A persistent major mutation in canonical jasmonate signaling is embedded in an herbivory-elicited gene network.

When insect herbivores attack plants, elicitors from oral secretions and regurgitants (OS) enter wounds during feeding, eliciting defense responses. These generally require plant jasmonate (JA) signaling, specifically, a jasmonoyl-L-isoleucine (JA-Ile) burst, for their activation and are well studied in the native tobacco Nicotiana attenuata. We used intraspecific diversity captured in a 26-parent MAGIC population planted in nature and an updated genome assembly to impute natural variation in the OS-elicited JA-Ile burst linked to a mutation in the JA-Ile biosynthetic gene NaJAR4. Experiments revealed that NaJAR4 variants were associated with higher fitness in the absence of herbivores but compromised foliar defenses, with two NaJAR homologues (4 and 6) complementing each other spatially and temporally. From decade-long seed collections of natural populations, we uncovered enzymatically inactive variants occurring at variable frequencies, consistent with a balancing selection regime maintaining variants. Integrative analyses of OS-induced transcriptomes and metabolomes of natural accessions revealed that NaJAR4 is embedded in a nonlinear complex gene coexpression network orchestrating responses to OS, which we tested by silencing four hub genes in two connected coexpressed networks and examining their OS-elicited metabolic responses. Lines silenced in two hub genes (NaGLR and NaFB67) co-occurring in the NaJAR4/6 module showed responses proportional to JA-Ile accumulations; two from an adjacent module (NaERF and NaFB61) had constitutively expressed defenses with high resistance. We infer that mutations with large fitness consequences can persist in natural populations due to compensatory responses from gene networks, which allow for diversification in conserved signaling pathways and are generally consistent with predictions of an omnigene model.

Independent regulation of strigolactones and blumenols during arbuscular mycorrhizal symbiosis in rice.

The apocarotenoid strigolactones (SLs) facilitate pre-symbiotic communication between arbuscular mycorrhizal (AM) fungi and plants. Related blumenol-C-glucosides (blumenols), have also been associated with symbiosis, but the cues that are involved in the regulation of blumenol accumulation during AM symbiosis remain unclear. In rice, our analyses demonstrated a strict correlation between foliar blumenol abundance and intraradical fungal colonisation. More specifically, rice mutants affected at distinct stages of the interaction revealed that fungal cortex invasion was required for foliar blumenol accumulation. Plant phosphate status and D14L hormone signalling had no effect, contrasting their known role in induction of SLs. This a proportion of the SL biosynthetic enzymes, D27 and D17, are equally required for blumenol production. These results importantly clarify that, while there is a partially shared biosynthetic pathway between SL and blumenols, the dedicated induction of the related apocarotenoids occurs in response to cues acting at distinct stages during the root colonisation process. However, we reveal that neither SLs nor blumenols are essential for fungal invasion of rice roots.

OXIDOSQUALENE CYCLASE 1 and 2 influence triterpene biosynthesis and defense in Nicotiana attenuata.

Triterpenes are a class of bioactive compounds with diverse biological functions, playing pivotal roles in plant defense against biotic stressors. Oxidosqualene cyclases (OSCs) serve as gatekeepers in the biosynthesis of triterpenes. In this study, we utilized a Nicotiana benthamiana heterologous expression system to characterize NaOSC1 from Nicotiana attenuata as a multifunctional enzyme capable of synthesizing lupeol, dammarenediol II, 3-alpha,20-lupanediol, and 7 other triterpene scaffolds. We also demonstrated that NaOSC2 is, in contrast, a selective enzyme, producing only the ß-amyrin scaffold. Through virus-induced gene silencing and in vitro toxicity assays, we elucidated the roles of NaOSC1 and NaOSC2 in the defense of N. attenuata against Manduca sexta larvae. Metabolomic and feature-based molecular network analyses of leaves with silenced NaOSC1 and NaOSC2 unveiled 3 potential triterpene glycoside metabolite clusters. Interestingly, features identified as triterpenes within these clusters displayed a significant negative correlation with larval mass. Our study highlights the pivotal roles of NaOSC1 and NaOSC2 from N. attenuata in the initial steps of triterpene biosynthesis, subsequently influencing defense against M. sexta through the modulation of downstream triterpene glycoside compounds.

Roles of cytochrome P450 monooxygenases in triterpene biosynthesis and their potential impact on growth and development.

Pentacyclic triterpenoids, recognized for their natural bioactivity, display complex spatiotemporal accumulation patterns within the ecological model plant Nicotiana attenuata. Despite their ecological importance, the underlying biosynthetic enzymes and functional attributes of triterpenoid synthesis in N. attenuata remain unexplored. Here, we show that three cytochrome P450 monooxygenases (NaCYP716A419, NaCYP716C87, and NaCYP716E107) from N. attenuata oxidize the pentacyclic triterpene skeleton, as evidenced by heterologous expression in Nicotiana benthamiana. NaCYP716A419 catalyzed a consecutive three-step oxidation reaction at the C28 position of ß-amyrin/lupeol/lupanediol, yielding the corresponding alcohol, aldehyde, and carboxylic acid. NaCYP716C87 hydroxylated the C2α position of ß-amyrin/lupeol/lupanediol/erythrodiol/oleanolic acid/betulinic acid, while NaCYP716E107 hydroxylated the C6ß position of ß-amyrin/oleanolic acid. The genes encoding these three CYP716 enzymes are highly expressed in flowers and respond to induction by ABA, MeJA, SA, GA3, and abiotic stress treatments. Using VIGS technology, we revealed that silencing of NaCYP716A419 affects the growth and reproduction of N. attenuata, suggesting the ecological significance of these specialized metabolite biosynthetic steps.

The downside of metabolic diversity: Postingestive rearrangements by specialized insects.

Deploying toxins in complex mixtures is thought to be advantageous and is observed during antagonistic interactions in nature. Toxin mixtures are widely utilized in medicine and pest control, as they are thought to slow the evolution of detoxification counterresponses in the targeted organisms. Here we show that caterpillars rearrange key constituents of two distinct plant defense pathways to postingestively disable the defensive properties of both pathways. Specifically, phenolic esters of quinic acid, chlorogenic acids (CAs), potent herbivore and ultraviolet (UV) defenses, are reesterified to decorate particular sugars of 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) and prevent their respective anti­herbivore defense functions. This was discovered through the employment of comparative metabolomics of the leaves of Nicotiana attenuata and the frass of this native tobacco's specialist herbivore, Manduca sexta larvae. Feeding caterpillars on leaves of transgenic plants abrogated in each of the two pathways, separately and together, revealed that one of the fully characterized frass conjugates, caffeoylated HGL-DTG, originated from ingested CA and HGL-DTGs and that both had negative effects on the defensive function of the other compound class, as revealed by rates of larval mass gain. This negative defensive synergy was further explored in 183 N. attenuata natural accessions, which revealed a strong negative covariance between the two defense pathways. Further mapping analyses in a biparental recombinant inbred line (RIL) population imputed quantitative trait loci (QTLs) for the two pathways at distinct genomic locations. The postingestive repurposing of defense metabolism constituents reveals a downside of deploying toxins in mixtures, a downside which plants in nature have evolved to counter.

Functional evidence supports adaptive plant chemical defense along a geographical cline.

Environmental clines in organismal defensive traits are usually attributed to stronger selection by enemies at lower latitudes or near the host's range center. Nonetheless, little functional evidence has supported this hypothesis, especially for coevolving plants and herbivores. We quantified cardenolide toxins in seeds of 24 populations of common milkweed (Asclepias syriaca) across 13 degrees of latitude, revealing a pattern of increasing cardenolide concentrations toward the host's range center. The unusual nitrogen-containing cardenolide labriformin was an exception and peaked at higher latitudes. Milkweed seeds are eaten by specialist lygaeid bugs that are even more tolerant of cardenolides than the monarch butterfly, concentrating most cardenolides (but not labriformin) from seeds into their bodies. Accordingly, whether cardenolides defend seeds against these specialist bugs is unclear. We demonstrate that Oncopeltus fasciatus (Lygaeidae) metabolized two major compounds (glycosylated aspecioside and labriformin) into distinct products that were sequestered without impairing growth. We next tested several isolated cardenolides in vitro on the physiological target of cardenolides (Na+/K+-ATPase); there was little variation among compounds in inhibition of an unadapted Na+/K+-ATPase, but tremendous variation in impacts on that of monarchs and Oncopeltus. Labriformin was the most inhibitive compound tested for both insects, but Oncopeltus had the greater advantage over monarchs in tolerating labriformin compared to other compounds. Three metabolized (and stored) cardenolides were less toxic than their parent compounds found in seeds. Our results suggest that a potent plant defense is evolving by natural selection along a geographical cline and targets specialist herbivores, but is met by insect tolerance, detoxification, and sequestration.

Specific decorations of 17-hydroxygeranyllinalool diterpene glycosides solve the autotoxicity problem of chemical defense in Nicotiana attenuata.

The native diploid tobacco Nicotiana attenuata produces abundant, potent anti-herbivore defense metabolites known as 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) whose glycosylation and malonylation biosynthetic steps are regulated by jasmonate signaling. To characterize the biosynthetic pathway of HGL-DTGs, we conducted a genome-wide analysis of uridine diphosphate glycosyltransferases (UGTs) and identified 107 family-1 UGT members. The transcript levels of three UGTs were highly correlated with the transcript levels two key HGL-DTG biosynthetic genes: geranylgeranyl diphosphate synthase (NaGGPPS) and geranyllinalool synthase (NaGLS). NaGLS's role in HGL-DTG biosynthesis was confirmed by virus-induced gene silencing. Silencing the Uridine diphosphate (UDP)-rhamnosyltransferase gene UGT91T1 demonstrated its role in the rhamnosylation of HGL-DTGs. In vitro enzyme assays revealed that UGT74P3 and UGT74P4 use UDP-glucose for the glucosylation of 17-hydroxygeranyllinalool (17-HGL) to lyciumoside I. Plants with stable silencing of UGT74P3 and UGT74P5 were severely developmentally deformed, pointing to a phytotoxic effect of the aglycone. The application of synthetic 17-HGL and silencing of the UGTs in HGL-DTG-free plants confirmed this phytotoxic effect. Feeding assays with tobacco hornworm (Manduca sexta) larvae revealed the defensive functions of the glucosylation and rhamnosylation steps in HGL-DTG biosynthesis. Glucosylation of 17-HGL is therefore a critical step that contributes to the resulting metabolites' defensive function and solves the autotoxicity problem of this potent chemical defense.

Exploring the metabolic basis of growth/defense trade-offs in complex environments with Nicotiana attenuata plants cosilenced in NaMYC2a/b expression.

In response to challenges from herbivores and competitors, plants use fitness-limiting resources to produce (auto)toxic defenses. Jasmonate signaling, mediated by MYC2 transcription factors (TF), is thought to reconfigure metabolism to minimize these formal costs of defense and optimize fitness in complex environments. To study the context-dependence of this metabolic reconfiguration, we cosilenced NaMYC2a/b by RNAi in Nicotiana attenuata and phenotyped plants in the field and increasingly realistic glasshouse setups with competitors and mobile herbivores. NaMYC2a/b had normal phytohormonal responses, and higher growth and fitness in herbivore-reduced environments, but were devastated in high herbivore-load environments in the field due to diminished accumulations of specialized metabolites. In setups with competitors and mobile herbivores, irMYC2a/b plants had lower fitness than empty vector (EV) in single-genotype setups but increased fitness in mixed-genotype setups. Correlational analyses of metabolic, resistance, and growth traits revealed the expected defense/growth associations for most sectors of primary and specialized metabolism. Notable exceptions were some HGL-DTGs and phenolamides that differed between single-genotype and mixed-genotype setups, consistent with expectations of a blurred functional trichotomy of metabolites. MYC2 TFs mediate the reconfiguration of primary and specialized metabolic sectors to allow plants to optimize their fitness in complex environments.

Arbuscular mycorrhizal fungi-indicative blumenol-C-glucosides predict lipid accumulations and fitness in plants grown without competitors.

Hydroxy- and carboxyblumenol C-glucosides specifically accumulate in roots and leaves of plants harboring arbuscular mycorrhizal fungi (AMF). To explore blumenol function in AMF relationships, we silenced an early key-gene in blumenol biosynthesis, CCD1 (carotenoid cleavage dioxygenase 1), in the ecological model plant, Nicotiana attenuata, and analyzed whole-plant performance in comparison with control and CCaMK-silenced plants, unable to form AMF associations. Root blumenol accumulations reflected a plant's Darwinian fitness, as estimated by capsule production, and were positively correlated with AMF-specific lipid accumulations in roots, with relationships that changed as plants matured when grown without competitors. When grown with wild-type competitors, transformed plants with decreased photosynthetic capacity or increased carbon flux to roots had blumenol accumulations that predicted plant fitness and genotype trends in AMF-specific lipids, but had similar levels of AMF-specific lipids between competing plants, likely reflecting AMF-networks. We propose that when grown in isolation, blumenol accumulations reflect AMF-specific lipid allocations and plant fitness. When grown with competitors, blumenol accumulations predict fitness outcomes, but not the more complicated AMF-specific lipid accumulations. RNA-seq analysis provided candidates for the final biosynthetic steps of these AMF-indicative blumenol C-glucosides; abrogation of these steps will provide valuable tools for understanding blumenol function in this context-dependent mutualism.

A single MYB transcription factor with multiple functions during flower development.

Members of the R2R3-MYB transcription factor subgroup 19 (SG19) have been extensively studied in multiple plant species using different silenced or mutated lines. Some studies have proposed a function in flower opening, others in floral organ development/maturation, or specialized metabolism production. While SG19 members are clearly key players during flower development and maturation, the resulting picture is complex, confusing our understanding in how SG19 genes function. To clarify the function of the SG19 transcription factors, we used a single system, Petunia axillaris, and targeted its two SG19 members (EOB1 and EOB2) by CRISPR-Cas9. Although EOB1 and EOB2 are highly similar, they display radically different mutant phenotypes. EOB1 has a specific role in scent emission while EOB2 has pleiotropic functions during flower development. The eob2 knockout mutants reveal that EOB2 is a repressor of flower bud senescence by inhibiting ethylene production. Moreover, partial loss-of-function mutants (transcriptional activation domain missing) show that EOB2 is also involved in both petal and pistil maturation through regulation of primary and secondary metabolism. Here, we provide new insights into the genetic regulation of flower maturation and senescence. It also emphasizes the function of EOB2 in the adaptation of plants to specific guilds of pollinators.

Strigolactone signaling regulates specialized metabolism in tobacco stems and interactions with stem-feeding herbivores.

Plants are attacked by herbivores, which often specialize on different tissues, and in response, have evolved sophisticated resistance strategies that involve different types of chemical defenses frequently targeted to different tissues. Most known phytohormones have been implicated in regulating these defenses, with jasmonates (JAs) playing a pivotal role in complex regulatory networks of signaling interactions, often generically referred to as "cross talk." The newly identified class of phytohormones, strigolactones (SLs), known to regulate the shoot architecture, remain unstudied with regard to plant-herbivore interactions. We explored the role of SL signaling in resistance to a specialist weevil (Trichobaris mucorea) herbivore of the native tobacco, Nicotiana attenuata, that attacks the root-shoot junction (RSJ), the part of the plant most strongly influenced by alterations in SL signaling (increased branching). As SL signaling shares molecular components, such as the core F-box protein MORE AXILLARY GROWTH 2 (MAX2), with another new class of phytohormones, the karrikins (KARs), which promote seed germination and seedling growth, we generated transformed lines, individually silenced in the expression of NaMAX2, DWARF 14 (NaD14: the receptor for SL) and CAROTENOID CLEAVAGE DIOXYGENASE 7 (NaCCD7: a key enzyme in SL biosynthesis), and KARRIKIN INSENSITIVE 2 (NaKAI2: the KAR receptor). The mature stems of all transgenic lines impaired in the SL, but not the KAR signaling pathway, overaccumulated anthocyanins, as did the stems of plants attacked by the larvae of weevil, which burrow into the RSJs to feed on the pith of N. attenuata stems. T. mucorea larvae grew larger in the plants silenced in the SL pathway, but again, not in the KAI2-silenced plants. These phenotypes were associated with elevated JA and auxin (indole-3-acetic acid [IAA]) levels and significant changes in the accumulation of defensive compounds, including phenolamides and nicotine. The overaccumulation of phenolamides and anthocyanins in the SL pathway-silenced plants likely resulted from antagonism between the SL and JA pathway in N. attenuata. We show that the repressors of SL signaling, suppressor of max2-like (NaSMXL6/7), and JA signaling, jasmonate zim-domain (NaJAZs), physically interact, promoting NaJAZb degradation and releasing JASMONATE INSENSITIVE 1 (JIN1/MYC2) (NaMYC2), a critical transcription factor promoting JA responses. However, the increased performance of T. mucorea larvae resulted from lower pith nicotine levels, which were inhibited by increased IAA levels in SL pathway-silenced plants. This inference was confirmed by decapitation and auxin transport inhibitor treatments that decreased pith IAA and increased nicotine levels. In summary, SL signaling tunes specific sectors of specialized metabolism in stems, such as phenylpropanoid and nicotine biosynthesis, by tailoring the cross talk among phytohormones, including JA and IAA, to mediate herbivore resistance of stems. The metabolic consequences of the interplay of SL, JA, and IAA signaling revealed here could provide a mechanism for the commonly observed pattern of herbivore tolerance/resistance trade-offs.

Heterologous expression of PtAAS1 reveals the metabolic potential of the common plant metabolite phenylacetaldehyde for auxin synthesis in planta.

Aromatic aldehydes and amines are common plant metabolites involved in several specialized metabolite biosynthesis pathways. Recently, we showed that the aromatic aldehyde synthase PtAAS1 and the aromatic amino acid decarboxylase PtAADC1 contribute to the herbivory-induced formation of volatile 2-phenylethanol and its glucoside 2-phenylethyl-ß-D-glucopyranoside in Populus trichocarpa. To unravel alternative metabolic fates of phenylacetaldehyde and 2-phenylethylamine beyond alcohol and alcohol glucoside formation, we heterologously expressed PtAAS1 and PtAADC1 in Nicotiana benthamiana and analyzed plant extracts using untargeted LC-qTOF-MS and targeted LC-MS/MS analysis. While the metabolomes of PtAADC1-expressing plants did not significantly differ from those of control plants, expression of PtAAS1 resulted in the accumulation of phenylacetic acid (PAA) and PAA-amino acid conjugates, identified as PAA-aspartate and PAA-glutamate. Herbivory-damaged poplar leaves revealed significantly induced accumulation of PAA-Asp, while levels of PAA remained unaltered upon herbivory. Transcriptome analysis showed that members of auxin-amido synthetase GH3 genes involved in the conjugation of auxins with amino acids were significantly upregulated upon herbivory in P. trichocarpa leaves. Overall, our data indicates that phenylacetaldehyde generated by poplar PtAAS1 serves as a hub metabolite linking the biosynthesis of volatile, non-volatile herbivory-induced specialized metabolites, and phytohormones, suggesting that plant growth and defense can be balanced on a metabolic level.

Sequestration of Defenses against Predators Drives Specialized Host Plant Associations in Preadapted Milkweed Bugs (Heteroptera: Lygaeinae).

AbstractHost plant specialization across herbivorous insects varies dramatically, but while the molecular mechanisms of host plant adaptations are increasingly known, we often lack a comprehensive understanding of the selective forces that favor specialization. The milkweed bugs (Heteroptera: Lygaeinae) are ancestrally associated with plants of the Apocynaceae from which they commonly sequester cardiac glycosides for defense, facilitated by resistant Na+/K+-ATPases and adaptations for transport, storage, and discharge of toxins. Here, we show that three Lygaeinae species independently colonized four novel nonapocynaceous hosts that convergently produce cardiac glycosides. A fourth species shifted to a new source of toxins by tolerating and sequestering alkaloids from meadow saffron (Colchicum autumnale, Colchicaceae). Across three milkweed bug species tested, feeding on seeds containing toxins did not improve growth or speed of development and even impaired growth and development in two species, but sequestration mediated protection of milkweed bugs against two natural predators: lacewing larvae and passerine birds. We conclude that physiological preadaptations and convergent phytochemistry facilitated novel specialized host associations. Since toxic seeds did not improve growth but either impaired growth or, at most, had neutral effects, selection by predators on sequestration of defenses, rather than the exploitation of additional profitable dietary resources, can lead to obligatory specialized host associations in otherwise generalist insects.

An unbiased approach elucidates variation in (S)-(+)-linalool, a context-specific mediator of a tri-trophic interaction in wild tobacco.

Plant volatile organic compounds (VOCs) mediate many interactions, and the function of common VOCs is especially likely to depend on ecological context. We used a genetic mapping population of wild tobacco, Nicotiana attenuata, originating from a cross of 2 natural accessions from Arizona and Utah, separated by the Grand Canyon, to dissect genetic variation controlling VOCs. Herbivory-induced leaf terpenoid emissions varied substantially, while green leaf volatile emissions were similar. In a field experiment, only emissions of linalool, a common VOC, correlated significantly with predation of the herbivore Manduca sexta by native predators. Using quantitative trait locus mapping and genome mining, we identified an (S)-(+)-linalool synthase (NaLIS). Genome resequencing, gene cloning, and activity assays revealed that the presence/absence of a 766-bp sequence in NaLIS underlies the variation of linalool emissions in 26 natural accessions. We manipulated linalool emissions and composition by ectopically expressing linalool synthases for both enantiomers, (S)-(+)- and (R)-(-)-linalool, reported to oppositely affect M. sexta oviposition, in the Arizona and Utah accessions. We used these lines to test ovipositing moths in increasingly complex environments. The enantiomers had opposite effects on oviposition preference, but the magnitude of the effect depended strongly both on plant genetic background, and complexity of the bioassay environment. Our study reveals that the emission of linalool, a common VOC, differs by orders-of-magnitude among geographically interspersed conspecific plants due to allelic variation in a linalool synthase, and that the response of a specialist herbivore to linalool depends on enantiomer, plant genotype, and environmental complexity.

ZEITLUPE facilitates the rhythmic movements of Nicotiana attenuata flowers.

Circadian organ movements are ubiquitous in plants. These rhythmic outputs are thought to be regulated by the circadian clock and auxin signalling, but the underlying mechanisms have not been clarified. Flowers of Nicotiana attenuata change their orientation during the daytime through a 140° arc to balance the need for pollinators and the protection of their reproductive organs. This rhythmic trait is under the control of the circadian clock and results from bending and re-straightening movements of the pedicel, stems that connect flowers to the inflorescence. Using an explant system that allowed pedicel growth and curvature responses to be characterized with high spatial and temporal resolution, we demonstrated that this movement is organ autonomous and mediated by auxin. Changes in the growth curvature of the pedicel are accompanied by an auxin gradient and dorsiventral asymmetry in auxin-dependent transcriptional responses; application of auxin transport inhibitors influenced the normal movements of this organ. Silencing the expression of the circadian clock component ZEITLUPE (ZTL) arrested changes in the growth curvature of the pedicel and altered auxin signalling and responses. IAA19-like, an Aux/IAA transcriptional repressor that is circadian regulated and differentially expressed between opposite tissues of the pedicel, and therefore possibly involved in the regulation of changes in organ curvature, physically interacted with ZTL. Together, these results are consistent with a direct link between the circadian clock and the auxin signalling pathway in the regulation of this rhythmic floral movement.

Light dominates the diurnal emissions of herbivore-induced volatiles in wild tobacco.

BACKGROUND: Timing is everything when it comes to the fitness outcome of a plant's ecological interactions, and accurate timing is particularly relevant for interactions with herbivores or mutualists that are based on ephemeral emissions of volatile organic compounds. Previous studies of the wild tobacco N. attenuata have found associations between the diurnal timing of volatile emissions, and daytime predation of herbivores by their natural enemies. RESULTS: Here, we investigated the role of light in regulating two biosynthetic groups of volatiles, terpenoids and green leaf volatiles (GLVs), which dominate the herbivore-induced bouquet of N. attenuata. Light deprivation strongly suppressed terpenoid emissions while enhancing GLV emissions, albeit with a time lag. Silencing the expression of photoreceptor genes did not alter terpenoid emission rhythms, but silencing expression of the phytochrome gene, NaPhyB1, disordered the emission of the GLV (Z)-3-hexenyl acetate. External abscisic acid (ABA) treatments increased stomatal resistance, but did not truncate the emission of terpenoid volatiles (recovered in the headspace). However, ABA treatment enhanced GLV emissions and leaf internal pools (recovered from tissue), and reduced internal linalool pools. In contrast to the pattern of diurnal terpenoid emissions and nocturnal GLV emissions, transcripts of herbivore-induced plant volatile (HIPV) biosynthetic genes peaked during the day. The promotor regions of these genes were populated with various cis-acting regulatory elements involved in light-, stress-, phytohormone- and circadian regulation. CONCLUSIONS: This research provides insights into the complexity of the mechanisms involved in the regulation of HIPV bouquets, a mechanistic complexity which rivals the functional complexity of HIPVs, which includes repelling herbivores, calling for body guards, and attracting pollinators.

Natural variation in linalool metabolites: One genetic locus, many functions?

The ubiquitous volatile linalool is metabolized in plants to nonvolatile derivatives. We studied Nicotiana attenuata plants which naturally vary in (S)-(+)-linalool contents, and lines engineered to produce either (R)-(-)- or (S)-(+)-linalool. Only (S)-(+)-linalool production was associated with slower growth of a generalist herbivore, and a large fraction was present as nonvolatile derivatives. We found that variation in volatile linalool and its nonvolatile glycosides mapped to the same genetic locus which harbored the biosynthetic gene, NaLIS, but that free linalool varied more in environmental responses. This study reveals how (S)-(+)-linalool and conjugates differ in their regulation and possible functions in resistance.

Using natural variation to achieve a whole-plant functional understanding of the responses mediated by jasmonate signaling.

The dramatic advances in our understanding of the molecular biology and biochemistry of jasmonate (JA) signaling have been the subject of several excellent recent reviews that have highlighted the phytohormonal function of this signaling pathway. Here, we focus on the responses mediated by JA signaling which have consequences for a plant's Darwinian fitness, i.e. the organism-level function of JA signaling. The most diverse module in the signaling cascade, the JAZ proteins, and their interactions with other proteins and transcription factors, allow this canonical signaling cascade to mediate a bewildering array of traits in different tissues at different times; the functional coherence of these diverse responses are best appreciated in an organismal/ecological context. From published work, it appears that jasmonates can function as the 'Swiss Army knife' of plant signaling, mediating many different biotic and abiotic stress and developmental responses that allow plants to contextualize their responses to their frequently changing local environments and optimize their fitness. We propose that a deeper analysis of the natural variation in both within-plant and within-population JA signaling components is a profitable means of attaining a coherent whole-plant functional perspective of this signaling cascade, and provide examples of this approach from the Nicotiana attenuata system.

TOC1 in Nicotiana attenuata regulates efficient allocation of nitrogen to defense metabolites under herbivory stress.

The circadian clock contextualizes plant responses to environmental signals. Plants use temporal information to respond to herbivory, but many of the functional roles of circadian clock components in these responses, and their contribution to fitness, remain unknown. We investigate the role of the central clock regulator TIMING OF CAB EXPRESSION 1 (TOC1) in Nicotiana attenuata's defense responses to the specialist herbivore Manduca sexta under both field and glasshouse conditions. We utilize 15 N pulse-labeling to quantify nitrogen incorporation into pools of three defense compounds: caffeoylputrescine (CP), dicaffeoyl spermidine (DCS) and nicotine. Nitrogen incorporation was decreased in CP and DCS and increased in nicotine pools in irTOC1 plants compared to empty vector (EV) under control conditions, but these differences were abolished after simulated herbivory. Differences between EV and irTOC1 plants in nicotine, but not phenolamide production, were abolished by treatment with the ethylene agonist 1-methylcyclopropene. Using micrografting, TOC1's effect on nicotine was isolated to the root and did not affect the fitness of heterografts under field conditions. These results suggest that the circadian clock contributes to plant fitness by balancing production of metabolically expensive nitrogen-rich defense compounds and mediating the allocation of resources between vegetative biomass and reproduction.

The Clock Gene TOC1 in Shoots, Not Roots, Determines Fitness of Nicotiana attenuata under Drought.

The highly conserved core circadian clock component TIMING OF CAB EXPRESSION1 (TOC1) contextualizes environmental stress responses in plants, for example by gating abscisic acid signaling and suppressing thermoresponsive growth. Selective interaction of TOC1 with PHYTOCHROME B under far-red-enriched light suggests a connection between circadian gating of light responses and sensitivity to ABA, an important regulator of growth and stress responses, including under drought. However, the fitness consequences of TOC1 function, particularly in the root, are poorly understood. Here, we used the desert annual, Nicotiana attenuata, to investigate the function of TOC1 in shoots and roots for maintaining fitness under drought, in both field and glasshouse experiments. Despite marked decreases in leaf water loss, TOC1-deficient lines failed to maintain fitness in response to drought stress as measured by total seed capsule production. Restoring TOC1 transcript levels in shoots via micrografting was sufficient to restore wild-type drought responses under field conditions. Microarrays identified a coexpression module in leaves strongly linking red and far-red light signaling to drought responses in a TOC1-dependent manner, but experiments with phytochrome-deficient lines revealed that the effects of TOC1 deficiency under drought cannot be attributed to changes in red/far-red light perception alone. Taken together, these results elucidate the sophisticated, tissue-dependent role of the circadian clock in maintaining fitness in the face of long-term abiotic stresses such as drought.