Jasmonic acid is an important regulator of plant growth, development and defense. The jasmonate-ZIM domain (JAZ) proteins are key regulators in jasmonate signaling ubiquitously present in flowering plants but their functional annotation remains largely incomplete. Recently, we identified 12 putative JAZ proteins in native tobacco, Nicotiana attenuata, and initiated systematic functional characterization of these proteins by reverse genetic approaches. In this report, Nicotiana attenuata plants silenced in the expression of NaJAZd (irJAZd) by RNA interference were used to characterize NaJAZd function. Although NaJAZd transcripts were strongly and transiently up-regulated in the rosette leaves by simulated herbivory treatment, we did not observe strong defense-related phenotypes, such as altered herbivore performance or the constitutive accumulation of defense-related secondary metabolites in irJAZd plants compared to wild type plants, both in the glasshouse and the native habitat of Nicotiana attenuata in the Great Basin Desert, Utah, USA. Interestingly, irJAZd plants produced fewer seed capsules than did wild type plants as a result of increased flower abscission in later stages of flower development. The early- and mid-developmental stages of irJAZd flowers had reduced levels of jasmonic acid and jasmonoyl-L-isoleucine, while fully open flowers had normal levels, but these were impaired in NaMYB305 transcript accumulations. Previously, NaMYB305-silenced plants were shown to have strong flower abscission phenotypes and contained lower NECTARIN 1 transcript levels, phenotypes which are copied in irJAZd plants. We propose that the NaJAZd protein is required to counteract flower abscission, possibly by regulating jasmonic acid and jasmonoyl-L-isoleucine levels and/or expression of NaMYB305 gene in Nicotiana attenuata flowers. This novel insight into the function of JAZ proteins in flower and seed development highlights the diversity of functions played by jasmonates and JAZ proteins.

Here we discuss opportunities for system-wide analysis of plant volatiles provided by the implementation of non-supervised data processing. We illustrate the value of such approaches by presenting recent findings on wild tobacco volatile emissions using two-dimensional gas chromatography.

Plant volatile organic compound (VOC) production requires a complex network of biochemical pathways, which, although well mapped from a biochemical point of view, remains only partly understood with regard to its physiological and genetic regulation. Additionally, although analytical procedures for plant VOC measurement have become increasingly faster and more sensitive in recent years, pinpointing relevant shifts in VOC production from the thousands of molecular fragments that are generated by modern mass spectrometer instruments remains challenging. Here we discuss novel opportunities for system-wide analysis provided by the implementation of non-targeted data processing and multivariate statistics in VOC analysis. We illustrate the value of implementing non-targeted data processing with examples of recent findings from our group on the interactive control exerted by salivary components of a lepidopteran herbivore, Manduca sexta, on herbivory-induced VOC emissions in the wild tobacco Nicotiana attenuata. Finally, we briefly discuss the use of multi-platform data integration for probing the nature of metabolic and regulatory systems underlying VOC emissions.

Background

Wound-inducible Pin-II Proteinase inhibitors (PIs) are one of the important plant serine PIs which have been studied extensively for their structural and functional diversity and relevance in plant defense against insect pests. To explore the functional specialization of an array of Capsicum annuum (L.) proteinase inhibitor (CanPIs) genes, we studied their expression, processing and tissue-specific distribution under steady-state and induced conditions. Inductions were performed by subjecting C. annuum leaves to various treatments, namely aphid infestation or mechanical wounding followed by treatment with either oral secretion (OS) of Helicoverpa armigera or water.

Results

The elicitation treatments regulated the accumulation of CanPIs corresponding to 4-, 3-, and 2-inhibitory repeat domains (IRDs). Fourty seven different CanPI genes composed of 28 unique IRDs were identified in total along with those reported earlier. The CanPI gene pool either from uninduced or induced leaves was dominated by 3-IRD PIs and trypsin inhibitory domains. Also a major contribution by 4-IRD CanPI genes possessing trypsin and chymotrypsin inhibitor domains was specifically revealed in wounded leaves treated with OS. Wounding displayed the highest number of unique CanPIs while wounding with OS treatment resulted in the high accumulation of specifically CanPI-4, -7 and −10. Characterization of the PI protein activity through two dimensional gel electrophoresis revealed tissue and induction specific patterns. Consistent with transcript abundance, wound plus OS or water treated C. annuum leaves exhibited significantly higher PI activity and isoform diversity contributed by 3- and 4-IRD CanPIs. CanPI accumulation and activity was weakly elicited by aphid infestation yet resulted in the higher expression of CanPI-26, -41 and −43.

Conclusions

Plants can differentially perceive various kinds of insect attacks and respond appropriately through activating plant defenses including regulation of PIs at transcriptional and post-translational levels. Based on the differentially elicited CanPI accumulation patterns, it is intriguing to speculate that generating sequence diversity in the form of multi-IRD PIs is a part of elaborative plant defense strategy to obtain a diverse pool of functional units to confine insect attack.

Background

Induced defense responses to herbivores are generally believed to have evolved as cost-saving strategies that defer the fitness costs of defense metabolism until these defenses are needed. The fitness costs of jasmonate (JA)-mediated defenses have been well documented. Those of the early signaling units mediating induced resistance to herbivores have yet to be examined. Early signaling components that mediate herbivore-induced defense responses in Nicotiana attenuata, have been well characterized and here we examine their growth and fitness costs during competition with conspecifics. Two mitogen-activated protein kinases (MAPKs), salicylic acid (SA)-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK) are rapidly activated after perception of herbivory and both kinases regulate herbivory-induced JA levels and JA-mediated defense metabolite accumulations. Since JA-induced defenses result in resource-based trade-offs that compromise plant productivity, we evaluated if silencing SIPK (irSIPK) and WIPK (irWIPK) benefits the growth and fitness of plants competiting with wild type (WT) plants, as has been shown for plants silenced in JA-signaling by the reduction of Lipoxygenase 3 (LOX3) levels.

Results

As expected, irWIPK and LOX3-silenced plants out-performed their competing WT plants. Surprisingly, irSIPK plants, which have the largest reductions in JA signaling, did not. Phytohormone profiling of leaves revealed that irSIPK plants accumulated higher levels of SA compared to WT. To test the hypothesis that these high levels of SA, and their presumed associated fitness costs of pathogen associated defenses in irSIPK plants had nullified the JA-deficiency-mediated growth benefits in these plants, we genetically reduced SA levels in irSIPK plants. Reducing SA levels partially recovered the biomass and fitness deficits of irSIPK plants. We also evaluated whether the increased fitness of plants with reduced SA or JA levels resulted from increased nitrogen or CO2 assimilation rates, and found no evidence that greater intake of these fitness-limiting resources were responsible.

Conclusions

Signaling mediated by WIPK, but not SIPK, is associated with large fitness costs in competing N. attenuata plants, demonstrating the contrasting roles that these two MAPKs play in regulating the plants’ growth-defense balance. We discuss the role of SIPK as an important regulator of plant fitness, possibly by modulating SA-JA crosstalk as mediated through ethylene signaling.

Background

Plant microRNAs (miRNAs) play key roles in the transcriptional responses to environmental stresses. However, the role of miRNAs in responses to insect herbivory has not been thoroughly explored. To identify herbivory-responsive miRNAs, we identified conserved miRNAs in the ecological model plant Nicotiana attenuata whose interactions with herbivores have been well-characterized in both laboratory and field studies.

Results

We identified 59 miRNAs from 36 families, and two endogenous trans-acting small interfering RNAs (tasiRNA) targeted by miRNAs. We characterized the response of the precursor and mature miRNAs to simulated attack from the specialist herbivore Manduca sexta by quantitative PCR analysis and used ir-aoc RNAi transformants, deficient in jasmonate biosynthesis, to identify jasmonate-dependent and -independent miRNA regulation. Expression analysis revealed that groups of miRNAs and tasiRNAs were specifically regulated by either mechanical wounding or wounding plus oral secretions from M. sexta larvae, and these small RNAs were accumulated in jasmonate-dependent or -independent manners. Moreover, cDNA microarray analysis indicated that the expression patterns of the corresponding target genes were correlated with the accumulation of miRNAs and tasiRNAs.

Conclusions

We show that a group of miRNAs and tasiRNAs orchestrates the expression of target genes involved in N. attenuata’s responses to herbivore attack.

From an herbivore's first bite, plants release herbivory-induced plant volatiles (HIPVs) which can attract enemies of herbivores. However, other animals and competing plants can intercept HIPVs for their own use, and it remains unclear whether HIPVs serve as an indirect defense by increasing fitness for the emitting plant. In a 2-year field study, HIPV-emitting N. attenuata plants produced twice as many buds and flowers as HIPV-silenced plants, but only when native Geocoris spp. predators reduced herbivore loads (by 50%) on HIPV-emitters. In concert with HIPVs, plants also employ antidigestive trypsin protease inhibitors (TPIs), but TPI-producing plants were not fitter than TPI-silenced plants. TPIs weakened a specialist herbivore's behavioral evasive responses to simulated Geocoris spp. attack, indicating that TPIs function against specialists by enhancing indirect defense.

DOI: http://dx.doi.org/10.7554/eLife.00007.001

eLife digest

As the population of the world continues to increase beyond 7 billion, and agricultural pests continue to rapidly evolve resistance to pesticides, it is becoming ever more important to cultivate arable land in a way that is sustainable for both humans and the environment. A better understanding of the different mechanisms used by wild plants to deter herbivores will help to increase crop production without harming the environment.

Plants use both direct and indirect methods to fend off herbivores. Direct defense methods include the production of chemicals that are toxic to herbivores or give them indigestion, and the growth of sticky prickles and spines that can injure or kill the herbivore. Indirect defense methods, on the other hand, generally rely on the plant attracting organisms that are either predators or parasites of the herbivore.

Plants produce odors known as herbivory-induced plant volatiles (HIPVs) that are thought to offer indirect defense against herbivores by betraying their location to predators and parasites. However, HIPVs also influence other members of the ecological community, sometimes in ways that are detrimental to plants. Moreover, despite 30 years of research, no study has demonstrated that HIPVs increase the fitness of a plant, so it is unclear what they have evolved to do.

Now, a 2-year field study by Schuman et al. has shown plants that emit green leaf volatiles (which are a type of HIPV) produce twice as many buds and flowers—a measure of fitness—as plants that have been genetically engineered not to emit green leaf volatiles. This study was conducted with Nicotiana attenuata, which is a wild tobacco plant that is often targeted by Manduca sexta, a type of moth that is also known as the tobacco hornworm. Green leaf volatiles only increased plants' fitness when various species of Geocoris—a bug that preys on Manduca sexta—reduced the number of herbivores by a factor of two. This is the first evidence that HIPVs offer indirect defense against herbivores.

Schuman et al. also studied the effects of molecules called protease inhibitors that are thought to function as direct defenses by making it difficult for herbivores to digest plants. They found that the ability to produce protease inhibitors did not increase the fitness of plants under herbivore attack; however, tobacco hornworms that had been fed plants containing protease inhibitors were found to be more sluggish in response to attack, which suggests that protease inhibitors can enhance the indirect defenses of plants. The results suggest that employing both direct and indirect defenses—such as a combination of biological pesticides and genetic engineering to produce both HIPVs and protease inhibitors—is the best approach for defending agricultural plants against pests.

DOI: http://dx.doi.org/10.7554/eLife.00007.002

Background

A plant’s endogenous clock (circadian clock) entrains physiological processes to light/dark and temperature cycles. Forward and reverse genetic approaches in Arabidopsis have revealed the mechanisms of the circadian clock and its components in the genome. Similar approaches have been used to characterize conserved clock elements in several plant species. A wild tobacco, Nicotiana attenuata has been studied extensively to understand responses to biotic or abiotic stress in the glasshouse and also in their native habitat. During two decades of field experiment, we observed several diurnal rhythmic traits of N. attenuata in nature. To expand our knowledge of circadian clock function into the entrainment of traits important for ecological processes, we here report three core clock components in N. attenuata.

Results

Protein similarity and transcript accumulation allowed us to isolate orthologous genes of the core circadian clock components, LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1/PSEUDO-RESPONSE REGULATOR 1 (TOC1/PRR1), and ZEITLUPE (ZTL). Transcript accumulation of NaLHY peaked at dawn and NaTOC1 peaked at dusk in plants grown under long day conditions. Ectopic expression of NaLHY and NaZTL in Arabidopsis resulted in elongated hypocotyl and late-flowering phenotypes. Protein interactions between NaTOC1 and NaZTL were confirmed by yeast two-hybrid assays. Finally, when NaTOC1 was silenced in N. attenuata, late-flowering phenotypes under long day conditions were clearly observed.

Conclusions

We identified three core circadian clock genes in N. attenuata and demonstrated the functional and biochemical conservation of NaLHY, NaTOC1, and NaZTL.

When attacked by herbivores, plants produce toxic secondary metabolites that function as direct defenses, as well as indirect defenses that attract and reward predators of the offending herbivores. These indirect defenses include both nutritive rewards such as extra floral nectar, as well as informational rewards, such as the production and release of volatile compounds that betray the location of feeding herbivores to predators. Herbivory of Nicotiana attenuata by the tobacco hornworm (Manduca larvae) alters the volatile profiles of both the plant and larval headspace. Herbivory-elicited specific changes in the volatile profiles are detected by arthropod predators of Manduca larvae. The known predators that perceive volatile cues induced by Manduca herbivory of N. attenuata are insects that target Manduca at early developmental stages, when the larvae are still small; large, late-instar larvae may have outgrown these predation risks. However, here we offer evidence that branched chain aliphatic acids derived from the digestion of plant O-acyl sugars from trichomes may betray Manduca larvae to lizard predators during late developmental stages as well.

Background

The N. attenuata HD20 gene belongs to the homeodomain-leucine zipper (HD-Zip) type I family of transcription factors and it has been previously associated with the regulation of ABA accumulation in leaves and the emission of benzyl acetone (BA; 4-phenyl-2-butanone) from night flowers. In this study, N. attenuata plants stably reduced in the expression of HD20 (ir-hd20) were generated to investigate the mechanisms controlling the emission of BA from night flowers.

Results

The expression of HD20 in corollas of ir-hd20 plants was reduced by 85 to 90% compared to wild-type plants (WT) without affecting flower morphology and development. Total BA emitted from flowers of ir-hd20 plants was reduced on average by 60%. This reduction occurred mainly at the late phase of BA emission and it was correlated with 2-fold higher levels of ABA in the corollas of ir-hd20 plants. When a 2-fold decline in ABA corolla levels of these plants was induced by salt stress, BA emissions recovered to WT levels. Supplying ABA to WT flowers either through the cuticle or by pedicle feeding reduced the total BA emissions by 25 to 50%; this reduction occurred primarily at the late phase of emission (similar to the reduction observed in corollas of ir-hd20 plants). Gene expression profiling of corollas collected at 12 pm (six hours before the start of BA emission) revealed that 274 genes changed expression levels significantly in ir-hd20 plants compared to WT. Among these genes, more than 35% were associated with metabolism and the most prominent group was associated with the metabolism of aromatic compounds and phenylpropanoid derivatives.

Conclusions

The results indicated that regulation of ABA levels in corollas is associated with the late phase of BA emission in N. attenuata plants and that HD20 affects this latter process by mediating changes in both ABA levels and metabolic gene expression.

SGT1 (suppressor of G-two allele of SKP1) is highly conserved among all eukaryotes. In plants, SGT1 interacts with various proteins, including molecular chaperones (HSP70 and HSP90) and certain SCF ubiquitin ligases, and hence SGT1 likely functions in protein folding and stability. Since these protein complexes are involved in many aspects of plant biology, plants with a defective SGT1 display a plethora of phenotypic alterations. In this mini-review we highlight the interaction between SGT1 with other protein complexes and summarize the function of SGT1 in plant defense responses and development, including the recent advancements in the understanding of the role of SGT1 in jasmonic acid (JA) biosynthesis and signaling.

Recent investigations showed that the model plant Arabidopsis thaliana specifically responds to herbivory-associated molecular patterns by activating a sophisticated signaling network. The lipase activity of insect oral secretions was shown to elevate oxylipin levels when applied to puncture wounds in leaves. The results also demonstrated that the oral secretions of the generalist Schistocerca gregaria contained other, probably non-proteinous, elicitors of plant defense responses which induced mitogen-activated protein kinases, calcium signaling and ethylene levels.1 This addendum presents data on the levels of additional phytohormones that are elevated after application of S. gregaria oral secretion to wounded leaves. Abscisic acid and salicylic acid levels are significantly elevated after elicitation with S. gregaria oral secretions, adding another layer of complexity to the herbivory-induced response of A. thaliana.

Almost a decade ago BRI1-associated kinase 1 (BAK1) was identified as a co-receptor of brassinosteroid (BR) insensitive 1 (BRI1), the receptor for BRs, which plays an essential role in transducing BR signaling to regulate plant development. BAK1 is also critical in resistance to various pathogens. BAK1 rapidly binds to certain receptors for pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) after the perception of pathogen elicitors and is required for the full elicitation of pathogen-induced defense responses, such as the activation of the mitogen-activated protein kinase 6 (MPK6) and production of reactive oxygen species. Thus, BAK1 functions in both BR signaling and PAMP-triggered immunity (PTI). Recently BAK1 was also found to play an important role in mediating defense responses against an insect herbivore (Manduca sexta) of Nicotiana attenuata. In this interaction, BAK1 positively modulates wound- or herbivore feeding-induced accumulation of jasmonic acid (JA) and JA-isoleucine (JA-Ile). This mini-review summarizes recent advances in our understanding of the functions of BAK1 in resistance to pathogens and herbivores.

Background

RNAi can be achieved in insect herbivores by feeding them host plants stably transformed to express double stranded RNA (dsRNA) of selected midgut-expressed genes. However, the development of stably transformed plants is a slow and laborious process and here we developed a rapid, reliable and transient method. We used viral vectors to produce dsRNA in the host plant Nicotiana attenuata to transiently silence midgut genes of the plant's lepidopteran specialist herbivore, Manduca sexta. To compare the efficacy of longer, undiced dsRNA for insect gene silencing, we silenced N. attenuata's dicer genes (NaDCL1- 4) in all combinations in a plant stably transformed to express dsRNA targeting an insect gene.

Methodology/Principal Findings

Stable transgenic N. attenuata plants harboring a 312 bp fragment of MsCYP6B46 in an inverted repeat orientation (ir-CYP6B46) were generated to produce CYP6B46 dsRNA. After consuming these plants, transcripts of CYP6B46 were significantly reduced in M. sexta larval midguts. The same 312 bp cDNA was cloned in an antisense orientation into a TRV vector and Agro-infiltrated into N. attenuata plants. When larvae ingested these plants, similar reductions in CYP6B46 transcripts were observed without reducing transcripts of the most closely related MsCYP6B45. We used this transient method to rapidly silence the expression of two additional midgut-expressed MsCYPs. CYP6B46 transcripts were further reduced in midguts, when the larvae fed on ir-CYP6B46 plants transiently silenced for two combinations of NaDCLs (DCL1/3/4 and DCL2/3/4) and contained higher concentrations of longer, undiced CYP6B46 dsRNA.

Conclusions

Both stable and transient expression of CYP6B46 dsRNA in host plants provides a specific and robust means of silencing this gene in M. sexta larvae, but the transient system is better suited for high throughput analyses. Transiently silencing NaDCLs in ir-CYP6B46 plants increased the silencing of MsCYP6B46, suggested that insect's RNAi machinery is more efficient with longer lengths of ingested dsRNA.

Plant protease inhibitors (PIs) are a diverse group of proteins which have been intensely investigated due to their potential function in protecting plants against herbivorous insects by inhibiting digestive proteases. Although this mechanism has been well documented for a number of single PIs and their target enzymes, whether this mechanism protects plants in nature remains unclear. Moreover, many plants express a number of different PIs and it was unknown if these proteins work synergistically as defenses or if they also have other functions. We recently identified four serine PIs (SPI) of Solanum nigrum and demonstrated that they differ substantially in substrate specificity, accumulation patterns, and their effect against different natural herbivorous insects in field- and glasshouse experiments. These differences suggest that SPIs have at least partially diversified to provide protection against different attackers. Although we could not detect effects on plant development or growth when silencing SPIs, gene- and tissue-specific expression patterns suggest multiple functions in generative tissues, including a possible involvement in development.

Many plants have intimate relationships with soil microbes, which improve the plant’s growth and fitness through a variety of mechanisms. Bacillus sp. isolates are natural root-associated bacteria, isolated from Nicotiana attenuata plant roots growing in native soils. A particular isolate B55, was found to have dramatic plant growth promotion (PGP) effects on wild type (WT) and transgenic plants impaired in ethylene (ET) perception (35S-etr1), the genotype from which this bacterium was first isolated. B55 not only improves N. attenuata growth under in vitro, glasshouse, and field conditions, but it also “rescues” many of the deleterious phenotypes associated with ET insensitivity. Most notably, B55 dramatically increases the growth and survival of 35S-etr1 plants under field conditions. To our knowledge, this is the first demonstration of a PGP effect in a native plant–microbe association under natural conditions. Our study demonstrates that this facultative mutualistic plant–microbe interaction should be viewed as part of the plant’s extended phenotype. Possible modalities of recruitment and mechanisms of PGP are discussed.

To adjust their development to the environment, plants rely on specific signals that travel from shoot to root and vice versa. Here we describe an efficient micrografting protocol for Nicotiana attenuata, a useful tool for identifying these signals and understanding their functions. Additionally we analyzed transcript accumulation profiles of scions and rootstocks of grafts performed with wild-type and stably transformed N. attenuata. Our results are consistent with the source-to-sink movement of an sRNA silencing signal.

Ecological performance is all about timing and the endogenous clock that allows the entrainment of rhythms and anticipation of fitness-determining events is being rapidly characterized. How plants anticipate daily abiotic stresses, such as cold in early mornings and drought at noon, as well as biotic stresses, such as the timing of pathogen infections, is being explored, but little is known about the clock's role in regulating responses to insect herbivores and mutualists, whose behaviors are known to be strongly diurnally regulated and whose attack is known to reconfigure plant metabolomes. We developed a liquid chromatography-mass spectrometry procedure and analyzed its output with model-based peak picking algorithms to identify metabolites with diurnal accumulation patterns in sink/source leaves and roots in an unbiased manner. The response of metabolites with strong diurnal patterns to simulated attack from the specialist herbivore, Manduca sexta larvae was analyzed and annotated with in-house and public databases. Roots and leaves had largely different rhythms and only 10 ions of 182 oscillating ions in leaves and 179 oscillating ions in roots were rhythmic in both tissues: root metabolites mainly peaked at dusk or night, while leaf metabolites peaked during the day. Many oscillating metabolites showed tissue-specific regulation by simulated herbivory of which systemic responses in unattacked tissues were particularly pronounced. Diurnal and herbivory-elicited accumulation patterns of disaccharide, phenylalanine, tyrosine, lyciumoside I, coumaroyl tyramine, 12-oxophytodienoic acid and jasmonic acid and those of their related biosynthetic transcripts were examined in detail. We conclude that oscillating metabolites of N. attenuata accumulate in a highly tissue-specific manner and the patterns reveal pronounced diurnal rhythms in the generalized and specialized metabolism that mediates the plant's responses to herbivores and mutualists. We propose that diurnal regulation will prove to an important element in orchestrating a plant's responses to herbivore attack.

A plant's inducible defenses against herbivores as well as certain developmental processes are known to be controlled by the jasmonic acid (JA) pathway. We have previously shown that ectopically expressing Arabidopsis thaliana JA O-methyltransferase in Nicotiana attenuata (35S-jmt) strongly reduces the herbivory-elicited jasmonate bursts by acting as metabolic sink that redirects free JA towards methylation; here we examine the consequences of this metabolic sink on N. attenuata's secondary metabolism and performance in nature. In the glasshouse, 35S-jmt plants produced fewer seed capsules due to shorter floral styles, which could be restored to wild type (WT) levels after hand-pollination, and were more susceptible to Manduca sexta larvae attack. When transplanted into the Great Basin Desert in Utah, 35S-jmt plants grew as well as WT empty vector, but were highly attacked by native herbivores of different feeding guilds: leaf chewers, miners, and single cell feeders. This greater susceptibility was strongly associated with reduced emissions of volatile organic compounds (hexenylesters, monoterpenes and sesquiterpenes) and profound alterations in the production of direct defenses (trypsin proteinase inhibitors [TPI], nicotine, diterpene glycosides [DTGs] and phenylpropanoid-polyamine conjugates) as revealed by a combination of targeted and metabolomics analyses of field collected samples. Complementation experiments with JA-Ile, whose formation is outcompeted in 35S-jmt plants by the methylation reaction, restored the local TPI activation to WT levels and partially complemented nicotine and DTG levels in elicited but not systemic leaves. These findings demonstrate that MeJA, the major JA metabolite in 35S-jmt plants, is not an active signal in defense activation and highlights the value of creating JA sinks to disrupt JA signaling, without interrupting the complete octadecanoid pathway, in order to investigate the regulation of plants' defense metabolism in nature.

Plants can distinguish mechanical damage from larval folivory through the recognition of specific constituents of larval oral secretions (OS) which are deposited on the surface of leaf wounds during feeding. Fatty acid-amino acid conjugates (FACs) are major constituents of the OS of Lepidopteran larvae and they are strong elicitors of herbivore-induced defense responses in several plant species, including the wild tobacco Nicotiana attenuata. When OS from Manduca sexta larvae is deposited on N. attenuata wounded leaves, the major FAC N-linolenoyl-glutamic acid (18:3-Glu) is modified within seconds by a heat labile process. Some of the major modified forms are oxygenated products derived from 13-lipoxygenase activity and one of these derivatives, 13-oxo-13:2-Glu, is an active elicitor of enhanced JA biosynthesis and differential monoterpene emission in N. attenuata leaves.

S-nitrosoglutathione reductase (GSNOR) reduces the nitric oxide (NO) adduct S-nitrosoglutathione (GSNO), an essential reservoir for NO bioactivity. In plants, GSNOR has been found to be important in resistance to bacterial and fungal pathogens, but whether it is also involved in plant–herbivore interactions was not known. Using a virus-induced gene silencing (VIGS) system, the activity of GSNOR in a wild tobacco species, Nicotiana attenuata, was knocked down and the function of GSNOR in defence against the insect herbivore Manduca sexta was examined. Silencing GSNOR decreased the herbivory-induced accumulation of jasmonic acid (JA) and ethylene, two important phytohormones regulating plant defence levels, without compromising the activity of two mitogen-activated protein kinases (MAPKs), salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK). Decreased activity of trypsin proteinase inhibitors (TPIs) were detected in GSNOR-silenced plants after simulated M. sexta feeding and bioassays indicated that GSNOR-silenced plants have elevated susceptibility to M. sexta attack. Furthermore, GSNOR is required for methyl jasmonate (MeJA)-induced accumulation of defence-related secondary metabolites (TPI, caffeoylputrescine, and diterpene glycosides) but is not needed for the transcriptional regulation of JAZ3 (jasmonate ZIM-domain 3) and TD (threonine deaminase), indicating that GSNOR mediates certain but not all jasmonate-inducible responses. This work highlights the important role of GSNOR in plant resistance to herbivory and jasmonate signalling and suggests the potential involvement of NO in plant–herbivore interactions. Our data also suggest that GSNOR could be a target of genetic modification for improving crop resistance to herbivores.

In a wild tobacco plant, Nicotiana attenuata, two mitogen-activated protein kinases (MAPKs), salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK), play central roles in modulating herbivory-induced phytohormone and anti-herbivore secondary metabolites. However, the identities of their upstream MAPK kinases (MAPKKs) were elusive. Ectopic overexpression studies in N. benthamiana and N. tabacum suggested that two MAPKKs, MKK1 and MEK2, may activate SIPK and WIPK. The homologues of MKK1 and MEK2 were cloned in N. attenuata (NaMKK1 and NaMEK2) and a virus-induced gene silencing approach was used to knock-down the transcript levels of these MAPKK genes. Plants silenced in NaMKK1 and NaMEK2 were treated with wounding or simulated herbivory by applying the oral secretions of the specialist herbivore Manduca sexta to wounds. MAPK activity assay indicated that after wounding or simulated herbivory NaMKK1 is not required for the phosphorylation of NaSIPK and NaWIPK; in contrast, NaMEK2 and other unknown MAPKKs are important for simulated herbivory-elicited activation of NaSIPK and NaWIPK, and after wounding NaMEK2 probably does not activate NaWIPK but plays a minor role in activating NaSIPK. Consistently, NaMEK2 and certain other MAPKKs, but not NaMKK1, are needed for wounding- and simulated herbivory-elicited accumulation of jasmonic acid (JA), JA–isoleucine, and ethylene. Furthermore, both NaMEK2 and NaMKK1 regulate the levels of trypsin proteinase inhibitors. The findings underscore the complexity of MAPK signalling pathways and highlight the importance of MAPKKs in regulating wounding- and herbivory-induced responses.

We examined performance of herbivores on plants lacking either jasmonate (JA, asLOX3) or ethylene (ET, mETR1) signaling or both (mETR1asLOX3). Plant defenses against Manduca sexta caterpillars were strongly impaired in JA-deficient asLOX3 plants; however, making asLOX3 plants ethylene insensitive did not further increase the performance of the larvae on a mETR1asLOX3 genetic cross. This result demonstrates the dominant role of JA over ET in the regulation of plant defenses against herbivores. However, ET-insensitivity combined with otherwise normal levels of JA in mETR1 plants promoted faster caterpillar growth, which correlated with reduced accumulation of the alkaloidal direct defense nicotine in mETR1 compared to WT plants. Our data points to an important accessory function of ET in the activation of JA-regulated plant defenses against herbivores at the level of alkaloid biosynthesis in the roots and/or accumulation in the leaves.

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.

In plants, herbivore attack elicits the rapid accumulation of jasmonic acid (JA) which results from the activation of constitutively expressed biosynthetic enzymes. The molecular mechanisms controlling the activation of JA biosynthesis remain largely unknown however new research has elucidated some of the early regulatory components involved in this process. Nicotiana attenuata plants, a wild tobacco species, responds to fatty acid amino acid conjuguates (FAC) elicitors in the oral secretion of its natural herbivore, Manduca sexta, by triggering specific defense and tolerance responses against it; all of the defense responses known to date require the amplification of the wound-induced JA increase. We recently demonstrated that this FAC-elicited JA burst requires an increased flux of free linolenic acid (18:3) likely originating from the activation of a plastidial glycerolipase (GLA1) which is activated by an abundant FAC found in insect oral secretions, N-linolenoyl-glutamate (18:3-Glu). The lack of accumulation of free 18:3 after elicitation suggests a tight physical association between GLA1 and LOX3 in N. attenuata leaves. In addition, the salicylate-induced protein kinase (SIPK) and the nonexpressor of PR-1 (NPR1) participate in this activation mechanism that controls the supply of 18:3. In contrast, the wound-induced protein kinase (WIPK) does not but instead regulates the conversion of 13(S)-hydroperoxy-18:3 into 12-oxo-phytodienoic acid (OPDA). These results open new perspectives on the complex network of signals and regulatory components inducing the JA biosynthetic pathway.

Homeodomain-leucine zipper type I (HD-Zip I) proteins are plant-specific transcription factors associated with the regulation of growth and development in response to changes in the environment. Nicotiana attenuata NaHD20 was identified as an HD-Zip I-coding gene whose expression was induced by multiple stress-associated stimuli including drought and wounding. To study the role of NaHD20 in the integration of stress responses with changes in growth and development, its expression was silenced by virus-induced gene silencing (VIGS), and control and silenced plants were metabolically and developmentally characterized. Phytohormone profiling showed that NaHD20 plays a positive role in abscisic acid (ABA) accumulation in leaves during water stress and in the expression of some dehydration-responsive genes including ABA biosynthetic genes. Moreover, consistent with the high levels of NaHD20 expression in corollas, the emission of benzylacetone from flowers was reduced in NaHD20-silenced plants. Additionally, bolting time and the opening of the inflorescence buds was decelerated in these plants in a specific developmental stage without affecting the total number of flowers produced. Water stress potentiated these effects; however, after plants recovered from this condition, the opening of the inflorescence buds was accelerated in NaHD20-silenced plants. In summary, NaHD20 plays multiple roles in N. attenuata and among these are the coordination of responses to dehydration and its integration with changes in flower transitions.