Integrated RNA-seq and sRNA-seq analysis identifies novel nitrate-responsive genes in Arabidopsis thaliana roots

Background:Nitrate and other nitrogen metabolites can act as signals that regulate global gene expression in plants. Adaptive changes in plant morphology and physiology triggered by changes in nitrate availability are partly explained by these changes in gene expression. Despite several genome-wide efforts to identify nitrate-regulated genes, no comprehensive study of the Arabidopsis root transcriptome under contrasting nitrate conditions has been carried out. Results:In this work, we employed the Illumina high throughput sequencing technology to perform an integrated analysis of the poly-A + enriched and the small RNA fractions of the Arabidopsis thaliana root transcriptome in response to nitrate treatments. Our sequencing strategy identified new nitrate-regulated genes including 40 genes not represented in the ATH1 Affymetrix GeneChip, a novel nitrate-responsive antisense transcript and a new nitrate responsive miRNA/TARGET module consisting of a novel microRNA, miR5640 and its target, AtPPC3. Conclusions:Sequencing of small RNAs and mRNAs uncovered new genes, and enabled us to develop new hypotheses for nitrate regulation and coordination of carbon and nitrogen metabolism

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

Hit-and-run transcriptional control by bZIP1 mediates rapid nutrient signaling in Arabidopsis

The dynamic nature of gene regulatory networks allows cells to rapidly respond to environmental change. However, the underlying temporal connections are missed, even in kinetic studies, as transcription factor (TF) binding within at least one time point is required to identify primary targets. The TF-regulated but unbound genes are dismissed as secondary targets. Instead, we report that these genes comprise transient TF-target interactions most relevant to rapid signal transduction. We temporally perturbed a master TF (Basic Leucine Zipper 1, bZIP1) and the nitrogen (N) signal it transduces and integrated TF regulation and binding data from the same cell samples. Our enabling approach could identify primary TF targets based solely on gene regulation, in the absence of TF binding. We uncovered three classes of primary TF targets: (i) poised (TF-bound but not TF-regulated), (ii) stable (TF-bound and TF-regulated), and (iii) transient (TF-regulated but not TF-bound), the largest class. Unexpectedly, the transient bZIP1 targets are uniquely relevant to rapid N signaling in planta, enriched in dynamic N-responsive genes, and regulated by TF and N signal interactions. These transient targets include early N responders nitrate transporter 2.1 and NIN-like protein 3, bound by bZIP1 at 1-5 min, but not at later time points following TF perturbation. Moreover, promoters of these transient targets are uniquely enriched with cis-regulatory motifs coinherited with bZIP1 binding sites, suggesting a recruitment role for bZIP1. This transient mode of TF action supports a classic, but forgotten, "hit-and-run" transcription model, which enables a "catalyst TF" to activate a large set of targets within minutes of signal perturbation

Expressed sequence tag analysis in Cycas, the most primitive living seed plant

BACKGROUND: Cycads are ancient seed plants (living fossils) with origins in the Paleozoic. Cycads are sometimes considered a 'missing link' as they exhibit characteristics intermediate between vascular non-seed plants and the more derived seed plants. Cycads have also been implicated as the source of 'Guam's dementia', possibly due to the production of S(+)-beta-methyl-alpha, beta-diaminopropionic acid (BMAA), which is an agonist of animal glutamate receptors. RESULTS: A total of 4,200 expressed sequence tags (ESTs) were created from Cycas rumphii and clustered into 2,458 contigs, of which 1,764 had low-stringency BLAST similarity to other plant genes. Among those cycad contigs with similarity to plant genes, 1,718 cycad 'hits' are to angiosperms, 1,310 match genes in gymnosperms and 734 match lower (non-seed) plants. Forty-six contigs were found that matched only genes in lower plants and gymnosperms. Upon obtaining the complete sequence from the clones of 37/46 contigs, 14 still matched only gymnosperms. Among those cycad contigs common to higher plants, ESTs were discovered that correspond to those involved in development and signaling in present-day flowering plants. We purified a cycad EST for a glutamate receptor (GLR)-like gene, as well as ESTs potentially involved in the synthesis of the GLR agonist BMAA. CONCLUSIONS: Analysis of cycad ESTs has uncovered conserved and potentially novel genes. Furthermore, the presence of a glutamate receptor agonist, as well as a glutamate receptor-like gene in cycads, supports the hypothesis that such neuroactive plant products are not merely herbivore deterrents but may also serve a role in plant signaling

A Systems Approach Uncovers Restrictions for Signal Interactions Regulating Genome-wide Responses to Nutritional Cues in Arabidopsis

As sessile organisms, plants must cope with multiple and combined variations of signals in their environment. However, very few reports have studied the genome-wide effects of systematic signal combinations on gene expression. Here, we evaluate a high level of signal integration, by modeling genome-wide expression patterns under a factorial combination of carbon (C), light (L), and nitrogen (N) as binary factors in two organs (O), roots and leaves. Signal management is different between C, N, and L and in shoots and roots. For example, L is the major factor controlling gene expression in leaves. However, in roots there is no obvious prominent signal, and signal interaction is stronger. The major signal interaction events detected genome wide in Arabidopsis roots are deciphered and summarized in a comprehensive conceptual model. Surprisingly, global analysis of gene expression in response to C, N, L, and O revealed that the number of genes controlled by a signal is proportional to the magnitude of the gene expression changes elicited by the signal. These results uncovered a strong constraining structure in plant cell signaling pathways, which prompted us to propose the existence of a “code” of signal integration

The Zea mays mutants opaque-2 and opaque-7 disclose extensive changes in endosperm metabolism as revealed by protein, amino acid, and transcriptome-wide analyses

<p>Abstract</p> <p>Background</p> <p>The changes in storage reserve accumulation during maize (<it>Zea mays </it>L.) grain maturation are well established. However, the key molecular determinants controlling carbon flux to the grain and the partitioning of carbon to starch and protein are more elusive. The <it>Opaque-2 </it>(<it>O2</it>) gene, one of the best-characterized plant transcription factors, is a good example of the integration of carbohydrate, amino acid and storage protein metabolisms in maize endosperm development. Evidence also indicates that the <it>Opaque-7 </it>(<it>O7</it>) gene plays a role in affecting endosperm metabolism. The focus of this study was to assess the changes induced by the <it>o2 </it>and <it>o7 </it>mutations on maize endosperm metabolism by evaluating protein and amino acid composition and by transcriptome profiling, in order to investigate the functional interplay between these two genes in single and double mutants.</p> <p>Results</p> <p>We show that the overall amino acid composition of the mutants analyzed appeared similar. Each mutant had a high Lys and reduced Glx and Leu content with respect to wild type. Gene expression profiling, based on a unigene set composed of 7,250 ESTs, allowed us to identify a series of mutant-related down (17.1%) and up-regulated (3.2%) transcripts. Several differentially expressed ESTs homologous to genes encoding enzymes involved in amino acid synthesis, carbon metabolism (TCA cycle and glycolysis), in storage protein and starch metabolism, in gene transcription and translation processes, in signal transduction, and in protein, fatty acid, and lipid synthesis were identified. Our analyses demonstrate that the mutants investigated are pleiotropic and play a critical role in several endosperm-related metabolic processes. Pleiotropic effects were less evident in the <it>o7 </it>mutant, but severe in the <it>o2 </it>and <it>o2o7 </it>backgrounds, with large changes in gene expression patterns, affecting a broad range of kernel-expressed genes.</p> <p>Conclusion</p> <p>Although, by necessity, this paper is descriptive and more work is required to define gene functions and dissect the complex regulation of gene expression, the genes isolated and characterized to date give us an intriguing insight into the mechanisms underlying endosperm metabolism.</p