase-inhibitors, which represent an efficient defense against herbivores.^6,25 Methyl salicylate has been reported to be an important elicitor of resistance responses directed towards pathogens and herbivores^26,27 and as a carnivore-attractant.^28,29 Thus, the quantitative variation or a complete lacking of single biological active compounds of volatile blends may have dramatic effects on ecological interactions. We suggest that different defense strategies may be realized in these lima bean accessions: some genotypes have evolved strong cyanogenesis in secondary leaves and now ‘pay’ for this efficient direct defense with reduced or lost abilities for indirect defense and/or plant-plant communication, while others invest less in cyanogenesis and are more “communicative” concerning the third trophic level and conspecifics.     

Plant Host Finding by Parasitic Plants: A New Perspective on Plant to Plant Communication

Plants release airborne chemicals that can convey ecologically relevant information to other organisms. These plant volatiles are known to mediate a large array of, often complex, interactions between plants and insects. It has been suggested that plant volatiles may have similar importance in mediating interactions among plant species, but there are few well-documented examples of plant-to-plant communication via volatiles, and the ecological significance of such interactions has been much debated. To date, nearly all studies of volatile-mediated interactions among plant species have focused on the reception of herbivore-induced volatiles by neighboring plants. We recently documented volatile effects in another system, demonstrating that the parasitic plant Cuscuta pentagona uses volatile cues to locate its hosts. This finding may broaden the discussion regarding plant-to-plant communication, and suggests that new classes of volatile-meditated interactions among plant species await discovery.Key Words: chemical communication, Cuscuta pentagona, host fiding, host selection, plant-plant communication, plant volatiles, parasitic plantsFor nearly 25 years, the ecological importance of plant-to-plant communication through volatiles has remained an open and much debated question. Plants exchange gases with the atmosphere and, in so doing, release plumes of volatile chemicals that can convey ecologically important information to other organisms. The potential ecological significance of these volatile cues is demonstrated by the large and growing array of interactions between plants and arthropods known to be mediated by plant volatiles. Volatiles serve as foraging cues both for insects that are beneficial to plants, such as pollinators,¹ and those that are harmful such as herbivores.^2,3 Because the volatile blends released by plants exhibit variation in response to environmental stimuli, volatiles can convey detailed information about the status of the emitting plant. Predatory and parasitic insects that feed on herbivorous insects respond preferentially to plant volatiles that are induced by insect feeding,⁴ while female herbivores use such cues to avoid laying their eggs on already-infested plants.^3,5 Moreover, the volatile blends released in response to herbivory can differ between individual herbivore species, providing highly specific cues to specialist parasitoids.⁶ Thus, plant volatiles are known to mediate complex interactions among plants and insects across multiple trophic levels.It has long been speculated that plant volatiles might have similar significance for interactions among plant species, yet there are few well-documented examples of communication between plants by way of volatile signals. Essentially all previous work on plant-to-plant communication has focused on the reception of herbivore-induced volatile signals by neighboring plants, which may use them as early warning signals to initiate their own direct and indirect defense responses. The first studies claiming to document such effects were published almost 25 years ago.^7,8 But issues with the experimental design of these early experiments and the availability of alternative explanations for their results led many ecologists to disregard the phenomenon.^9–11 Later, a number of studies demonstrated that direct and indirect plant defenses could be elicited by exposure to certain induced plant volatiles.^12–15 But many of these effects were demonstrated in airtight chambers with volatile concentrations far higher than those likely experienced in natural settings, again raising doubts about the ecological significance of plant-plant communication.^16–18 Still more recently, some researchers have provided evidence that more realistic volatile concentrations likely induce priming of the defenses of receiving plants, rather than the initiation of full scale responses,¹⁵ while others have documented volatile effects under natural conditions.^19–21 Thus, despite continuing caution about the interpretation of experiments in this area,^17,18 there is mounting evidence that plant herbivore-induced volatiles can serve as early warning signals to neighboring plants.We recently documented an entirely new class of volatile mediated interactions among plants: the role of plant volatiles in host location by parasitic plants that attach to above ground shoots of other plants. Plant parasites are important components of natural and agricultural ecosystems and play important roles in determining community structure and dynamics.^22,23 We are exploring the mechanisms of host-location and other interactions between parasitic plants in the genus Cuscuta (dodder) and their host plants. Dodder vines germinate from seeds containing limited energy reserves and, as the parasites have no roots and little photosynthetic ability, must quickly locate and attach to suitable hosts in order to survive (Fig. 1). Thus, there is presumably significant selection pressure for dodder vines to employ efficient strategies for host location, and host plant volatiles may be expected to provide relevant directional cues. Dodder seedlings exhibit a rotational growth habit (circumnutation) following germination and previous researchers have suggested that host-finding might involve random growth²⁴ or the exploitation of light cues.²⁵Open in a separate windowFigure 1Seedling of Cuscuta pentagona (A) foraging toward a 20-day-old tomato plant, (B) attaching to and beginning to grow from stems of tomato seedlings and (C) close up of C. pentagona attachment.Using a very simple experimental design, we explored the possibility that host-plant volatiles might mediate host-location by seedlings of C. pentagona. We placed a germinated seedling in a vial of water located at the center of a dry filter paper disk. A host plant (a 20-day old tomato seedling) was placed near the edge of the disk and the dodder seedling was allowed to forage for four days. By the end of the experiment the seedling would lay horizontally on the disk and we traced its position on the filter paper in order to assess the directionality of growth relative to the host plant. This experiment was replicated 30 times and our results clearly indicated directional growth toward the tomato plant (80% of the tested seedlings grew into the disk half nearest the host) demonstrating that C. pentagona seedlings were perceiving some host-derived cue.We did not observe directed growth when we tested dodder seedling response to alternative targets including pots of moist soil, artificial plants, and vials of colored water intended to mimic possible light cues. In order to confirm a role for plant volatiles in host location by C. pentagona, we tested seedling response to host plant volatiles extracted from filtered air in a volatile collection system and then released from rubber septa in the absence of any other host-derived cues. Here we observed a directed growth response similar to that exhibited toward an intact tomato seedling, confirming that host plant volatiles do provide a cue used for host location by C. pentagona. In subsequent experiments we found directed growth toward impatiens and alfalfa plants, which are attractive hosts for C. pentagona and also toward wheat plants which are poor hosts, suggesting that the host-location mechanisms operate over a wide range of host species.Since discriminating between more and less desirable host species is likely to be important in natural settings, we next explored whether dodder seedlings could distinguish volatile signals from host and nonhost plants. Cuscuta pentagona seedlings exhibited directional growth toward tomato plants in preference to wheat plants and also to extracted volatiles from tomato in preference to those from wheat, demonstrating an ability to distinguish and choose among volatiles from more and less preferred hosts.When we tested seedling responses to individual compounds from the wheat and tomato blends, we found that three compounds from tomato, α-pinene, β-myrcene, and β-phellandrene elicited directed growth. β-myrcene was also present in the wheat blend. Unexpectedly, we also found that one compound present in the wheat blend, (Z)-3-hexenyl acetate, was repellent, providing a plausible explanation for the lower attractiveness of the wheat blend. It is interesting to note that (Z)-3-hexenyl acetate is also released by tomato in response to feeding by herbivores, and we have some data suggesting that C. pentagona seedlings may find tomato seedlings infested by Heliothis virescens caterpillars less attractive than un-attacked plants (unpublished data).The discovery that some parasitic plants exploit host plant volatiles for host location provides a new perspective on volatile mediated interactions among plant species, demonstrating that plant volatiles play a role in mediating ecologically significant interactions in at least one system other than the transfer of herbivore-induced warning signals. We think it is quite likely that plant volatiles will be found to play a role in host location by other parasitic plants and perhaps even by vining plants generally. Moreover, we think it is more likely than not that more classes of volatile mediated interactions among plants remain to be discovered given the potential availability of volatile cues and the fitness benefits to be derived by plants using such cues to gather information about the identity and condition of their neighbors.     

Salicylhydroxamic acid (SHAM) negatively mediates tea herbivore-induced direct and indirect defense against the tea geometrid Ectropis obliqua

We investigated the effect of the SHAM treatment of tea plants on their induced defense on a tea geometrid (TG), Ectropis obliqua Prout. Treatment of tea leaves with SHAM reduced the performance of TG and TG-elicited level of the lipoxygenase gene CsiLOX1 and the putative allene oxide synthase gene CsiAOS1. The release of wound-induced green leaf volatiles (GLVs) and the expression of the hydroperoxide lyase (HPL) gene CsiHPL1 were also reduced by SHAM treatment. The negative effect of SHAM dramatically reduced the total hebivore-induced plant volatiles (HIPVs) and the attractiveness to the parasitoid wasp Apanteles sp. These results indicated that SHAM may negatively mediate tea defense response against TG by modulating the wound-induced emission of GLVs, the expression of genes involved in oxylipin pathway, and the emission of other HIPV compounds that mediate direct and indirect defenses.     

Emerging roles in plant defense for cis-jasmone-induced cytochrome P450 CYP81D11

Cis-jasmone is a volatile organic compound emitted constitutively by flowers or leaves of several plant species where it acts as an attractant for pollinators and as a chemical cue for host localization (or avoidance) for insects.^1–3 It is also released by some plant species after feeding damage inflicted by herbivorous insects and in this case might serve as a chemical cue for parasitoids to guide them to their prey (so called “indirect defense”).^4,5 Moreover, we have recently shown that plants can perceive cis-jasmone and that it acts as a signaling molecule in A. thaliana, inducing a discrete and distinctive suite of genes, of which a large subset is putatively involved in metabolism and defense responses.⁶ Cytochrome P450s feature prominently in these functional subsets and of these the highest fold change upon cis-jasmone treatment occurred with the cytochrome CYP81D11 (At3g28740).⁶ Hence this gene was chosen for a more thorough analysis of the potential biological relevance of the cis-jasmone induced defense response. Although the precise function of CYP81D11 remains to be determined, we could previously demonstrate its involvement in the indirect defense response in Arabidopsis, as plants exposed to cis-jasmone ceased to be attractive to the aphid parasitoid Aphidius ervi when this P450 was inactivated by T-DNA insertion mutagenesis.⁶ Here we report additional experiments which give further support to a role of CYP81D11 in the direct or indirect defense response of A. thaliana.Key words: cis-Jasmone, Cytochrome P450, indirect defense, tritrophic interactions, volatile signaling     

Phytohormones in plant root-Piriformospora indica mutualism

Piriformospora indica is a mutualistic root-colonising basidiomycete that tranfers various benefits to colonized host plants including growth promotion, yield increases as well as abiotic and biotic stress tolerance. The fungus is characterized by a broad host spectrum encompassing various monocots and dicots.^1,2 Our recent microarray-based studies indicate a general plant defense suppression by P. indica and significant changes in the GA biosynthesis pathway.³ Furthermore, barley plants impaired in GA synthesis and perception showed a significant reduction in mutualistic colonization, which was associated with an elevated expression of defense-related genes. Here, we discuss the importance of plant hormones for compatibility in plant root-P. indica associations. Our data might provide a first explanation for the colonization success of the fungus in a wide range of higher plants.Key words: compatibility, plant defense, gibberellic acid, symbiosis, plant hormones     

Increasing insight into induced plant defense mechanisms using elicitors and inhibitors

One of the strategies that plants employ to defend themselves against herbivore attack is the induced production of carnivore-attracting volatiles. Using elicitors and inhibitors of different steps of the signal-transduction pathways can improve our understanding of the mechanisms underlying induced plant defenses. For instance, we recently showed that application of jasmonic acid, a key hormone in the octadecanoid pathway involved in herbivore-induced defense, to Brassica oleracea affects gene expression, hormone levels, and volatile emission, as well as oviposition by herbivores and host location behavior by parasitoids. Such defense responses vary with the dose of the elicitor and with time since application. This addendum describes how the use of inhibitors, in addition to the use of elicitors like jasmonic acid, can be applied in bio-assays to investigate the role of signal-transduction pathways involved in induced plant defense. We show how inhibition of different steps of the octadecanoid pathway affects host location behavior by parasitoids.Key words: Brassica oleracea, Cotesia glomerata, elicitor, herbivore-induced plant defense, inhibitor, jasmonic acid, octadecanoid pathway, phenidone, propyl gallate, diethyldithiocarbamic acid (DIECA)Chemical information plays an important role in the interactions between plants and insects. When a plant is damaged, it can respond with the production of specific volatiles and toxins.¹ Insects associated with these plants can use the resulting chemical information to find their host plants and to determine the suitability of a plant for feeding or oviposition. Application of chemicals acting as elicitors can be used to mimic such plant responses, while knowledge of the signal transduction pathways involved can be used to select potential inhibitors of induced plant response. Compared to exposing plants to herbivores, the application of elicitors and inhibitors allows for manipulation of defined steps in signal-transduction pathways, as well as to induce plants in a dose-controlled manner.² However, also with elicitors and inhibitors it is often difficult to link the applied dose to the strength of the induction of the plant, as the plant may use alternatives routes to express certain traits and the manipulation can result in unwanted effects on other processes in the plant, such as flowering or senescence.^3,4 Hence, experiments using elicitors or inhibitors should preferably use rather short incubation times (hours to days), to avoid developmental differences due to treatment.⁵JA is a key hormone in the octadecanoid pathway, involved in direct as well as indirect plant defenses against herbivores. Application of this phytohormone is known to mediate induction of volatile emission, increase toxin levels and to upregulate defense gene expression. In turn, the changes in these plant traits affect members of the insect community associated with the plant and may result in higher parasitism rates of herbivores, attraction of predators, and reduced oviposition and development of herbivores.^6–12JA is often used to mimic herbivory in studies on induced plant responses. However, recent studies on JA-application to e.g., Brassica oleracea var. gemmifera L. (Brussels sprouts) also indicate that the JA-induced volatile emission differs from volatile emission induced by herbivores.¹² More nectar was secreted by flowers of herbivore-infested Brassica nigra L. (black mustard) than by flowers from JA-induced plants.⁶ The intensity of the behavioral responses of herbivores and parasitoids differs between JA- and herbivore-induced plants, but compared to non-induced plants, both treatments are favored by parasitoids on both Brussels sprouts and black mustard plants,^6,12 while Pieris butterflies avoid oviposition on induced Brussels sprouts plants.¹¹ The results indicate that JA-mediated responses do play an important role in plant defense against herbivorous insects, and can be used to induce defense responses in many plant species. However, cross-talk with other phytohormones, as well as visual cues will also affect plant defense responses.While JA application induces the octadecanoid pathway, inhibitors of steps in this pathway are also available (Fig. 1). This approach allows including visual cues of feeding damage while eliminating or reducing chemical cues. Three inhibitors of different steps of the octadecanoid pathway are phenidone (1-phenyl-pyrazolidinone), DIECA (diethyldithiocarbamic acid) and n-propyl gallate (3,4,5-trihydroxybenzoic acid propyl ester; all obtained from Sigma-Aldrich, St. Louis, MO; Fig. 1). The redox-active compound phenidone is known to inhibit the activity of lipoxygenases (LOXs),^13–15 by reducing the active form of LOX to an inactive form. Therefore, phenidone is an effective inhibitor of an early step in the octadecanoid pathway, and thus of the plant’s induced defense system.^16,17 DIECA reduces 13-hydroperoxylinolenic acid to its corresponding alcohol 13-hydroxylinolenic acid, which is not a signaling intermediate and cannot be converted into JA.^18–20 Propyl gallate is a less specific inhibitor inhibiting both LOX and allene oxide cyclase (AOC), an enzyme catalyzing the step to 12-oxo-phytodienoic acid (OPDA) in the octadecanoid pathway.^14,21,22 We investigated the effects of these three inhibitors on herbivore-induced parasitoid attraction. For all three inhibitors 2 mM aqueous solutions with 0.1% Tween were applied to the plants.Open in a separate windowFigure 1Representation of the octadecanoid pathway with indication of which step of the signal-transduction pathway is affected by the different elicitors (+) and inhibitors (−).The response of the parasitoid Cotesia glomerata was tested to Pieris brassicae-infested plants (15 2^nd instar larvae) treated with inhibitor solution, Pieris brassicae-infested plants treated with a solution without inhibitor or intact plants sprayed with inhibitor solution. Recently, Bruinsma et al.¹⁷ showed that Pieris brassicae- infested plants treated with phenidone were less attractive to C. glomerata than infested plants treated with control solution (binomial test, N = 42, p = 0.008, Fig. 2). However, infested plants treated with phenidone were still more attractive than intact plants sprayed with phenidone (binomial test, N = 39, p < 0.001, Fig. 2). Thus, phenidone did reduce the induction of parasitoid attractants, but did not eliminate the induction completely. Here, we present additional experiments with the inhibitors DIECA and propyl gallate. DIECA application shows similar results as phenidone application; infested plants treated with DIECA are less attractive to C. glomerata than infested plants treated with control solution, but are more attractive than uninfested plants treated with DIECA (binomial test, N = 46, p = 0.026 and N = 26, p < 0.001, respectively, Fig. 2). Treatment with propyl gallate resulted in lower attractiveness of infested inhibitor-treated plants compared to infested control plants, but not significantly so (binomial test, N = 45, p = 0.072, Fig. 2), and propyl gallate-treated infested plants were more attractive than propyl gallate-treated intact plants (binomial test, N = 28, p < 0.001; Fig. 2). Summarizing, phenidone and DIECA treatment of Brussels sprouts plants resulted in a reduced attractiveness of caterpillar-infested B. oleracea plants to C. glomerata. Although propyl gallate-treated plants also attracted fewer parasitoids, this difference was marginally insignificant. Of the three inhibitors, the LOX inhibitor phenidone had the largest effect on the attraction of the parasitoid C. glomerata.Open in a separate windowFigure 2Attraction of Cotesia glomerata to plants sprayed with the inhibitors phenidone, DIECA, or propyl gallate, or sprayed with a control solution, with or without infestation with Pieris brassicae. Numbers to the left of the bars indicate the total number of parasitoids tested, numbers between brackets the number of parasitoids that landed on a plant (binomial test, ***p < 0.001, **p < 0.01, *p < 0.05).Our data show that both elicitors and inhibitors can be used in bio-assays to demonstrate the importance of certain steps in defense pathways.^5,23 Although application of the inhibitors to herbivore-infested plants did not abolish the response of the plants and the parasitoids still preferred them over non-induced plants, the inhibition of the octadecanoid pathway did reduce the attractiveness of the plants to the parasitoids. This implies that the octadecanoid pathway is involved in attracting parasitoids, but it is not the only factor determining parasitoid host location. This shows that use of inhibitors can provide interesting opportunities to comparatively investigate ecological interactions of genetically identical plants that differ in the degree of defense expression. Integrating knowledge on mechanisms with studies on ecological interactions and applying this to studies of increasingly complex interactions will further promote the understanding of induced defense in a community ecology context.^24,25     

A cry for help from leaf to root: Aboveground insect feeding leads to the recruitment of rhizosphere microbes for plant self-protection against subsequent diverse attacks

Plants have evolved general and specific defense mechanisms to protect themselves from diverse enemies, including herbivores and pathogens. To maintain fitness in the presence of enemies, plant defense mechanisms are aimed at inducing systemic resistance: in response to the attack of pathogens or herbivores, plants initiate extensive changes in gene expression to activate “systemic acquired resistance” against pathogens and “indirect defense” against herbivores. Recent work revealed that leaf infestation by whiteflies, stimulated systemic defenses against both an airborne pathogen and a soil-borne pathogen, which was confirmed by the detection of the systemic expression of pathogenesis-related genes in response to salicylic acid and jasmonic acid-signaling pathway activation. Further investigation revealed that plants use self protection mechanisms against subsequent herbivore attacks by recruiting beneficial microorganisms called plant growth-promoting rhizobacteria/fungi, which are capable of reducing whitefly populations. Our results provide new evidence that plant-mediated aboveground to belowground communication and vice versa are more common than expected.Key words: aboveground, induced systemic resistance, pepper, plant growth-promoting rhizobacteria, underground, whiteflyAs sessile organisms, plants are unable to actively avoid the attack of predators. To overcome this, plants have evolved a multilayer immune system against herbivores and pathogens.¹ Plants, unlike animals, lack adaptive immunity. Instead, plants are dependent on a heritable, innate immunity based on the recognition by receptors of the presence of microbial triggers (cues) including effector proteins and microbe-associated molecular patterns.¹ The perception of microbial cues leads to the induction of a broad spectrum of plant defenses called systemic acquired resistance (SAR).² Until recently, SAR was thought to be limited to the induction of plant defenses against foliar microbial pathogens. However, recent results suggested that plants can activate signal exchanges between aboveground (AG) and belowground (BG) responses.³ Three phenomena indicate that plants can make use of cues that are systemically indicative of future enemy attack: (1) induced resistance against AG pathogens by BG microbes and vice versa, (2) indirect defenses against AG insects by AG herbivore infestation and (3) BG pathogen infection leading to root exudate-mediated recruitment of BG bacteria. First, many strains of rhizosphere microbes referred to as plant growth-promoting rhizobacteria/fungi (PGPR/PGPF) have beneficial effects by positively affecting plant growth and resistance against foliar plant pathogens—a process known as induced systemic resistance (ISR).⁴ Inducible defense responses triggered by the foliar pathogen Pseudomonas syringae pv. tomato DC3000 included the induction of root secretions such as L-malic acid that effectively recruited a PGPR strain, Bacillus subtilis FB17, in Arabidopsis roots.⁵ Second, herbivore attacks on plants trigger the induction of distinct resistance responses referred to as “indirect defenses.”⁶ In addition to the “direct defense” reaction mediated by the de novo production of toxic secondary compounds against enemies, plants also defend themselves by releasing volatile organic compounds (VOCs) or extrafloral nectar (EFN) to attract natural enemies (carnivores) of the herbivores AG.⁷ Third, as plant root exudates function as BG signaling molecules that affect the composition of rhizosphere microbial populations,⁸ certain rhizobacteria express antifungal-associated genes such as the 2,4-diacetylphloroglucinol biosynthesis gene phlA. The expression of these genes is in turn influenced by root exudates, which are modulated by soilborne fungal infections.⁹In prior studies, only one-way signal transduction was considered, such as AG to BG, AG to AG or BG to BG (Fig. 1).^10–13 The above three examples provide evidence of induced resistance against the same or a similar group of organisms, such as resistance against insects by insects, or against microbes by microbes. However, there are few studies addressing insect-microbe combinations during the elicitation of induced resistance. More specifically, indirect defenses by symbiotic root interactions AG were found, such as the volatile blends released by plants with arbuscular mycorrhizal fungi, which were more attractive to aphid parasitoids than the blends from plants without mycorrhiza.¹⁴ The BG to AG defense responses of plants are not limited to arbuscular mycorrhizal fungi against herbivores. In addition to mycorrhiza-altered insect feeding preferences, a combination of Pseudomonas spp. strains affected the development of leaffolder pest and actively enhanced resistance against leaffolder attack by triggering the synthesis of systemic defense enzymes such as chitinase and proteinase inhibitors in rice plants.¹⁵ Bacillus sp. PGPR strain treatment of tomato triggered ISR to Tomato mottle virus under natural conditions by reducing the population of the silverleaf whitefly vector.¹⁶Open in a separate windowFigure 1Putative model of plant-mediated aboveground to belowground communication and vice versa during the induction of systemic resistance via tritrophic (insect-plant-rhizobacteria) interactions. (A) A plant under normal condition. (B) Whitefly infestation elicits plant systemic defenses against leaf and root pathogens. Chemical cues from root exudates secreted from AG whitefly infestation trigger the recruitment of beneficial microbes including saprophytic fungi, Gram-positive bacteria and actinomycetes. (C) The induction of systemic resistance by colonization by beneficial microbes confers plant self-protection against subsequent herbivore attacks.Recently, we found another type of induced resistance response: bidirectional signal exchanges between AG and BG (Fig. 1).¹⁷ Our study demonstrated that the phloem feeding whiteflies can induce systemic resistance against both a leaf bacterial pathogen and a soil-borne bacterial pathogen. A similar study using the whitefly as an AG feeding insect to test the induction of plant defenses only observed its effects against conspecific insect herbivore competitors AG.¹⁸ However, in our study, foliar attack by the whitefly not only elicited AG resistance against a leaf pathogenic bacterium, Xanthomonas axonopodis pv. vesicatoria, but also enhanced resistance against the soil-borne pathogenic bacterium, Ralstonia solanacearum. The induction of systemic resistance was confirmed by significant upregulation of the SA and JA defense signaling pathway marker genes, Capsicum annuum pathogenesis-related protein (CaPR)1, CaPR4, CaPR10 and Ca protease inhibitor (CaPIN) in both leaves (AG) and roots (BG) after whitefly feeding. Interestingly, AG white-fly feeding significantly increased the population density of beneficial BG microflora including Gram-positive bacteria, actinomycetes and saprophytic fungi that may induce systemic resistance (Fig 1).⁴ Among BG microbial groups, several Grampositive Bacillus sp. strains significantly elicited plant systemic defenses against the whitefly population in the tomato field.¹⁶ Our studies provide a new understanding of tritrophic (insect-plant-PGPR) interactions and their role in the induction of defense mechanisms. In the near future, it will be important to define plant defense signaling molecules from AG to BG and to dissect the signaling transduction pathways using “omics” technology to reveal the mechanisms by which plants protect themselves against enemy attacks.     

Dissection of salicylic acid-mediated defense signaling networks

The small phenolic molecule salicylic acid (SA) plays a key role in plant defense. Significant progress has been made recently in understanding SA-mediated defense signaling networks. Functional analysis of a large number of genes involved in SA biosynthesis and regulation of SA accumulation and signal transduction has revealed distinct but interconnecting pathways that orchestrate the control of plant defense. Further studies utilizing combinatorial approaches in genetics, molecular biology, biochemistry and genomics will uncover finer details of SA-mediated defense networks as well as further insights into the crosstalk of SA with other defense signaling pathways. The complexity of defense networks illustrates the capacity of plants to integrate multiple developmental and environmental signals into a tight control of the costly defense responses.Key words: salicylic acid, disease resistance, signal transduction, Arabidopsis, Pseudomonas syringaePlants have evolved sophisticated defense mechanisms to ward off attacks from pathogens. In addition to pre-formed physical/chemical barriers, plants can actively monitor the presence of pathogens and subsequently activate defense signaling networks, which in turn restrict the further growth and spread of pathogens.The small phenolic compound salicylic acid (SA) plays a central role in plant defense signaling. It is required for recognition of pathogen-derived components and subsequent establishment of local resistance in the infected region as well as systemic resistance at the whole plant level.^1–3 SA accumulation is inducible upon infections of various pathogens, treatment with elicitors from pathogens, and stress conditions.^3–5 Exogenous application of SA and its synthetic analogs to plants is sufficient to invoke disease resistance.^6–9 Disruption of SA accumulation and/or signaling by mutations or by a transgenic SA hydrolase encoded by the bacterial gene nahG greatly compromises defense against pathogens.¹⁰ In addition, the phytohormones jasmonic acid (JA) and ethylene (ET) regulate SA-mediated defense as well as many aspects of plant development. Emerging evidence also implicates additional phytohormones in plant defense, two of which, auxin and abscisic acid, were recently shown to impact the SA pathway.^11,12The past two decades have witnessed exciting progress made towards a comprehensive understanding of defense networks in the model plant Arabidopsis, especially those regulated by SA. The discovery of an expanding array of genes involved in SA-mediated defense suggests the complexity of defense networks. Surprisingly, information on functional relationships among many defense genes is sparse. Connecting the dots (genes) on the defense map to form pathways, which are further interconnected into complex defense networks, still remains a challenging task. This review focuses on our current understanding of the interactions among genes that regulate three key sub-circuits of the SA pathway: SA biosynthesis, SA accumulation and SA signal transduction. Discussions of the crosstalk between components involved in the SA pathway and those in other defense pathways can be found in some excellent reviews.^13–17     

Plant defenses against parasitic plants show similarities to those induced by herbivores and pathogens

Herbivores and pathogens come quickly to mind when one thinks of the biotic challenges faced by plants. Important but less appreciated enemies are parasitic plants, which can have important consequences for the fitness and survival of their hosts. Our knowledge of plant perception, signaling and response to herbivores and pathogens has expanded rapidly in recent years, but information is generally lacking for parasitic species. In a recent paper we reported that some of the same defense responses induced by herbivores and pathogens—notably increases in jasmonic acid (JA), salicylic acid (SA), and a hypersensitive-like response (HLR)—also occur in tomato plants upon attack by the parasitic plant Cuscuta pentagona (field dodder). Parasitism induced a distinct pattern of JA and SA accumulation, and growth trials using genetically-altered tomato hosts suggested that both JA and SA govern effective defenses against the parasite, though the extent of the response varied with host plant age. Here we discuss similarities between the induced responses we observed in response to Cuscuta parasitism to those previously described for herbivores and pathogens and present new data showing that trichomes should be added to the list of plant defenses that act against multiple enemies and across kingdoms.Key words: Cuscuta, induced defenses, parasitic plant, jasmonic acid, salicylic acid, phytohormones, hypersensitive response, trichomes, defense signalingSeveral thousand species of plants are parasitic, stealing water and nutrients from other plants through a specialized feeding structure, the haustorium.¹ Haustoria are thought to be modified roots that grow into tissues and fuse with the vascular system of their photosynthetic hosts.¹ Considering that these parasites include some of the world''s most devastating agricultural pests² and are influential, fascinating components of natural communities,^1,3 surprisingly little is known about host defenses induced by parasitic plants. To address this shortcoming, we used a metabolomics approach to track biochemical changes induced in tomato shoots by invasion of C. pentagona haustoria.⁴We found that parasitism induced large increases in both JA and SA beginning about 24 hr after formation of haustoria began, but that production of JA and SA was largely separated in time. Host production of JA was transitory and reached a maximum at 36 hr, whereas SA peaked 12 hr later and remained elevated 5 d later. We also found that C. pentagona grew larger on mutant tomato plants in which the SA (NahG) or JA (jasmonic acid-insensitive1) pathways were disrupted, suggesting that these hormones can act independently to reduce parasite growth. Taken together, these findings suggest the staggered production of JA and SA may be an adaptive response to parasitism—by sequentially activating the JA and SA pathways, tomato plants may minimize the potential for cross-talk between these sometimes antagonistic pathways^5,6 and utilize both signaling molecules.^6,7 Thus, defenses against C. pentagona contain elements characteristic of responses to both herbivores (primarily JA-mediated⁸) and pathogens (primarily SA-mediated⁹)—though it should be noted that some herbivores induce SA¹⁰ and some pathogens JA.¹¹ It is worth noting that parasitism induced predominately cis-JA, the same jasmonate isomer induced by herbivore feeding.¹² Host responses to Cuscuta seem to most resemble that of known plant responses to some pathogens in which a similar sequence of JA and SA production is required to limit disease.¹³C. pentagona also triggered a hypersensitive-like response (HLR) localized around the points of parasite attachment. Using a trypan blue staining technique, we verified host cell death in these parasite-induced lesions. The deposition of eggs by some insect herbivores can elicit the formation of necrotic tissue,¹⁴ but localized cell death is most widely associated with the hypersensitive response (HR) of plants to pathogens. This complex early defense response can restrict the growth and spread of viruses, fungi and bacteria.⁹ Our work adds to existing evidence¹⁵ that the Cuscuta-induced HLR can play a similar role by preventing or limiting the growth of the parasite.An interesting discovery was that the first attachment by C. pentagona elicited almost no response from young 10-day-old hosts, whereas a subsequent attachment after 10 days induced the wholesale changes discussed above (we also found changes in abscisic acid and free fatty acids). Trials in which we varied the age of the host and parasite indicated that host age, rather than a priming effect on defenses, determined the magnitude of response. We have previously observed that Cuscuta spp. in natural populations germinate very early in the growing season, and hypothesized that this tactic promotes successful parasitism by ensuring the presence of young hosts; recent field work seems to corroborate this.¹⁶ As with the response to Cuscuta parasitism, levels of host plant defenses against insects¹⁷ and pathogens¹⁸ are known to be vary with host age.In an earlier paper we reported that tomato plants parasitized by C. pentagona released greater amounts of volatiles than did unparasitized control plants.¹⁹ The production and release of volatiles is a hallmark of plant responses to feeding by herbivores.²⁰ Herbivore-induced volatiles serve as an indirect plant defense by attracting herbivores'' natural enemies,²¹ repelling herbivores,²² or acting as intra-plant signals that prime systemic responses.²³ Although less well documented, pathogen attack can also induce emissions of volatile compounds,²⁴ some of which are antimicrobial and may serve as a direct defense against infection.²⁵ The same volatile compounds induced by Cuscuta (e.g., 2-carene, α-pinene, limonene, β-phellandrene) were also induced by caterpillar feeding and application of JA.¹⁹ Like herbivores, the JA induced by C. pentagona may regulate the emissions of plant volatiles. Whether or how parasitic plant-induced volatiles might function in defense is unknown, but they presumably could affect host plant choice by Cuscuta seedlings, which use plant volatiles to locate and select hosts.²⁶Following on from our previous studies we examined the potential role of host trichomes in resistance to parasitism by C. pentagona. Plant trichomes have been long appreciated as the first line of defense against insect herbivores^27,28 and more recently pathogens.²⁹ We hypothesized that trichomes could also defend against parasitic plants based on our observations that (1) tomato trichomes become denser with age (Fig. 1), notably on hypocotyls which is the first area contacted by Cuscuta seedlings, and (2) these trichomes can act as a physical barrier to C. pentagona seedlings. To test this hypothesis we allowed seedlings of C. pentagona to attach to 25-day-old tomato plants (Solanum lycopersicum ‘Halley 3155’) in a climate controlled growth chamber. Of 20 trials conducted, in six (30%) the parasite seedling was completely blocked by trichomes and was unable to reach the host stem—the parasite perished in each of these. Type I glandular trichomes, which are several millimeters long with a glandular tip,³⁰ were primarily responsible for the blocking effect. Thus, trichomes can defend against parasitic plants in a manner analogous to herbivores by physically obstructing their movement. Interestingly, the effectiveness of trichomes is also dependent on age of the host since those on younger tomato plants (<20 days old) are too sparse to impede Cuscuta seedlings (Fig. 1).Open in a separate windowFigure 1A newly germinated Cuscuta pentagona seedling encircles and attaches to the hypocotyl of a 10-day-old tomato seedling; the early development of haustoria are visible as nod-like swellings. The trichomes on hypocotyls of young tomato seedlings are not dense enough to affect C. pentagona seedlings, but the increased density of trichomes on 25-day-old plants can act as a physical barrier that blocks parasite seedlings (inset).Considering that the majority of plant defenses are mediated by only a small number of master regulators (e.g., JA, SA, ethylene),⁷ it is not surprising that plant responses to parasitic plants share commonalities with those induced by herbivores and pathogens. These few molecules mediate complex, interacting signaling networks that can be variously activated and modified by plants to tune defenses against a seemingly endless variety of attackers.⁷ Our finding that JA and SA act to defend plants from attack by other plants, further support these phytohormones as ‘global’ defense signals. It is also apparent that constitutive defenses, such as trichomes, can be effective against diverse antagonists (e.g., herbivores and parasitic plants). These new insights into host defenses against parasitic plants suggest many avenues of needed research including the molecular events induced by parasitic plant attack, the parasite-derived cues that elicit responses, and the ways in which JA and SA act to reduce parasite growth. Finally, our findings suggest it might be possible to manipulate induced responses or host plant age by varying planting date to control parasitic plants in agriculture.     

Perception of volatiles produced by UVC-irradiated plants alters the response to viral infection in na?ve neighboring plants

Interplant communication of stress via volatile signals is a well-known phenomenon. It has been shown that plants undergoing stress caused by pathogenic bacteria or insects generate volatile signals that elicit defense response in neighboring naïve plants.¹ Similarly, we have recently shown that naïve plants sharing the same gaseous environment with UVC-exposed plants exhibit similar changes in genome instability as UVC-exposed plants.² We found that methyl salicylate (MeSA) and methyl jasmonate (MeJA) serve as volatile signals communicating genome instability (as measured by an increase in the homologous recombination frequency). UVC-exposed plants produce high levels of MeSA and MeJA, a response that is missing in an npr1 mutant. Concomitantly, npr1 mutants are impaired in communicating the signal leading to genome instability, presumably because this mutant does not develop new necrotic lesion after UVC irradiation as observed in wt plants.² To analyze the potential biological significance of such plant-plant communication, we have now determined whether bystander plants that receive volatile signals from UVC-irradiated plants, become more resistant to UVC irradiation or infection with oilseed rape mosaic virus (ORMV). Specifically, we analyzed the number of UVC-elicited necrotic lesions, the level of anthocyanin pigments, and the mRNA levels corresponding to ORMV coat protein and the NPR1-regulated pathogenesis-related protein PR1 in the irradiated or virus-infected bystander plants that have been previously exposed to volatiles produced by UVC-irradiated plants. These experiments showed that the bystander plants responded similarly to control plants following UVC irradiation. Interestingly, however, the bystander plants appeared to be more susceptible to ORMV infection, even though PR1 mRNA levels in systemic tissue were significantly higher than in the control plants, which indicates that bystander plants could be primed to strongly respond to bacterial infection.     

Is crypsis a common defensive strategy in plants?: Speculation on signal deception in the New Zealand flora

Color is a common feature of animal defense. Herbivorous insects are often colored in shades of green similar to their preferred food plants, making them difficult for predators to locate. Other insects advertise their presence with bright colors after they sequester enough toxins from their food plants to make them unpalatable. Some insects even switch between cryptic and aposomatic coloration during development.¹ Although common in animals, quantitative evidence for color-based defense in plants is rare. After all, the primary function of plant leaves is to absorb light for photosynthesis, rather than reflect light in ways that alter their appearance to herbivores. However, recent research is beginning to challenge the notion that color-based defence is restricted to animals.Key words: aposomatic colouration, cryptic colouration, herbivory, moa, plant defenseTemperate deciduous forests provide what is arguably the most extraordinary display of color in nature. Prior to leaf-fall in autumn, the leaves of many deciduous tree species in Asia, Europe and North America turn red, leading to brilliantly colored landscapes. Once thought to be a by-product of chlorophyll re-absorption prior to leaf abscission, autumn flushes in red leaf colors are now known to result from the active synthesis of red-colored pigments.^2,3 Although the exact reason for the production of red-colored pigments prior to leaf-fall is unknown, it has recently been hypothesized to be a form of defense.⁴ Aphids are common phloem-feeding herbivores in deciduous forests, which disperse from the forest floor into tree crowns in autumn, and the synthesis of red pigments could signal the timing of leaf fall and the reduction in the supply of photosynthate.^5,6 Although there are also physiological explanations,⁷ red leaf colors could be a reliable signal of unpalatably to herbivores.⁸Lev-Yadun and Holopainen⁹ recently showed that there are fewer red-colored deciduous tree species in Europe than in North America, and they speculate that historical processes are the cause. During the advance and retreat of glaciers in the Pleistocene, the European Alps would have hindered the movement of plants and their herbivores in response to long-term climate change. Mountain ranges in North America run perpendicular to the equator, which would facilitate these migrations. Therefore, if red leaves are signals to herbivores, geographic differences in leaf pigmentation may be ‘anachronistic’.¹⁰ In other words, they may result from historical coevolutionary dynamics between plants and their herbivores, rather than present day selection pressures alone.Despite these important insights, our understanding of color-based defense in plants is in its infancy and progress hinges on quantitative tests in other parts of the globe. Previous work is also restricted largely to aposomatic, or warning colors.¹¹ Given that cryptic coloration is widespread in animals, it might also be common in plants. Yet we are far from determining if this is true.Here, I discuss several New Zealand plant species that seem to be colored in ways that would make them difficult for herbivores to locate. I suggest that these plants are anachronisms; their unusual appearance is the result of selection from flightless browsing birds called moa, which went extinct following the arrival of humans in New Zealand 750 years ago. I also discuss the difficulties associated with testing for cypsis in plants and finish by outlining a methodological approach to test for color-based defense in plants when the putative herbivores are either unknown or extinct.     

Analyzing plant defenses in nature

A broad range of chemical plant defenses against herbivores has been studied extensively under laboratory conditions. In many of these cases there is still little understanding of their relevance in nature. In natural systems, functional analyses of plant traits are often complicated by an extreme variability, which affects the interaction with higher trophic levels. Successful analyses require consideration of the numerous sources of variation that potentially affect the plant trait of interest. In our recent study on wild lima bean (Phaseolus lunatus L.) in South Mexico, we applied an integrative approach combining analyses for quantitative correlations of cyanogenic potential (HCNp; the maximum amount of cyanide that can be released from a given tissue) and herbivory in the field with subsequent feeding trials under controlled conditions. This approach allowed us to causally explain the consequences of quantitative variation of HCNp on herbivore-plant interactions in nature and highlights the importance of combining data obtained in natural systems with analyses under controlled conditions.Key words: natural systems, plant defensive traits, optimal defense hypothesis (ODH), cyanogenesis, lima bean, Phaseolus lunatus L., plant-herbivore interaction, plant-pathogen interaction, multiple defense syndromesAnalyzing plant defenses against herbivores in nature is often complicated by an extreme variability in multiple factors. Plant populations generally show high genetic variability resulting in substantial intraspecific variation of plant traits.¹ In addition to genotypic variability, phenotypic plasticity of plants is a source of variation.² At the level of individual plants, expression of defensive traits strongly depends on plant organ and ontogeny of plants or plant parts. Within an individual plant, it is quite common for reproductive structures and young leaves to be better chemically defended than older leaf tissues. To explain these within-plant variations of defenses, the optimal defense hypothesis (ODH) was formulated. Concerning the variability of chemical defenses of leaves, the ODH predicts that within the total foliage of a plant, young leaves make a larger contribution to plant fitness than old leaves as they have a higher potential photosynthetic value resulting from a longer expected life-time.^3–5 In addition, younger leaves are often more nutritious and thus more attractive to herbivores⁶ and should consequently be better defended.⁷ In this line, the basic assumption of the ODH is that three main factors—cost of defense, risk of attack and value of the respective plant organ—determine the investment in defensive secondary metabolites.^8,9 Thus, the higher the risk of a given plant tissue to be consumed by herbivores and the higher its value for plant fitness, the more energy should be allocated to its defense.^10,11 Beyond genotypic and ontogenetic variability of a given defense, potential co-variation with other defensive or nutritive traits expressed by the same plant individual can strongly affect its efficiency as defense against herbivores.^12,13 In addition to these endogenous sources of defensive variability, the expression of plant traits strongly depends on multiple external factors such as temperature or availability of plant nutrients, water or light (Fig. 1).¹⁴ At the same time, the outcome of herbivore-plant interactions is crucially determined by biotic interactions. Plant interactions with mutualistic microorganisms such as Rhizobia, mycorrhiza and above-ground fungal endophytes as well as tri-trophic interactions with predators and parasitoids of herbivores can all strongly impact plant fitness.¹⁵Open in a separate windowFigure 1Factors influencing variability of plant defenses. Plant defensive traits are affected by various endogenous and external factors. Endogenous factors comprise plant genotype and ontogeny of plants or plant parts. External factors can be categorized as abiotic or biotic. Important abiotic factors that can influence plant defenses are light exposure, temperature, soil salinity, as well as water and nutrient availability. Biotic factors that can have an effect on plant defense are interspecific interaction with Rhizobia (in the case of legumes), mycorrhizal and endophytic fungi, pathogens as well as the interaction with conspecifics or different plant species.Variability in herbivore-plant interactions can also be associated with herbivore variation. Different attackers of a particular plant species might be affected in different ways by toxins in food plants (Fig. 1). The efficiency of a specific defensive compound can also depend on the feeding mode, i.e., sucking or chewing, as well as on the degree of specialization of the herbivore to the respective plant.¹⁶ Defenses mediated by secondary plant compounds are generally believed not to affect specialist herbivores, because of their capacity to tolerate or to detoxify defensive compounds of their hosts by behavioral or physiological adaptations.^17–20 In this context, the specialist herbivore paradigm predicts that adapted herbivores are less affected by a given chemical defense than generalists,^21,22 although exceptions have been noted.^23–25While it is important to consider these numerous sources of variation affecting the outcome of herbivore-plant interactions when designing functional studies, a significant fraction of the variability in natural systems will always remain unidentified. Consequently, approaches combining field observations with experiments under controlled conditions provide a powerful tool to uncover functional interactions between plants and their multiple antagonists in nature.In a recent study, we analyzed the importance of wild lima bean''s cyanogenesis—i.e., the release of toxic hydrogen cyanide from preformed precursors in response to cell damage—as plant defense at a natural site in South Mexico.²⁵ Although cyanogenesis is generally considered an efficient direct defense against herbivores, in numerous studies plant cyanide production had little or no effect on herbivores.^26–28 One would like to think that most of these inconsistencies in cyanogenesis-based herbivore defense efficiency could be explained by one or more sources of variation mentioned above. Nevertheless, field studies analyzing the action of plant cyanogenesis on a quantitative basis have been scarce. In our study, a two-step approach was used to gain insight into the function of cyanogenesis in nature.²⁵ First, cyanide concentration and herbivore damage were quantified by measuring removed leaf area of individual leaves derived from different individual plants while considering microclimate conditions. Significant negative correlations between cyanogenesis and leaf damage were observed. Second, since existing correlations do not necessarily indicate causal associations, we conducted consecutive feeding experiments under controlled conditions. To consider natural variability of lima beans'' cyanogenesis observed in nature in our analysis, we prepared clones from field-grown plants with different but defined cyanogenic features. These clonal plants showed high constancy of cyanogenic traits compared to their respective mother plants and thus, could be used in comparative analyses. Every effort was made to duplicate natural conditions and so herbivore species selected for feeding trials represented those identified in the field as the most important plant consumers at the respective site (pers. observ.). Feeding trials supported our hypothesis that cyanogenesis has quantitative effects on herbivore behavior in nature and explained the negative correlation of lima bean''s cyanogenesis and herbivory observed in the field.Analytical approaches combining field observations with controlled experiments help to explain natural patterns and may represent a powerful methodological approach for functional analyses of herbivore-plant interactions.     

Shared signals -'alarm calls' from plants increase apparency to herbivores and their enemies in nature

The attraction of natural enemies of herbivores by volatile organic compounds as an induced indirect defence has been studied in several plant systems. The evidence for their defensive function originates mainly from laboratory studies with trained parasitoids and predators; the defensive function of these emissions for plants in natural settings has been rarely demonstrated. In native populations and laboratory Y-tube choice experiments with transgenic Nicotiana attenuata plants unable to release particular volatiles, we demonstrate that predatory bugs use terpenoids and green leaf volatiles (GLVs) to locate their prey on herbivore-attacked plants. By attracting predators with volatile signals, this native plant reduces its herbivore load – demonstrating the defensive function of herbivore-induced volatile emissions. However, plants producing GLVs are also damaged more by flea beetles. The implications of these conflicting ecological effects for the evolution of induced volatile emissions and for the development of sustainable agricultural practices are discussed.