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SummaryLupins are promising legume crops that accumulate toxic alkaloids in the seeds, complicating their use as high-protein crops. The alkaloids are synthesized in green organs (leaves, stems, and pods) and a subset of them is transported to the seeds during fruit development. The exact sites of biosynthesis and accumulation remain unknown, however mesophyll cells have been proposed as sources, and epidermal cells have been suggested as sinks. We examined the spatial localization of the alkaloids in biosynthetic organs of narrow-leafed lupin using mass spectrometry-based imaging (MSI). The alkaloids that accumulate in seeds (“core” alkaloids) were evenly distributed across tissues, however their esterified versions accumulated primarily in the epidermis. In addition, we generated a tissue-specific RNAseq dataset of biosynthetic organs using laser-capture microdissection. The dataset revealed that alkaloid biosynthetic genes are strongly expressed in the epidermis. To confirm the biosynthetic capacity of the leaf epidermis, we combined precursor feeding studies with mass spectrometry imaging, which showed that the lower epidermis is highly biosynthetic. Our work challenges the current assumptions on the precise sites of lupin alkaloid biosynthesis, with direct implications for the elucidation of the alkaloid biosynthesis pathway and the long-distance transport network from source to seed.

We report a public resource for examining the spatiotemporal RNA expression of 54,893M. truncatulagenes during the first 72 hours of response to rhizobial inoculation. Using a methodology that allows synchronous inoculation and growth of over 100 plants in a single media container, we harvested the same segment of each root responding to rhizobia in the initial inoculation over a time course, collected individual tissues from these segments with laser capture microdissection, and created and sequenced RNA libraries generated from these tissues. We demonstrate the utility of the resource by examining the expression patterns of a set of genes induced very early in nodule signaling, as well as two gene families (CLE peptides and nodule specific PLAT-domain proteins) and show that despite similar whole root expression patterns, there are tissue differences in expression between the genes. Using a rhizobial response data set generated from transcriptomics on intact root segments, we also examined differential temporal expression patterns and determined that, after nodule tissue, the epidermis and cortical cells contained the most temporally patterned genes. We circumscribed gene lists for each time and tissue examined and developed an expression pattern visualization tool. Finally, we explored transcriptomic differences between the inner cortical cells that become nodules and those that do not, confirming that the expression of ACC synthases distinguishes inner cortical cells that become nodules and provide and describe potential downstream genes involved in early nodule cell division.

Reproductive success hinges on precisely coordinated meiosis, yet our understanding of how structural rearrangements of chromatin and phase transitions during meiosis are transcriptionally regulated is limited. In crop plants, detailed analysis of the meiotic transcriptome could identify regulatory genes and epigenetic regulators that can be targeted to increase recombination rates and broaden genetic variation, as well as provide a resource for comparison among eukaryotes of different taxa to answer outstanding questions about meiosis. We conducted a meiotic stage-specific analysis of messenger RNA (mRNA), small non-coding RNA (sncRNA), and long intervening/intergenic non-coding RNA (lincRNA) in wheat (Triticum aestivum L.) and revealed novel mechanisms of meiotic transcriptional regulation and meiosis-specific transcripts. Amidst general repression of mRNA expression, significant enrichment of ncRNAs was identified during prophase I relative to vegetative cells. The core meiotic transcriptome was comprised of 9309 meiosis-specific transcripts, 48 134 previously unannotated meiotic transcripts, and many known and novel ncRNAs differentially expressed at specific stages. The abundant meiotic sncRNAs controlled the reprogramming of central metabolic pathways by targeting genes involved in photosynthesis, glycolysis, hormone biosynthesis, and cellular homeostasis, and lincRNAs enhanced the expression of nearby genes. Alternative splicing was not evident in this polyploid species, but isoforms were switched at phase transitions. The novel, stage-specific regulatory controls uncovered here challenge the conventional understanding of this crucial biological process and provide a new resource of requisite knowledge for those aiming to directly modulate meiosis to improve crop plants. The wheat meiosis transcriptome dataset can be queried for genes of interest using an eFP browser located at https://bar.utoronto.ca/efp_wheat/cgi-bin/efpWeb.cgi?dataSource=Wheat_Meiosis.

The economically valuable Brassica species include the six related members of U’s Triangle. Despite the agronomic and economic importance of these Brassicas, the impacts of evolution and relatively recent domestication events on the genetic landscape of seed development have not been comprehensively examined in these species. Here we present a 3D transcriptome atlas for the six species of U’s Triangle, producing a unique resource that captures gene expression data for the major subcompartments of the seed, from the unfertilized ovule to the mature embryo and seed coat. This comprehensive dataset for seed development in tetraploid and ancestral diploid Brassicas provides new insights into evolutionary divergence and expression bias at the gene and subgenome levels during the domestication of these valued crop species. Comparisons of gene expression associated with regulatory networks and metabolic pathways operating in the embryo and seed coat during seed development reveal differences in storage reserve accumulation and fatty acid metabolism among the six Brassica species. This study illustrates the genetic underpinnings of seed traits and the selective pressures placed on seed production, providing an immense resource for continued investigation of Brassica polyploid biology, genomics and evolution.

Euphorbia peplus(petty spurge) is a small, fast-growing plant that is native to Eurasia and has become a naturalized weed in North America and Australia.E. peplusis not only medicinally valuable, serving as a source for the skin cancer drug ingenol mebutate, but also has great potential as a model for latex production owing to its small size, ease of manipulation in the laboratory, and rapid reproductive cycle. To help establishE. peplusas a new model, we generated a 267.2 Mb HiC-anchored PacBio HiFi nuclear genome assembly with an embryophyta BUSCO score of 98.5%, a genome annotation based on RNA-seq data from six tissues, and publicly accessible tools including a genome browser and an interactive organ-specific expression atlas. Chromosome number is highly variable acrossEuphorbiaspecies. Using a comparative analysis of our newly sequencedE. peplusgenome with other Euphorbiaceae genomes, we show that variation inEuphorbiachromosome number is likely due to fragmentation and rearrangement rather than aneuploidy. Moreover, we found that theE. peplusgenome is relatively compact compared to related members of the genus in part due to restricted expansion of the Ty3 transposon family. Finally, we identify a large gene cluster that contains many previously identified enzymes in the putative ingenol mebutate biosynthesis pathway, along with additional gene candidates for this biosynthetic pathway. The genomic resources we have created forE. pepluswill help advance research on latex production and ingenol mebutate biosynthesis in the commercially important Euphorbiaceae family.Significance statementEuphorbiais one of the five largest genera in the plant kingdom. Despite an impressive phenotypic and metabolic diversity in this genus, only oneEuphorbiagenome has been sequenced so far, restricting insights intoEuphorbiabiology.Euphorbia peplushas excellent potential as a model species due to its latex production, fast growth rate and production of the anticancer drug ingenol mebutate. Here, we present a chromosome-levelE. peplusgenome assembly and publicly accessible resources to support molecular research for this unique species and the broader genus. We also provide an explanation of one reason the genome is so small, and identify more candidate genes for the anticancer drug and related compounds.

Summary Mass spectrometry is the predominant analytical tool used in the field of plant lipidomics. However, there are many challenges associated with the mass spectrometric detection and identification of lipids because of the highly complex nature of plant lipids. Studies into lipid biosynthetic pathways, gene functions in lipid metabolism, lipid changes during plant growth and development, and the holistic examination of the role of plant lipids in environmental stress responses are often hindered. Here, we leveraged a robust pipeline that we previously established to extract and analyze lipid profiles of different tissues and developmental stages from the model plant Arabidopsis thaliana. We analyzed seven tissues at several different developmental stages and identified more than 200 lipids from each tissue analyzed. The data were used to create a web‐accessible in silico lipid map that has been integrated into an electronic Fluorescent Pictograph (eFP) browser. This in silico library of Arabidopsis lipids allows the visualization and exploration of the distribution and changes of lipid levels across selected developmental stages. Furthermore, it provides information on the characteristic fragments of lipids and adducts observed in the mass spectrometer and their retention times, which can be used for lipid identification. The Arabidopsis tissue lipid map can be accessed at http://bar.utoronto.ca/efp_arabidopsis_lipid/cgi‐bin/efpWeb.cgi., Significance Statement With the complexity of plant lipids, a web database collecting information on plant lipids is of great value. Here the most comprehensive and detailed tissue‐specific lipid profile of Arabidopsis thaliana across developmental stages is provided in the form of an open‐source, electronic Fluorescent Pictograph (eFP) browser, along with insights into lipid dynamics across plant growth and development. This resource will significantly advance future research into the Arabidopsis lipidome and facilitate research into other plants.

While root diseases are among the most devastating stresses in global crop production, our understanding of root immunity is still limited relative to our knowledge of immune responses in leaves. Considering that root performance is based on the concerted functions of its different cell types, we undertook a cell type-specific transcriptome analysis to identify gene networks activated in epidermis, cortex, and pericycle cells of Arabidopsis (Arabidopsis thaliana) roots challenged with two immunity elicitors, the bacterial flagellin-derived flg22 and the endogenous Pep1 peptide. Our analyses revealed distinct immunity gene networks in each cell type. To further substantiate our understanding of regulatory patterns underlying these cell type-specific immunity networks, we developed a tool to analyze paired transcription factor binding motifs in the promoters of cell type-specific genes. Our study points toward a connection between cell identity and cell type-specific immunity networks that might guide cell types in launching immune response according to the functional capabilities of each cell type.

Summary Improvements in next‐generation sequencing technologies have resulted in dramatically reduced sequencing costs. This has led to an explosion of ‘‐seq’‐based methods, of which RNA sequencing (RNA‐seq) for generating transcriptomic data is the most popular. By analysing global patterns of gene expression in organs/tissues/cells of interest or in response to chemical or environmental perturbations, researchers can better understand an organism's biology. Tools designed to work with large RNA‐seq data sets enable analyses and visualizations to help generate hypotheses about a gene's function. We present here a user‐friendly RNA‐seq data exploration tool, called the ‘eFP‐Seq Browser’, that shows the read map coverage of a gene of interest in each of the samples along with ‘electronic fluorescent pictographic’ (eFP) images that serve as visual representations of expression levels. The tool also summarizes the details of each RNA‐seq experiment, providing links to archival databases and publications. It automatically computes the reads per kilobase per million reads mapped expression‐level summaries and point biserial correlation scores to sort the samples based on a gene's expression level or by how dissimilar the read map profile is from a gene splice variant, to quickly identify samples with the strongest expression level or where alternative splicing might be occurring. Links to the Integrated Genome Browser desktop visualization tool allow researchers to visualize and explore the details of RNA‐seq alignments summarized in eFP‐Seq Browser as coverage graphs. We present four cases of use of the eFP‐Seq Browser for ABI3,SR34,SR45a and U2AF65B, where we examine expression levels and identify alternative splicing. The URL for the browser is https://bar.utoronto.ca/eFP-Seq_Browser/. Open research badges This article has earned an Open Data Badge for making publicly available the digitally‐shareable data necessary to reproduce the reported results. Tool is at http://sps:urlprefix::https; RNA‐seq data at http://sps:urlprefix::https and http://sps:urlprefix::https. Code is available at http://sps:urlprefix::https, Significance Statement We present a tool, the eFP‐Seq Browser, for rapidly identifying RNA sequencing samples with strong expression levels of a given gene, or where the read maps for a given gene/sample best match a particular gene model. Details can be called up with convenient links to the Integrated Genome Browser.

The early maize (Zea mays) seed undergoes several developmental stages after double fertilization to become fully differentiated within a short period of time, but the genetic control of this highly dynamic and complex developmental process remains largely unknown. Here, we report a high temporal-resolution investigation of transcriptomes using 31 samples collected at an interval of 4 or 6 h within the first six days of seed development. These time-course transcriptomes were clearly separated into four distinct groups corresponding to the stages of double fertilization, coenocyte formation, cellularization, and differentiation. A total of 22,790 expressed genes including 1415 transcription factors (TFs) were detected in early stages of maize seed development. In particular, 1093 genes including 110 TFs were specifically expressed in the seed and displayed high temporal specificity by expressing only in particular period of early seed development. There were 160, 22, 112, and 569 seed-specific genes predominantly expressed in the first 16 h after pollination, coenocyte formation, cellularization, and differentiation stage, respectively. In addition, network analysis predicted 31,256 interactions among 1317 TFs and 14,540 genes. The high temporal-resolution transcriptome atlas reported here provides an important resource for future functional study to unravel the genetic control of seed development.

Multiple genes in sugarcane control sucrose accumulation and the biosynthesis of cell wall components; however, it is unclear how these genes are expressed in its apical culms. To better understand this process, we sequenced mRNA from +1 stem internodes collected from four genotypes with different concentrations of soluble solids. Culms were collected at four different time points, ranging from six to 12-month-old plants. Here we show differentially expressed genes related to sucrose metabolism and cell wall biosynthesis, including genes encoding invertases, sucrose synthase and cellulose synthase. Our results showed increased expression of invertases in IN84-58, the genotype with lower sugar and higher fiber content, as well as delayed expression of secondary cell wall-related cellulose synthase for the other genotypes. Interestingly, genes involved with hormone metabolism were differentially expressed across time points in the three genotypes with higher soluble solids content. A similar result was observed for genes controlling maturation and transition to reproductive stages, possibly a result of selection against flowering in sugarcane breeding programs. These results indicate that carbon partitioning in apical culms of contrasting genotypes is mainly associated with differential cell wall biosynthesis, and may include early modifications for subsequent sucrose accumulation. Co-expression network analysis identified transcription factors related to growth and development, showing a probable time shift for carbon partitioning occurred in 10-month-old plants.

High-resolution spatial transcriptomics identifies previously unknown regulators for barley floral meristem development., Organ development in plants predominantly occurs postembryonically through combinatorial activity of meristems; therefore, meristem and organ fate are intimately connected. Inflorescence morphogenesis in grasses (Poaceae) is complex and relies on a specialized floral meristem, called spikelet meristem, that gives rise to all other floral organs and ultimately the grain. The fate of the spikelet determines reproductive success and contributes toward yield-related traits in cereal crops. Here, we examined the transcriptional landscapes of floral meristems in the temperate crop barley (Hordeum vulgare L.) using RNA-seq of laser capture microdissected tissues from immature, developing floral structures. Our unbiased, high-resolution approach revealed fundamental regulatory networks, previously unknown pathways, and key regulators of barley floral fate and will equally be indispensable for comparative transcriptional studies of grass meristems.

Gene regulatory networks (GRNs) are complex networks that capture multi-level regulatory events between one or more regulatory macromolecules, such as transcription factors (TFs), and their target genes. Advancements in screening technologies such as enhanced yeast-one-hybrid screens have allowed for high throughput determination of GRNs. However, visualization of GRNs in Arabidopsis has been limited to ad hoc networks and are not interactive. Here, we describe the Arabidopsis GEne Network Tool (AGENT) that houses curated GRNs and provides tools to visualize and explore them. AGENT features include expression overlays, subnetwork motif scanning, and network analysis. We show how to use AGENT’s multiple built-in tools to identify key genes that are involved in flowering and seed development along with identifying temporal multi-TF control of a key transporter in nitrate signaling. AGENT can be accessed at https://bar.utoronto.ca/AGENT.

ePlant was introduced in 2017 for exploring large Arabidopsis thaliana data sets from the kilometre to nanometre scales. In the past four years we have used the ePlant framework to develop ePlants for 15 agronomically-important species: maize, poplar, tomato, Camelina sativa, soybean, potato, barley, Medicago truncatula, eucalyptus, rice, willow, sunflower, Cannabis sativa, wheat and sugarcane. We also updated the interface to improve performance and accessibility, and added two new views to the Arabidopsis ePlant – the Navigator and Pathways viewers. The former shows phylogenetic relationships between homologs in other species and their expression pattern similarities, with links to view data for those genes in the respective ePlants. The latter shows Plant Reactome metabolic reactions. We also describe new Arabidopsis data sets including single cell RNA-seq data from roots, and how to embed ePlant eFP expression pictographs into any web page.

High throughput sequencing has opened the doors for investigators to probe genetic variation present in large populations of organisms. In plants, the 1001 Genomes Project (1001genomes.org) is one such effort that sought to characterize the extant worldwide variation in Arabidopsis thaliana for future analyses to compare and draw upon. We developed a web application that accesses the 1001 Genomes database called The Variant Viewer, for investigators to view variants in any A. thaliana gene and within gene families. These variants may be visualized in the context of alignments of queried genes, across splice isoforms of these genes and in relation to conserved domains.

Drought is an important environmental stress that limits crop production. Guard cells (GC) act to control the rate of water loss. To better understand how GCs change their gene expression during a progressive drought we generated guard cell-specific RNA-seq transcriptomes during mild, moderate, and severe drought stress. We additionally sampled re-watered plants that had experienced severe drought stress. These transcriptomes were generated using the INTACT system to capture the RNA from GC nuclei. We optimized the INTACT protocol for Arabidopsis thaliana leaf tissue, incorporating fixation to preserve RNA during nuclear isolation. To be able to identify gene expression changes unique to GCs, we additionally generated transcriptomes from all cell types, using a 35S viral promoter to capture the nuclei of all cell types in leaves. These data sets highlight shared and unique gene expression changes between GCs and the bulk leaf tissue. The timing of gene expression changes is different between GCs and other cell types: we found that only GCs had detectable gene expression changes at the earliest drought time point. The drought responsive GC and leaf RNA-seq transcriptomes are available in the Arabidopsis ePlant at the Bio-Analytic Resource for Plant Biology website.

Conceived as a replacement for the anticipated retirement of The Arabidopsis Information Resource (TAIR), the Araport project was funded by the U.S. National Science Foundation (NSF) in 2013 to develop a new, extensible framework for Arabidopsis ( Arabidopsis thaliana ) bioinformatics that would

Determining the complete Arabidopsis (Arabidopsis thaliana) protein-protein interaction network is essential for understanding the functional organization of the proteome. Numerous small-scale studies and a couple of large-scale ones have elucidated a fraction of the estimated 300,000 binary protein-protein interactions in Arabidopsis. In this study, we provide evidence that a docking algorithm has the ability to identify real interactions using both experimentally determined and predicted protein structures. We ranked 0.91 million interactions generated by all possible pairwise combinations of 1,346 predicted structure models from an Arabidopsis predicted “structure-ome” and found a significant enrichment of real interactions for the top-ranking predicted interactions, as shown by cosubcellular enrichment analysis and yeast two-hybrid validation. Our success rate for computationally predicted, structure-based interactions was 63% of the success rate for published interactions naively tested using the yeast two-hybrid system and 2.7 times better than for randomly picked pairs of proteins. This study provides another perspective in interactome exploration and biological network reconstruction using protein structural information. We have made these interactions freely accessible through an improved Arabidopsis Interactions Viewer and have created community tools for accessing these and ∼2.8 million other protein-protein and protein-DNA interactions for hypothesis generation by researchers worldwide. The Arabidopsis Interactions Viewer is freely available at http://bar.utoronto.ca/interactions2/.

Plant species have evolved myriads of solutions to adapt to dynamic environments, including complex cell type development and regulation. To understand this diversity, we profiled tomato root cell type translatomes and chromatin accessibility. Using xylem differentiation in tomato, relative to Arabidopsis, examples of functional innovation, repurposing and conservation of transcription factors are described. Repurposing and innovation of genes are further observed within an exodermis regulatory network and illustrate its function. Translatome analyses of rice, tomato and Arabidopsis tissues suggest that root meristems are more conserved, and that 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 conserved between plants and animals.One Sentence SummaryPan-species cell type translatome and chromatin accessibility data reveal novelty, conservation and repurposing of gene function.

International audience; A population of endosperm cells adjacent to the embryo scutellum shows transcriptomic enrichment in transport functions and is influenced by embryo development.Seeds are complex biological systems comprising three genetically distinct tissues nested one inside another (embryo, endosperm, and maternal tissues). However, the complexity of the kernel makes it difficult to understand intercompartment interactions without access to spatially accurate information. Here, we took advantage of the large size of the maize (Zea mays) kernel to characterize genome-wide expression profiles of tissues at different embryo/endosperm interfaces. Our analysis identifies specific transcriptomic signatures in two interface tissues compared with whole seed compartments: the scutellar aleurone layer and the newly named endosperm adjacent to scutellum (EAS). The EAS, which appears around 9 d after pollination and persists for around 11 d, is confined to one to three endosperm cell layers adjacent to the embryonic scutellum. Its transcriptome is enriched in genes encoding transporters. The absence of the embryo in an embryo specific mutant can alter the expression pattern of EAS marker genes. The detection of cell death in some EAS cells together with an accumulation of crushed cell walls suggests that the EAS is a dynamic zone from which cell layers in contact with the embryo are regularly eliminated and to which additional endosperm cells are recruited as the embryo grows.

A big challenge in current systems biology research arises when different types of data must be accessed from separate sources and visualized using separate tools. The high cognitive load required to navigate such a workflow is detrimental to hypothesis generation. Accordingly, there is a need for a robust research platform that incorporates all data and provides integrated search, analysis, and visualization features through a single portal. Here, we present ePlant (http://bar.utoronto.ca/eplant), a visual analytic tool for exploring multiple levels of Arabidopsis thaliana data through a zoomable user interface. ePlant connects to several publicly available web services to download genome, proteome, interactome, transcriptome, and 3D molecular structure data for one or more genes or gene products of interest. Data are displayed with a set of visualization tools that are presented using a conceptual hierarchy from big to small, and many of the tools combine information from more than one data type. We describe the development of ePlant in this article and present several examples illustrating its integrative features for hypothesis generation. We also describe the process of deploying ePlant as an “app” on Araport. Building on readily available web services, the code for ePlant is freely available for any other biological species research.

Selaginella moellendorffii is a representative of the lycophyte lineage that is studied to understand the evolution of land plant traits such as the vasculature, leaves, stems, roots, and secondary metabolism. However, only a few studies have investigated the expression and transcriptional coordination of Selaginella genes, precluding us from understanding the evolution of the transcriptional programs behind these traits. We present a gene expression atlas comprising all major organs, tissue types, and the diurnal gene expression profiles for S. moellendorffii. We show that the transcriptional gene module responsible for the biosynthesis of lignocellulose evolved in the ancestor of vascular plants and pinpoint the duplication and subfunctionalization events that generated multiple gene modules involved in the biosynthesis of various cell wall types. We demonstrate how secondary metabolism is transcriptionally coordinated and integrated with other cellular pathways. Finally, we identify root-specific genes and show that the evolution of roots did not coincide with an increased appearance of gene families, suggesting that the development of new organs does not coincide with increased fixation of new gene functions. Our updated database at conekt.plant.tools represents a valuable resource for studying the evolution of genes, gene families, transcriptomes, and functional gene modules in the Archaeplastida kingdom.

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 tissues suggest increased expression conservation of root meristems compared with other homologous tissues. 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.

Tung tree (Vernicia fordii) is an economically important woody oil plant that produces tung oil containing a high proportion of eleostearic acid (∼80%). Here we report a high-quality, chromosome-scale tung tree genome sequence of 1.12 Gb with 28,422 predicted genes and over 73% repeat sequences. Tung tree genome was assembled by combining Illumina short reads, PacBio single-molecule real-time long reads and Hi-C sequencing data. Insertion time analysis revealed that the repeat-driven tung tree genome expansion might be due to long standing long terminal repeat (LTR) retrotransposon bursts and lack of efficient DNA deletion mechanisms. An electronic fluorescent pictographic (eFP) browser was generated based on genomic and RNA-seq data from 17 various tissues and developmental stages. We identified 88 nucleotide-binding site (NBS)-encoding resistance genes, of which 17 genes may help the tung tree resist the Fusarium wilt shortly after infection. A total of 651 oil-related genes were identified and 88 of them were predicted to be directly involved in tung oil biosynthesis. The fewer phosphoenolpyruvate carboxykinase (PEPC) genes, and synergistic effects between transcription factors and oil biosynthesis-related genes may contribute to high oil content in tung seeds. The tung tree genome should provide valuable resources for molecular breeding and genetic improvement.

SummaryThe lycophyte Selaginella moellendorffii represents early vascular plants and is studied to understand the evolution of higher plant traits such as the vasculature, leaves, stems, roots, and secondary metabolism. However, little is known about the gene expression and transcriptional coordination of Selaginella genes, which precludes us from understanding the evolution of transcriptional programs behind these traits.We here present a gene expression atlas comprising all major organs, tissue types, and the diurnal gene expression profiles for S. moellendorffii. The atlas is part of the CoNekT-Plants database (conekt.plant.tools), which enables comparative transcriptomic analyses across two algae and seven land plants.We show that the transcriptional gene module responsible for the biosynthesis of lignocellulose evolved in the ancestor of vascular plants, and pinpoint the duplication and subfunctionalization events that generated multiple gene modules involved in the biosynthesis of various cell wall types. We further demonstrate how secondary metabolism is transcriptionally coordinated and integrated with other cellular pathways. Finally, we identify root-specific genes in vascular plants and show that the evolution of roots did not coincide with an increased appearance of gene families, suggesting that the existing genetic material was sufficient to generate new organs.Our updated database at conekt.plant.tools provides a unique resource to study the evolution of genes, gene families, transcriptomes, and functional gene modules in the Archaeplastida kingdom.

Background Crassulacean acid metabolism (CAM), a specialized mode of photosynthesis, enables plant adaptation to water-limited environments and improves photosynthetic efficiency via an inorganic carbon-concentrating mechanism. Kalanchoë fedtschenkoi is an obligate CAM model featuring a relatively small genome and easy stable transformation. However, the molecular responses to light quality and intensity in CAM plants remain understudied. Results Here we present a genome-wide expression atlas of K. fedtschenkoi plants grown under 12 h/12 h photoperiod with different light quality (blue, red, far-red, white light) and intensity (0, 150, 440, and 1,000 μmol m–2 s–1) based on RNA sequencing performed for mature leaf samples collected at dawn (2 h before the light period) and dusk (2 h before the dark period). An eFP web browser was created for easy access of the gene expression data. Based on the expression atlas, we constructed a light-responsive co-expression network to reveal the potential regulatory relationships in K. fedtschenkoi. Measurements of leaf titratable acidity, soluble sugar, and starch turnover provided metabolic indicators of the magnitude of CAM under the different light treatments and were used to provide biological context for the expression dataset. Furthermore, CAM-related subnetworks were highlighted to showcase genes relevant to CAM pathway, circadian clock, and stomatal movement. In comparison with white light, monochrome blue/red/far-red light treatments repressed the expression of several CAM-related genes at dusk, along with a major reduction in acid accumulation. Increasing light intensity from an intermediate level (440 μmol m−2 s−1) of white light to a high light treatment (1,000 μmol m–2 s–1) increased expression of several genes involved in dark CO2 fixation and malate transport at dawn, along with an increase in organic acid accumulation. Conclusions This study provides a useful genomics resource for investigating the molecular mechanism underlying the light regulation of physiology and metabolism in CAM plants. Our results support the hypothesis that both light intensity and light quality can modulate the CAM pathway through regulation of CAM-related genes in K. fedtschenkoi.

Identifying sets of genes that are specifically expressed in certain tissues or in response to an environmental stimulus is useful for designing reporter constructs, generating gene expression markers, or for understanding gene regulatory networks. We have developed an easy-to-use online tool for defining a desired expression profile (a modification of our Expression Angler program), which can then be used to identify genes exhibiting patterns of expression that match this profile as closely as possible. Further, we have developed another online tool, Cistome, for predicting or exploring cis-elements in the promoters of sets of co-expressed genes identified by such a method, or by other methods. We present two use cases for these tools, which are freely available on the Bio-Analytic Resource at http://BAR.utoronto.ca.

Plants use surface receptors to perceive information about many aspects of their local environment. These receptors physically interact to form both steady state and signalling competent complexes. The signalling events downstream of receptor activation impact both plant developmental and immune responses. Here, we present a comprehensive study of the physical interactions between the extracellular domains of leucine-rich repeat receptor kinases (LRR-RKs) in Arabidopsis. Using a sensitized assay, we tested reciprocal interactions among 200 of the 225 Arabidopsis LRR-RKs for a total search space of 40,000 interactions. Applying a stringent statistical cut-off and requiring that interactions performed well in both bait-prey and prey-bait orientations resulted in a high-confidence set of 567 bidirectional interactions. Additionally, we identified a total of 2,586 unidirectional interactions, which passed our stringent statistical cut-off in only one orientation. These datasets will guide further investigation into the regulatory roles of LRR-RKs in plant developmental and immune signalling decisions.

Modern wheat production comes from two polyploid species, Triticum aestivum and Triticumturgidum (var durum), which putatively arose from diploid ancestors Triticumurartu, Aegilops speltoides, and Aegilopstauschii How gene expression during embryogenesis and grain development in wheats has been shaped by the differing contributions of diploid genomes through hybridization, polyploidization, and breeding selection is not well understood. This study describes the global landscape of gene activities during wheat embryogenesis and grain development. Using comprehensive transcriptomic analyses of two wheat cultivars and three diploid grasses, we investigated gene expression at seven stages of embryo development, two endosperm stages, and one pericarp stage. We identified transcriptional signatures and developmental similarities and differences among the five species, revealing the evolutionary divergence of gene expression programs and the contributions of A, B, and D subgenomes to grain development in polyploid wheats. The characterization of embryonic transcriptional programming in hexaploid wheat, tetraploid wheat, and diploid grass species provides insight into the landscape of gene expression in modern wheat and its ancestral species. This study presents a framework for understanding the evolution of domesticated wheat and the selective pressures placed on grain production, with important implications for future performance and yield improvements.plantcell;31/12/2888/FX1F1fx1.

Summary Maize (Zea mays L.), a model species for genetic studies, is one of the two most important crop species worldwide. The genome sequence of the reference genotype, B73, representative of the stiff stalk heterotic group was recently updated (AGPv4) using long‐read sequencing and optical mapping technology. To facilitate the use of AGPv4 and to enable functional genomic studies and association of genotype with phenotype, we determined expression abundances for replicated mRNA‐sequencing datasets from 79 tissues and five abiotic/biotic stress treatments revealing 36 207 expressed genes. Characterization of the B73 transcriptome across six organs revealed 4154 organ‐specific and 7704 differentially expressed (DE) genes following stress treatment. Gene co‐expression network analyses revealed 12 modules associated with distinct biological processes containing 13 590 genes providing a resource for further association of gene function based on co‐expression patterns. Presence−absence variants (PAVs) previously identified using whole genome resequencing data from 61 additional inbred lines were enriched in organ‐specific and stress‐induced DE genes suggesting that PAVs may function in phenological variation and adaptation to environment. Relative to core genes conserved across the 62 profiled inbreds, PAVs have lower expression abundances which are correlated with their frequency of dispersion across inbreds and on average have significantly fewer co‐expression network connections suggesting that a subset of PAVs may be on an evolutionary path to pseudogenization. To facilitate use by the community, we developed the Maize Genomics Resource website (maize.plantbiology.msu.edu) for viewing and data‐mining these resources and deployed two new views on the maize electronic Fluorescent Pictograph Browser (bar.utoronto.ca/efp_maize)., Significance Statement We developed a comprehensive transcriptome resource for the maize reference genotype B73 based on the AGPv4 long‐read assembly using replicated and publicly available RNA‐sequencing datasets. Gene expression analyses were performed on a robust B73 developmental and stress gene expression atlas, utilizing core and dispensable genes of the maize pan‐genome as a case study to demonstrate the utility of the data.

Insights from the annotated wheat genome Wheat is one of the major sources of food for much of the world. However, because bread wheat's genome is a large hybrid mix of three separate subgenomes, it has been difficult to produce a high-quality reference sequence. Using recent advances in sequencing, the International Wheat Genome Sequencing Consortium presents an annotated reference genome with a detailed analysis of gene content among subgenomes and the structural organization for all the chromosomes. Examples of quantitative trait mapping and CRISPR-based genome modification show the potential for using this genome in agricultural research and breeding. Ramírez-González et al. exploited the fruits of this endeavor to identify tissue-specific biased gene expression and coexpression networks during development and exposure to stress. These resources will accelerate our understanding of the genetic basis of bread wheat.; International audience; The coordinated expression of highly related homoeologous genes in polyploid species underlies the phenotypes of many of the world's major crops. Here we combine extensive gene expression datasets to produce a comprehensive, genome-wide analysis of homoeolog expression patterns in hexaploid bread wheat. Bias in homoeolog expression varies between tissues, with ~30% of wheat homoeologs showing nonbalanced expression. We found expression asymmetries along wheat chromosomes, with homoeologs showing the largest inter-tissue, inter-cultivar, and coding sequence variation, most often located in high-recombination distal ends of chromosomes. These transcriptionally dynamic genes potentially represent the first steps toward neo- or subfunctionalization of wheat homoeologs. Coexpression networks reveal extensive coordination of homoeologs throughout development and, alongside a detailed expression atlas, provide a framework to target candidate genes underpinning agronomic traits in wheat.nonbalanced expression patterns, with higher or lower expression from a single homoeolog with respect to the other two. These differences between homoeologs were associated with epigenetic changes affecting DNA methylation and histone modifications. Although nonbalanced homoeolog expression could be partially predicted by expression in diploid ancestors, large changes in relative homoeolog expression were observed owing to polyploidization. Our results suggest that the transposable elements in promoters relate more closely to the variation in the relative expression of homoeologs across tissues than to a ubiquitous effect across all tissues. We found that homoeologs with the highest inter-tissue variation had promoters with more frequent transposable element insertions and more varied cis-regulatory elements than homoeologs that were stable across tissues. We also identified expression asymmetry along wheat chromosomes. Homoeologs with the largest inter-tissue, inter-cultivar, and coding sequence variation were most often located in the highly recombinogenic distal ends of chromosomes. These transcriptionally dynamic homoeologs are under more relaxed selection pressure, potentially representing the first steps toward functional innovation through neo- or subfunctionalization. We generated tissue- and stress-specific coexpression networks that reveal extensive coordination of homoeolog expression throughout development. These networks, alongside detailed gene expression atlases (www.wheat-expression.com and http://bar.utoronto.ca), lay the groundwork to identify candidate genes influencing agronomic traits in wheat. CONCLUSION This study provides detailed insights into the transcriptional landscape of bread wheat, an evolutionarily young polyploid. Our work shows that homoeolog expression patterns in bread wheat have been shaped by polyploidy and are associated with both epigenetic modifications and variation in transposable elements within promoters of homoeologous genes. The extensive datasets and analyses presented here provide a framework that can help researchers and breeders develop strategies to improve crops by manipulating individual or multiple homoeologs to modulate trait responses.

Highlight This transcriptome study highlights genes exclusively and preferentially expressed in maize root hairs and their suggested functions, and provides a starting point for future genetic analyses., Root hairs are tubular extensions of epidermis cells. Transcriptome profiling demonstrated that the single cell-type root hair transcriptome was less complex than the transcriptome of multiple cell-type primary roots without root hairs. In total, 831 genes were exclusively and 5585 genes were preferentially expressed in root hairs [false discovery rate (FDR) ≤1%]. Among those, the most significantly enriched Gene Ontology (GO) functional terms were related to energy metabolism, highlighting the high energy demand for the development and function of root hairs. Subsequently, the maize homologs for 138 Arabidopsis genes known to be involved in root hair development were identified and their phylogenetic relationship and expression in root hairs were determined. This study indicated that the genetic regulation of root hair development in Arabidopsis and maize is controlled by common genes, but also shows differences which need to be dissected in future genetic experiments. Finally, a maize root view of the eFP browser was implemented including the root hair transcriptome of the present study and several previously published maize root transcriptome data sets. The eFP browser provides color-coded expression levels for these root types and tissues for any gene of interest, thus providing a novel resource to study gene expression and function in maize roots.

While Brachypodium distachyon (Brachypodium) is an emerging model for grasses, no expression atlas or gene coexpression network is available. Such tools are of high importance to provide insights into the function of Brachypodium genes. We present a detailed Brachypodium expression atlas, capturing gene expression in its major organs at different developmental stages. The data were integrated into a large-scale coexpression database ( www.gene2function.de), enabling identification of duplicated pathways and conserved processes across 10 plant species, thus allowing genome-wide inference of gene function. We highlight the importance of the atlas and the platform through the identification of duplicated cell wall modules, and show that a lignin biosynthesis module is conserved across angiosperms. We identified and functionally characterised a putative ferulate 5-hydroxylase gene through overexpression of it in Brachypodium, which resulted in an increase in lignin syringyl units and reduced lignin content of mature stems, and led to improved saccharification of the stem biomass. Our Brachypodium expression atlas thus provides a powerful resource to reveal functionally related genes, which may advance our understanding of important biological processes in grasses.

Using both pharmacological and transcriptomic approaches, it is shown that Nod factors and auxin interact synergistically to control lateral root formation and gene regulatory responses., Nodulation (Nod) factors (NFs) are symbiotic molecules produced by rhizobia that are essential for establishment of the rhizobium–legume endosymbiosis. Purified NFs can stimulate lateral root formation (LRF) in Medicago truncatula, but little is known about the molecular mechanisms involved. Using a combination of reporter constructs, pharmacological and genetic approaches, we show that NFs act on early steps of LRF in M. truncatula, independently of the ethylene signaling pathway and of the cytokinin receptor MtCRE1, but in interaction with auxin. We conducted a whole-genome transcriptomic study upon NF and/or auxin treatments, using a lateral root inducible system adapted for M. truncatula. This revealed a large overlap between NF and auxin signaling and, more interestingly, synergistic interactions between these molecules. Three groups showing interaction effects were defined: group 1 contained more than 1500 genes responding specifically to the combinatorial treatment of NFs and auxin; group 2 comprised auxin-regulated genes whose expression was enhanced or antagonized by NFs; and in group 3 the expression of NF regulated genes was antagonized by auxin. Groups 1 and 2 were enriched in signaling and metabolic functions, which highlights important crosstalk between NF and auxin signaling for both developmental and symbiotic processes.

Summary Camelina sativa is currently being embraced as a viable industrial bio-platform crop due to a number of desirable agronomic attributes and the unique fatty acid profile of the seed oil that has applications for food, feed and biofuel. The recent completion of the reference genome sequence of C. sativa identified a young hexaploid genome. To complement this work, we have generated a genome-wide developmental transcriptome map by RNA sequencing of 12 different tissues covering major developmental stages during the life cycle of C. sativa. We have generated a digital atlas of this comprehensive transcriptome resource that enables interactive visualization of expression data through a searchable database of electronic fluorescent pictographs (eFP browser). An analysis of this dataset supported expression of 88% of the annotated genes in C. sativa and provided a global overview of the complex architecture of temporal and spatial gene expression patterns active during development. Conventional differential gene expression analysis combined with weighted gene expression network analysis uncovered similarities as well as differences in gene expression patterns between different tissues and identified tissue-specific genes and network modules. A high quality census of transcription factors, analysis of alternative splicing and tissue-specific genome dominance provided insight into the transcriptional dynamics and sub-genome interplay among the well preserved triplicated repertoire of homeologous loci. The comprehensive transcriptome atlas in combination with the reference genome sequence provides a powerful resource for genomics research which can be leveraged to identify functional associations between genes and understand the regulatory networks underlying developmental processes. This article is protected by copyright. All rights reserved.

Seeds respond to multiple different environmental stimuli that regulate germination. Nitrate stimulates germination in many plants but how it does so remains unclear. Here we show that the Arabidopsis NIN-like protein 8 (NLP8) is essential for nitrate-promoted seed germination. Seed germination in nlp8 loss-of-function mutants does not respond to nitrate. NLP8 functions even in a nitrate reductase-deficient mutant background, and the requirement for NLP8 is conserved among Arabidopsis accessions. NLP8 reduces abscisic acid levels in a nitrate-dependent manner and directly binds to the promoter of CYP707A2, encoding an abscisic acid catabolic enzyme. Genetic analysis shows that NLP8-mediated promotion of seed germination by nitrate requires CYP707A2. Finally, we show that NLP8 localizes to nuclei and unlike NLP7, does not appear to be activated by nitrate-dependent nuclear retention of NLP7, suggesting that seeds have a unique mechanism for nitrate signalling., Nitrate stimulates seed germination in many plant species. Here, Yan et al. show that the Arabidopsis transcription factor NIN-like protein 8 is required to stimulate germination in response to nitrate and induces expression of an enzyme involved in ABA catabolism.

Summary: Gene Slider helps visualize the conservation and entropy of orthologous DNA and protein sequences by presenting them as one long sequence logo that can be zoomed in and out of, from an overview of the entire sequence down to just a few residues at a time. A search function enables users to find motifs such as cis-elements in promoter regions by simply ‘drawing’ a sequence logo representation of the desired motif as a query. In addition to displaying user-supplied FASTA files, our demonstration version of Gene Slider loads and displays a rich database of 90 000+ conserved non-coding regions across the Brassicaceae indexed to the TAIR10 Col-0 Arabidopsis thaliana sequence. It also displays transcription factor binding sites, enabling easy identification of regions that are both conserved across multiple species and may contain transcription factor binding sites. Availability and Implementation: Freely available on the web at: http://www.bar.utoronto.ca/GeneSlider and also as an app on http://araport.org. Website implemented in JavaScript and Processing.js with all major browsers supported. Source code available under GNU GPLv2 at SourceForge: https://sourceforge.net/projects/geneslider/. Contact: nicholas.provart@utoronto.ca

International audience; Seed longevity, the maintenance of viability during storage, is a crucial factor for preservation of genetic resources and ensuring proper seedling establishment and high crop yield. We used a systems biology approach to identify key genes regulating the acquisition of longevity during seed maturation of Medicago truncatula. Using 104 transcriptomes from seed developmental time courses obtained in five growth environments, we generated a robust, stable coexpression network (MatNet), thereby capturing the conserved backbone of maturation. Using a trait-based gene significance measure, a coexpression module related to the acquisition of longevity was inferred from MatNet. Comparative analysis of the maturation processes in M. truncatula and Arabidopsis thaliana seeds and mining Arabidopsis interaction databases revealed conserved connectivity for 87% of longevity module nodes between both species. Arabidopsis mutant screening for longevity and maturation phenotypes demonstrated high predictive power of the longevity cross-species network. Overrepresentation analysis of the network nodes indicated biological functions related to defense, light, and auxin. Characterization of defense-related wrky3 and nf-x1-like1 (nfxl1) transcription factor mutants demonstrated that these genes regulate some of the network nodes and exhibit impaired acquisition of longevity during maturation. These data suggest that seed longevity evolved by co-opting existing genetic pathways regulating the activation of defense against pathogens.