TOR acts as a metabolic gatekeeper for auxin-dependent lateral root initiation in Arabidopsis thaliana.

Plant organogenesis requires matching the available metabolic resources to developmental programs. In Arabidopsis, the root system is determined by primary root-derived lateral roots (LRs), and adventitious roots (ARs) formed from non-root organs. Lateral root formation entails the auxin-dependent activation of transcription factors ARF7, ARF19, and LBD16. Adventitious root formation relies on LBD16 activation by auxin and WOX11. The allocation of shoot-derived sugar to the roots influences branching, but how its availability is sensed for LRs formation remains unknown. We combine metabolic profiling with cell-specific interference to show that LRs switch to glycolysis and consume carbohydrates. The target-of-rapamycin (TOR) kinase is activated in the lateral root domain. Interfering with TOR kinase blocks LR initiation while promoting AR formation. The target-of-rapamycin inhibition marginally affects the auxin-induced transcriptional response of the pericycle but attenuates the translation of ARF19, ARF7, and LBD16. TOR inhibition induces WOX11 transcription in these cells, yet no root branching occurs as TOR controls LBD16 translation. TOR is a central gatekeeper for root branching that integrates local auxin-dependent pathways with systemic metabolic signals, modulating the translation of auxin-induced genes.

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.

Synthesis of Non-natural Aza-Iridoids via Ynamides and Molecular Networking-Based Tracing of Their In Planta Bioconversion.

A synthesis of new-to-nature aza-iridoids via ynamides is presented. ZrCl4 proved to be the best acid to perform this transformation. Various ynamides were accommodated, and seco-iridoids could be obtained as well. Aza-iridoids were infiltrated into leaves of Scrophularia Nodosa, an iridoid-producing plant species. High-resolution mass spectrometry coupled to computational metabolomic approaches was employed for the detection of aza-iridoid bioconversion products.

Ethylene is a local modulator of jasmonate-dependent phenolamide accumulation during Manduca sexta herbivory in Nicotiana attenuata.

Rapid reconfigurations of interconnected phytohormone signalling networks allow plants to tune their physiology to constantly varying ecological conditions. During insect herbivory, most of the induced changes in defence-related leaf metabolites are controlled by jasmonate (JA) signalling, which, in the wild tobacco Nicotiana attenuata, recruits MYB8, a transcription factor controlling the accumulation of phenolic-polyamine conjugates (phenolamides). In this and other plant species, herbivory also locally triggers ethylene (ET) production but the outcome of the JA-ET cross-talk at the level of secondary metabolism regulation has remained only superficially investigated. Here, we analysed local and systemic herbivory-induced changes by mass spectrometry-based metabolomics in leaves of transgenic plants impaired in JA, ET and MYB8 signalling. Parsing deregulations in this factorial data-set identified a network of JA/MYB8-dependent phenolamides for which impairment of ET signalling attenuated their accumulation only in locally damaged leaves. Further experiments revealed that ET, albeit biochemically interrelated to polyamine metabolism via the intermediate S-adenosylmethionine, does not alter the free polyamine levels, but instead significantly modulates phenolamide levels with marginal modulations of transcript levels. The work identifies ET as a local modulator of phenolamide accumulations and provides a metabolomics data-platform with which to mine associations among herbivory-induced signalling and specialized metabolites in N. attenuata.

Toward a Systematic Nomenclature for (Neo)Lignanamides.

Phenylalkenoic acid amides, often referred to as phenol amides or hydroxycinnamic acid amides, are bioactive phytochemicals, whose bioactivity can be enhanced by coupling to form dimers or oligomers. Phenylalkenoic acid amides consist of a (hydroxy)cinnamic acid derivative (i.e., the phenylalkenoic acid subunit) linked to an amine-containing compound (i.e., the amine subunit) via an amide bond. The phenylalkenoic acid moiety can undergo oxidative coupling, either catalyzed by oxidative enzymes or due to autoxidation, which leads to the formation of (neo)lignanamides. Dimers described in the literature are often named after the species in which the compound was first discovered; however, the naming of these compounds lacks a systematic approach. We propose a new nomenclature, inspired by the existing system used for hydroxycinnamic acid dimers and lignin. In the proposed systematic nomenclature for (neo)lignanamides, compound names will be composed of three-letter codes and prefixes denoting the subunits, and numbers that indicate the carbon atoms involved in the linkage between the monomeric precursors. The proposed nomenclature is consistent, future-proof, and systematic.

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.

Wild tobacco genomes reveal the evolution of nicotine biosynthesis.

Nicotine, the signature alkaloid of Nicotiana species responsible for the addictive properties of human tobacco smoking, functions as a defensive neurotoxin against attacking herbivores. However, the evolution of the genetic features that contributed to the assembly of the nicotine biosynthetic pathway remains unknown. We sequenced and assembled genomes of two wild tobaccos, Nicotiana attenuata (2.5 Gb) and Nicotiana obtusifolia (1.5 Gb), two ecological models for investigating adaptive traits in nature. We show that after the Solanaceae whole-genome triplication event, a repertoire of rapidly expanding transposable elements (TEs) bloated these Nicotiana genomes, promoted expression divergences among duplicated genes, and contributed to the evolution of herbivory-induced signaling and defenses, including nicotine biosynthesis. The biosynthetic machinery that allows for nicotine synthesis in the roots evolved from the stepwise duplications of two ancient primary metabolic pathways: the polyamine and nicotinamide adenine dinucleotide (NAD) pathways. In contrast to the duplication of the polyamine pathway that is shared among several solanaceous genera producing polyamine-derived tropane alkaloids, we found that lineage-specific duplications within the NAD pathway and the evolution of root-specific expression of the duplicated Solanaceae-specific ethylene response factor that activates the expression of all nicotine biosynthetic genes resulted in the innovative and efficient production of nicotine in the genus Nicotiana Transcription factor binding motifs derived from TEs may have contributed to the coexpression of nicotine biosynthetic pathway genes and coordinated the metabolic flux. Together, these results provide evidence that TEs and gene duplications facilitated the emergence of a key metabolic innovation relevant to plant fitness.

Illuminating a plant's tissue-specific metabolic diversity using computational metabolomics and information theory.

Secondary metabolite diversity is considered an important fitness determinant for plants' biotic and abiotic interactions in nature. This diversity can be examined in two dimensions. The first one considers metabolite diversity across plant species. A second way of looking at this diversity is by considering the tissue-specific localization of pathways underlying secondary metabolism within a plant. Although these cross-tissue metabolite variations are increasingly regarded as important readouts of tissue-level gene function and regulatory processes, they have rarely been comprehensively explored by nontargeted metabolomics. As such, important questions have remained superficially addressed. For instance, which tissues exhibit prevalent signatures of metabolic specialization? Reciprocally, which metabolites contribute most to this tissue specialization in contrast to those metabolites exhibiting housekeeping characteristics? Here, we explore tissue-level metabolic specialization in Nicotiana attenuata, an ecological model with rich secondary metabolism, by combining tissue-wide nontargeted mass spectral data acquisition, information theory analysis, and tandem MS (MS/MS) molecular networks. This analysis was conducted for two different methanolic extracts of 14 tissues and deconvoluted 895 nonredundant MS/MS spectra. Using information theory analysis, anthers were found to harbor the most specialized metabolome, and most unique metabolites of anthers and other tissues were annotated through MS/MS molecular networks. Tissue-metabolite association maps were used to predict tissue-specific gene functions. Predictions for the function of two UDP-glycosyltransferases in flavonoid metabolism were confirmed by virus-induced gene silencing. The present workflow allows biologists to amortize the vast amount of data produced by modern MS instrumentation in their quest to understand gene function.

MetCirc: navigating mass spectral similarity in high-resolution MS/MS metabolomics data.

SUMMARY: Among the main challenges in metabolomics are the rapid dereplication of previously characterized metabolites across a range of biological samples and the structural prediction of unknowns from MS/MS data. Here, we developed MetCirc to comprehensively align and calculate pairwise similarity scores among MS/MS spectral data and visualize these across a range of biological samples. MetCirc comprises functionalities to interactively organize these data according to compound familial groupings and to accelerate the discovery of shared metabolites and hypothesis formulation for unknowns. As such, MetCirc provides a significant advance to address biological questions in areas where chemodiversity plays a role. AVAILABILITY AND IMPLEMENTATION: MetCirc , implemented in the open-source R language, together with its vignette are available in the Bioconductor project and at https://github.com/PlantDefenseMetabolism/MetCirc . CONTACT: thomasnaake@googlemail.com or emmanuel.gaquerel@cos.uni-heidelberg.de. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

O-Acyl Sugars Protect a Wild Tobacco from Both Native Fungal Pathogens and a Specialist Herbivore.

O-Acyl sugars (O-AS) are abundant trichome-specific metabolites that function as indirect defenses against herbivores of the wild tobacco Nicotiana attenuata; whether they also function as generalized direct defenses against herbivores and pathogens remains unknown. We characterized natural variation in O-AS among 26 accessions and examined their influence on two native fungal pathogens, Fusarium brachygibbosum U4 and Alternaria sp. U10, and the specialist herbivore Manduca sexta At least 15 different O-AS structures belonging to three classes were found in N. attenuata leaves. A 3-fold quantitative variation in total leaf O-AS was found among the natural accessions. Experiments with natural accessions and crosses between high- and low-O-AS accessions revealed that total O-AS levels were associated with resistance against herbivores and pathogens. Removing O-AS from the leaf surface increased M. sexta growth rate and plant fungal susceptibility. O-AS supplementation in artificial diets and germination medium reduced M. sexta growth and fungal spore germination, respectively. Finally, silencing the expression of a putative branched-chain α-ketoacid dehydrogenase E1 ß-subunit-encoding gene (NaBCKDE1B) in the trichomes reduced total leaf O-AS by 20% to 30% and increased susceptibility to Fusarium pathogens. We conclude that O-AS function as direct defenses to protect plants from attack by both native pathogenic fungi and a specialist herbivore and infer that their diversification is likely shaped by the functional interactions among these biotic stresses.

Navigating natural variation in herbivory-induced secondary metabolism in coyote tobacco populations using MS/MS structural analysis.

Natural variation can be extremely useful in unraveling the determinants of phenotypic trait evolution but has rarely been analyzed with unbiased metabolic profiling to understand how its effects are organized at the level of biochemical pathways. Native populations of Nicotiana attenuata, a wild tobacco species, have been shown to be highly genetically diverse for traits important for their interactions with insects. To resolve the chemodiversity existing in these populations, we developed a metabolomics and computational pipeline to annotate leaf metabolic responses to Manduca sexta herbivory. We selected seeds from 43 accessions of different populations from the southwestern United States--including the well-characterized Utah 30th generation inbred accession--and grew 183 plants in the glasshouse for standardized herbivory elicitation. Metabolic profiles were generated from elicited leaves of each plant using a high-throughput ultra HPLC (UHPLC)-quadrupole TOFMS (qTOFMS) method, processed to systematically infer covariation patterns among biochemically related metabolites, as well as unknown ones, and finally assembled to map natural variation. Navigating this map revealed metabolic branch-specific variations that surprisingly only partly overlapped with jasmonate accumulation polymorphisms and deviated from canonical jasmonate signaling. Fragmentation analysis via indiscriminant tandem mass spectrometry (idMS/MS) was conducted with 10 accessions that spanned a large proportion of the variance found in the complete accession dataset, and compound spectra were computationally assembled into spectral similarity networks. The biological information captured by this networking approach facilitates the mining of the mass spectral data of unknowns with high natural variation, as demonstrated by the annotation of a strongly herbivory-inducible phenolic derivative, and can guide pathway analysis.

Current Challenges in Plant Eco-Metabolomics.

The relatively new research discipline of Eco-Metabolomics is the application of metabolomics techniques to ecology with the aim to characterise biochemical interactions of organisms across different spatial and temporal scales. Metabolomics is an untargeted biochemical approach to measure many thousands of metabolites in different species, including plants and animals. Changes in metabolite concentrations can provide mechanistic evidence for biochemical processes that are relevant at ecological scales. These include physiological, phenotypic and morphological responses of plants and communities to environmental changes and also interactions with other organisms. Traditionally, research in biochemistry and ecology comes from two different directions and is performed at distinct spatiotemporal scales. Biochemical studies most often focus on intrinsic processes in individuals at physiological and cellular scales. Generally, they take a bottom-up approach scaling up cellular processes from spatiotemporally fine to coarser scales. Ecological studies usually focus on extrinsic processes acting upon organisms at population and community scales and typically study top-down and bottom-up processes in combination. Eco-Metabolomics is a transdisciplinary research discipline that links biochemistry and ecology and connects the distinct spatiotemporal scales. In this review, we focus on approaches to study chemical and biochemical interactions of plants at various ecological levels, mainly plant⁻organismal interactions, and discuss related examples from other domains. We present recent developments and highlight advancements in Eco-Metabolomics over the last decade from various angles. We further address the five key challenges: (1) complex experimental designs and large variation of metabolite profiles; (2) feature extraction; (3) metabolite identification; (4) statistical analyses; and (5) bioinformatics software tools and workflows. The presented solutions to these challenges will advance connecting the distinct spatiotemporal scales and bridging biochemistry and ecology.

Using the knowns to discover the unknowns: MS-based dereplication uncovers structural diversity in 17-hydroxygeranyllinalool diterpene glycoside production in the Solanaceae.

Exploring the diversity of plant secondary metabolism requires efficient methods to obtain sufficient structural insights to discriminate previously known from unknown metabolites. De novo structure elucidation and confirmation of known metabolites (dereplication) remain a major bottleneck for mass spectrometry-based metabolomic workflows, and few systematic dereplication strategies have been developed for the analysis of entire compound classes across plant families, partly due to the complexity of plant metabolic profiles that complicates cross-species comparisons. 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) are abundant defensive secondary metabolites whose malonyl and glycosyl decorations are induced by jasmonate signaling in the ecological model plant Nicotiana attenuata. The multiple labile glycosidic bonds of HGL-DTGs result in extensive in-source fragmentation (IS-CID) during ionization. To reconstruct these IS-CID clusters from profiling data and identify precursor ions, we applied a deconvolution algorithm and created an MS/MS library from positive-ion spectra of purified HGL-DTGs. From this library, 251 non-redundant fragments were annotated, and a workflow to characterize leaf, flower and fruit extracts of 35 solanaceous species was established. These analyses predicted 105 novel HGL-DTGs that were restricted to Nicotiana, Capsicum and Lycium species. Interestingly, malonylation is a highly conserved step in HGL-DTG metabolism, but is differentially affected by jasmonate signaling among Nicotiana species. This MS-based workflow is readily applicable for cross-species re-identification/annotation of other compound classes with sufficient fragmentation knowledge, and therefore has the potential to support hypotheses regarding secondary metabolism diversification.

Nicotiana attenuata Data Hub (NaDH): an integrative platform for exploring genomic, transcriptomic and metabolomic data in wild tobacco.

BACKGROUND: Nicotiana attenuata (coyote tobacco) is an ecological model for studying plant-environment interactions and plant gene function under real-world conditions. During the last decade, large amounts of genomic, transcriptomic and metabolomic data have been generated with this plant which has provided new insights into how native plants interact with herbivores, pollinators and microbes. However, an integrative and open access platform that allows for the efficient mining of these -omics data remained unavailable until now. DESCRIPTION: We present the Nicotiana attenuata Data Hub (NaDH) as a centralized platform for integrating and visualizing genomic, phylogenomic, transcriptomic and metabolomic data in N. attenuata. The NaDH currently hosts collections of predicted protein coding sequences of 11 plant species, including two recently sequenced Nicotiana species, and their functional annotations, 222 microarray datasets from 10 different experiments, a transcriptomic atlas based on 20 RNA-seq expression profiles and a metabolomic atlas based on 895 metabolite spectra analyzed by mass spectrometry. We implemented several visualization tools, including a modified version of the Electronic Fluorescent Pictograph (eFP) browser, co-expression networks and the Interactive Tree Of Life (iTOL) for studying gene expression divergence among duplicated homologous. In addition, the NaDH allows researchers to query phylogenetic trees of 16,305 gene families and provides tools for analyzing their evolutionary history. Furthermore, we also implemented tools to identify co-expressed genes and metabolites, which can be used for predicting the functions of genes. Using the transcription factor NaMYB8 as an example, we illustrate that the tools and data in NaDH can facilitate identification of candidate genes involved in the biosynthesis of specialized metabolites. CONCLUSION: The NaDH provides interactive visualization and data analysis tools that integrate the expression and evolutionary history of genes in Nicotiana, which can facilitate rapid gene discovery and comparative genomic analysis. Because N. attenuata shares many genome-wide features with other Nicotiana species including cultivated tobacco, and hence NaDH can be a resource for exploring the function and evolution of genes in Nicotiana species in general. The NaDH can be accessed at: http://nadh.ice.mpg.de/ .

Jasmonoyl-L-isoleucine coordinates metabolic networks required for anthesis and floral attractant emission in wild tobacco (Nicotiana attenuata).

Jasmonic acid and its derivatives (jasmonates [JAs]) play central roles in floral development and maturation. The binding of jasmonoyl-L-isoleucine (JA-Ile) to the F-box of CORONATINE INSENSITIVE1 (COI1) is required for many JA-dependent physiological responses, but its role in anthesis and pollinator attraction traits remains largely unexplored. Here, we used the wild tobacco Nicotiana attenuata, which develops sympetalous flowers with complex pollination biology, to examine the coordinating function of JA homeostasis in the distinct metabolic processes that underlie flower maturation, opening, and advertisement to pollinators. From combined transcriptomic, targeted metabolic, and allometric analyses of transgenic N. attenuata plants for which signaling deficiencies were complemented with methyl jasmonate, JA-Ile, and its functional homolog, coronatine (COR), we demonstrate that (1) JA-Ile/COR-based signaling regulates corolla limb opening and a JA-negative feedback loop; (2) production of floral volatiles (night emissions of benzylacetone) and nectar requires JA-Ile/COR perception through COI1; and (3) limb expansion involves JA-Ile-induced changes in limb fresh mass and carbohydrate metabolism. These findings demonstrate a master regulatory function of the JA-Ile/COI1 duet for the main function of a sympetalous corolla, that of advertising for and rewarding pollinator services. Flower opening, by contrast, requires JA-Ile signaling-dependent changes in primary metabolism, which are not compromised in the COI1-silenced RNA interference line used in this study.

Revealing insect herbivory-induced phenolamide metabolism: from single genes to metabolic network plasticity analysis.

The phenylpropanoid metabolic space comprises a network of interconnected metabolic branches that contribute to the biosynthesis of a large array of compounds with functions in plant development and stress adaptation. During biotic challenges, such as insect attack, a major rewiring of gene networks associated with phenylpropanoid metabolism is observed. This rapid reconfiguration of gene expression allows prioritized production of metabolites that help the plant solve ecological problems. Phenolamides are a group of phenolic derivatives that originate from diversion of hydroxycinnamoyl acids from the main phenylpropanoid pathway after N-acyltransferase-dependent conjugation to polyamines or aryl monoamines. These structurally diverse metabolites are abundant in the reproductive organs of many plants, and have recently been shown to play roles as induced defenses in vegetative tissues. In the wild tobacco, Nicotiana attenuata, in which herbivory-induced regulation of these metabolites has been studied, rapid elevations of the levels of phenolamides that function as induced defenses result from a multi-hormonal signaling network that re-shapes connected metabolic pathways. In this review, we summarize recent findings in the regulation of phenolamides obtained by mass spectrometry-based metabolomics profiling, and outline a conceptual framework for gene discovery in this pathway. We also introduce a multifactorial approach that is useful in deciphering metabolic pathway reorganizations among tissues in response to stress.

The roots of plant defenses: integrative multivariate analyses uncover dynamic behaviors of gene and metabolic networks of roots elicited by leaf herbivory.

High-throughput analyses have frequently been used to characterize herbivory-induced reconfigurations in plant primary and secondary metabolism in above- and below-ground tissues, but the conclusions drawn from these analyses are often limited by the univariate methods used to analyze the data. Here we use our previously described multivariate time-series data analysis to evaluate leaf herbivory-elicited transcriptional and metabolic dynamics in the roots of Nicotiana attenuata. We observed large, but transient, systemic responses in the roots that contrasted with the pattern of co-linearity observed in the up- and downregulation of genes and metabolites across the entire time series in treated and systemic leaves. Using this newly developed approach for the analysis of whole-plant molecular responses in a time-course multivariate data set, we simultaneously analyzed stress responses in leaves and roots in response to the elicitation of a leaf. We found that transient systemic responses in roots resolved into two principal trends characterized by: (i) an inversion of root-specific semi-diurnal (12 h) transcript oscillations and (ii) transcriptional changes with major amplitude effects that translated into a distinct suite of root-specific secondary metabolites (e.g. alkaloids synthesized in the roots of N. attenuata). These findings underscore the importance of understanding tissue-specific stress responses in the correct day-night phase context and provide a holistic framework for the important role played by roots in above-ground stress responses.

Improving the accuracy of expression data analysis in time course experiments using resampling.

BACKGROUND: As time series experiments in higher eukaryotes usually obtain data from different individuals collected at the different time points, a time series sample itself is not equivalent to a true biological replicate but is, rather, a combination of several biological replicates. The analysis of expression data derived from a time series sample is therefore often performed with a low number of replicates due to budget limitations or limitations in sample availability. In addition, most algorithms developed to identify specific patterns in time series dataset do not consider biological variation in samples collected at the same conditions. RESULTS: Using artificial time course datasets, we show that resampling considerably improves the accuracy of transcripts identified as rhythmic. In particular, the number of false positives can be greatly reduced while at the same time the number of true positives can be maintained in the range of other methods currently used to determine rhythmically expressed genes. CONCLUSIONS: The resampling approach described here therefore increases the accuracy of time series expression data analysis and furthermore emphasizes the importance of biological replicates in identifying oscillating genes. Resampling can be used for any time series expression dataset as long as the samples are acquired from independent individuals at each time point.

Deciphering herbivory-induced gene-to-metabolite dynamics in Nicotiana attenuata tissues using a multifactorial approach.

In response to biotic stresses, such as herbivore attack, plants reorganize their transcriptomes and reconfigure their physiologies not only in attacked tissues but throughout the plant. These whole-organismic reconfigurations are coordinated by a poorly understood network of signal transduction cascades. To explore tissue-based interdependencies in the resistance of Nicotiana attenuata to insect attack, we conducted time-series transcriptome and metabolome profiling of herbivory-elicited source leaves and unelicited sink leaves and roots. To probe the multidimensionality of these molecular responses, we designed a novel approach of combining an extended self-organizing maps-based dimensionality reduction method with bootstrap-based nonparametric analysis of variance models to identify the onset and context of signaling and metabolic pathway activations. We illustrate the value of this analysis by revisiting dynamic changes in the expression of regulatory and structural genes of the oxylipin pathway and by studying nonlinearities in gene-metabolite associations involved in the acyclic diterpene glucoside pathway after selectively extracting modules based on their dynamic response patterns. This novel dimensionality reduction approach is broadly applicable to capture the dynamic rewiring of gene and metabolite networks in experimental design with multiple factors.

NaMYC2 transcription factor regulates a subset of plant defense responses in Nicotiana attenuata.

BACKGROUND: To survive herbivore attack, plants have evolved potent mechanisms of mechanical or chemical defense that are either constitutively present or inducible after herbivore attack. Due to the costs of defense deployment, plants often regulate their biosynthesis using various transcription factors (TFs). MYC2 regulators belong to the bHLH family of transcription factors that are involved in many aspects of plant defense and development. In this study, we identified a novel MYC2 TF from N. attenuata and characterized its regulatory function using a combination of molecular, analytic and ecological methods. RESULTS: The transcript and targeted metabolite analyses demonstrated that NaMYC2 is mainly involved in the regulation of the biosynthesis of nicotine and phenolamides in N. attenuata. In addition, using broadly-targeted metabolite analysis, we identified a number of other metabolite features that were regulated by NaMYC2, which, after full annotation, are expected to broaden our understanding of plant defense regulation. Unlike previous reports, the biosynthesis of jasmonates and some JA-/NaCOI1-dependent metabolites (e.g. HGL-DTGs) were not strongly regulated by NaMYC2, suggesting the involvement of other independent regulators. No significant differences were observed in the performance of M. sexta on MYC2-silenced plants, consistent with the well-known ability of this specialist insect to tolerate nicotine. CONCLUSION: By regulating the biosynthesis of nicotine, NaMYC2 is likely to enhance plant resistance against non-adapted herbivores and contribute to plant fitness; however, multiple JA/NaCOI1-dependent mechanisms (perhaps involving other MYCs) that regulate separate defense responses are likely to exist in N. attenuata. The considerable variation observed amongst different plant families in the responses regulated by jasmonate signaling highlights the sophistication with which plants craft highly specific and fine-tuned responses against the herbivores that attack them.