Cross-Species Network Analysis Uncovers Conserved Nitrogen-Regulated Network Modules in Rice

In this study, we used a cross-species network approach to uncover nitrogen-regulated network modules conserved across a model and a crop species. By translating gene “network knowledge” from the data-rich model Arabidopsis (Arabidopsis thaliana) to a crop (Oryza sativa), we identified evolutionarily conserved N-regulatory modules as targets for translational studies to improve N-use efficiency in transgenic plants. To uncover such conserved N-regulatory network modules, we first generated a N-regulatory network based solely on rice (O. sativa) transcriptome and gene interaction data. Next, we enhanced the “network knowledge” in the rice N-regulatory network using transcriptome and gene interaction data from Arabidopsis and new data from Arabidopsis and rice plants exposed to the same N-treatment conditions. This cross-species network analysis uncovered a set of N-regulated transcription factors (TFs) predicted to target the same genes and network modules in both species. Supernode analysis of the TFs and their targets in these conserved network modules uncovered genes directly related to nitrogen use (e.g. N-assimilation) and to other shared biological processes indirectly related to nitrogen. This cross-species network approach was validated with members of two TF families in the supernode network, bZIP-TGA and HRS1/HHO family, have recently been experimentally validated to mediate the N-response in Arabidopsis.Fil: Obertello, Mariana. University of New York; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular ; ArgentinaFil: Shrivastava, Stuti. University of New York; Estados UnidosFil: Katari, Manpreet S.. University of New York; Estados UnidosFil: Coruzzi, Gloria M.. University of New York; Estados Unido

In Silico Evaluation of Predicted Regulatory Interactions in Arabidopsis thaliana

<p>Abstract</p> <p>Background</p> <p>Prediction of transcriptional regulatory mechanisms in <it>Arabidopsis </it>has become increasingly critical with the explosion of genomic data now available for both gene expression and gene sequence composition. We have shown in previous work <abbrgrp><abbr bid="B1">1</abbr></abbrgrp>, that a combination of correlation measurements and <it>cis</it>-regulatory element (CRE) detection methods are effective in predicting targets for candidate transcription factors for specific case studies which were validated. However, to date there has been no quantitative assessment as to which correlation measures or CRE detection methods used alone or in combination are most effective in predicting TF→target relationships on a genome-wide scale.</p> <p>Results</p> <p>We tested several widely used methods, based on correlation (Pearson and Spearman Rank correlation) and <it>cis-</it>regulatory element (CRE) detection (≥1 CRE or CRE over-representation), to determine which of these methods individually or in combination is the most effective by various measures for making regulatory predictions. To predict the regulatory targets of a transcription factor (TF) of interest, we applied these methods to microarray expression data for genes that were regulated over treatment and control conditions in wild type (WT) plants. Because the chosen data sets included identical experimental conditions used on TF over-expressor or T-DNA knockout plants, we were able to test the TF→target predictions made using microarray data from WT plants, with microarray data from mutant/transgenic plants. For each method, or combination of methods, we computed sensitivity, specificity, positive and negative predictive value and the F-measure of balance between sensitivity and positive predictive value (precision). This analysis revealed that the ≥1 CRE and Spearman correlation (used alone or in combination) were the most balanced CRE detection and correlation methods, respectively with regard to their power to accurately predict regulatory-target interactions.</p> <p>Conclusion</p> <p>These findings provide an approach and guidance for researchers interested in predicting transcriptional regulatory mechanisms using microarray data that they generate (or microarray data that is publically available) combined with CRE detection in promoter sequence data.</p

Modeling the global effect of the basic-leucine zipper transcription factor 1 (bZIP1) on nitrogen and light regulation in Arabidopsis

Background: Nitrogen and light are two major regulators of plant metabolism and development. While genes involved in the control of each of these signals have begun to be identified, regulators that integrate gene responses to nitrogen and light signals have yet to be determined. Here, we evaluate the role of bZIP1, a transcription factor involved in light and nitrogen sensing, by exposing wild-type (WT) and bZIP1 T-DNA null mutant plants to a combinatorial space of nitrogen (N) and light (L) treatment conditions and performing transcriptome analysis. We use ANOVA analysis combined with clustering and Boolean modeling, to evaluate the role of bZIP1 in mediating L and N signaling genome-wide. Results: This transcriptome analysis demonstrates that a mutation in the bZIP1 gene can alter the L and/or N-regulation of several gene clusters. More surprisingly, the bZIP1 mutation can also trigger N and/or L regulation of genes that are not normally controlled by these signals in WT plants. This analysis also reveals that bZIP1 can, to a large extent, invert gene regulation (e. g., several genes induced by N in WT plants are repressed by N in the bZIP1 mutant). Conclusion: These findings demonstrate that the bZIP1 mutation triggers a genome-wide de-regulation in response to L and/or N signals that range from i) a reduction of the L signal effect, to ii) unlocking gene regulation in response to L and N combinations. This systems biology approach demonstrates that bZIP1 tunes L and N signaling relationships genome-wide, and can suppress regulatory mechanisms hypothesized to be needed at different developmental stages and/or environmental conditions

An integrated genetic, genomic and systems approach defines gene networks regulated by the interaction of light and carbon signaling pathways in Arabidopsis

<p>Abstract</p> <p>Background</p> <p>Light and carbon are two important interacting signals affecting plant growth and development. The mechanism(s) and/or genes involved in sensing and/or mediating the signaling pathways involving these interactions are unknown. This study integrates genetic, genomic and systems approaches to identify a genetically perturbed gene network that is regulated by the interaction of carbon and light signaling in Arabidopsis.</p> <p>Results</p> <p>Carbon and light insensitive (<it>cli</it>) mutants were isolated. Microarray data from <it>cli186 </it>is analyzed to identify the genes, biological processes and gene networks affected by the integration of light and carbon pathways. Analysis of this data reveals 966 genes regulated by light and/or carbon signaling in wild-type. In <it>cli186</it>, 216 of these light/carbon regulated genes are misregulated in response to light and/or carbon treatments where 78% are misregulated in response to light and carbon interactions. Analysis of the gene lists show that genes in the biological processes "energy" and "metabolism" are over-represented among the 966 genes regulated by carbon and/or light in wild-type, and the 216 misregulated genes in <it>cli186</it>. To understand connections among carbon and/or light regulated genes in wild-type and the misregulated genes in <it>cli186</it>, the microarray data is interpreted in the context of metabolic and regulatory networks. The network created from the 966 light/carbon regulated genes in wild-type, reveals that <it>cli186 </it>is affected in the light and/or carbon regulation of a network of 60 connected genes, including six transcription factors. One transcription factor, HAT22 appears to be a regulatory "hub" in the <it>cli186 </it>network as it shows regulatory connections linking a metabolic network of genes involved in "amino acid metabolism", "C-compound/carbohydrate metabolism" and "glycolysis/gluconeogenesis".</p> <p>Conclusion</p> <p>The global misregulation of gene networks controlled by light and carbon signaling in <it>cli186 </it>indicates that it represents one of the first Arabidopsis mutants isolated that is specifically disrupted in the integration of both carbon and light signals to control the regulation of metabolic, developmental and regulatory genes. The network analysis of misregulated genes suggests that <it>CLI186 </it>acts to integrate light and carbon signaling interactions and is a master regulator connecting the regulation of a host of downstream metabolic and regulatory processes.</p

ESTimating plant phylogeny: lessons from partitioning

BACKGROUND: While Expressed Sequence Tags (ESTs) have proven a viable and efficient way to sample genomes, particularly those for which whole-genome sequencing is impractical, phylogenetic analysis using ESTs remains difficult. Sequencing errors and orthology determination are the major problems when using ESTs as a source of characters for systematics. Here we develop methods to incorporate EST sequence information in a simultaneous analysis framework to address controversial phylogenetic questions regarding the relationships among the major groups of seed plants. We use an automated, phylogenetically derived approach to orthology determination called OrthologID generate a phylogeny based on 43 process partitions, many of which are derived from ESTs, and examine several measures of support to assess the utility of EST data for phylogenies. RESULTS: A maximum parsimony (MP) analysis resulted in a single tree with relatively high support at all nodes in the tree despite rampant conflict among trees generated from the separate analysis of individual partitions. In a comparison of broader-scale groupings based on cellular compartment (ie: chloroplast, mitochondrial or nuclear) or function, only the nuclear partition tree (based largely on EST data) was found to be topologically identical to the tree based on the simultaneous analysis of all data. Despite topological conflict among the broader-scale groupings examined, only the tree based on morphological data showed statistically significant differences. CONCLUSION: Based on the amount of character support contributed by EST data which make up a majority of the nuclear data set, and the lack of conflict of the nuclear data set with the simultaneous analysis tree, we conclude that the inclusion of EST data does provide a viable and efficient approach to address phylogenetic questions within a parsimony framework on a genomic scale, if problems of orthology determination and potential sequencing errors can be overcome. In addition, approaches that examine conflict and support in a simultaneous analysis framework allow for a more precise understanding of the evolutionary history of individual process partitions and may be a novel way to understand functional aspects of different kinds of cellular classes of gene products

EST analysis in Ginkgo biloba: an assessment of conserved developmental regulators and gymnosperm specific genes

BACKGROUND: Ginkgo biloba L. is the only surviving member of one of the oldest living seed plant groups with medicinal, spiritual and horticultural importance worldwide. As an evolutionary relic, it displays many characters found in the early, extinct seed plants and extant cycads. To establish a molecular base to understand the evolution of seeds and pollen, we created a cDNA library and EST dataset from the reproductive structures of male (microsporangiate), female (megasporangiate), and vegetative organs (leaves) of Ginkgo biloba. RESULTS: RNA from newly emerged male and female reproductive organs and immature leaves was used to create three distinct cDNA libraries from which 6,434 ESTs were generated. These 6,434 ESTs from Ginkgo biloba were clustered into 3,830 unigenes. A comparison of our Ginkgo unigene set against the fully annotated genomes of rice and Arabidopsis, and all available ESTs in Genbank revealed that 256 Ginkgo unigenes match only genes among the gymnosperms and non-seed plants – many with multiple matches to genes in non-angiosperm plants. Conversely, another group of unigenes in Gingko had highly significant homology to transcription factors in angiosperms involved in development, including MADS box genes as well as post-transcriptional regulators. Several of the conserved developmental genes found in Ginkgo had top BLAST homology to cycad genes. We also note here the presence of ESTs in G. biloba similar to genes that to date have only been found in gymnosperms and an additional 22 Ginkgo genes common only to genes from cycads. CONCLUSION: Our analysis of an EST dataset from G. biloba revealed genes potentially unique to gymnosperms. Many of these genes showed homology to fully sequenced clones from our cycad EST dataset found in common only with gymnosperms. Other Ginkgo ESTs are similar to developmental regulators in higher plants. This work sets the stage for future studies on Ginkgo to better understand seed and pollen evolution, and to resolve the ambiguous phylogenetic relationship of G. biloba among the gymnosperms

Using Phylogenomic Patterns and Gene Ontology to Identify Proteins of Importance in Plant Evolution

We use measures of congruence on a combined expressed sequenced tag genome phylogeny to identify proteins that have potential significance in the evolution of seed plants. Relevant proteins are identified based on the direction of partitioned branch and hidden support on the hypothesis obtained on a 16-species tree, constructed from 2,557 concatenated orthologous genes. We provide a general method for detecting genes or groups of genes that may be under selection in directions that are in agreement with the phylogenetic pattern. Gene partitioning methods and estimates of the degree and direction of support of individual gene partitions to the overall data set are used. Using this approach, we correlate positive branch support of specific genes for key branches in the seed plant phylogeny. In addition to basic metabolic functions, such as photosynthesis or hormones, genes involved in posttranscriptional regulation by small RNAs were significantly overrepresented in key nodes of the phylogeny of seed plants. Two genes in our matrix are of critical importance as they are involved in RNA-dependent regulation, essential during embryo and leaf development. These are Argonaute and the RNA-dependent RNA polymerase 6 found to be overrepresented in the angiosperm clade. We use these genes as examples of our phylogenomics approach and show that identifying partitions or genes in this way provides a platform to explain some of the more interesting organismal differences among species, and in particular, in the evolution of plants

A Functional Phylogenomic View of the Seed Plants

A novel result of the current research is the development and implementation of a unique functional phylogenomic approach that explores the genomic origins of seed plant diversification. We first use 22,833 sets of orthologs from the nuclear genomes of 101 genera across land plants to reconstruct their phylogenetic relationships. One of the more salient results is the resolution of some enigmatic relationships in seed plant phylogeny, such as the placement of Gnetales as sister to the rest of the gymnosperms. In using this novel phylogenomic approach, we were also able to identify overrepresented functional gene ontology categories in genes that provide positive branch support for major nodes prompting new hypotheses for genes associated with the diversification of angiosperms. For example, RNA interference (RNAi) has played a significant role in the divergence of monocots from other angiosperms, which has experimental support in Arabidopsis and rice. This analysis also implied that the second largest subunit of RNA polymerase IV and V (NRPD2) played a prominent role in the divergence of gymnosperms. This hypothesis is supported by the lack of 24nt siRNA in conifers, the maternal control of small RNA in the seeds of flowering plants, and the emergence of double fertilization in angiosperms. Our approach takes advantage of genomic data to define orthologs, reconstruct relationships, and narrow down candidate genes involved in plant evolution within a phylogenomic view of species' diversification