Ecophysiological comparison of direct and indirect defenses in Nicotiana attenuata

After herbivore attack, plants launch a suite of direct and indirect defense responses that must be coordinated if plants are to realize a fitness benefit from these responses. Here we characterize the volatile emissions in the native tobacco plant, Nicotiana attenuata Torr. ex Wats., that are elicited by tobacco hornworm (Manduca sexta L.) attack and are known to function as attractants for parasitoids. To provide the first ecophysiological comparison of examples of both types of defense in the same species, we characterize the elicitation and signaling mechanisms, the resources required, and the potential costs and benefits of the volatile release and compare these traits with those of the well-described induced direct defense in this species, nicotine production. The release of (E)-β-ocimene, cis-α-bergamotene and linalool is dramatically induced within 24 h by application of methyl jasmonate (MeJA), caterpillar feeding, and the treatment of mechanical wounds with larval oral secretions (OS), but not by mechanical damage alone. Plants from different geographic locations produce volatile blends that differ in composition. The most consistently released component from all genotypes, cis-α-berga-motene, is positively related to the amount of MeJA and the level of wounding if OS are applied to the wounds. The volatile release is strongly light dependent, dropping to undetectable quantities during dark periods, even when temperatures are elevated to match those of the light period. Inhibitors of wound-induced jasmonate accumulation (salicylates and auxins), which are known to inhibit wound-induced nicotine production, do not inhibit the release of volatiles. By individually inducing different leaf positions with OS and, on other plants, excising them after induction, we demonstrate that the emission is largely a systemic, whole-plant response, which is maximally triggered when the second fully expanded leaf is induced. We conclude that while both are whole-plant, systemic responses that utilize recently acquired resources for their production and are activated by the jasmonate cascade, the elicitation of the volatile release exhibits greater tissue sensitivity and utilizes additional signaling components than does nicotine production. In contrast to the large investment of fitness-limiting resources required for induced nicotine production or the resources used in benzyl acetone release from flowers for pollinator attraction, the resource requirements for the volatile release are minor. Hence the argument that the volatile release incurs comparatively large physiological costs cannot be supported in this system. Received: 4 November 1999 / Accepted: 1 March 2000     

Ectopic expression of AtJMT in Nicotiana attenuata: creating a metabolic sink has tissue-specific consequences for the jasmonate metabolic network and silences downstream gene expression

To create a metabolic sink in the jasmonic acid (JA) pathway, we generated transgenic Nicotiana attenuata lines ectopically expressing Arabidopsis (Arabidopsis thaliana) jasmonic acid O-methyltransferase (35S-jmt) and additionally silenced in other lines the N. attenuata methyl jasmonate esterase (35S-jmt/ir-mje) to reduce the deesterification of methyl jasmonate (MeJA). Basal jasmonate levels did not differ between transgenic and wild-type plants; however, after wounding and elicitation with Manduca sexta oral secretions, the bursts of JA, jasmonoyl-isoleucine (JA-Ile), and their metabolites that are normally observed in the lamina, midvein, and petiole of elicited wild-type leaves were largely absent in both transformants but replaced by a burst of endogenous MeJA that accounted for almost half of the total elicited jasmonate pools. In these plants, MeJA became a metabolic sink that affected the jasmonate metabolic network and its spread to systemic leaves, with major effects on 12-oxo-phytodieonic acid, JA, and hydroxy-JA in petioles and on JA-Ile in laminas. Alterations in the size of jasmonate pools were most obvious in systemic tissues, especially petioles. Expression of threonine deaminase and trypsin proteinase inhibitor, two JA-inducible defense genes, was strongly decreased in both transgenic lines without influencing the expression of JA biosynthesis genes that were uncoupled from the wounding and elicitation with M. sexta oral secretions-elicited JA-Ile gradient in elicited leaves. Taken together, this study provides support for a central role of the vasculature in the propagation of jasmonates and new insights into the versatile spatiotemporal characteristics of the jasmonate metabolic network.     

Consequences of sequential attack for resistance to herbivores when plants have specific induced responses

Plants in nature are attacked sequentially by herbivores, and theory predicts that herbivore-specific responses allow plants to tailor their defenses. We present a novel field test of this hypothesis, and find that specific responses of Solanum dulcamara lead to season-long consequences for two naturally colonizing herbivores, irrespective of the second herbivore to attack plants. This result indicates that responses induced by the initial herbivore made plants less responsive to subsequent attack. We show that initial herbivory by flea beetles and tortoise beetles induce distinct plant chemical responses. Initial herbivory by flea beetles lowered the occurrence of conspecifics and tortoise beetles relative to controls. Conversely, initial herbivory by tortoise beetles did not influence future herbivory. Remarkably, the experimentally imposed second herbivore to feed on plants did not modify consequences (induced resistance or lack thereof) of the first attacker. Induction of plant chemical responses was consistent with these ecological effects; i.e. the second herbivore did not modify the plant's initial induced response. Thus, canalization of the plant resistance phenotype may constrain defensive responses in a rapidly changing environment.     

Jasmonic acid signalling and herbivore resistance traits constrain regrowth after herbivore attack in Nicotiana attenuata

Because traits conferring resistance on herbivores can reduce fitness-associated traits, trade-offs may occur between tolerance and resistance responses. We examined these trade-offs in genotypes of Nicotiana attenuata that were transformed to silence trypsin proteinase inhibitor (TPI) production (AS-Natpi), an antiherbivore defense associated with (14%) reductions in seed production, and the jasmonate signal cascade that elicits these defenses (AS-Nalox3), by measuring stalk and axillary branch growth and seed production after two defoliation regimes and Manduca sexta larval attack to bottom or middle and top stalk leaves. Larval attack and defoliation at middle and top leaves depressed seed production and increased axillary branching more than at bottom leaves. AS-Nalox3 and AS-Natpi plants produced significantly longer (two- to fourfold) branches than did wild-type (WT) plants, results that are consistent with resource-based trade-offs between resistance and regrowth. Methyl jasmonate (MeJA) treatment of AS-Nalox3 plants restored WT branch growth, suggesting that jasmonic acid (JA) signalling suppresses regrowth and contributes to apical dominance. These results are consistent with the existence of JA- and resource-mediated trade-offs between regrowth and herbivore resistance traits.     

Differential elicitation of two processing proteases controls the processing pattern of the trypsin proteinase inhibitor precursor in Nicotiana attenuata

Trypsin proteinase inhibitors (TPIs) of Nicotiana attenuata are major antiherbivore defenses that increase dramatically in leaves after attack or methyl jasmonate (MeJA) elicitation. To understand the elicitation process, we characterized the proteolytic fragmentation and release of TPIs from a multidomain precursor by proteases in MeJA-elicited and unelicited plants. A set of approximately 6-kD TPI peptides was purified from leaves, and their posttranslational modifications were characterized. In MeJA-elicited plants, the diversity of TPI structures was greater than the precursor gene predicted. This elicited structural heterogeneity resulted from differential fragmentation of the linker peptide (LP) that separates the seven-domain TPI functional domains. Using an in vitro fluorescence resonance energy transfer assay and synthetic substrates derived from the LP sequence, we characterized proteases involved in both the processing of the TPI precursor and its vacuolar targeting sequence. Although both a vacuolar processing enzyme and a subtilisin-like protease were found to participate in a two-step processing of LP, only the activity of the subtilisin-like protease was significantly increased by MeJA elicitation. We propose that MeJA elicitation increases TPI precursor production and saturates the proteolytic machinery, changing the processing pattern of TPIs. To test this hypothesis, we elicited a TPI-deficient N. attenuata genotype that had been transformed with a functional NaTPI gene under control of a constitutive promoter and characterized the resulting TPIs. We found no alterations in the processing pattern predicted from the sequence: a result consistent with the saturation hypothesis.     

Jasmonate-mediated induced plant resistance affects a community of herbivores

1. The negative effect of induced plant resistance on the preference and performance of herbivores is a well‐documented ecological phenomenon that is thought to be important for both plants and herbivores. This study links the well‐developed mechanistic understanding of the biochemistry of induced plant resistance in the tomato system with an examination of how these mechanisms affect the community of herbivores in the field. 2. Several proteins that are induced in tomato foliage following herbivore damage have been linked causally to reductions in herbivore performance under laboratory conditions. Application of jasmonic acid, a natural elicitor of these defensive proteins, to tomato foliage stimulates induced responses to herbivory. 3. Jasmonic acid was sprayed on plants in three doses to generate plants with varying levels of induced responses, which were measured as increases in the activities of proteinase inhibitors and polyphenol oxidase. 4. Field experiments conducted over 3 years indicated that induction of these defensive proteins is associated with decreases in the abundance of all four naturally abundant herbivores, including insects in three feeding guilds, caterpillars, flea beetles, aphids, and thrips. Induced resistance killed early instars of noctuid caterpillars. Adult flea beetles strongly preferred control plants over induced plants, and this effect on host plant preference probably contributed to differences in the natural abundance of flea beetles. 5. The general nature of the effects observed in this study suggests that induced resistance will suppress many members of the herbivore community. By linking plant biochemistry, insect preference, performance, and abundance, tools can be developed to manipulate plant resistance sensibly and to predict its outcome under field conditions.     

Jasmonate-induced defenses: a tale of intelligence, collaborators and rascals

Plants have sophisticated defense systems to protect their tissues against the attack of herbivorous organisms. Many of these defenses are orchestrated by the oxylipin jasmonate. A growing body of evidence indicates that the expression of jasmonate-induced responses is tightly regulated by the ecological context of the plant. Ecological information is provided by molecular signals that indicate the nature of the attacker, the value of the attacked organs, phytochrome status and thereby proximity of competing plants, association with beneficial organisms and history of plant interactions with pathogens and herbivores. This review discusses recent advances in this field and highlights the need to map the activities of informational modulators to specific control points within our emerging model of jasmonate signaling.     

Diffuse interactions between two herbivores and constraints on the evolution of resistance in horsenettle (<Emphasis Type="Italic">Solanum carolinense</Emphasis>)

Interactions between plants and herbivores are considered “diffuse” if the ecological interactions between two species, or their evolutionary responses, differ depending on the presence of a third species. I looked for evidence of diffuse interactions among horsenettle (Solanum carolinense) and two common herbivores. Plants experimentally protected from lace bugs (Gargaphia solani) were twice as susceptible to attack from flea beetles (Epitrix fuscula) once transplanted into the field. Thus, the selective benefit to horsenettle of resistance against lace bugs in nature would likely be diminished by increased attack from flea beetles. Moreover, the relative rank among horsenettle genotypes for resistance against flea beetles was changed by prior exposure to lace bugs. Thus, the genetic expression of resistance to flea beetles would depend on the “lace bug environment.” By affecting selective pressures or response to selection, these sorts of diffuse interactions in a plant-herbivore community can slow the host plant’s evolution of resistance to its herbivores.     

Ecological trade-offs between jasmonic acid-dependent direct and indirect plant defences in tritrophic interactions

Recent studies on plants genetically modified in jasmonic acid (JA) signalling support the hypothesis that the jasmonate family of oxylipins plays an important role in mediating direct and indirect plant defences. However, the interaction of two modes of defence in tritrophic systems is largely unknown. In this study, we examined the preference and performance of a herbivorous leafminer (Liriomyza huidobrensis) and its parasitic wasp (Opius dissitus) on three tomato genotypes: a wild-type (WT) plant, a JA biosynthesis (spr2) mutant, and a JA-overexpression 35S::prosys plant. Their proteinase inhibitor production and volatile emission were used as direct and indirect defence factors to evaluate the responses of leafminers and parasitoids. Here, we show that although spr2 mutant plants are compromised in direct defence against the larval leafminers and in attracting parasitoids, they are less attractive to adult flies compared with WT plants. Moreover, in comparison to other genotypes, the 35S::prosys plant displays greater direct and constitutive indirect defences, but reduced success of parasitism by parasitoids. Taken together, these results suggest that there are distinguished ecological trade-offs between JA-dependent direct and indirect defences in genetically modified plants whose fitness should be assessed in tritrophic systems and under natural conditions.     

Costs of jasmonate-induced responses in plants competing for limited resources

We present a design to quantify fitness consequences of jasmonate-induced responses in plants that are competing for limited resources with a conspecific. Under both high and low nitrogen supply rates, uninduced (control) Nicotiana attenuata plants growing next to a plant induced with 250 μg methyl jasmonate (MJ) yielded more seed capsules than control plants competing with another control plant. We conclude that there is a opportunity benefit for control plants growing next to an induced plant. Initially, MJ-induced plants grew more slowly, but by senescence they had produced the same number of seed capsules as control plants that had competed with another control plant. Replacement series showed that the fitness of MJ-induced plants is not influenced by the competitive status of their neighbour plant. We argue that competitive designs are useful tools for evaluating the phenotypic costs of ecologically important traits.     

Herbivore-induced plant vaccination. Part I. The orchestration of plant defenses in nature and their fitness consequences in the wild tobacco Nicotiana attenuata

A plant's responses to attack from particular pathogens and herbivores may result in resistance to subsequent attack from the same species, but may also affect different species. Such a cross-resistance, called immunization or vaccination, can benefit the plant, if the fitness consequences of attack from the initial attacker are less than those from subsequent attackers. Here, we report an example of naturally occurring vaccination of the native tobacco plant, Nicotiana attenuata, against Manduca hornworms by prior attack from the mirid bug, Tupiocoris notatus (Dicyphus minimus), which results from the elicitation of two categories of induced plant responses. First, attack from both herbivore species causes the plants in nature to release predator-attracting volatile organic compounds (VOCs), and the attracted generalist predator, Geocoris pallens, preferentially attacks the less mobile hornworm larvae. Second, attack from both mirids and hornworms increases the accumulation of secondary metabolites and proteinase inhibitors (PIs) in the leaf tissue, which is correlated with the slow growth of Manduca larvae. Mirid damage does not significantly reduce the fitness of the plant in nature, whereas attack from the hornworm reduces lifetime seed production. Consequently, plants that are attacked by mirids realize a significant fitness advantage in environments with both herbivores. The combination of growth-slowing direct defenses and predator-attracting indirect defenses results in greater hornworm mortality on mirid-attacked plants and provides the mechanism of the vaccination phenomenon.     

BAK1 regulates the accumulation of jasmonic acid and the levels of trypsin proteinase inhibitors in Nicotiana attenuata's responses to herbivory

BAK1 is a co-receptor of brassinosteroid (BR) receptor BRI1, and plays a well-characterized role in BR signalling. BAK1 also physically interacts with the flagellin receptor FLS2 and regulates pathogen resistance. The role of BAK1 in mediating Nicotiana attenuata's resistance responses to its specialist herbivore, Manduca sexta, was examined here. A virus-induced gene-silencing system was used to generate empty vector (EV) and NaBAK1-silenced plants. The wounding- and herbivory-induced responses were examined on EV and NaBAK1-silenced plants by wounding plants or simulating herbivory by treating wounds with larval oral secretions (OS). After wounding or OS elicitation, NaBAK1-silenced plants showed attenuated jasmonic acid (JA) and JA-isoleucine bursts, phytohormone responses important in mediating plant defences against herbivores. However, these decreased JA and JA-Ile levels did not result from compromised MAPK activity or elevated SA levels. After simulated herbivory, NaBAK1-silenced plants had EV levels of defensive secondary metabolites, namely, trypsin proteinase inhibitors (TPIs), and similar levels of resistance to Manduca sexta larvae. Additional experiments demonstrated that decreased JA levels in NaBAK1-VIGS plants, rather than the enzymatic activity of JAR proteins or Ile levels, were responsible for the reduced JA-Ile levels observed in these plants. Methyl jasmonate application elicited higher levels of TPI activity in NaBAK1-silenced plants than in EV plants, suggesting that silencing NaBAK1 enhances the accumulation of TPIs induced by a given level of JA. Thus NaBAK1 is involved in modulating herbivory-induced JA accumulation and how JA levels are transduced into TPI levels in N. attenuata.