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Analysis of the nitrate-responsive transcriptome of the Arabidopsis root

ABSTRACT: 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. 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. This nitrate-responsive miRNA/target module might be involved in controlling carbon flux to assimilate nitrate into amino acids, suggesting that microRNAs can have both developmental and metabolic functions in the nitrate response of Arabidopsis roots.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE44062 | GEO | 2013/07/01

SECONDARY ACCESSION(S): PRJNA188503

REPOSITORIES: GEO

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Analysis of the nitrate-responsive transcriptome of the Arabidopsis root

Project description: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. 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. This nitrate-responsive miRNA/target module might be involved in controlling carbon flux to assimilate nitrate into amino acids, suggesting that microRNAs can have both developmental and metabolic functions in the nitrate response of Arabidopsis roots. Arabidopsis thaliana wild-type Col-0 plants were grown in hydroponic nitrate-free medium with 0.5 mM ammonium succinate as the only N-source for two weeks and were treated with 5 mM KNO3, or 5 mM KCl as control, for 2 hours. Total RNA from two independent sets of plants (biological replicates) was extracted from roots, and poly-A+ enriched and sRNA fractions were used to construct libraries for Illumina sequencing.

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Project description:Nitrate regulates plant growth and development and acts as a potent signal to control gene expression in Arabidopsis. Using an integrative bioinformatics approach we identified TGA1 and TGA4 as putative regulatory factors that mediate nitrate responses in Arabidopsis thaliana roots. We showed that both TGA1 and TGA4 mRNAs accumulate strongly and quickly after nitrate treatments in root organs in a tissue-specific manner. Phenotypic analysis of tga1/tga4 double mutant plants indicated that TGA1 and TGA4 are necessary for nitrate modulation of both primary and lateral root growth. Global gene expression analysis revealed that 97% of the genes with altered expression in tga1/tga4 double mutant plants are regulated by nitrate treatments indicating these transcription factors have a specific role in nitrate responses in Arabidopsis root organs. Among the nitrate-responsive genes that depend on TGA1/TGA4 for normal regulation of gene expression, we found nitrate transporters NRT2.1, NRT2.2 and nitrite reductase (NIR) genes. Specific binding of TGA1 to its cognate DNA sequence on the target gene promoters was confirmed by chromatin immunoprecipitation assays. These results identify TGA1 and TGA4 as important regulatory factors of the nitrate response in Arabidopsis roots. Arabidopsis seedlings of the Col-0 and tga1/tga4 genotypes were grown on hydroponic medium containing 1X MS salts without Nitrogen, supplemented with 0.5 mM ammonium succinate as Nitrogen source and 3 mM sucrose on a Percival chamber under a photoperiod of 16 hours of light (100 ?E/m2/sec) and 8 hours of dark at 22°C for 14 days. The plants were treated at the onset of the light cycle with 5 mM KNO3 or 5 mM KCl as control for 2 hours. Whole roots were cut from seedlings and frozen on liquid Nitrogen. Total RNA was extracted using the TriZol reagent. 3 independent biological replicates were performed.

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