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.

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.

An integrative statistical method to explore herbivory-specific responses in plants.

Spatial-temporal coordination between multiple processes/pathways is a key determinant of whole-organism transcriptome and metabolome reconfigurations in plants' response to biotic stresses. To explore tissue-based interdependencies in Nicotiana attenuata's resistance to insect attack, we performed time course analyses of the plant's transcriptome and metabolome in herbivory-elicited source leaves and unelicited sink leaves and roots. To dissect the multidimensionality of these responses, we have recently designed a novel approach of constructing interactive motifs by combining an extended self-organizing maps (SOM) based dimensionality reduction method with bootstrap-based non-parametric AN OVA models. In this previous study, we used this method to study nonlinearities in gene-metabolite associations involved in the acyclic diterpene glucoside pathway. Here, we extend the application of this method to the extraction of genes showing herbivory-elicitation specifically in systemic (distal from the treatment sites) tissues using motif analysis for different combinations of treatment applied to Nicotiana attenuata.

The CCHamide 1 receptor modulates sensory perception and olfactory behavior in starved Drosophila.

The olfactory response of the vinegar fly Drosophila melanogaster to food odor is modulated by starvation. Here we show that this modulation is not restricted to food odors and their detecting sensory neurons but rather increases the behavioral response to odors as different as food odors, repellents and pheromones. The increased behavioral responsiveness is paralleled by an increased physiological sensitivity of sensory neurons regardless whether they express olfactory or ionotropic receptors and regardless whether they are housed in basiconic, coeloconic, or trichoid sensilla. Silencing several genes that become up-regulated under starvation confirmed the involvement of the short neuropeptide f receptor in the starvation effect. In addition it revealed that the CCHamide-1 receptor is another important factor governing starvation-induced olfactory modifications.

Tissue specific diurnal rhythms of metabolites and their regulation during herbivore attack in a native tobacco, Nicotiana attenuata.

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.