Nicholas J. Provart

An “Electronic Fluorescent Pictograph” Browser for Exploring and Analyzing Large-Scale Biological Data Sets

Background The exploration of microarray data and data from other high-throughput projects for hypothesis generation has become a vital aspect of post-genomic research. For the non-bioinformatics specialist, however, many of the currently available tools provide overwhelming amounts of data that are presented in a non-intuitive way. Methodology/Principal Findings In order to facilitate the interpretation and analysis of microarray data and data from other large-scale data sets, we have developed a tool, which we have dubbed the electronic Fluorescent Pictograph – or eFP – Browser, available at http://www.bar.utoronto.ca/, for exploring microarray and other data for hypothesis generation. This eFP Browser engine paints data from large-scale data sets onto pictographic representations of the experimental samples used to generate the data sets. We give examples of using the tool to present Arabidopsis gene expression data from the AtGenExpress Consortium (Arabidopsis eFP Browser), data for subcellular localization of Arabidopsis proteins (Cell eFP Browser), and mouse tissue atlas microarray data (Mouse eFP Browser). Conclusions/Significance The eFP Browser software is easily adaptable to microarray or other large-scale data sets from any organism and thus should prove useful to a wide community for visualizing and interpreting these data sets for hypothesis generation.

Abscisic Acid Inhibits Type 2C Protein Phosphatases via the PYR/PYL Family of START Proteins

ABA Receptor Rumbled? The plant hormone abscisic acid (ABA) is critical for normal development and for mediating plant responses to stressful environmental conditions. Now, two papers present analyses of candidate ABA receptors (see the news story by Pennisi). Ma et al. (p. 1064; published online 30 April) and Park et al. (p. 1068, published online 30 April) used independent strategies to search for proteins that physically interact with ABI family phosphatase components of the ABA response signaling pathway. Both groups identified different members of the same family of proteins, which appear to interact with ABI proteins to form a heterocomplex that can act as the ABA receptor. The variety of both families suggests that the ABA receptor may not be one entity, but rather a class of closely related complexes, which may explain previous difficulties in establishing its identity. Links between two ancient multimember protein families signal responses to the plant hormone abscisic acid. Type 2C protein phosphatases (PP2Cs) are vitally involved in abscisic acid (ABA) signaling. Here, we show that a synthetic growth inhibitor called pyrabactin functions as a selective ABA agonist. Pyrabactin acts through PYRABACTIN RESISTANCE 1 (PYR1), the founding member of a family of START proteins called PYR/PYLs, which are necessary for both pyrabactin and ABA signaling in vivo. We show that ABA binds to PYR1, which in turn binds to and inhibits PP2Cs. We conclude that PYR/PYLs are ABA receptors functioning at the apex of a negative regulatory pathway that controls ABA signaling by inhibiting PP2Cs. Our results illustrate the power of the chemical genetic approach for sidestepping genetic redundancy.

The developmental dynamics of the maize leaf transcriptome

We have analyzed the maize leaf transcriptome using Illumina sequencing. We mapped more than 120 million reads to define gene structure and alternative splicing events and to quantify transcript abundance along a leaf developmental gradient and in mature bundle sheath and mesophyll cells. We detected differential mRNA processing events for most maize genes. We found that 64% and 21% of genes were differentially expressed along the developmental gradient and between bundle sheath and mesophyll cells, respectively. We implemented Gbrowse, an electronic fluorescent pictograph browser, and created a two-cell biochemical pathway viewer to visualize datasets. Cluster analysis of the data revealed a dynamic transcriptome, with transcripts for primary cell wall and basic cellular metabolism at the leaf base transitioning to transcripts for secondary cell wall biosynthesis and C4 photosynthetic development toward the tip. This dataset will serve as the foundation for a systems biology approach to the understanding of photosynthetic development.

Expansion and diversification of the Populus R2R3-MYB family of transcription factors

The R2R3-MYB proteins comprise one of the largest families of transcription factors in plants. R2R3-MYB family members regulate plant-specific processes, such as the elaboration of specialized cell types, including xylem, guard cells, trichomes, and root hairs, and the biosynthesis of specialized branches of metabolism, including phenylpropanoid biosynthesis. As such, R2R3-MYB family members are hypothesized to contribute to the emergence of evolutionary innovations that have arisen in specific plant lineages. As a first step in determining the role played by R2R3-MYB family members in the emergence of lineage-specific innovations in the genus Populus, the entire Populus trichocarpa R2R3-MYB family was characterized. The Populus R2R3-MYB complement is much larger than that found in other angiosperms with fully sequenced genomes. Phylogenetic analyses, together with chromosome placement, showed that the expansion of the Populus R2R3-MYB family was not only attributable to whole genome duplication but also involved selective expansion of specific R2R3-MYB clades. Expansion of the Populus R2R3-MYB family prominently involved members with expression patterns that suggested a role in specific components of Populus life history, including wood formation and reproductive development. An expandable compendium of microarray-based expression data (PopGenExpress) and associated Web-based tools were developed to better enable within- and between-species comparisons of Populus R2R3-MYB gene expression. This resource, which includes intuitive graphic visualization of gene expression data across multiple tissues, organs, and treatments, is freely available to, and expandable by, scientists wishing to better understand the genome biology of Populus, an ecologically dominant and economically important forest tree genus.

A Predicted Interactome for Arabidopsis

The complex cellular functions of an organism frequently rely on physical interactions between proteins. A map of all protein-protein interactions, an interactome, is thus an invaluable tool. We present an interactome for Arabidopsis (Arabidopsis thaliana) predicted from interacting orthologs in yeast (Saccharomyces cerevisiae), nematode worm (Caenorhabditis elegans), fruitfly (Drosophila melanogaster), and human (Homo sapiens). As an internal quality control, a confidence value was generated based on the amount of supporting evidence for each interaction. A total of 1,159 high confidence, 5,913 medium confidence, and 12,907 low confidence interactions were identified for 3,617 conserved Arabidopsis proteins. There was significant coexpression of genes whose proteins were predicted to interact, even among low confidence interactions. Interacting proteins were also significantly more likely to be found within the same subcellular location, and significantly less likely to be found in conflicting localizations than randomly paired proteins. A notable exception was that proteins located in the Golgi were more likely to interact with Golgi, vacuolar, or endoplasmic reticulum sorted proteins, indicating possible docking or trafficking interactions. These predictions can aid researchers by extending known complexes and pathways with candidate proteins. In addition we have predicted interactions for many previously unknown proteins in known pathways and complexes. We present this interactome, and an online Web interface the Arabidopsis Interactions Viewer, as a first step toward understanding global signaling in Arabidopsis, and to whet the appetite for those who are awaiting results from high-throughput experimental approaches.

Cloning and electrophysiological analysis of KST1, an inward rectifying K+ channel expressed in potato guard cells.

Potassium uptake by guard cells represents part of the osmotic motor which drives stomatal opening. Patch‐clamp measurements have identified inward rectifying K+ channels capable of mediating K+ uptake in guard cells and various other plant cell types. Here we report the molecular cloning and characterization of a voltage‐dependent K+ channel (KST1) from potato (Solanum tuberosum L.) guard cells. In situ hybridization shows expression of kst1 in guard cells. Two‐electrode voltage‐clamp and patch‐clamp studies of the gene product after cRNA injection into Xenopus oocytes identified KST1 as a slowly activating, voltage‐dependent, inward rectifying K+ channel. The single channel current voltage curve was linear in the range ‐160 to +20 mV, with a deduced single channel conductance of 7 pS in symmetrical 100 mM K+. This channel type, modulated by pH changes within the physiological range, required ATP for activation. In line with the properties of a K(+)‐selective channel, KST1 was permeable to K+, Rb+ and NH4+ and excluded Na+ and Li+. Cs+ at submillimolar concentrations blocked the channel in a voltage‐dependent manner. Related studies on potato guard cell protoplasts confirmed the biophysical characteristics of the kst1 gene product (KST1) in the heterologous expression system. Therefore, KST1 represents a major K+ uptake channel in potato guard cells.

Next-generation mapping of Arabidopsis genes

Next-generation genomic sequencing technologies have made it possible to directly map mutations responsible for phenotypes of interest via direct sequencing. However, most mapping strategies proposed to date require some prior genetic analysis, which can be very time-consuming even in genetically tractable organisms. Here we present a de novo method for rapidly and robustly mapping the physical location of EMS mutations by sequencing a small pooled F₂ population. This method, called Next Generation Mapping (NGM), uses a chastity statistic to quantify the relative contribution of the parental mutant and mapping lines to each SNP in the pooled F₂ population. It then uses this information to objectively localize the candidate mutation based on its exclusive segregation with the mutant parental line. A user-friendly, web-based tool for performing NGM analysis is available at http://bar.utoronto.ca/NGM. We used NGM to identify three genes involved in cell-wall biology in Arabidopsis thaliana, and, in a power analysis, demonstrate success in test mappings using as few as ten F₂ lines and a single channel of Illumina Genome Analyzer data. This strategy can easily be applied to other model organisms, and we expect that it will also have utility in crops and any other eukaryote with a completed genome sequence.

Genome-wide network model capturing seed germination reveals coordinated regulation of plant cellular phase transitions

Seed germination is a complex trait of key ecological and agronomic significance. Few genetic factors regulating germination have been identified, and the means by which their concerted action controls this developmental process remains largely unknown. Using publicly available gene expression data from Arabidopsis thaliana, we generated a condition-dependent network model of global transcriptional interactions (SeedNet) that shows evidence of evolutionary conservation in flowering plants. The topology of the SeedNet graph reflects the biological process, including two state-dependent sets of interactions associated with dormancy or germination. SeedNet highlights interactions between known regulators of this process and predicts the germination-associated function of uncharacterized hub nodes connected to them with 50% accuracy. An intermediate transition region between the dormancy and germination subdomains is enriched with genes involved in cellular phase transitions. The phase transition regulators SERRATE and EARLY FLOWERING IN SHORT DAYS from this region affect seed germination, indicating that conserved mechanisms control transitions in cell identity in plants. The SeedNet dormancy region is strongly associated with vegetative abiotic stress response genes. These data suggest that seed dormancy, an adaptive trait that arose evolutionarily late, evolved by coopting existing genetic pathways regulating cellular phase transition and abiotic stress. SeedNet is available as a community resource (http://vseed.nottingham.ac.uk) to aid dissection of this complex trait and gene function in diverse processes.

AtMetExpress development: a phytochemical atlas of Arabidopsis development.

Plants possess many metabolic genes for the production of a wide variety of phytochemicals in a tissue-specific manner. However, the metabolic systems behind the diversity and tissue-dependent regulation still remain unknown due to incomplete characterization of phytochemicals produced in a single plant species. Thus, having a metabolome dataset in addition to the genome and transcriptome information resources would enrich our knowledge of plant secondary metabolism. Here we analyzed phytochemical accumulation during development of the model plant Arabidopsis (Arabidopsis thaliana) using liquid chromatography-mass spectrometry in samples covering many growth stages and organs. We also obtained tandem mass spectrometry spectral tags of many metabolites as a resource for elucidation of metabolite structure. These are part of the AtMetExpress metabolite accumulation atlas. Based on the dataset, we detected 1,589 metabolite signals from which the structures of 167 metabolites were elucidated. The integrated analyses with transcriptome data demonstrated that Arabidopsis produces various phytochemicals in a highly tissue-specific manner, which often accompanies the expression of key biosynthesis-related genes. We also found that a set of biosynthesis-related genes is coordinately expressed among the tissues. These data suggested that the simple mode of regulation, transcript to metabolite, is an origin of the dynamics and diversity of plant secondary metabolism.

Gene Expression Phenotypes of Arabidopsis Associated with Sensitivity to Low Temperatures

Chilling is a common abiotic stress that leads to economic losses in agriculture. By comparing the transcriptome of Arabidopsis under normal (22°C) and chilling (13°C) conditions, we have surveyed the molecular responses of a chilling-resistant plant to acclimate to a moderate reduction in temperature. The mRNA accumulation of approximately 20% of the approximately 8,000 genes analyzed was affected by chilling. In particular, a highly significant number of genes involved in protein biosynthesis displayed an increase in transcript abundance. We have analyzed the molecular phenotypes of 12 chilling-sensitive mutants exposed to 13°C before any visible phenotype could be detected. The number and pattern of expression of chilling-responsive genes in the mutants were consistent with their final degree of chilling injury. The mRNA accumulation profiles for the chilling-lethal mutants chs1, chs2, and chs3 were highly similar and included extensive chilling-induced and mutant-specific alterations in gene expression. The expression pattern of the mutants upon chilling suggests that the normal function of the mutated loci prevents a damaging widespread effect of chilling on transcriptional regulation. In addition, we have identified 634 chilling-responsive genes with aberrant expression in all of the chilling-lethal mutants. This reference gene list, including genes related to lipid metabolism, chloroplast function, carbohydrate metabolism and free radical detoxification, represents a potential source for genes with a critical role in plant acclimation to suboptimal temperatures. The comparison of transcriptome profiles after transfer of Arabidopsis plants from 22°C to 13°C versus transfer to 4°C suggests that quantitative and temporal differences exist between these molecular responses.