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.

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.

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.

Background

Plant fatty acid α-dioxygenases (α-DOX) are oxylipin-forming enzymes induced by biotic and abiotic stresses, which also participate in developmental processes. In Nicotiana attenuata, herbivory strongly induces the expression of an α-dox1 gene. To determine its role, we silenced its expression using Agrobacterium-mediated plant transformation with an inverted repeat construct. More than half of the transformed lines showed a severe dwarf growth phenotype that was very similar to the phenotype of tomato plants mutated at a second α-dox isoform. This led us to identify the corresponding α-dox2 gene in N. attenuata and examine the regulation of both α-dox genes as well as the consequences of their silencing in plant development and anti-herbivore defense.

Results

The transformed lines exhibiting a dwarf growth phenotype are co-silenced for both α-dox genes resulting in a nearly complete suppression of α-DOX activity, which is associated with increases in ABA, JA and anthocyanin levels, all metabolic signatures of oxidative stress. The other lines, only silenced for α-dox1, developed similarly to wild-type plants, exhibited a 40% reduction of α-DOX activity resulting in a 50% reduction of its main product in planta (2-HOT) and showed no signs of oxidative stress. In contrast to α-dox1, the expression of α-dox2 gene is not induced by wounding or elicitors in the oral secretions of Manduca sexta. Instead, α-dox2 is expressed in roots and flowers which lack α-dox1 expression, but both genes are equally regulated during leaf maturation. We transiently silenced α-dox gene copies with gene-specific constructs using virus induced gene silencing and determined the consequences for plant development and phytohormone and 2-HOT levels. While individual silencing of α-dox1 or α-dox2 had no effects on plant growth, the co-suppression of both α-dox genes decreased plant growth. Plants transiently silenced for both α-dox genes had increased constitutive levels of JA and ABA but silencing α-dox1 alone resulted in lower M. sexta-induced levels of JA, 2-HOT and ABA.

Conclusions

Thus, both α-dox isoforms function in the development of N. attenuata. In leaf maturation, the two α-dox genes have overlapping functions, but only α-dox2 is involved in root and flower development and only α-dox1 functions in anti-herbivore defense.

Background

Some plants distinguish mechanical wounding from herbivore attack by recognizing specific constituents of larval oral secretions (OS) which are introduced into plant wounds during feeding. Fatty acid-amino acid conjugates (FACs) are major constituents of Manduca sexta OS and strong elicitors of herbivore-induced defense responses in Nicotiana attenuata plants.

Results

The metabolism of one of the major FACs in M. sexta OS, N-linolenoyl-glutamic acid (18:3-Glu), was analyzed on N. attenuata wounded leaf surfaces. Between 50 to 70% of the 18:3-Glu in the OS or of synthetic 18:3-Glu were metabolized within 30 seconds of application to leaf wounds. This heat-labile process did not result in free α-linolenic acid (18:3) and glutamate but in the biogenesis of metabolites both more and less polar than 18:3-Glu. Identification of the major modified forms of this FAC showed that they corresponded to 13-hydroxy-18:3-Glu, 13-hydroperoxy-18:3-Glu and 13-oxo-13:2-Glu. The formation of these metabolites occurred on the wounded leaf surface and it was dependent on lipoxygenase (LOX) activity; plants silenced in the expression of NaLOX2 and NaLOX3 genes showed more than 50% reduced rates of 18:3-Glu conversion and accumulated smaller amounts of the oxygenated derivatives compared to wild-type plants. Similar to 18:3-Glu, 13-oxo-13:2-Glu activated the enhanced accumulation of jasmonic acid (JA) in N. attenuata leaves whereas 13-hydroxy-18:3-Glu did not. Moreover, compared to 18:3-Glu elicitation, 13-oxo-13:2-Glu induced the differential emission of two monoterpene volatiles (β-pinene and an unidentified monoterpene) in irlox2 plants.

Conclusions

The metabolism of one of the major elicitors of herbivore-specific responses in N. attenuata plants, 18:3-Glu, results in the formation of oxidized forms of this FAC by a LOX-dependent mechanism. One of these derivatives, 13-oxo-13:2-Glu, is an active elicitor of JA biosynthesis and differential monoterpene emission.

Background

Plants trigger and tailor defense responses after perception of the oral secretions (OS) of attacking specialist lepidopteran larvae. Fatty acid-amino acid conjugates (FACs) in the OS of the Manduca sexta larvae are necessary and sufficient to elicit the herbivory-specific responses in Nicotiana attenuata, an annual wild tobacco species. How FACs are perceived and activate signal transduction mechanisms is unknown.

Results

We used SuperSAGE combined with 454 sequencing to quantify the early transcriptional changes elicited by the FAC N-linolenoyl-glutamic acid (18:3-Glu) and virus induced gene silencing (VIGS) to examine the function of candidate genes in the M. sexta-N. attenuata interaction. The analysis targeted mRNAs encoding regulatory components: rare transcripts with very rapid FAC-elicited kinetics (increases within 60 and declines within 120 min). From 12,744 unique Tag sequences identified (UniTags), 430 and 117 were significantly up- and down-regulated ≥ 2.5-fold, respectively, after 18:3-Glu elicitation compared to wounding. Based on gene ontology classification, more than 25% of the annotated UniTags corresponded to putative regulatory components, including 30 transcriptional regulators and 22 protein kinases. Quantitative PCR analysis was used to analyze the FAC-dependent regulation of a subset of 27 of these UniTags and for most of them a rapid and transient induction was confirmed. Six FAC-regulated genes were functionally characterized by VIGS and two, a putative lipid phosphate phosphatase (LPP) and a protein of unknown function, were identified as important mediators of the M. sexta-N. attenuata interaction.

Conclusions

The analysis of the early changes in the transcriptome of N. attenuata after FAC elicitation using SuperSAGE/454 has identified regulatory genes involved in insect-specific mediated responses in plants. Moreover, it has provided a foundation for the identification of additional novel regulators associated with this process.

Background

The adage from Shakespeare, "troubles, not as single spies, but in battalions come," holds true for Nicotiana attenuata, which is commonly attacked by both pathogens (Pseudomonas spp.) and herbivores (Manduca sexta) in its native habitats. Defense responses targeted against the pathogens can directly or indirectly influence the responses against the herbivores. Nadefensin is an effective induced defense gene against the bacterial pathogen Pseudomonas syringae pv tomato (PST DC3000), which is also elicited by attack from M. sexta larvae, but whether this defense protein influences M. sexta's growth and whether M. sexta-induced Nadefensin directly or indirectly influences PST DC3000 resistance are unknown.

Results

M. sexta larvae consumed less on WT and on Nadefensin-silenced N. attenuata plants that had previously been infected with PST DC3000 than on uninfected plants. WT plants infected with PST DC3000 showed enhanced resistance to PST DC3000 and decreased leaf consumption by M. sexta larvae, but larval mass gain was unaffected. PST DC3000-infected Nadefensin-silenced plants were less resistant to subsequent PST DC3000 challenge, and on these plants, M. sexta larvae consumed less and gained less mass. WT and Nadefensin-silenced plants previously damaged by M. sexta larvae were better able to resist subsequent PST DC3000 challenges than were undamaged plants.

Conclusion

These results demonstrate that Na-defensin directly mediates defense against PST DC3000 and indirectly against M. sexta in N. attenuata. In plants that were previously infected with PST DC3000, the altered leaf chemistry in PST DC3000-resistant WT plants and PST DC3000-susceptible Nadefensin-silenced plants differentially reduced M. sexta's leaf consumption and mass gain. In plants that were previously damaged by M. sexta, the combined effect of the altered host plant chemistry and a broad spectrum of anti-herbivore induced metabolomic responses was more effective than Nadefensin alone in resisting PST DC3000.