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.

The transcriptome landscape of developing barley seeds.

Cereal grains are an important source of food and feed. To provide comprehensive spatiotemporal information about biological processes in developing seeds of cultivated barley (Hordeum vulgare L. subsp. vulgare), we performed a transcriptomic study of the embryo, endosperm, and seed maternal tissues collected from grains 4-32 days after pollination. Weighted gene co-expression network and motif enrichment analyses identified specific groups of genes and transcription factors (TFs) potentially regulating barley seed tissue development. We defined a set of tissue-specific marker genes and families of TFs for functional studies of the pathways controlling barley grain development. Assessing selected groups of chromatin regulators revealed that epigenetic processes are highly dynamic and likely play a major role during barley endosperm development. The repressive H3K27me3 modification is globally reduced in endosperm tissues and at specific genes related to development and storage compounds. Altogether, this atlas uncovers the complexity of developmentally regulated gene expression in developing barley grains.

Mapping the signaling network of BIN2 kinase using TurboID-mediated biotin labeling and phosphoproteomics.

Elucidating enzyme-substrate relationships in posttranslational modification (PTM) networks is crucial for understanding signal transduction pathways but is technically difficult because enzyme-substrate interactions tend to be transient. Here, we demonstrate that TurboID-based proximity labeling (TbPL) effectively and specifically captures the substrates of kinases and phosphatases. TbPL-mass spectrometry (TbPL-MS) identified over 400 proximal proteins of Arabidopsis thaliana BRASSINOSTEROID-INSENSITIVE2 (BIN2), a member of the GLYCOGEN SYNTHASE KINASE 3 (GSK3) family that integrates signaling pathways controlling diverse developmental and acclimation processes. A large portion of the BIN2-proximal proteins showed BIN2-dependent phosphorylation in vivo or in vitro, suggesting that these are BIN2 substrates. Protein-protein interaction network analysis showed that the BIN2-proximal proteins include interactors of BIN2 substrates, revealing a high level of interactions among the BIN2-proximal proteins. Our proteomic analysis establishes the BIN2 signaling network and uncovers BIN2 functions in regulating key cellular processes such as transcription, RNA processing, translation initiation, vesicle trafficking, and cytoskeleton organization. We further discovered significant overlap between the GSK3 phosphorylome and the O-GlcNAcylome, suggesting an evolutionarily ancient relationship between GSK3 and the nutrient-sensing O-glycosylation pathway. Our work presents a powerful method for mapping PTM networks, a large dataset of GSK3 kinase substrates, and important insights into the signaling network that controls key cellular functions underlying plant growth and acclimation.

Nutri-cereal tissue-specific transcriptome atlas during development: Functional integration of gene expression to identify mineral uptake pathways in little millet (Panicum sumatrense).

Little millet (Panicum sumatrense Roth ex Roem. & Schult.) is an essential minor millet of southeast Asia and Africa's temperate and subtropical regions. The plant is stress-tolerant, has a short life cycle, and has a mineral-rich nutritional profile associated with unique health benefits. We report the developmental gene expression atlas of little millet (genotype JK-8) from ten tissues representing different stages of its life cycle, starting from seed germination and vegetative growth to panicle maturation. The developmental transcriptome atlas led to the identification of 342 827 transcripts. The BUSCO analysis and comparison with the transcriptomes of related species confirm that this study presents high-quality, in-depth coverage of the little millet transcriptome. In addition, the eFP browser generated here has a user-friendly interface, allowing interactive visualizations of tissue-specific gene expression. Using these data, we identified transcripts, the orthologs of which in Arabidopsis and rice are involved in nutrient acquisition, transport, and response pathways. The comparative analysis of the expression levels of these transcripts holds great potential for enhancing the mineral content in crops, particularly zinc and iron, to address the issue of "hidden hunger" and to attain nutritional security, making it a valuable asset for translational research.

Information theory and machine learning illuminate large-scale metabolomic responses of Brachypodium distachyon to environmental change.

Plant responses to environmental change are mediated via changes in cellular metabolomes. However, <5% of signals obtained from liquid chromatography tandem mass spectrometry (LC-MS/MS) can be identified, limiting our understanding of how metabolomes change under biotic/abiotic stress. To address this challenge, we performed untargeted LC-MS/MS of leaves, roots, and other organs of Brachypodium distachyon (Poaceae) under 17 organ-condition combinations, including copper deficiency, heat stress, low phosphate, and arbuscular mycorrhizal symbiosis. We found that both leaf and root metabolomes were significantly affected by the growth medium. Leaf metabolomes were more diverse than root metabolomes, but the latter were more specialized and more responsive to environmental change. We found that 1 week of copper deficiency shielded the root, but not the leaf metabolome, from perturbation due to heat stress. Machine learning (ML)-based analysis annotated approximately 81% of the fragmented peaks versus approximately 6% using spectral matches alone. We performed one of the most extensive validations of ML-based peak annotations in plants using thousands of authentic standards, and analyzed approximately 37% of the annotated peaks based on these assessments. Analyzing responsiveness of each predicted metabolite class to environmental change revealed significant perturbations of glycerophospholipids, sphingolipids, and flavonoids. Co-accumulation analysis further identified condition-specific biomarkers. To make these results accessible, we developed a visualization platform on the Bio-Analytic Resource for Plant Biology website (https://bar.utoronto.ca/efp_brachypodium_metabolites/cgi-bin/efpWeb.cgi), where perturbed metabolite classes can be readily visualized. Overall, our study illustrates how emerging chemoinformatic methods can be applied to reveal novel insights into the dynamic plant metabolome and stress adaptation.

Multi-omics atlas of combinatorial abiotic stress responses in wheat.

Field-grown crops rarely experience growth conditions in which yield can be maximized. Environmental stresses occur in combination, with advancements in crop tolerance further complicated by its polygenic nature. Strategic targeting of causal genes is required to meet future crop production needs. Here, we employed a systems biology approach in wheat (Triticum aestivum L.) to investigate physio-metabolic adjustments and transcriptome reprogramming involved in acclimations to heat, drought, salinity and all combinations therein. A significant shift in magnitude and complexity of plant response was evident across stress scenarios based on the agronomic losses, increased proline concentrations and 8.7-fold increase in unique differentially expressed transcripts (DETs) observed under the triple stress condition. Transcriptome data from all stress treatments were assembled into an online, open access eFP browser for visualizing gene expression during abiotic stress. Weighted gene co-expression network analysis revealed 152 hub genes of which 32% contained the ethylene-responsive element binding factor-associated amphiphilic repression (EAR) transcriptional repression motif. Cross-referencing against the 31 DETs common to all stress treatments isolated TaWRKY33 as a leading candidate for greater plant tolerance to combinatorial stresses. Integration of our findings with available literature on gene functional characterization allowed us to further suggest flexible gene combinations for future adaptive gene stacking in wheat. Our approach demonstrates the strength of robust multi-omics-based data resources for gene discovery in complex environmental conditions. Accessibility of such datasets will promote cross-validation of candidate genes across studies and aid in accelerating causal gene validation for crop resiliency.

The coordinated regulation of early meiotic stages is dominated by non-coding RNAs and stage-specific transcription in wheat.

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 cacao gene atlas: a transcriptome developmental atlas reveals highly tissue-specific and dynamically-regulated gene networks in Theobroma cacao L.

BACKGROUND: Theobroma cacao, the cocoa tree, is a tropical crop grown for its highly valuable cocoa solids and fat which are the basis of a 200-billion-dollar annual chocolate industry. However, the long generation time and difficulties associated with breeding a tropical tree crop have limited the progress of breeders to develop high-yielding disease-resistant varieties. Development of marker-assisted breeding methods for cacao requires discovery of genomic regions and specific alleles of genes encoding important traits of interest. To accelerate gene discovery, we developed a gene atlas composed of a large dataset of replicated transcriptomes with the long-term goal of progressing breeding towards developing high-yielding elite varieties of cacao. RESULTS: We describe the creation of the Cacao Transcriptome Atlas, its global characterization and define sets of genes co-regulated in highly organ- and temporally-specific manners. RNAs were extracted and transcriptomes sequenced from 123 different tissues and stages of development representing major organs and developmental stages of the cacao lifecycle. In addition, several experimental treatments and time courses were performed to measure gene expression in tissues responding to biotic and abiotic stressors. Samples were collected in replicates (3-5) to enable statistical analysis of gene expression levels for a total of 390 transcriptomes. To promote wide use of these data, all raw sequencing data, expression read mapping matrices, scripts, and other information used to create the resource are freely available online. We verified our atlas by analyzing the expression of genes with known functions and expression patterns in Arabidopsis (ACT7, LEA19, AGL16, TIP13, LHY, MYB2) and found their expression profiles to be generally similar between both species. We also successfully identified tissue-specific genes at two thresholds in many tissue types represented and a set of genes highly conserved across all tissues. CONCLUSION: The Cacao Gene Atlas consists of a gene expression browser with graphical user interface and open access to raw sequencing data files as well as the unnormalized and CPM normalized read count data mapped to several cacao genomes. The gene atlas is a publicly available resource to allow rapid mining of cacao gene expression profiles. We hope this resource will be used to help accelerate the discovery of important genes for key cacao traits such as disease resistance and contribute to the breeding of elite varieties to help farmers increase yields.

A high-resolution gene expression map of the medial and lateral domains of the gynoecium of Arabidopsis.

Angiosperms are characterized by the formation of flowers, and in their inner floral whorl, one or various gynoecia are produced. These female reproductive structures are responsible for fruit and seed production, thus ensuring the reproductive competence of angiosperms. In Arabidopsis (Arabidopsis thaliana), the gynoecium is composed of two fused carpels with different tissues that need to develop and differentiate to form a mature gynoecium and thus the reproductive competence of Arabidopsis. For these reasons, they have become the object of study for floral and fruit development. However, due to the complexity of the gynoecium, specific spatio-temporal tissue expression patterns are still scarce. In this study, we used precise laser-assisted microdissection and high-throughput RNA sequencing to describe the transcriptional profiles of the medial and lateral domain tissues of the Arabidopsis gynoecium. We provide evidence that the method used is reliable and that, in addition to corroborating gene expression patterns of previously reported regulators of these tissues, we found genes whose expression dynamics point to being involved in cytokinin and auxin homeostasis and in cell cycle progression. Furthermore, based on differential gene expression analyses, we functionally characterized several genes and found that they are involved in gynoecium development. This resource is available via the Arabidopsis eFP browser and will serve the community in future studies on developmental and reproductive biology.

Toward a data infrastructure for the Plant Cell Atlas.

We review how a data infrastructure for the Plant Cell Atlas might be built using existing infrastructure and platforms. The Human Cell Atlas has developed an extensive infrastructure for human and mouse single cell data, while the European Bioinformatics Institute has developed a Single Cell Expression Atlas, that currently houses several plant data sets. We discuss issues related to appropriate ontologies for describing a plant single cell experiment. We imagine how such an infrastructure will enable biologists and data scientists to glean new insights into plant biology in the coming decades, as long as such data are made accessible to the community in an open manner.

Profiling of the gene expression and alternative splicing landscapes of Eucalyptus grandis.

Eucalyptus is a widely planted hardwood tree species due to its fast growth, superior wood properties and adaptability. However, the post-transcriptional regulatory mechanisms controlling tissue development and stress responses in Eucalyptus remain poorly understood. In this study, we performed a comprehensive analysis of the gene expression profile and the alternative splicing (AS) landscape of E. grandis using strand-specific RNA-Seq, which encompassed 201 libraries including different organs, developmental stages, and environmental stresses. We identified 10 416 genes (33.49%) that underwent AS, and numerous differentially expressed and/or differential AS genes involved in critical biological processes, such as primary-to-secondary growth transition of stems, adventitious root formation, aging and responses to phosphorus- or boron-deficiency. Co-expression analysis of AS events and gene expression patterns highlighted the potential upstream regulatory role of AS events in multiple processes. Additionally, we highlighted the lignin biosynthetic pathway to showcase the potential regulatory functions of AS events in the KNAT3 and IRL3 genes within this pathway. Our high-quality expression atlas and AS landscape serve as valuable resources for unravelling the genetic control of woody plant development, long-term adaptation, and understanding transcriptional diversity in Eucalyptus. Researchers can conveniently access these resources through the interactive ePlant browser (https://bar.utoronto.ca/eplant_eucalyptus).

Anno genominis XX: 20 years of Arabidopsis genomics.

Twenty years ago, the Arabidopsis thaliana genome sequence was published. This was an important moment as it was the first sequenced plant genome and explicitly brought plant science into the genomics era. At the time, this was not only an outstanding technological achievement, but it was characterized by a superb global collaboration. The Arabidopsis genome was the seed for plant genomic research. Here, we review the development of numerous resources based on the genome that have enabled discoveries across plant species, which has enhanced our understanding of how plants function and interact with their environments.

Publisher Correction: An extracellular network of Arabidopsis leucine-rich repeat receptor kinases.

In this Letter, an incorrect version of the Supplementary Information file was inadvertently used, which contained several errors. The details of references 59-65 were missing from the end of the Supplementary Discussion section on page 4. In addition, the section 'Text 3. Y2H on ICD interactions' incorrectly referred to 'Extended Data Fig. 4d' instead of 'Extended Data Fig. 3d' on page 3. Finally, the section 'Text 4. Interaction network analysis' incorrectly referred to 'Fig. 1b and Extended Data Fig. 6' instead of 'Fig. 2b and Extended Data Fig. 7' on page 3. These errors have all been corrected in the Supplementary Information.

An extracellular network of Arabidopsis leucine-rich repeat receptor kinases.

The cells of multicellular organisms receive extracellular signals using surface receptors. The extracellular domains (ECDs) of cell surface receptors function as interaction platforms, and as regulatory modules of receptor activation. Understanding how interactions between ECDs produce signal-competent receptor complexes is challenging because of their low biochemical tractability. In plants, the discovery of ECD interactions is complicated by the massive expansion of receptor families, which creates tremendous potential for changeover in receptor interactions. The largest of these families in Arabidopsis thaliana consists of 225 evolutionarily related leucine-rich repeat receptor kinases (LRR-RKs), which function in the sensing of microorganisms, cell expansion, stomata development and stem-cell maintenance. Although the principles that govern LRR-RK signalling activation are emerging, the systems-level organization of this family of proteins is unknown. Here, to address this, we investigated 40,000 potential ECD interactions using a sensitized high-throughput interaction assay, and produced an LRR-based cell surface interaction network (CSILRR) that consists of 567 interactions. To demonstrate the power of CSILRR for detecting biologically relevant interactions, we predicted and validated the functions of uncharacterized LRR-RKs in plant growth and immunity. In addition, we show that CSILRR operates as a unified regulatory network in which the LRR-RKs most crucial for its overall structure are required to prevent the aberrant signalling of receptors that are several network-steps away. Thus, plants have evolved LRR-RK networks to process extracellular signals into carefully balanced responses.

Elevated tyrosine results in the cytosolic retention of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase in Arabidopsis thaliana.

The shikimate pathway plays a central role in the biosynthesis of aromatic amino acids and specialized metabolites in plants. The first enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS) serves as a key regulatory point for the pathway in various organisms. These enzymes are important in regulating the shikimate pathway in multiple microbial systems. The mechanism of regulation of DAHPS is poorly understood in plants, and the role of tyrosine (Tyr) with respect to the three DAHPS isozymes from Arabidopsis thaliana was investigated. In vitro enzymatic analyses established that Tyr does not function as an allosteric regulator for the A. thaliana DAHPS isozymes. In contrast, Arabidopsis T-DNA insertional mutants for the DAHPS1 locus, dahps1, are hypersensitive to elevated Tyr. Tyr hypersensitivity can be reversed with tryptophan and phenylalanine supplementation, indicating that Tyr is affecting the shikimate pathway flux in the dahps1 mutant. Tyr treatment of Arabidopsis seedlings showed reduced accumulation of overexpressed DAHPS2 in the chloroplast. Further, bimolecular fluorescence complementation studies revealed that DAHPS2 interacts with a 14-3-3 protein in the cytosol, and this interaction is enhanced with Tyr treatment. This interaction with 14-3-3 may retain DAHPS2 in the cytosol, which prevents its ability to function in the chloroplast with elevated Tyr.

Laser Capture Microdissection Transcriptome Reveals Spatiotemporal Tissue Gene Expression Patterns of Medicago truncatula Roots Responding to Rhizobia.

We report a public resource for examining the spatiotemporal RNA expression of 54,893 Medicago truncatula genes during the first 72 h of response to rhizobial inoculation. Using a methodology that allows synchronous inoculation and growth of more than 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 dataset 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 1-aminocyclopropane-1-carboxylate synthases distinguishes inner cortical cells that become nodules and provide and describe potential downstream genes involved in early nodule cell division. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Expression Atlas of Selaginella moellendorffii Provides Insights into the Evolution of Vasculature, Secondary Metabolism, and Roots.

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.

Regulation of Cell Type-Specific Immunity Networks in Arabidopsis Roots.

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.

Transcriptomics at Maize Embryo/Endosperm Interfaces Identifies a Transcriptionally Distinct Endosperm Subdomain Adjacent to the Embryo Scutellum.

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.