GXP: Analyze and Plot Plant Omics Data in Web Browsers.

Next-generation sequencing and metabolomics have become very cost and work efficient and are integrated into an ever-growing number of life science research projects. Typically, established software pipelines analyze raw data and produce quantitative data informing about gene expression or concentrations of metabolites. These results need to be visualized and further analyzed in order to support scientific hypothesis building and identification of underlying biological patterns. Some of these tools already exist, but require installation or manual programming. We developed "Gene Expression Plotter" (GXP), an RNAseq and Metabolomics data visualization and analysis tool entirely running in the user's web browser, thus not needing any custom installation, manual programming or uploading of confidential data to third party servers. Consequently, upon receiving the bioinformatic raw data analysis of RNAseq or other omics results, GXP immediately enables the user to interact with the data according to biological questions by performing knowledge-driven, in-depth data analyses and candidate identification via visualization and data exploration. Thereby, GXP can support and accelerate complex interdisciplinary omics projects and downstream analyses. GXP offers an easy way to publish data, plots, and analysis results either as a simple exported file or as a custom website. GXP is freely available on GitHub (see introduction).

MapMan Visualization of RNA-Seq Data Using Mercator4 Functional Annotations.

Plant omics research has advanced to the stage where it is feasible to generate data from multiple samples and multiple time points to gain insight into biological processes. This impressive array of data can prove challenging to interpret. In this chapter, we describe a solution to this problem, consisting of the MapMan transcript visualization application and the associated MapMan4 ontology and Mercator4 online annotation process.

Genomes of the Venus Flytrap and Close Relatives Unveil the Roots of Plant Carnivory.

Most plants grow and develop by taking up nutrients from the soil while continuously under threat from foraging animals. Carnivorous plants have turned the tables by capturing and consuming nutrient-rich animal prey, enabling them to thrive in nutrient-poor soil. To better understand the evolution of botanical carnivory, we compared the draft genome of the Venus flytrap (Dionaea muscipula) with that of its aquatic sister, the waterwheel plant Aldrovanda vesiculosa, and the sundew Drosera spatulata. We identified an early whole-genome duplication in the family as source for carnivory-associated genes. Recruitment of genes to the trap from the root especially was a major mechanism in the evolution of carnivory, supported by family-specific duplications. Still, these genomes belong to the gene poorest land plants sequenced thus far, suggesting reduction of selective pressure on different processes, including non-carnivorous nutrient acquisition. Our results show how non-carnivorous plants evolved into the most skillful green hunters on the planet.

MapMan4: A Refined Protein Classification and Annotation Framework Applicable to Multi-Omics Data Analysis.

Genome sequences from over 200 plant species have already been published, with this number expected to increase rapidly due to advances in sequencing technologies. Once a new genome has been assembled and the genes identified, the functional annotation of their putative translational products, proteins, using ontologies is of key importance as it places the sequencing data in a biological context. Furthermore, to keep pace with rapid production of genome sequences, this functional annotation process must be fully automated. Here we present a redesigned and significantly enhanced MapMan4 framework, together with a revised version of the associated online Mercator annotation tool. Compared with the original MapMan, the new ontology has been expanded almost threefold and enforces stricter assignment rules. This framework was then incorporated into Mercator4, which has been upgraded to reflect current knowledge across the land plant group, providing protein annotations for all embryophytes with a comparably high quality. The annotation process has been optimized to allow a plant genome to be annotated in a matter of minutes. The output results continue to be compatible with the established MapMan desktop application.

Footprints of parasitism in the genome of the parasitic flowering plant Cuscuta campestris.

A parasitic lifestyle, where plants procure some or all of their nutrients from other living plants, has evolved independently in many dicotyledonous plant families and is a major threat for agriculture globally. Nevertheless, no genome sequence of a parasitic plant has been reported to date. Here we describe the genome sequence of the parasitic field dodder, Cuscuta campestris. The genome contains signatures of a fairly recent whole-genome duplication and lacks genes for pathways superfluous to a parasitic lifestyle. Specifically, genes needed for high photosynthetic activity are lost, explaining the low photosynthesis rates displayed by the parasite. Moreover, several genes involved in nutrient uptake processes from the soil are lost. On the other hand, evidence for horizontal gene transfer by way of genomic DNA integration from the parasite's hosts is found. We conclude that the parasitic lifestyle has left characteristic footprints in the C. campestris genome.

Identification and Characterization of Plant Membrane Proteins Using ARAMEMNON.

Membrane proteins are estimated to constitute a quarter of all proteins encoded in plant genomes, yet only a limited number have been experimentally characterized. This is mainly due to the large variation in particular physical properties coupled with purification difficulties. Computational methods are therefore very helpful for the initial characterization of a candidate membrane protein. Individual prediction tools can, with varying levels of success, predict the occurrence of transmembrane spans, the subcellular location, and lipid posttranslational modifications. Since it can be tedious to consult each prediction tool separately, ARAMEMNON has been designed to compile various computational predictions for plant membrane proteins and to present the results via a user-friendly web interface. This protocol describes how to use ARAMEMNON to identify and characterize plant membrane proteins.

From plant genomes to phenotypes.

Recent advances in sequencing technologies have greatly accelerated the rate of plant genome and applied breeding research. Despite this advancing trend, plant genomes continue to present numerous difficulties to the standard tools and pipelines not only for genome assembly but also gene annotation and downstream analysis. Here we give a perspective on tools, resources and services necessary to assemble and analyze plant genomes and link them to plant phenotypes.

Cytokinin Response Factor 5 has transcriptional activity governed by its C-terminal domain.

Cytokinin Response Factors (CRFs) are AP2/ERF transcription factors involved in cytokinin signal transduction. CRF proteins consist of a N-terminal dimerization domain (CRF domain), an AP2 DNA-binding domain, and a clade-specific C-terminal region of unknown function. Using a series of sequential deletions in yeast-2-hybrid assays, we provide evidence that the C-terminal region of Arabidopsis CRF5 can confer transactivation activity. Although comparative analyses identified evolutionarily conserved protein sequence within the C-terminal region, deletion experiments suggest that this transactivation domain has a partially redundant modular structure required for activation of target gene transcription.

DNA Damage-Induced Transcription of Transposable Elements and Long Non-coding RNAs in Arabidopsis Is Rare and ATM-Dependent.

Induction and mobilization of transposable elements (TEs) following DNA damage or other stresses has been reported in prokaryotes and eukaryotes. Recently it was discovered that eukaryotic TEs are frequently associated with long non-coding RNAs (lncRNAs), many of which are also upregulated by stress. Yet, it is unknown whether DNA damage-induced transcriptional activation of TEs and lncRNAs occurs sporadically or is a synchronized, genome-wide response. Here we investigated the transcriptome of Arabidopsis wild-type (WT) and ataxia telangiectasia mutated (atm) mutant plants 3 h after induction of DNA damage. In WT, expression of 5.2% of the protein-coding genes is ≥2-fold changed, whereas in atm plants, only 2.6% of these genes are regulated, and the response of genes associated with DNA repair, replication, and cell cycle is largely lost. In contrast, only less than 0.6% of TEs and lncRNAs respond to DNA damage in WT plants, and the regulation of ≥95% of them is ATM-dependent. The ATM-downstream factors BRCA1, DRM1, JMJ30, AGO2, and the ATM-independent AGO4 participate in the regulation of individual TEs and lncRNAs. Remarkably, protein-coding genes located adjacent to DNA damage-responsive TEs and lncRNAs are frequently coexpressed, which is consistent with the hypothesis that TEs and lncRNAs located close to genes commonly function as controlling elements.

Getting ready for host invasion: elevated expression and action of xyloglucan endotransglucosylases/hydrolases in developing haustoria of the holoparasitic angiosperm Cuscuta.

Changes in cell walls have been previously observed in the mature infection organ, or haustorium, of the parasitic angiosperm Cuscuta, but are not equally well charted in young haustoria. In this study, we focused on the molecular processes in the early stages of developing haustoria; that is, before the parasite engages in a physiological contact with its host. We describe first the identification of differentially expressed genes in young haustoria whose development was induced by far-red light and tactile stimuli in the absence of a host plant by suppression subtractive hybridization. To improve sequence information and to aid in the identification of the obtained candidates, reference transcriptomes derived from two species of Cuscuta, C. gronovii and C. reflexa, were generated. Subsequent quantitative gene expression analysis with different tissues of C. reflexa revealed that among the genes that were up-regulated in young haustoria, two xyloglucan endotransglucosylase/hydrolase (XTH) genes were highly expressed almost exclusively at the onset of haustorium development. The same expression pattern was also found for the closest XTH homologues from C. gronovii. In situ assays for XTH-specific action suggested that xyloglucan endotransglucosylation was most pronounced in the cell walls of the swelling area of the haustorium facing the host plant, but was also detectable in later stages of haustoriogenesis. We propose that xyloglucan remodelling by Cuscuta XTHs prepares the parasite for host infection and possibly aids the invasive growth of the haustorium.

The genome of the stress-tolerant wild tomato species Solanum pennellii.

Solanum pennellii is a wild tomato species endemic to Andean regions in South America, where it has evolved to thrive in arid habitats. Because of its extreme stress tolerance and unusual morphology, it is an important donor of germplasm for the cultivated tomato Solanum lycopersicum. Introgression lines (ILs) in which large genomic regions of S. lycopersicum are replaced with the corresponding segments from S. pennellii can show remarkably superior agronomic performance. Here we describe a high-quality genome assembly of the parents of the IL population. By anchoring the S. pennellii genome to the genetic map, we define candidate genes for stress tolerance and provide evidence that transposable elements had a role in the evolution of these traits. Our work paves a path toward further tomato improvement and for deciphering the mechanisms underlying the myriad other agronomic traits that can be improved with S. pennellii germplasm.

Organic carbon transformations in high-Arctic peat soils: key functions and microorganisms.

A substantial part of the Earths' soil organic carbon (SOC) is stored in Arctic permafrost peatlands, which represent large potential sources for increased emissions of the greenhouse gases CH(4) and CO(2) in a warming climate. The microbial communities and their genetic repertoire involved in the breakdown and mineralisation of SOC in these soils are, however, poorly understood. In this study, we applied a combined metagenomic and metatranscriptomic approach on two Arctic peat soils to investigate the identity and the gene pool of the microbiota driving the SOC degradation in the seasonally thawed active layers. A large and diverse set of genes encoding plant polymer-degrading enzymes was found, comparable to microbiotas from temperate and subtropical soils. This indicates that the metabolic potential for SOC degradation in Arctic peat is not different from that of other climatic zones. The majority of these genes were assigned to three bacterial phyla, Actinobacteria, Verrucomicrobia and Bacteroidetes. Anaerobic metabolic pathways and the fraction of methanogenic archaea increased with peat depth, evident for a gradual transition from aerobic to anaerobic lifestyles. A population of CH(4)-oxidising bacteria closely related to Methylobacter tundripaludum was the dominating active group of methanotrophs. Based on the in-depth characterisation of the microbes and their genes, we conclude that these Arctic peat soils will turn into CO(2) sources owing to increased active layer depth and prolonged growing season. However, the extent of future CH(4) emissions will critically depend on the response of the methanotrophic bacteria.

GabiPD - The GABI Primary Database integrates plant proteomic data with gene-centric information.

GabiPD is an integrative plant "omics" database that has been established as part of the German initiative for Genome Analysis of the Plant Biological System (GABI). Data from different "omics" disciplines are integrated and interactively visualized. Proteomics is represented by data and tools aiding studies on the identification of post-translational modification and function of proteins. Annotated 2D electrophoresis-gel images are offered to inspect protein sets expressed in different tissues of Arabidopsis thaliana and Brassica napus. From a given protein spot, a link will direct the user to the related GreenCard Gene entry where detailed gene-centric information will support the functional annotation. Beside MapMan- and GO-classification, information on conserved protein domains and on orthologs is integrated in this GreenCard service. Moreover, all other GabiPD data related to the gene, including transcriptomic data, as well as gene-specific links to external resources are provided. Researches interested in plant protein phosphorylation will find information on potential MAP kinase substrates identified in different protein microarray studies integrated in GabiPD's Phosphoproteomics page. These data can be easily compared to experimentally identified or predicted phosphorylation sites in PhosPhAt via the related Gene GreenCard. This will allow the selection of interesting candidates for further experimental validation of their phosphorylation.

Cyanophora paradoxa genome elucidates origin of photosynthesis in algae and plants.

The primary endosymbiotic origin of the plastid in eukaryotes more than 1 billion years ago led to the evolution of algae and plants. We analyzed draft genome and transcriptome data from the basally diverging alga Cyanophora paradoxa and provide evidence for a single origin of the primary plastid in the eukaryote supergroup Plantae. C. paradoxa retains ancestral features of starch biosynthesis, fermentation, and plastid protein translocation common to plants and algae but lacks typical eukaryotic light-harvesting complex proteins. Traces of an ancient link to parasites such as Chlamydiae were found in the genomes of C. paradoxa and other Plantae. Apparently, Chlamydia-like bacteria donated genes that allow export of photosynthate from the plastid and its polymerization into storage polysaccharide in the cytosol.

A membrane protein/signaling protein interaction network for Arabidopsis version AMPv2.

Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs) out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway-compatible vector. The mating-based split ubiquitin system was used to screen for potential protein-protein interactions (pPPIs) among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases (RLKs), 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions, and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 386) pPPIs between 179 proteins, yielding a scale-free network (r(2) = 0.863). Eighty of 142 transmembrane RLKs tested positive, identifying 3 homomers, 63 heteromers, and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs) had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G-protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1;1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa.

Comparative survey of plastid and mitochondrial targeting properties of transcription factors in Arabidopsis and rice.

A group of nuclear transcription factors, the Whirly proteins, were recently shown to be targeted also to chloroplasts and mitochondria. In order to find out whether other proteins might share this feature, an in silico-based screening of transcription factors from Arabidopsis and rice was carried out with the aim of identifying putative N-terminal chloroplast and mitochondrial targeting sequences. For this, the individual predictions of several independent programs were combined to a consensus prediction using a naïve Bayes method. This consensus prediction shows a higher specificity at a given sensitivity value than each of the single programs. In both species, transcription factors from a variety of protein families that possess putative N-terminal plastid or mitochondrial target peptides as well as nuclear localization sequences, were found. A search for homologues within members of the AP2/EREBP protein family revealed that target peptide-containing proteins are conserved among monocotyledonous and dicotyledonous species. Fusion of one of these proteins to GFP revealed, indeed, a dual targeting activity of this protein. We propose that dually targeted transcription factors might be involved in the communication between the nucleus and the organelles in plant cells. We further discuss how recent results on the physical interaction between the organelles and the nucleus could have significance for the regulation of the localization of these proteins.

Transcription analysis of arabidopsis membrane transporters and hormone pathways during developmental and induced leaf senescence.

A comparative transcriptome analysis for successive stages of Arabidopsis (Arabidopsis thaliana) developmental leaf senescence (NS), darkening-induced senescence of individual leaves attached to the plant (DIS), and senescence in dark-incubated detached leaves (DET) revealed many novel senescence-associated genes with distinct expression profiles. The three senescence processes share a high number of regulated genes, although the overall number of regulated genes during DIS and DET is about 2 times lower than during NS. Consequently, the number of NS-specific genes is much higher than the number of DIS- or DET-specific genes. The expression profiles of transporters (TPs), receptor-like kinases, autophagy genes, and hormone pathways were analyzed in detail. The Arabidopsis TPs and other integral membrane proteins were systematically reclassified based on the Transporter Classification system. Coordinate activation or inactivation of several genes is observed in some TP families in all three or only in individual senescence types, indicating differences in the genetic programs for remobilization of catabolites. Characteristic senescence type-specific differences were also apparent in the expression profiles of (putative) signaling kinases. For eight hormones, the expression of biosynthesis, metabolism, signaling, and (partially) response genes was investigated. In most pathways, novel senescence-associated genes were identified. The expression profiles of hormone homeostasis and signaling genes reveal additional players in the senescence regulatory network.

Solute transporters of the plastid envelope membrane.

Plastids are metabolically extraordinarily active and versatile organelles that are found in all plant cells with the exception of angiosperm pollen grains. Many of the plastid-localized biochemical pathways depend on precursors from the cytosol and, in turn, many cytosolic pathways depend on the supply of precursor molecules from the plastid stroma. Hence, a massive traffic of metabolites occurs across the permeability barrier between plastids and cytosol that is called the plastid envelope membrane. Many of the known plastid envelope solute transporters have been identified by biochemical purification and peptide sequencing. This approach is of limited use for less abundant proteins and for proteins of plastid subtypes that are difficult to isolate in preparative amounts. Hence, the majority of plastid envelope membrane transporters are not yet identified at the molecular level. The availability of fully sequenced plant genomes, the progress in bioinformatics to predict membrane transporters localized in plastids, and the development of highly sensitive proteomics techniques open new avenues toward identifying additional, to date unknown, plastid envelope membrane transporters.

Plant membrane proteome databases.

In all living organisms transmembrane (TM) proteins are crucially involved in many physiological processes and constitute 20-30% of the proteome. An important class of TM proteins are transporters that interconnect biochemical pathways across the plasma membrane and intracellular membranes, e.g. the mitochondrial membranes and chloroplast envelope membranes. In recent years, bioinformatical tools to predict TM domains and subcellular localization were developed and used to analyze the first complete plant genomes of Arabidopsis and rice. This review focuses on plant TM proteome databases that compile topology and intracellular targeting predictions and different kinds of experimental data. In addition, other web sites are discussed that contribute useful experimental and/or bioinformatical data.

Identification of an Arabidopsis mitochondrial succinate-fumarate translocator.

Complementation of a yeast acr1 mutant carrying a deletion of the succinate/fumarate carrier gene enabled functional identification of a mitochondrial succinate translocator from Arabidopsis thaliana (AtmSFC1). Thus complementation of yeast mutants is applicable also for identification and characterization of organellar transporters. Reverse transcription polymerase chain reaction and promoter-GUS fusion showed expression of AtmSFC1 in 2 day old dark grown seedlings, which declined in cotyledons during further development, consistent with a role in export of fumarate for gluconeogenesis during lipid mobilization at early germination of Arabidopsis seeds. In mature plants, expression was found in developing and germinating pollen, suggesting a role in ethanolic fermentation.