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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.

ORGANISM(S): Oryza sativa

PROVIDER: GSE43243 | GEO | 2013/01/03

SECONDARY ACCESSION(S): PRJNA185141

REPOSITORIES: GEO

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Project description:In comparison with provision of either ammonium or nitrate alone, simultaneously supplying both forms of N results in superior growth and yield for the majority of plants including rice. Using a rice 22K oligo-array, we performed transcriptome analysis to identify genes of rice (Oryza sativa L. ssp. japonica) responsive to change of N-supply forms and N-starvation. Using the supply of ammonium nitrate (one to one molar ratio) as control, the total number of root genes that were equal or more than two fold up- or down- regulated was 445, 324, and 781 by upon supply of either ammonium or nitrate or continuous N starvation, respectively for 96 h. In the shoot the equivalent numbers were much smaller only 32, 58, and 165, respectively. Clustering of the rice genes associated with different environmental stresses revealed substantial organ specificity of the root and shoot to N starvation, and also to the N supply form. Genes encoding transporters for ammonium and nitrate, nitrate reductase, glutamate dehydrogenase, and aspartate amino transferase, showed great response to change of the N supply form, especially to N starvation. Some of the genes involved in chlorophyll metabolism, carbon fixation and assimilation, were enhanced by ammonium supply only, but significantly suppressed by N-starvation. In the shoot there was increased expression of more general stress genes under nitrate when compared to ammonium nutrition. In the root the reverse situation was true with more apparent stress under ammonium nutrition. The microarray approach has revealed new levels of complexity in the response of rice to the form of N supply. Experiment Overall Design: Rice seedlings were grown in a hydroponic system in a growth chamber with control of both temperature and light. After normal growth for two weeks and for one week with completely avoiding any N source, the plants at four and a half leaf stage were re-supplied with ammonium nitrate (equal amount of either form of N), or only ammonium (ammonium sulphate and ammonium chloride), or only nitrate (calcium nitrate and magnesium nitrate), and continuing at zero N (N starvation). All the other essential nutrients were the same for all four treatments, except for sulfate and chloride which ranged between 1.1 â 2.6 mM and 0.5 â 2.5 mM, respectively. In Arabidopsis, addition of 3 mM Cl to âcontrolsâ did not produce any changes in the expression of genes in an experiment investigating the re-supply of 3 mM nitrate (Scheible et al., 2004). In other microarray experiments, extra addition of 5 mM Cl for Arabidopsis (Wang et al., 2000; Wang et al., 2004) and 1.4 mM sulphate for tomato (Wang et al., 2001) were used to replace nitrate for identifying the N deprivation responsive genes. Therefore, we assume that the relative smaller difference in sulfate and chloride concentrations among the four treatments in the normal supply range for plants would not significantly affect the expression profile of genes responsible for changes in N supply form and starvation in our experiments. Experiment Overall Design: For identifying the genes responding to the various N conditions by micro-array analysis, we extracted total RNA respectively from the roots and shoots at the 96 h after initiation of the four respective treatments. We used amplified and labeled cRNA from ammonium nitrate treatment as control, we performed array-hybridization for the cRNA from the treatment of single ammonium form, single nitrate form, or continuous N depletion, respectively. Agilent 60-mer oligonucleotide arrays containing 21,938 unique transcription units (Agilent Technologies, Tokyo, Japan) were used. Two biological replicates of each treatment were performed for microarray analyses.

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

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