Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Effect of overexpression of a nitrate-inducible GARP-type transcriptional repressor NIGT1 in rice

ABSTRACT: Nitrogen is the most important macronutrient in plants and its supply induces responses in gene expression, metabolism and developmental processes. However, the molecular mechanisms underlying the nitrogen responses remain poorly understood. Here we show that the supply of nitrate but not ammonium immediately induces the expression of a transcriptional repressor gene in rice, designated NIGT1 (Nitrate-Inducible, GARP-type Transcriptional Repressor 1). The results of DNA-binding site selection experiments and electrophoretic mobility shift assays indicated that NIGT1 binds to DNA containing either of two consensus sequences, GAATC and GAATATTC. In transient reporter assays, NIGT1 was found to repress transcription from the promoters containing the identified NIGT1-binding sequences in vivo. Furthermore, NIGT1 repressed the activity of its own promoter, suggesting an auto-repression mechanism. Consistently, nitrate-induced NIGT1 expression was found to be downregulated after a transient peak during nitrate treatment, and the nitrate-induced expression of NIGT1 decreased in transgenic rice plants in which this gene was constitutively overexpressed. Furthermore, the chlorophyll content that could be a marker of nitrogen utilization was found to be decreased in NIGT1 overexpressors of rice grown with nitrate medium but not with ammonium medium. Thus, we propose NIGT1 as a nitrate-inducible and auto-repressible transcriptional repressor that may play a role in the nitrogen response in rice. Taken together with the fact that the NIGT1-binding sites are conserved in promoter sequences of Arabidopsis NIGT1 homologs, our findings imply the presence of a time-dependent complex system for nitrate-responsive transcriptional regulation that is conserved in both monocots and dicots. Nitrate-induced gene expression in rice shoots was monitored 2 hours after the treatment with 10 mM potassium nitrate in the NIGT1-overexpressing lines and vector control lines.

ORGANISM(S): Oryza sativa

SUBMITTER: Shuichi Yanagisawa

PROVIDER: E-GEOD-43243 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Plants take up nitrate in soils and utilize it both for nitrogen assimilation and as a signaling molecule. Thus, an essential role of nitrate in plants is triggering changes in gene expression patterns, including immediate induction of the expression of genes involved in nitrate transport and assimilation as well as several transcription factor genes and genes related with carbon metabolism and cytokinin biosynthesis and response. Recently we identified NIN-like proteins (NLPs) as transcription factors governing nitrate-inducible gene expression in Arabidopsis. Because of their similar DNA-binding property, nine NLP proteins may play redundant roles in controlling nitrate-regulated gene expression in Arabidopsis. The physiological roles of NLPs were therefore assessed by generating transgenic Arabidopsis plants expressing NLP6 fused to the transcriptional repression domain of SUPERMAN (SUPRD), which has an ability to convert transcriptional activators into transcriptional repressors. Our transcriptome analysis revealed that levels of nitrate-inducible expression of nitrate transporter genes (NRT1.1 and NRT2.1), nitrate reduction enzyme genes (NIA1, NIA2 and NIR1), the genes associated with ammonium assimilation (GLT1 for glutamine-2-oxoglutarate aminotransferase and ASN2 for asparagine synthetase), LBD37-39 transcription factor genes and genes encoding homologs of rice nitrate-inducible NIGT1 transcriptional repressor, were reduced in the NLP6-SUPRD lines. Furthermore, levels of nitrate-inducible expression of the genes associated with the oxidative pentose phosphate pathway (G6PD2 and G6PD3 for glucose-6-phosphate dehydrogenases, and 6-phosphogluconate dehydrogenase genes), cytokinin biosynthesis (IPT3) and signal transduction (A-type ARR genes), a gene encoding a Ser/Thr protein kinase associated with a calcineurin BM-bM-^@M-^Slike calcium sensor (CIPK3) were also found to be largely decreased by reductions in NLP activity. Because induction folds by nitrate and reduction folds by decreases in the NLP activity were well proportional, the NLP activity appeared to be responsible for most of nitrate responsiveness of the genes whose expression occurs immediately after nitrate treatment. Overall design: Gene expression in 3.5-day-old seedlings treated with or without 10 mM potassium nitrate was compared between the transgenic lines expressing a chimeric repressor form of NLP6 (NLP6-SUPRD) and the parental control line.

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Effect of overexpression of a nitrate-inducible GARP-type transcriptional repressor NIGT1 in rice

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