Innovation, conservation, and repurposing of gene function in root cell type development.

Plant species have evolved myriads of solutions, including complex cell type development and regulation, to adapt to dynamic environments. To understand this cellular diversity, we profiled tomato root cell type translatomes. Using xylem differentiation in tomato, examples of functional innovation, repurposing, and conservation of transcription factors are described, relative to the model plant Arabidopsis. Repurposing and innovation of genes are further observed within an exodermis regulatory network and illustrate its function. Comparative translatome analyses of rice, tomato, and Arabidopsis cell populations suggest increased expression conservation of root meristems compared with other homologous populations. In addition, the functions of constitutively expressed genes are more conserved than those of cell type/tissue-enriched genes. These observations suggest that higher order properties of cell type and pan-cell type regulation are evolutionarily conserved between plants and animals.

Retrograde signaling by the plastidial metabolite MEcPP regulates expression of nuclear stress-response genes.

Plastid-derived signals are known to coordinate expression of nuclear genes encoding plastid-localized proteins in a process termed retrograde signaling. To date, the identity of retrograde-signaling molecules has remained elusive. Here, we show that methylerythritol cyclodiphosphate (MEcPP), a precursor of isoprenoids produced by the plastidial methylerythritol phosphate (MEP) pathway, elicits the expression of selected stress-responsive nuclear-encoded plastidial proteins. Genetic and pharmacological manipulations of the individual MEP pathway metabolite levels demonstrate the high specificity of MEcPP as an inducer of these targeted stress-responsive genes. We further demonstrate that abiotic stresses elevate MEcPP levels, eliciting the expression of the aforementioned genes. We propose that the MEP pathway, in addition to producing isoprenoids, functions as a stress sensor and a coordinator of expression of targeted stress-responsive nuclear genes via modulation of the levels of MEcPP, a specific and critical retrograde-signaling metabolite.

Complementing model species with model clades.

Model species continue to underpin groundbreaking plant science research. At the same time, the phylogenetic resolution of the land plant Tree of Life continues to improve. The intersection of these two research paths creates a unique opportunity to further extend the usefulness of model species across larger taxonomic groups. Here we promote the utility of the Arabidopsis thaliana model species, especially the ability to connect its genetic and functional resources, to species across the entire Brassicales order. We focus on the utility of using genomics and phylogenomics to bridge the evolution and diversification of several traits across the Brassicales to the resources in Arabidopsis, thereby extending scope from a model species by establishing a "model clade". These Brassicales-wide traits are discussed in the context of both the model species Arabidopsis thaliana and the family Brassicaceae. We promote the utility of such a "model clade" and make suggestions for building global networks to support future studies in the model order Brassicales.

Genetic variation underlying differential ammonium and nitrate responses in Arabidopsis thaliana.

Nitrogen is an essential element required for plant growth and productivity. Understanding the mechanisms and natural genetic variation underlying nitrogen use in plants will facilitate the engineering of plant nitrogen use to maximize crop productivity while minimizing environmental costs. To understand the scope of natural variation that may influence nitrogen use, we grew 1,135 Arabidopsis thaliana natural genotypes on two nitrogen sources, nitrate and ammonium, and measured both developmental and defense metabolite traits. By using different environments and focusing on multiple traits, we identified a wide array of different nitrogen responses. These responses are associated with numerous genes, most of which were not previously associated with nitrogen responses. Only a small portion of these genes appear to be shared between environments or traits, while most are predominantly specific to a developmental or defense trait under a specific nitrogen source. Finally, by using a large population, we were able to identify unique nitrogen responses, such as preferring ammonium or nitrate, which appear to be generated by combinations of loci rather than a few large-effect loci. This suggests that it may be possible to obtain novel phenotypes in complex nitrogen responses by manipulating sets of genes with small effects rather than solely focusing on large-effect single gene manipulations.

Integrated omics reveal novel functions and underlying mechanisms of the receptor kinase FERONIA in Arabidopsis thaliana.

The receptor kinase FERONIA (FER) is a versatile regulator of plant growth and development, biotic and abiotic stress responses, and reproduction. To gain new insights into the molecular interplay of these processes and to identify new FER functions, we carried out quantitative transcriptome, proteome, and phosphoproteome profiling of Arabidopsis (Arabidopsis thaliana) wild-type and fer-4 loss-of-function mutant plants. Gene ontology terms for phytohormone signaling, abiotic stress, and biotic stress were significantly enriched among differentially expressed transcripts, differentially abundant proteins, and/or misphosphorylated proteins, in agreement with the known roles for FER in these processes. Analysis of multiomics data and subsequent experimental evidence revealed previously unknown functions for FER in endoplasmic reticulum (ER) body formation and glucosinolate biosynthesis. FER functions through the transcription factor NAI1 to mediate ER body formation. FER also negatively regulates indole glucosinolate biosynthesis, partially through NAI1. Furthermore, we found that a group of abscisic acid (ABA)-induced transcription factors is hypophosphorylated in the fer-4 mutant and demonstrated that FER acts through the transcription factor ABA INSENSITIVE5 (ABI5) to negatively regulate the ABA response during cotyledon greening. Our integrated omics study, therefore, reveals novel functions for FER and provides new insights into the underlying mechanisms of FER function.

Microbial terroir: associations between soil microbiomes and the flavor chemistry of mustard (Brassica juncea).

Here, we characterized the independent role of soil microbiomes (bacterial and fungal communities) in determining the flavor chemistry of harvested mustard seed (Brassica juncea). Given the known impacts of soil microbial communities on various plant characteristics, we hypothesized that differences in rhizosphere microbiomes would result in differences in seed flavor chemistry (glucosinolate content). In a glasshouse study, we introduced distinct soil microbial communities to mustard plants growing in an otherwise consistent environment. At the end of the plant life cycle, we characterized the rhizosphere and root microbiomes and harvested produced mustard seeds for chemical characterization. Specifically, we measured the concentrations of glucosinolates, secondary metabolites known to create spicy and bitter flavors. We examined associations between rhizosphere microbial taxa or genes and seed flavor chemistry. We identified links between the rhizosphere microbial community composition and the concentration of the main glucosinolate, allyl, in seeds. We further identified specific rhizosphere taxa predictive of seed allyl concentration and identified bacterial functional genes, namely genes for sulfur metabolism, which could partly explain the observed associations. Together, this work offers insight into the potential influence of the belowground microbiome on the flavor of harvested crops.

Large-scale identification of novel transcriptional regulators of the aliphatic glucosinolate pathway in Arabidopsis.

Aliphatic glucosinolates are a large group of plant secondary metabolites characteristic of Brassicaceae, including the model plant Arabidopsis. The diverse and complex degradation products of aliphatic glucosinolates contribute to plant responses to herbivory, pathogen attack, and environmental stresses. Most of the biosynthesis genes in the aliphatic glucosinolate pathway have been cloned in Arabidopsis, and the research focus has recently shifted to the regulatory mechanisms controlling aliphatic glucosinolate accumulation. Up till now, more than 40 transcriptional regulators have been identified as regulating the aliphatic glucosinolate pathway, but many more novel regulators likely remain to be discovered based on research evidence over the past decade. In the current study, we took a systemic approach to functionally test 155 candidate transcription factors in Arabidopsis identified by yeast one-hybrid assay, and successfully validated at least 30 novel regulators that could significantly influence the accumulation of aliphatic glucosinolates in our experimental set-up. We also showed that the regulators of the aliphatic glucosinolate pathway have balanced positive and negative effects, and glucosinolate metabolism and plant development can be coordinated. Our work is the largest scale effort so far to validate transcriptional regulators of a plant secondary metabolism pathway, and provides new insights into how the highly diverse plant secondary metabolism is regulated at the transcriptional level.

Transcriptional regulation of nitrogen-associated metabolism and growth.

Nitrogen is an essential macronutrient for plant growth and basic metabolic processes. The application of nitrogen-containing fertilizer increases yield, which has been a substantial factor in the green revolution1. Ecologically, however, excessive application of fertilizer has disastrous effects such as eutrophication2. A better understanding of how plants regulate nitrogen metabolism is critical to increase plant yield and reduce fertilizer overuse. Here we present a transcriptional regulatory network and twenty-one transcription factors that regulate the architecture of root and shoot systems in response to changes in nitrogen availability. Genetic perturbation of a subset of these transcription factors revealed coordinate transcriptional regulation of enzymes involved in nitrogen metabolism. Transcriptional regulators in the network are transcriptionally modified by feedback via genetic perturbation of nitrogen metabolism. The network, genes and gene-regulatory modules identified here will prove critical to increasing agricultural productivity.

Red-light is an environmental effector for mutualism between begomovirus and its vector whitefly.

Environments such as light condition influence the spread of infectious diseases by affecting insect vector behavior. However, whether and how light affects the host defense which further affects insect preference and performance, remains unclear, nor has been demonstrated how pathogens co-adapt light condition to facilitate vector transmission. We previously showed that begomoviral ßC1 inhibits MYC2-mediated jasmonate signaling to establish plant-dependent mutualism with its insect vector. Here we show red-light as an environmental catalyzer to promote mutualism of whitefly-begomovirus by stabilizing ßC1, which interacts with PHYTOCHROME-INTERACTING FACTORS (PIFs) transcription factors. PIFs positively control plant defenses against whitefly by directly binding to the promoter of terpene synthase genes and promoting their transcription. Moreover, PIFs interact with MYC2 to integrate light and jasmonate signaling and regulate the transcription of terpene synthase genes. However, begomovirus encoded ßC1 inhibits PIFs' and MYC2' transcriptional activity via disturbing their dimerization, thereby impairing plant defenses against whitefly-transmitted begomoviruses. Our results thus describe how a viral pathogen hijacks host external and internal signaling to enhance the mutualistic relationship with its insect vector.

Epigenomic divergence correlates with sequence polymorphism in Arabidopsis paralogs.

Processes affecting rates of sequence polymorphism are fundamental to the evolution of gene duplicates. The relationship between gene activity and sequence polymorphism can influence the likelihood that functionally redundant gene copies are co-maintained in stable evolutionary equilibria vs other outcomes such as neofunctionalization. Here, we investigate genic variation in epigenome-associated polymorphism rates in Arabidopsis thaliana and consider whether these affect the evolution of gene duplicates. We compared the frequency of sequence polymorphism and patterns of genetic differentiation between genes classified by exon methylation patterns: unmethylated (unM), gene-body methylated (gbM), and transposon-like methylated (teM) states, which reflect divergence in gene expression. We found that the frequency of polymorphism was higher in teM (transcriptionally repressed, tissue-specific) genes and lower in gbM (active, constitutively expressed) genes. Comparisons of gene duplicates were largely consistent with genome-wide patterns - gene copies that exhibit teM accumulate more variation, evolve faster, and are in chromatin states associated with reduced DNA repair. This relationship between expression, the epigenome, and polymorphism may lead to the breakdown of equilibrium states that would otherwise maintain genetic redundancies. Epigenome-mediated polymorphism rate variation may facilitate the evolution of novel gene functions in duplicate paralogs maintained over evolutionary time.

The rhizosphere microbiome and host plant glucosinolates exhibit feedback cycles in Brassica rapa.

The rhizosphere microbiome influences many aspects of plant fitness, including production of secondary compounds and defence against insect herbivores. Plants also modulate the composition of the microbial community in the rhizosphere via secretion of root exudates. We tested both the effect of the rhizosphere microbiome on plant traits, and host plant effects on rhizosphere microbes using recombinant inbred lines (RILs) of Brassica rapa that differ in production of glucosinolates (GLS), secondary metabolites that contribute to defence against insect herbivores. First, we investigated the effect of genetic variation in GLS production on the composition of the rhizosphere microbiome. Using a Bayesian Dirichlet-multinomial regression model (DMBVS), we identified both negative and positive associations between bacteria from six genera and the concentration of five GLS compounds produced in plant roots. Additionally, we tested the effects of microbial inoculation (an intact vs. disrupted soil microbiome) on GLS production and insect damage in these RILs. We found a significant microbial treatment × genotype interaction, in which total GLS was higher in the intact relative to the disrupted microbiome treatment in some RILs. However, despite differences in GLS production between microbial treatments, we observed no difference in insect damage between treatments. Together, these results provide evidence for a full feedback cycle of plant-microbe interactions mediated by GLS; that is, GLS compounds produced by the host plant "feed-down" to influence rhizosphere microbial community and rhizosphere microbes "feed-up" to influence GLS production.

Is specialized metabolite regulation specialized?

Recent technical and theoretical advances have generated an explosion in the identification of specialized metabolite pathways. In comparison, our understanding of how these pathways are regulated is relatively lagging. This and the relatively young age of specialized metabolite pathways has partly contributed to a default and common paradigm whereby specialized metabolite regulation is theorized as relatively simple with a few key transcription factors and the compounds are non-regulatory end-products. In contrast, studies into model specialized metabolites, such as glucosinolates, are beginning to identify a new understanding whereby specialized metabolites are highly integrated into the plants' core metabolic, physiological, and developmental pathways. This model includes a greatly extended compendium of transcription factors controlling the pathway, key transcription factors that co-evolve with the pathway and simultaneously control core metabolic and developmental components, and finally the compounds themselves evolve regulatory connections to integrate into the plants signaling machinery. In this review, these concepts are illustrated using studies in the glucosinolate pathway within the Brassicales. This suggests that the broader community needs to reconsider how they do or do not integrate specialized metabolism into the regulatory network of their study species.

Fine mapping identifies NAD-ME1 as a candidate underlying a major locus controlling temporal variation in primary and specialized metabolism in Arabidopsis.

Plant metabolism is modulated by a complex interplay between internal signals and external cues. A major goal of all quantitative metabolomic studies is to clone the underlying genes to understand the mechanistic basis of this variation. Using fine-scale genetic mapping, in this work we report the identification and initial characterization of NAD-DEPENDENT MALIC ENZYME 1 (NAD-ME1) as the candidate gene underlying the pleiotropic network Met.II.15 quantitative trait locus controlling variation in plant metabolism and circadian clock outputs in the Bay × Sha Arabidopsis population. Transcript abundance and promoter analysis in NAD-ME1Bay-0 and NAD-ME1Sha alleles confirmed allele-specific expression that appears to be due a polymorphism disrupting a putative circadian cis-element binding site. Analysis of transfer DNA insertion lines and heterogeneous inbred families showed that transcript variation of the NAD-ME1 gene led to temporal shifts of tricarboxylic acid cycle intermediates, glucosinolate (GSL) accumulation, and altered regulation of several GSL biosynthesis pathway genes. Untargeted metabolomic analyses revealed complex regulatory networks of NAD-ME1 dependent upon the daytime. The mutant led to shifts in plant primary metabolites, cell wall components, isoprenoids, fatty acids, and plant immunity phytochemicals, among others. Our findings suggest that NAD-ME1 may act as a key gene to coordinate plant primary and secondary metabolism in a time-dependent manner.

A genome-scale TF-DNA interaction network of transcriptional regulation of Arabidopsis primary and specialized metabolism.

Plant metabolism is more complex relative to individual microbes. In single-celled microbes, transcriptional regulation by single transcription factors (TFs) is sufficient to shift primary metabolism. Corresponding genome-level transcriptional regulatory maps of metabolism reveal the underlying design principles responsible for these shifts as a model in which master regulators largely coordinate specific metabolic pathways. Plant primary and specialized metabolism occur within innumerable cell types, and their reactions shift depending on internal and external cues. Given the importance of plants and their metabolites in providing humanity with food, fiber, and medicine, we set out to develop a genome-scale transcriptional regulatory map of Arabidopsis metabolic genes. A comprehensive set of protein-DNA interactions between Arabidopsis thaliana TFs and gene promoters in primary and specialized metabolic pathways were mapped. To demonstrate the utility of this resource, we identified and functionally validated regulators of the tricarboxylic acid (TCA) cycle. The resulting network suggests that plant metabolic design principles are distinct from those of microbes. Instead, metabolism appears to be transcriptionally coordinated via developmental- and stress-conditional processes that can coordinate across primary and specialized metabolism. These data represent the most comprehensive resource of interactions between TFs and metabolic genes in plants.

Interactions of Tomato and Botrytis cinerea Genetic Diversity: Parsing the Contributions of Host Differentiation, Domestication, and Pathogen Variation.

Although the impacts of crop domestication on specialist pathogens are well known, less is known about the interaction of crop variation and generalist pathogens. To study how genetic variation within a crop affects plant resistance to generalist pathogens, we infected a collection of wild and domesticated tomato accessions with a genetically diverse population of the generalist pathogen Botrytis cinerea We quantified variation in lesion size of 97 B. cinerea genotypes (isolates) on six domesticated tomato genotypes (Solanum lycopersicum) and six wild tomato genotypes (Solanum pimpinellifolium). Lesion size was significantly affected by large effects of the host and pathogen's genotype, with a much smaller contribution of domestication. This pathogen collection also enables genome-wide association mapping of B. cinerea Genome-wide association mapping of the pathogen showed that virulence is highly polygenic and involves a diversity of mechanisms. Breeding against this pathogen would likely require the use of diverse isolates to capture all possible mechanisms. Critically, we identified a subset of B. cinerea genes where allelic variation was linked to altered virulence against wild versus domesticated tomato, as well as loci that could handle both groups. This generalist pathogen already has a large collection of allelic variation that must be considered when designing a breeding program.

flasher, a novel mutation in a glucosinolate modifying enzyme, conditions changes in plant architecture and hormone homeostasis.

Meristem function is underpinned by numerous genes that affect hormone levels, ultimately controlling phyllotaxy, the transition to flowering and general growth properties. Class I KNOX genes are major contributors to this process, promoting cytokinin biosynthesis but repressing gibberellin production to condition a replication competent state. We identified a suppressor mutant of the KNOX1 mutant brevipedicellus (bp) that we termed flasher (fsh), which promotes stem and pedicel elongation, suppresses early senescence, and negatively affects reproductive development. Map-based cloning and complementation tests revealed that fsh is due to an E40K change in the flavin monooxygenase GS-OX5, a gene encoding a glucosinolate (GSL) modifying enzyme. In vitro enzymatic assays revealed that fsh poorly converts substrate to product, yet the levels of several GSLs are higher in the suppressor line, implicating FSH in feedback control of GSL flux. FSH is expressed predominantly in the vasculature in patterns that do not significantly overlap those of BP, implying a non-cell autonomous mode of meristem control via one or more GSL metabolites. Hormone analyses revealed that cytokinin levels are low in bp, but fsh restores cytokinin levels to near normal by activating cytokinin biosynthesis genes. In addition, jasmonate levels in the fsh suppressor are significantly lower than in bp, which is likely due to elevated expression of JA inactivating genes. These observations suggest the involvement of the GSL pathway in generating one or more negative effectors of growth that influence inflorescence architecture and fecundity by altering the balance of hormonal regulators.

Plant Secondary Metabolites as Defenses, Regulators, and Primary Metabolites: The Blurred Functional Trichotomy.

The plant kingdom produces hundreds of thousands of low molecular weight organic compounds. Based on the assumed functions of these compounds, the research community has classified them into three overarching groups: primary metabolites, which are directly required for plant growth; secondary (or specialized) metabolites, which mediate plant-environment interactions; and hormones, which regulate organismal processes and metabolism. For decades, this functional trichotomy of plant metabolism has shaped theory and experimentation in plant biology. However, exact biochemical boundaries between these different metabolite classes were never fully established. A new wave of genetic and chemical studies now further blurs these boundaries by demonstrating that secondary metabolites are multifunctional; they can function as potent regulators of plant growth and defense as well as primary metabolites sensu lato. Several adaptive scenarios may have favored this functional diversity for secondary metabolites, including signaling robustness and cost-effective storage and recycling. Secondary metabolite multifunctionality can provide new explanations for ontogenetic patterns of defense production and can refine our understanding of plant-herbivore interactions, in particular by accounting for the discovery that adapted herbivores misuse plant secondary metabolites for multiple purposes, some of which mirror their functions in plants. In conclusion, recent work unveils the limits of our current functional classification system for plant metabolites. Viewing secondary metabolites as integrated components of metabolic networks that are dynamically shaped by environmental selection pressures and transcend multiple trophic levels can improve our understanding of plant metabolism and plant-environment interactions.

Diverse Allyl Glucosinolate Catabolites Independently Influence Root Growth and Development.

Glucosinolates (GSLs) are sulfur-containing defense metabolites produced in the Brassicales, including the model plant Arabidopsis (Arabidopsis thaliana). Previous work suggests that specific GSLs may function as signals to provide direct feedback regulation within the plant to calibrate defense and growth. These GSLs include allyl-GSL, a defense metabolite that is one of the most widespread GSLs in Brassicaceae and has also been associated with growth inhibition. Here we show that at least three separate potential catabolic products of allyl-GSL or closely related compounds affect growth and development by altering different mechanisms influencing plant development. Two of the catabolites, raphanusamic acid and 3-butenoic acid, differentially affect processes downstream of the auxin signaling cascade. Another catabolite, acrylic acid, affects meristem development by influencing the progression of the cell cycle. These independent signaling events propagated by the different catabolites enable the plant to execute a specific response that is optimal to any given environment.

mGWAS Uncovers Gln-Glucosinolate Seed-Specific Interaction and its Role in Metabolic Homeostasis.

Gln is a key player in plant metabolism. It is one of the major free amino acids that is transported into the developing seed and is central for nitrogen metabolism. However, Gln natural variation and its regulation and interaction with other metabolic processes in seeds remain poorly understood. To investigate the latter, we performed a metabolic genome-wide association study (mGWAS) of Gln-related traits measured from the dry seeds of the Arabidopsis (Arabidopsis thaliana) diversity panel using all potential ratios between Gln and the other members of the Glu family as traits. This semicombinatorial approach yielded multiple candidate genes that, upon further analysis, revealed an unexpected association between the aliphatic glucosinolates (GLS) and the Gln-related traits. This finding was confirmed by an independent quantitative trait loci mapping and statistical analysis of the relationships between the Gln-related traits and the presence of specific GLS in seeds. Moreover, an analysis of Arabidopsis mutants lacking GLS showed an extensive seed-specific impact on Gln levels and composition that manifested early in seed development. The elimination of GLS in seeds was associated with a large effect on seed nitrogen and sulfur homeostasis, which conceivably led to the Gln response. This finding indicates that both Gln and GLS play key roles in shaping the seed metabolic homeostasis. It also implies that select secondary metabolites might have key functions in primary seed metabolism. Finally, our study shows that an mGWAS performed on dry seeds can uncover key metabolic interactions that occur early in seed development.