A genome scale metabolic network for rice and accompanying analysis of tryptophan, auxin and serotonin biosynthesis regulation under biotic stress.

BACKGROUND: Functional annotations of large plant genome projects mostly provide information on gene function and gene families based on the presence of protein domains and gene homology, but not necessarily in association with gene expression or metabolic and regulatory networks. These additional annotations are necessary to understand the physiology, development and adaptation of a plant and its interaction with the environment. RESULTS: RiceCyc is a metabolic pathway networks database for rice. It is a snapshot of the substrates, metabolites, enzymes, reactions and pathways of primary and intermediary metabolism in rice. RiceCyc version 3.3 features 316 pathways and 6,643 peptide-coding genes mapped to 2,103 enzyme-catalyzed and 87 protein-mediated transport reactions. The initial functional annotations of rice genes with InterPro, Gene Ontology, MetaCyc, and Enzyme Commission (EC) numbers were enriched with annotations provided by KEGG and Gramene databases. The pathway inferences and the network diagrams were first predicted based on MetaCyc reference networks and plant pathways from the Plant Metabolic Network, using the Pathologic module of Pathway Tools. This was enriched by manually adding metabolic pathways and gene functions specifically reported for rice. The RiceCyc database is hierarchically browsable from pathway diagrams to the associated genes, metabolites and chemical structures. Through the integrated tool OMICs Viewer, users can upload transcriptomic, proteomic and metabolomic data to visualize expression patterns in a virtual cell. RiceCyc, along with additional species-specific pathway databases hosted in the Gramene project, facilitates comparative pathway analysis. CONCLUSIONS: Here we describe the RiceCyc network development and discuss its contribution to rice genome annotations. As a case study to demonstrate the use of RiceCyc network as a discovery environment we carried out an integrated bioinformatic analysis of rice metabolic genes that are differentially regulated under diurnal photoperiod and biotic stress treatments. The analysis of publicly available rice transcriptome datasets led to the hypothesis that the complete tryptophan biosynthesis and its dependent metabolic pathways including serotonin biosynthesis are induced by taxonomically diverse pathogens while also being under diurnal regulation. The RiceCyc database is available online for free access at http://www.gramene.org/pathway/.

Gramene: a growing plant comparative genomics resource.

Gramene (www.gramene.org) is a curated resource for genetic, genomic and comparative genomics data for the major crop species, including rice, maize, wheat and many other plant (mainly grass) species. Gramene is an open-source project. All data and software are freely downloadable through the ftp site (ftp.gramene.org/pub/gramene) and available for use without restriction. Gramene's core data types include genome assembly and annotations, other DNA/mRNA sequences, genetic and physical maps/markers, genes, quantitative trait loci (QTLs), proteins, ontologies, literature and comparative mappings. Since our last NAR publication 2 years ago, we have updated these data types to include new datasets and new connections among them. Completely new features include rice pathways for functional annotation of rice genes; genetic diversity data from rice, maize and wheat to show genetic variations among different germplasms; large-scale genome comparisons among Oryza sativa and its wild relatives for evolutionary studies; and the creation of orthologous gene sets and phylogenetic trees among rice, Arabidopsis thaliana, maize, poplar and several animal species (for reference purpose). We have significantly improved the web interface in order to provide a more user-friendly browsing experience, including a dropdown navigation menu system, unified web page for markers, genes, QTLs and proteins, and enhanced quick search functions.

A rapid and versatile combined DNA/RNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsis thaliana ecotypes Col-0 and Landsberg erecta.

BACKGROUND: Many established PCR-based approaches in plant molecular biology rely on lengthy and expensive methods for isolation of nucleic acids. Although several rapid DNA isolation protocols are available, they have not been tested for simultaneous RNA isolation for RT-PCR applications. In addition, traditional map-based cloning technologies often use ill-proportioned marker regions even when working with the model plant Arabidopsis thaliana, where the availability of the full genome sequence can now be exploited for the creation of a high-density marker systems. RESULTS: We designed a high-density polymorphic marker set between two frequently used ecotypes. This new polymorphic marker set allows size separation of PCR products on agarose gels and provides an initial resolution of 10 cM in linkage mapping experiments, facilitated by a rapid plant nucleic acid extraction protocol using minimal amounts of A. thaliana tissue. Using this extraction protocol, we have also characterized segregating T-DNA insertion mutations. In addition, we have shown that our rapid nucleic acid extraction protocol can also be used for monitoring transcript levels by RT-PCR amplification. Finally we have demonstrated that our nucleic acid isolation method is also suitable for other plant species, such as tobacco and barley. CONCLUSION: To facilitate high-throughput linkage mapping and other genomic applications, our nucleic acid isolation protocol yields sufficient quality of DNA and RNA templates for PCR and RT-PCR reactions, respectively. This new technique requires considerably less time compared to other purification methods, and in combination with a new polymorphic PCR marker set dramatically reduces the workload required for linkage mapping of mutations in A. thaliana utilizing crosses between Col-0 and Landsberg erecta (Ler) ecotypes.

Photoreceptor regulation of CONSTANS protein in photoperiodic flowering.

Many plants flower in response to seasonal fluctuations in day length. The CONSTANS (CO) gene of Arabidopsis promotes flowering in long days. Flowering is induced when CO messenger RNA expression coincides with the exposure of plants to light. However, how this promotes CO activity is unknown. We show that light stabilizes nuclear CO protein in the evening, whereas in the morning or in darkness the protein is degraded by the proteasome. Photoreceptors regulate CO stability and act antagonistically to generate daily rhythms in CO abundance. This layer of regulation refines the circadian rhythm in CO messenger RNA and is central to the mechanism by which day length controls flowering.

Antagonistic regulation of flowering-time gene SOC1 by CONSTANS and FLC via separate promoter motifs.

Flowering in Arabidopsis is controlled by endogenous and environmental signals relayed by distinct genetic pathways. The MADS-box flowering-time gene SOC1 is regulated by several pathways and is proposed to co-ordinate responses to environmental signals. SOC1 is directly activated by CONSTANS (CO) in long photoperiods and is repressed by FLC, a component of the vernalization (low-temperature) pathway. We show that in transgenic plants overexpressing CO and FLC, these proteins regulate flowering time antagonistically and FLC blocks transcriptional activation of SOC1 by CO. A series of SOC1::GUS reporter genes identified a 351 bp promoter sequence that mediates activation by CO and repression by FLC. A CArG box (MADS-domain protein binding element) within this sequence was recognized specifically by FLC in vitro and mediated repression by FLC in vivo, suggesting that FLC binds directly to the SOC1 promoter. We propose that CO is recruited to a separate promoter element by a DNA-binding factor and that activation by CO is impaired when FLC is bound to an adjacent CArG motif.