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Approximately 60% of the genes and gene products in the model species Arabidopsis thaliana have been functionally characterized. In non-model plant species, the functional annotation of the gene space is largely based on homology, with the assumption that genes with shared common ancestry have conserved functions. However, the wide variety in possible morphological, physiological, and ecological differences between plant species gives rise to many species- and clade-specific genes, for which this transfer of knowledge is not possible. Other complications, such as difficulties with genetic transformation, the absence of large-scale mutagenesis methods, and long generation times, further lead to the slow characterization of genes in non-model species. Here, we discuss different resources that integrate plant gene function information. Different approaches that support the functional annotation of gene products, based on orthology or network biology, are described. While sequence-based tools to characterize the functional landscape in non-model species are maturing and becoming more readily available, easy-to-use network-based methods inferring plant gene functions are not as prevalent and have limited functionality., (© 2024 Scandinavian Plant Physiology Society.)

Sexual reproduction is a major driver of adaptation and speciation in eukaryotes. In diatoms, siliceous microalgae with a unique cell size reduction-restitution life cycle and among the world's most prolific primary producers, sex also acts as the main mechanism for cell size restoration through the formation of an expanding auxospore. However, the molecular regulators of the different stages of sexual reproduction and size restoration are poorly explored. Here, we combined RNA sequencing with the assembly of a 55 Mbp reference genome for Cylindrotheca closterium to identify patterns of gene expression during different stages of sexual reproduction. These were compared with a corresponding transcriptomic time series of Seminavis robusta to assess the degree of expression conservation. Integrative orthology analysis revealed 138 one-to-one orthologues that are upregulated during sex in both species, among which 56 genes consistently upregulated during cell pairing and gametogenesis, and 11 genes induced when auxospores are present. Several early, sex-specific transcription factors and B-type cyclins were also upregulated during sex in other pennate and centric diatoms, pointing towards a conserved core regulatory machinery for meiosis and gametogenesis across diatoms. Furthermore, we find molecular evidence that the pheromone-induced cell cycle arrest is short-lived in benthic diatoms, which may be linked to their active mode of mate finding through gliding. Finally, we exploit the temporal resolution of our comparative analysis to report the first marker genes for auxospore identity called AAE1-3 ("Auxospore-Associated Expression"). Altogether, we introduce a multi-species model of the transcriptional dynamics during size restoration in diatoms and highlight conserved gene expression dynamics during different stages of sexual reproduction., (© 2024 John Wiley & Sons Ltd.)

Understanding the underlying mechanisms of plant development is crucial to successfully steer or manipulate plant growth in a targeted manner. Leaves, the primary sites of photosynthesis, are vital organs for many plant species, and leaf growth is controlled by a tight temporal and spatial regulatory network. In this review, we focus on the genetic networks governing leaf cell proliferation, one major contributor to final leaf size. First, we provide an overview of six regulator families of leaf growth in Arabidopsis: DA1, PEAPODs, KLU, GRFs, the SWI/SNF complexes, and DELLAs, together with their surrounding genetic networks. Next, we discuss their evolutionary conservation to highlight similarities and differences among species, because knowledge transfer between species remains a big challenge. Finally, we focus on the increase in knowledge of the interconnectedness between these genetic pathways, the function of the cell cycle machinery as their central convergence point, and other internal and environmental cues., (© 2023 Society for Experimental Biology and John Wiley & Sons Ltd.)

Plant roots originated independently in lycophytes and euphyllophytes, whereas early vascular plants were rootless. The organization of the root apical meristem in euphyllophytes is well documented, especially in the model plant Arabidopsis. However, little is known about lycophyte roots and their molecular innovations during evolution. In this study, spatial transcriptomics was used to detect 97 root-related genes in the roots of the lycophyte Selaginella moellendorffii. A high number of genes showed expression patterns similar to what has been reported for seed plants, supporting the idea of a highly convergent evolution of mechanisms to control root development. Interaction and complementation data of SHORTROOT (SHR) and SCARECROW (SCR) homologs, furthermore, support a comparable regulation of the ground tissue (GT) between euphyllophytes and lycophytes. Root cap formation, in contrast, appears to be differently regulated. Several experiments indicated an important role of the WUSCHEL-RELATED HOMEOBOX13 gene SmWOX13a in Selaginella root cap formation. In contrast to multiple Arabidopsis WOX paralogs, SmWOX13a is able to induce root cap cells in Arabidopsis and has functionally conserved homologs in the fern Ceratopteris richardii. Lycophytes and a part of the euphyllophytes, therefore, may share a common mechanism regulating root cap formation, which was diversified or lost during seed plant evolution. In summary, we here provide a new spatial data resource for the Selaginella root, which in general advocates for conserved mechanisms to regulate root development but shows a clear divergence in the control of root cap formation, with a novel putative role of WOX genes in root cap formation in non-seed plants., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

PLAZA is a platform for comparative, evolutionary, and functional plant genomics. It makes a broad set of genomes, data types and analysis tools available to researchers through a user-friendly website, an API, and bulk downloads. In this latest release of the PLAZA platform, we are integrating a record number of 134 high-quality plant genomes, split up over two instances: PLAZA Dicots 5.0 and PLAZA Monocots 5.0. This number of genomes corresponds with a massive expansion in the number of available species when compared to PLAZA 4.0, which offered access to 71 species, a 89% overall increase. The PLAZA 5.0 release contains information for 5 882 730 genes, and offers pre-computed gene families and phylogenetic trees for 5 274 684 protein-coding genes. This latest release also comes with a set of new and updated features: a new BED import functionality for the workbench, improved interactive visualizations for functional enrichments and genome-wide mapping of gene sets, and a fully redesigned and extended API. Taken together, this new version offers extended support for plant biologists working on different families within the green plant lineage and provides an efficient and versatile toolbox for plant genomics. All PLAZA releases are accessible from the portal website: https://bioinformatics.psb.ugent.be/plaza/., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Most plants rely on specialized root-associated microbes to obtain essential nitrogen (N), yet not much is known about the evolutionary history of the rhizosphere-plant interaction. We conducted a common garden experiment to investigate the plant root-rhizosphere microbiome association using chloridoid grasses sampled from around the world and grown from seed in a greenhouse. We sought to test whether plants that are more closely related phylogenetically have more similar root bacterial microbiomes than plants that are more distantly related. Using metagenome sequencing, we found that there is a conserved core and a variable rhizosphere bacterial microbiome across the chloridoid grasses. Additionally, phylogenetic distance among the host plant species was correlated with bacterial community composition, suggesting the plant hosts prefer specific bacterial lineages. The functional potential for N utilization across microbiomes fluctuated extensively and mirrored variation in the microbial community composition across host plants. Variation in the bacterial potential for N fixation was strongly affected by the host plants' phylogeny, whereas variation in N recycling, nitrification, and denitrification was unaffected. This study highlights the evolutionary linkage between the N fixation traits of the microbial community and the plant host and suggests that not all functional traits are equally important for plant-microbe associations.

Advances in high-throughput sequencing have resulted in a massive increase of RNA-Seq transcriptome data. However, the promise of rapid gene expression profiling in a specific tissue, condition, unicellular organism or microbial community comes with new computational challenges. Owing to the limited availability of well-resolved reference genomes, de novo assembled (meta)transcriptomes have emerged as popular tools for investigating the gene repertoire of previously uncharacterized organisms. Yet, despite their potential, these datasets often contain fragmented or contaminant sequences, and their analysis remains difficult. To alleviate some of these challenges, we developed TRAPID 2.0, a web application for the fast and efficient processing of assembled transcriptome data. The initial processing phase performs a global characterization of the input data, providing each transcript with several layers of annotation, comprising structural, functional, and taxonomic information. The exploratory phase enables downstream analyses from the web application. Available analyses include the assessment of gene space completeness, the functional analysis and comparison of transcript subsets, and the study of transcripts in an evolutionary context. A comparison with similar tools highlights TRAPID's unique features. Finally, analyses performed within TRAPID 2.0 are complemented by interactive data visualizations, facilitating the extraction of new biological insights, as demonstrated with diatom community metatranscriptomes., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Condensins are best known for their role in shaping chromosomes. Other functions such as organizing interphase chromatin and transcriptional control have been reported in yeasts and animals, but little is known about their function in plants. To elucidate the specific composition of condensin complexes and the expression of CAP-D2 (condensin I) and CAP-D3 (condensin II), we performed biochemical analyses in Arabidopsis. The role of CAP-D3 in interphase chromatin organization and function was evaluated using cytogenetic and transcriptome analysis in cap-d3 T-DNA insertion mutants. CAP-D2 and CAP-D3 are highly expressed in mitotically active tissues. In silico and pull-down experiments indicate that both CAP-D proteins interact with the other condensin I and II subunits. In cap-d3 mutants, an association of heterochromatic sequences occurs, but the nuclear size and the general histone and DNA methylation patterns remain unchanged. Also, CAP-D3 influences the expression of genes affecting the response to water, chemicals, and stress. The expression and composition of the condensin complexes in Arabidopsis are similar to those in other higher eukaryotes. We propose a model for the CAP-D3 function during interphase in which CAP-D3 localizes in euchromatin loops to stiffen them and consequently separates centromeric regions and 45S rDNA repeats., (© 2021 The Authors New Phytologist © 2021 New Phytologist Foundation.)

Gene regulation is a dynamic process in which transcription factors (TFs) play an important role in controlling spatiotemporal gene expression. To enhance our global understanding of regulatory interactions in Arabidopsis thaliana, different regulatory input networks capturing complementary information about DNA motifs, open chromatin, TF-binding and expression-based regulatory interactions were combined using a supervised learning approach, resulting in an integrated gene regulatory network (iGRN) covering 1,491 TFs and 31,393 target genes (1.7 million interactions). This iGRN outperforms the different input networks to predict known regulatory interactions and has a similar performance to recover functional interactions compared to state-of-the-art experimental methods. The iGRN correctly inferred known functions for 681 TFs and predicted new gene functions for hundreds of unknown TFs. For regulators predicted to be involved in reactive oxygen species (ROS) stress regulation, we confirmed in total 75% of TFs with a function in ROS and/or physiological stress responses. This includes 13 ROS regulators, previously not connected to any ROS or stress function, that were experimentally validated in our ROS-specific phenotypic assays of loss- or gain-of-function lines. In conclusion, the presented iGRN offers a high-quality starting point to enhance our understanding of gene regulation in plants by integrating different experimental data types.

The Neoproterozoic Era records the transition from a largely bacterial to a predominantly eukaryotic phototrophic world, creating the foundation for the complex benthic ecosystems that have sustained Metazoa from the Ediacaran Period onward. This study focuses on the evolutionary origins of green seaweeds, which play an important ecological role in the benthos of modern sunlit oceans and likely played a crucial part in the evolution of early animals by structuring benthic habitats and providing novel niches. By applying a phylogenomic approach, we resolve deep relationships of the core Chlorophyta (Ulvophyceae or green seaweeds, and freshwater or terrestrial Chlorophyceae and Trebouxiophyceae) and unveil a rapid radiation of Chlorophyceae and the principal lineages of the Ulvophyceae late in the Neoproterozoic Era. Our time-calibrated tree points to an origin and early diversification of green seaweeds in the late Tonian and Cryogenian periods, an interval marked by two global glaciations with strong consequent changes in the amount of available marine benthic habitat. We hypothesize that unicellular and simple multicellular ancestors of green seaweeds survived these extreme climate events in isolated refugia, and diversified in benthic environments that became increasingly available as ice retreated. An increased supply of nutrients and biotic interactions, such as grazing pressure, likely triggered the independent evolution of macroscopic growth via different strategies, including true multicellularity, and multiple types of giant-celled forms., Competing Interests: The authors declare no competing interest.

Leaf growth is a complex trait for which many similarities exist in different plant species, suggesting functional conservation of the underlying pathways. However, a global view of orthologous genes involved in leaf growth showing conserved expression in dicots and monocots is currently missing. Here, we present a genome-wide comparative transcriptome analysis between Arabidopsis and maize, identifying conserved biological processes and gene functions active during leaf growth. Despite the orthology complexity between these distantly related plants, 926 orthologous gene groups including 2829 Arabidopsis and 2974 maize genes with similar expression during leaf growth were found, indicating conservation of the underlying molecular networks. We found 65% of these genes to be involved in one-to-one orthology, whereas only 28.7% of the groups with divergent expression had one-to-one orthology. Within the pool of genes with conserved expression, 19 transcription factor families were identified, demonstrating expression conservation of regulators active during leaf growth. Additionally, 25 Arabidopsis and 25 maize putative targets of the TCP transcription factors with conserved expression were determined based on the presence of enriched transcription factor binding sites. Based on large-scale phenotypic data, we observed that genes with conserved expression have a higher probability to be involved in leaf growth and that leaf-related phenotypes are more frequently present for genes having orthologues between dicots and monocots than clade-specific genes. This study shows the power of integrating transcriptomic with orthology data to identify or select candidates for functional studies during leaf development in flowering plants., (© 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

In plants, methylation at cytosines often leads to changes in gene expression and inactivation of transposable elements. Changes in cytosine methylation (epimutations) might produce epialleles with distinct phenotypes. We present a genome-wide cytosine methylation profiling method based on bisulfite conversion and next-generation sequencing, which is applicable for plant species with available reference genomes. This so-called plant-RRBS profiling method reproducibly covers specific genomic regions and enriches for coverage of cytosine positions that are suitable for comparative analyses in multi-sample studies in basic biology and breeding studies. The plant-RRBS workflow consists of genomic DNA digestion with coverage-efficient restriction endonuclease combinations followed by a performant library generation and next-generation sequencing and a straightforward, publically available methylation data processing pipeline. Plant-RRBS has a twofold higher ratio of cytosine coverage per covered genome as compared to whole-genome bisulfite sequencing, covering tens of millions of cytosine positions, and allows detection of differential cytosine methylation, which was evaluated using rice epilines.

Genome annotations offer ample opportunities to study gene functions, biochemical and regulatory pathways, or quantitative trait loci in plants. Determining the quality and completeness of a genome annotation, and maintaining the balance between them, are major challenges, even for genomes of well-studied model organisms. In this review, we present a historical overview of the complexity in different plant genomes and discuss the hurdles and possible solutions in obtaining a complete and high-quality genome annotation. We illustrate there is no clear-cut answer to solve these challenges for different gene types, but provide tips on guiding the iterative process of generating a superior genome annotation, which is a moving target as our knowledge about plant genomics increases and additional data sources become available., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The target of rapamycin (TOR) kinase is a conserved regulatory hub that translates environmental and nutritional information into permissive or restrictive growth decisions. Despite the increased appreciation of the essential role of the TOR complex in plants, no large-scale phosphoproteomics or interactomics studies have been performed to map TOR signalling events in plants. To fill this gap, we combined a systematic phosphoproteomics screen with a targeted protein complex analysis in the model plant Arabidopsis thaliana. Integration of the phosphoproteome and protein complex data on the one hand shows that both methods reveal complementary subspaces of the plant TOR signalling network, enabling proteome-wide discovery of both upstream and downstream network components. On the other hand, the overlap between both data sets reveals a set of candidate direct TOR substrates. The integrated network embeds both evolutionarily-conserved and plant-specific TOR signalling components, uncovering an intriguing complex interplay with protein synthesis. Overall, the network provides a rich data set to start addressing fundamental questions about how TOR controls key processes in plants, such as autophagy, auxin signalling, chloroplast development, lipid metabolism, nucleotide biosynthesis, protein translation or senescence.

We report here the 98.5 Mbp haploid genome (12,924 protein coding genes) of Ulva mutabilis, a ubiquitous and iconic representative of the Ulvophyceae or green seaweeds. Ulva's rapid and abundant growth makes it a key contributor to coastal biogeochemical cycles; its role in marine sulfur cycles is particularly important because it produces high levels of dimethylsulfoniopropionate (DMSP), the main precursor of volatile dimethyl sulfide (DMS). Rapid growth makes Ulva attractive biomass feedstock but also increasingly a driver of nuisance "green tides." Ulvophytes are key to understanding the evolution of multicellularity in the green lineage, and Ulva morphogenesis is dependent on bacterial signals, making it an important species with which to study cross-kingdom communication. Our sequenced genome informs these aspects of ulvophyte cell biology, physiology, and ecology. Gene family expansions associated with multicellularity are distinct from those of freshwater algae. Candidate genes, including some that arose following horizontal gene transfer from chromalveolates, are present for the transport and metabolism of DMSP. The Ulva genome offers, therefore, new opportunities to understand coastal and marine ecosystems and the fundamental evolution of the green lineage., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The draft genome of the moss model, Physcomitrella patens, comprised approximately 2000 unordered scaffolds. In order to enable analyses of genome structure and evolution we generated a chromosome-scale genome assembly using genetic linkage as well as (end) sequencing of long DNA fragments. We find that 57% of the genome comprises transposable elements (TEs), some of which may be actively transposing during the life cycle. Unlike in flowering plant genomes, gene- and TE-rich regions show an overall even distribution along the chromosomes. However, the chromosomes are mono-centric with peaks of a class of Copia elements potentially coinciding with centromeres. Gene body methylation is evident in 5.7% of the protein-coding genes, typically coinciding with low GC and low expression. Some giant virus insertions are transcriptionally active and might protect gametes from viral infection via siRNA mediated silencing. Structure-based detection methods show that the genome evolved via two rounds of whole genome duplications (WGDs), apparently common in mosses but not in liverworts and hornworts. Several hundred genes are present in colinear regions conserved since the last common ancestor of plants. These syntenic regions are enriched for functions related to plant-specific cell growth and tissue organization. The P. patens genome lacks the TE-rich pericentromeric and gene-rich distal regions typical for most flowering plant genomes. More non-seed plant genomes are needed to unravel how plant genomes evolve, and to understand whether the P. patens genome structure is typical for mosses or bryophytes., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.)

PLAZA (https://bioinformatics.psb.ugent.be/plaza) is a plant-oriented online resource for comparative, evolutionary and functional genomics. The PLAZA platform consists of multiple independent instances focusing on different plant clades, while also providing access to a consistent set of reference species. Each PLAZA instance contains structural and functional gene annotations, gene family data and phylogenetic trees and detailed gene colinearity information. A user-friendly web interface makes the necessary tools and visualizations accessible, specific for each data type. Here we present PLAZA 4.0, the latest iteration of the PLAZA framework. This version consists of two new instances (Dicots 4.0 and Monocots 4.0) providing a large increase in newly available species, and offers access to updated and newly implemented tools and visualizations, helping users with the ever-increasing demands for complex and in-depth analyzes. The total number of species across both instances nearly doubles from 37 species in PLAZA 3.0 to 71 species in PLAZA 4.0, with a much broader coverage of crop species (e.g. wheat, palm oil) and species of evolutionary interest (e.g. spruce, Marchantia). The new PLAZA instances can also be accessed by a programming interface through a RESTful web service, thus allowing bioinformaticians to optimally leverage the power of the PLAZA platform., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In several organisms, particular functional categories of genes, such as regulatory and complex-forming genes, are preferentially retained after whole-genome multiplications but rarely duplicate through small-scale duplication, a pattern referred to as reciprocal retention. This peculiar duplication behavior is hypothesized to stem from constraints on the dosage balance between the genes concerned and their interaction context. However, the evidence for a relationship between reciprocal retention and dosage balance sensitivity remains fragmentary. Here, we identified which gene families are most strongly reciprocally retained in the angiosperm lineage and studied their functional and evolutionary characteristics. Reciprocally retained gene families exhibit stronger sequence divergence constraints and lower rates of functional and expression divergence than other gene families, suggesting that dosage balance sensitivity is a general characteristic of reciprocally retained genes. Gene families functioning in regulatory and signaling processes are much more strongly represented at the top of the reciprocal retention ranking than those functioning in multiprotein complexes, suggesting that regulatory imbalances may lead to stronger fitness effects than classical stoichiometric protein complex imbalances. Finally, reciprocally retained duplicates are often subject to dosage balance constraints for prolonged evolutionary times, which may have repercussions for the ease with which genome multiplications can engender evolutionary innovation., (© 2017 American Society of Plant Biologists. All rights reserved.)

Motivation: Comparative and evolutionary studies utilize phylogenetic trees to analyze and visualize biological data. Recently, several web-based tools for the display, manipulation and annotation of phylogenetic trees, such as iTOL and Evolview, have released updates to be compatible with the latest web technologies. While those web tools operate an open server access model with a multitude of registered users, a feature-rich open source solution using current web technologies is not available., Results: Here, we present an extension of the widely used PhyloXML standard with several new options to accommodate functional genomics or annotation datasets for advanced visualization. Furthermore, PhyD3 has been developed as a lightweight tool using the JavaScript library D3.js to achieve a state-of-the-art phylogenetic tree visualization in the web browser, with support for advanced annotations. The current implementation is open source, easily adaptable and easy to implement in third parties' web sites., Availability and Implementation: More information about PhyD3 itself, installation procedures and implementation links are available at http://phyd3.bits.vib.be and at http://github.com/vibbits/phyd3/ ., Contact: klaas.vandepoele@ugent.vib.be or michiel.vanbel@ugent.vib.be., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Background: Cytosine methylation in plant genomes is important for the regulation of gene transcription and transposon activity. Genome-wide methylomes are studied upon mutation of the DNA methyltransferases, adaptation to environmental stresses or during development. However, from basic biology to breeding programs, there is a need to monitor multiple samples to determine transgenerational methylation inheritance or differential cytosine methylation. Methylome data obtained by sodium hydrogen sulfite (bisulfite)-conversion and next-generation sequencing (NGS) provide genome-wide information on cytosine methylation. However, a profiling method that detects cytosine methylation state dispersed over the genome would allow high-throughput analysis of multiple plant samples with distinct epigenetic signatures. We use specific restriction endonucleases to enrich for cytosine coverage in a bisulfite and NGS-based profiling method, which was compared to whole-genome bisulfite sequencing of the same plant material., Methods: We established an effective methylome profiling method in plants, termed plant-reduced representation bisulfite sequencing (plant-RRBS), using optimized double restriction endonuclease digestion, fragment end repair, adapter ligation, followed by bisulfite conversion, PCR amplification and NGS. We report a performant laboratory protocol and a straightforward bioinformatics data analysis pipeline for plant-RRBS, applicable for any reference-sequenced plant species., Results: As a proof of concept, methylome profiling was performed using an Oryza sativa ssp. indica pure breeding line and a derived epigenetically altered line (epiline). Plant-RRBS detects methylation levels at tens of millions of cytosine positions deduced from bisulfite conversion in multiple samples. To evaluate the method, the coverage of cytosine positions, the intra-line similarity and the differential cytosine methylation levels between the pure breeding line and the epiline were determined. Plant-RRBS reproducibly covers commonly up to one fourth of the cytosine positions in the rice genome when using MspI-DpnII within a group of five biological replicates of a line. The method predominantly detects cytosine methylation in putative promoter regions and not-annotated regions in rice., Conclusions: Plant-RRBS offers high-throughput and broad, genome-dispersed methylation detection by effective read number generation obtained from reproducibly covered genome fractions using optimized endonuclease combinations, facilitating comparative analyses of multi-sample studies for cytosine methylation and transgenerational stability in experimental material and plant breeding populations.

Wheat is a cereal grain and one of the world's major food crops. Recent advances in wheat genome sequencing are by now facilitating its genomic and proteomic analyses. However, little is known about possible differences in total protein levels of hexaploid versus tetraploid wheat cultivars, and also knowledge of phosphorylated wheat proteins is still limited. Here, we performed a detailed analysis of the proteome of seedling leaves from two hexaploid wheat cultivars ( Triticum aestivum L. Pavon 76 and USU-Apogee) and one tetraploid wheat ( T. turgidum ssp. durum cv. Senatore Cappelli). Our shotgun proteomics data revealed that, whereas we observed some significant differences, overall a high similarity between hexaploid and tetraploid varieties with respect to protein abundance was observed. In addition, already at the seedling stage, a small set of proteins was differential between the small (USU-Apogee) and larger hexaploid wheat cultivars (Pavon 76), which could potentially act as growth predictors. Finally, the phosphosites identified in this study can be retrieved from the in-house developed plant PTM-Viewer (bioinformatics.psb.ugent.be/webtools/ptm_viewer/), making this the first searchable repository for phosphorylated wheat proteins. This paves the way for further in depth, quantitative (phospho)proteome-wide differential analyses upon a specific trigger or environmental change.

Selecting and filtering a reference expression and interaction dataset when studying specific pathways and regulatory interactions can be a very time-consuming and error-prone task. In order to reduce the duplicated efforts required to amass such datasets, we have created the CORNET (CORrelation NETworks) platform which allows for easy access to a wide variety of data types: coexpression data, protein-protein interactions, regulatory interactions, and functional annotations. The CORNET platform outputs its results in either text format or through the Cytoscape framework, which is automatically launched by the CORNET website.CORNET 3.0 is the third iteration of the web platform designed for the user exploration of the coexpression space of plant genomes, with a focus on the model species Arabidopsis thaliana. Here we describe the platform: the tools, data, and best practices when using the platform. We indicate how the platform can be used to infer networks from a set of input genes, such as upregulated genes from an expression experiment. By exploring the network, new target and regulator genes can be discovered, allowing for follow-up experiments and more in-depth study. We also indicate how to avoid common pitfalls when evaluating the networks and how to avoid over interpretation of the results.All CORNET versions are available at http://bioinformatics.psb.ugent.be/cornet/ .

Protein phosphorylation is one of the most common post-translational modifications (PTMs), which can regulate protein activity and localization as well as protein-protein interactions in numerous cellular processes. Phosphopeptide enrichment techniques enable plant researchers to acquire insight into phosphorylation-controlled signaling networks in various plant species. Most phosphoproteome analyses of plant samples still involve stable isotope labeling, peptide fractionation, and demand a lot of mass spectrometry (MS) time. Here, we present a simple workflow to probe, map, and catalogue plant phosphoproteomes, requiring relatively low amounts of starting material, no labeling, no fractionation, and no excessive analysis time. Following optimization of the different experimental steps on Arabidopsis thaliana samples, we transferred our workflow to maize, a major monocot crop, to study signaling upon drought stress. In addition, we included normalization to protein abundance to identify true phosphorylation changes. Overall, we identified a set of new phosphosites in both Arabidopsis thaliana and maize, some of which are differentially phosphorylated upon drought. All data are available via ProteomeXchange with identifier PXD003634, but to provide easy access to our model plant and crop data sets, we created an online database, Plant PTM Viewer ( bioinformatics.psb.ugent.be/webtools/ptm_viewer/ ), where all phosphosites identified in our study can be consulted.

Transcription factors (TFs) regulate gene expression by binding cis-regulatory elements, of which the identification remains an ongoing challenge owing to the prevalence of large numbers of nonfunctional TF binding sites. Powerful comparative genomics methods, such as phylogenetic footprinting, can be used for the detection of conserved noncoding sequences (CNSs), which are functionally constrained and can greatly help in reducing the number of false-positive elements. In this study, we applied a phylogenetic footprinting approach for the identification of CNSs in 10 dicot plants, yielding 1,032,291 CNSs associated with 243,187 genes. To annotate CNSs with TF binding sites, we made use of binding site information for 642 TFs originating from 35 TF families in Arabidopsis (Arabidopsis thaliana). In three species, the identified CNSs were evaluated using TF chromatin immunoprecipitation sequencing data, resulting in significant overlap for the majority of data sets. To identify ultraconserved CNSs, we included genomes of additional plant families and identified 715 binding sites for 501 genes conserved in dicots, monocots, mosses, and green algae. Additionally, we found that genes that are part of conserved mini-regulons have a higher coherence in their expression profile than other divergent gene pairs. All identified CNSs were integrated in the PLAZA 3.0 Dicots comparative genomics platform (http://bioinformatics.psb.ugent.be/plaza/versions/plaza_v3_dicots/) together with new functionalities facilitating the exploration of conserved cis-regulatory elements and their associated genes. The availability of this data set in a user-friendly platform enables the exploration of functional noncoding DNA to study gene regulation in a variety of plant species, including crops., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

In plants, the generation of new cell types and tissues depends on coordinated and oriented formative cell divisions. The plasma membrane-localized receptor kinase ARABIDOPSIS CRINKLY 4 (ACR4) is part of a mechanism controlling formative cell divisions in the Arabidopsis root. Despite its important role in plant development, very little is known about the molecular mechanism with which ACR4 is affiliated and its network of interactions. Here, we used various complementary proteomic approaches to identify ACR4-interacting protein candidates that are likely regulators of formative cell divisions and that could pave the way to unraveling the molecular basis behind ACR4-mediated signaling. We identified PROTEIN PHOSPHATASE 2A-3 (PP2A-3), a catalytic subunit of PP2A holoenzymes, as a previously unidentified regulator of formative cell divisions and as one of the first described substrates of ACR4. Our in vitro data argue for the existence of a tight posttranslational regulation in the associated biochemical network through reciprocal regulation between ACR4 and PP2A-3 at the phosphorylation level.

Ubiquitination, the covalent binding of the small protein modifier ubiquitin to a target protein, is an important and frequently studied posttranslational protein modification. Multiple reports provide useful insights into the plant ubiquitinome, but mostly at the protein level without comprehensive site identification. Here, we implemented ubiquitin combined fractional diagonal chromatography (COFRADIC) for proteome-wide ubiquitination site mapping on Arabidopsis thaliana cell cultures. We identified 3009 sites on 1607 proteins, thereby greatly increasing the number of known ubiquitination sites in this model plant. Finally, The Ubiquitination Site tool (http://bioinformatics.psb.ugent.be/webtools/ubiquitin_viewer/) gives access to the obtained ubiquitination sites, not only to consult the ubiquitination status of a given protein, but also to conduct intricate experiments aiming to study the roles of specific ubiquitination events. Together with the antibodies recognizing the ubiquitin remnant motif, ubiquitin COFRADIC represents a powerful tool to resolve the ubiquitination maps of numerous cellular processes in plants., (© 2016 American Society of Plant Biologists. All rights reserved.)

A plethora of diverse programmed cell death (PCD) processes has been described in living organisms. In animals and plants, different forms of PCD play crucial roles in development, immunity, and responses to the environment. While the molecular control of some animal PCD forms such as apoptosis is known in great detail, we still know comparatively little about the regulation of the diverse types of plant PCD. In part, this deficiency in molecular understanding is caused by the lack of reliable reporters to detect PCD processes. Here, we addressed this issue by using a combination of bioinformatics approaches to identify commonly regulated genes during diverse plant PCD processes in Arabidopsis (Arabidopsis thaliana). Our results indicate that the transcriptional signatures of developmentally controlled cell death are largely distinct from the ones associated with environmentally induced cell death. Moreover, different cases of developmental PCD share a set of cell death-associated genes. Most of these genes are evolutionary conserved within the green plant lineage, arguing for an evolutionary conserved core machinery of developmental PCD. Based on this information, we established an array of specific promoter-reporter lines for developmental PCD in Arabidopsis. These PCD indicators represent a powerful resource that can be used in addition to established morphological and biochemical methods to detect and analyze PCD processes in vivo and in planta., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Motivation: The accurate discovery and annotation of regulatory elements remains a challenging problem. The growing number of sequenced genomes creates new opportunities for comparative approaches to motif discovery. Putative binding sites are then considered to be functional if they are conserved in orthologous promoter sequences of multiple related species. Existing methods for comparative motif discovery usually rely on pregenerated multiple sequence alignments, which are difficult to obtain for more diverged species such as plants. As a consequence, misaligned regulatory elements often remain undetected., Results: We present a novel algorithm that supports both alignment-free and alignment-based motif discovery in the promoter sequences of related species. Putative motifs are exhaustively enumerated as words over the IUPAC alphabet and screened for conservation using the branch length score. Additionally, a confidence score is established in a genome-wide fashion. In order to take advantage of a cloud computing infrastructure, the MapReduce programming model is adopted. The method is applied to four monocotyledon plant species and it is shown that high-scoring motifs are significantly enriched for open chromatin regions in Oryza sativa and for transcription factor binding sites inferred through protein-binding microarrays in O.sativa and Zea mays. Furthermore, the method is shown to recover experimentally profiled ga2ox1-like KN1 binding sites in Z.mays., Availability and Implementation: BLSSpeller was written in Java. Source code and manual are available at http://bioinformatics.intec.ugent.be/blsspeller, Contact: Klaas.Vandepoele@psb.vib-ugent.be or jan.fostier@intec.ugent.be., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2015. Published by Oxford University Press.)

Most molecular processes during plant development occur with a particular spatio-temporal specificity. Thus far, it has remained technically challenging to capture dynamic protein-protein interactions within a growing organ, where the interplay between cell division and cell expansion is instrumental. Here, we combined high-resolution sampling of the growing maize (Zea mays) leaf with tandem affinity purification followed by mass spectrometry. Our results indicate that the growth-regulating SWI/SNF chromatin remodeling complex associated with ANGUSTIFOLIA3 (AN3) was conserved within growing organs and between dicots and monocots. Moreover, we were able to demonstrate the dynamics of the AN3-interacting proteins within the growing leaf, since copurified GROWTH-REGULATING FACTORs (GRFs) varied throughout the growing leaf. Indeed, GRF1, GRF6, GRF7, GRF12, GRF15, and GRF17 were significantly enriched in the division zone of the growing leaf, while GRF4 and GRF10 levels were comparable between division zone and expansion zone in the growing leaf. These dynamics were also reflected at the mRNA and protein levels, indicating tight developmental regulation of the AN3-associated chromatin remodeling complex. In addition, the phenotypes of maize plants overexpressing miRNA396a-resistant GRF1 support a model proposing that distinct associations of the chromatin remodeling complex with specific GRFs tightly regulate the transition between cell division and cell expansion. Together, our data demonstrate that advancing from static to dynamic protein-protein interaction analysis in a growing organ adds insights in how developmental switches are regulated., (© 2015 American Society of Plant Biologists. All rights reserved.)

Comparative sequence analysis has significantly altered our view on the complexity of genome organization and gene functions in different kingdoms. PLAZA 3.0 is designed to make comparative genomics data for plants available through a user-friendly web interface. Structural and functional annotation, gene families, protein domains, phylogenetic trees and detailed information about genome organization can easily be queried and visualized. Compared with the first version released in 2009, which featured nine organisms, the number of integrated genomes is more than four times higher, and now covers 37 plant species. The new species provide a wider phylogenetic range as well as a more in-depth sampling of specific clades, and genomes of additional crop species are present. The functional annotation has been expanded and now comprises data from Gene Ontology, MapMan, UniProtKB/Swiss-Prot, PlnTFDB and PlantTFDB. Furthermore, we improved the algorithms to transfer functional annotation from well-characterized plant genomes to other species. The additional data and new features make PLAZA 3.0 (http://bioinformatics.psb.ugent.be/plaza/) a versatile and comprehensible resource for users wanting to explore genome information to study different aspects of plant biology, both in model and non-model organisms., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Sugarcane interacts with particular types of beneficial nitrogen-fixing bacteria that provide fixed-nitrogen and plant growth hormones to host plants, promoting an increase in plant biomass. Other benefits, as enhanced tolerance to abiotic stresses have been reported to some diazotrophs. Here we aim to study the effects of the association between the diazotroph Gluconacetobacter diazotrophicus PAL5 and sugarcane cv. SP70-1143 during water depletion by characterizing differential transcriptome profiles of sugarcane. RNA-seq libraries were generated from roots and shoots of sugarcane plants free of endophytes that were inoculated with G. diazotrophicus and subjected to water depletion for 3 days. A sugarcane reference transcriptome was constructed and used for the identification of differentially expressed transcripts. The differential profile of non-inoculated SP70-1143 suggests that it responds to water deficit stress by the activation of drought-responsive markers and hormone pathways, as ABA and Ethylene. qRT-PCR revealed that root samples had higher levels of G. diazotrophicus 3 days after water deficit, compared to roots of inoculated plants watered normally. With prolonged drought only inoculated plants survived, indicating that SP70-1143 plants colonized with G. diazotrophicus become more tolerant to drought stress than non-inoculated plants. Strengthening this hypothesis, several gene expression responses to drought were inactivated or regulated in an opposite manner, especially in roots, when plants were colonized by the bacteria. The data suggests that colonized roots would not be suffering from stress in the same way as non-inoculated plants. On the other hand, shoots specifically activate ABA-dependent signaling genes, which could act as key elements in the drought resistance conferred by G. diazotrophicus to SP70-1143. This work reports for the first time the involvement of G. diazotrophicus in the promotion of drought-tolerance to sugarcane cv. SP70-1143, and it describes the initial molecular events that may trigger the increased drought tolerance in the host plant.

Transcriptome analysis through next-generation sequencing technologies allows the generation of detailed gene catalogs for non-model species, at the cost of new challenges with regards to computational requirements and bioinformatics expertise. Here, we present TRAPID, an online tool for the fast and efficient processing of assembled RNA-Seq transcriptome data, developed to mitigate these challenges. TRAPID offers high-throughput open reading frame detection, frameshift correction and includes a functional, comparative and phylogenetic toolbox, making use of 175 reference proteomes. Benchmarking and comparison against state-of-the-art transcript analysis tools reveals the efficiency and unique features of the TRAPID system. TRAPID is freely available at http://bioinformatics.psb.ugent.be/webtools/trapid/.

With the advent of next generation genome sequencing, the number of sequenced algal genomes and transcriptomes is rapidly growing. Although a few genome portals exist to browse individual genome sequences, exploring complete genome information from multiple species for the analysis of user-defined sequences or gene lists remains a major challenge. pico-PLAZA is a web-based resource (http://bioinformatics.psb.ugent.be/pico-plaza/) for algal genomics that combines different data types with intuitive tools to explore genomic diversity, perform integrative evolutionary sequence analysis and study gene functions. Apart from homologous gene families, multiple sequence alignments, phylogenetic trees, Gene Ontology, InterPro and text-mining functional annotations, different interactive viewers are available to study genome organization using gene collinearity and synteny information. Different search functions, documentation pages, export functions and an extensive glossary are available to guide non-expert scientists. To illustrate the versatility of the platform, different case studies are presented demonstrating how pico-PLAZA can be used to functionally characterize large-scale EST/RNA-Seq data sets and to perform environmental genomics. Functional enrichments analysis of 16 Phaeodactylum tricornutum transcriptome libraries offers a molecular view on diatom adaptation to different environments of ecological relevance. Furthermore, we show how complementary genomic data sources can easily be combined to identify marker genes to study the diversity and distribution of algal species, for example in metagenomes, or to quantify intraspecific diversity from environmental strains., (© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.)

The analysis of gene expression data generated by high-throughput microarray transcript profiling experiments has shown that transcriptionally coordinated genes are often functionally related. Based on large-scale expression compendia grouping multiple experiments, this guilt-by-association principle has been applied to study modular gene programmes, identify cis-regulatory elements or predict functions for unknown genes in different model plants. Recently, several studies have demonstrated how, through the integration of gene homology and expression information, correlated gene expression patterns can be compared between species. The incorporation of detailed functional annotations as well as experimental data describing protein-protein interactions, phenotypes or tissue specific expression, provides an invaluable source of information to identify conserved gene modules and translate biological knowledge from model organisms to crops. In this review, we describe the different steps required to systematically compare expression data across species. Apart from the technical challenges to compute and display expression networks from multiple species, some future applications of plant comparative transcriptomics are highlighted., (© 2012 Blackwell Publishing Ltd.)

Background: Bathycoccus prasinos is an extremely small cosmopolitan marine green alga whose cells are covered with intricate spider's web patterned scales that develop within the Golgi cisternae before their transport to the cell surface. The objective of this work is to sequence and analyze its genome, and to present a comparative analysis with other known genomes of the green lineage., Research: Its small genome of 15 Mb consists of 19 chromosomes and lacks transposons. Although 70% of all B. prasinos genes share similarities with other Viridiplantae genes, up to 428 genes were probably acquired by horizontal gene transfer, mainly from other eukaryotes. Two chromosomes, one big and one small, are atypical, an unusual synapomorphic feature within the Mamiellales. Genes on these atypical outlier chromosomes show lower GC content and a significant fraction of putative horizontal gene transfer genes. Whereas the small outlier chromosome lacks colinearity with other Mamiellales and contains many unknown genes without homologs in other species, the big outlier shows a higher intron content, increased expression levels and a unique clustering pattern of housekeeping functionalities. Four gene families are highly expanded in B. prasinos, including sialyltransferases, sialidases, ankyrin repeats and zinc ion-binding genes, and we hypothesize that these genes are associated with the process of scale biogenesis., Conclusion: The minimal genomes of the Mamiellophyceae provide a baseline for evolutionary and functional analyses of metabolic processes in green plants.

With the arrival of low-cost, next-generation sequencing, a multitude of new plant genomes are being publicly released, providing unseen opportunities and challenges for comparative genomics studies. Here, we present PLAZA 2.5, a user-friendly online research environment to explore genomic information from different plants. This new release features updates to previous genome annotations and a substantial number of newly available plant genomes as well as various new interactive tools and visualizations. Currently, PLAZA hosts 25 organisms covering a broad taxonomic range, including 13 eudicots, five monocots, one lycopod, one moss, and five algae. The available data consist of structural and functional gene annotations, homologous gene families, multiple sequence alignments, phylogenetic trees, and colinear regions within and between species. A new Integrative Orthology Viewer, combining information from different orthology prediction methodologies, was developed to efficiently investigate complex orthology relationships. Cross-species expression analysis revealed that the integration of complementary data types extended the scope of complex orthology relationships, especially between more distantly related species. Finally, based on phylogenetic profiling, we propose a set of core gene families within the green plant lineage that will be instrumental to assess the gene space of draft or newly sequenced plant genomes during the assembly or annotation phase.

The number of sequenced genomes of representatives within the green lineage is rapidly increasing. Consequently, comparative sequence analysis has significantly altered our view on the complexity of genome organization, gene function, and regulatory pathways. To explore all this genome information, a centralized infrastructure is required where all data generated by different sequencing initiatives is integrated and combined with advanced methods for data mining. Here, we describe PLAZA, an online platform for plant comparative genomics (http://bioinformatics.psb.ugent.be/plaza/). This resource integrates structural and functional annotation of published plant genomes together with a large set of interactive tools to study gene function and gene and genome evolution. Precomputed data sets cover homologous gene families, multiple sequence alignments, phylogenetic trees, intraspecies whole-genome dot plots, and genomic colinearity between species. Through the integration of high confidence Gene Ontology annotations and tree-based orthology between related species, thousands of genes lacking any functional description are functionally annotated. Advanced query systems, as well as multiple interactive visualization tools, are available through a user-friendly and intuitive Web interface. In addition, detailed documentation and tutorials introduce the different tools, while the workbench provides an efficient means to analyze user-defined gene sets through PLAZA's interface. In conclusion, PLAZA provides a comprehensible and up-to-date research environment to aid researchers in the exploration of genome information within the green plant lineage.

Motivation: Many problems in genome annotation are tackled by using a classification model to predict functional sites such as splice sites, translation start sites or stop codons. Locating the correct position of these sites remains one of the most important but also one of the most difficult issues in the structural annotation of genomes. Most of the software currently in use is written for a very specific problem, thereby limiting the possibilities for reuse., Summary: We developed a software platform that uses a very general approach towards the classification of functional sites in DNA sequences. The program uses an ab initio approach towards the identification of these sites, and extends SpliceMachine, a previously developed splice site predictor that shows state-of-the-art performance for both donor and acceptor splice site recognition in the human and Arabidopsis thaliana genome., Availability: The program is developed as a stand-alone Java application, and is available as GPLv3 open-source software. The program, source and documentation can be obtained from the 'Software' section at http://bioinformatics.psb.ugent.be/., Supplementary Information: Supplementary data is available at Bioinformatics online.