Biotechnology Research Center, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657 Japan.
Plant Cell Physiol. 2013 Apr;54(4):506-17. doi: 10.1093/pcp/pct007. Epub 2013 Jan 16.
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 or 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 autorepression mechanism. Consistently, nitrate-induced NIGT1 expression was found to be down-regulated 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 autorepressible 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.
氮是植物最重要的大量营养素，其供应会诱导基因表达、代谢和发育过程的反应。然而，氮响应的分子机制仍知之甚少。在这里，我们表明，硝酸盐的供应而不是铵盐会立即诱导水稻中一种转录抑制剂基因的表达，该基因被命名为 NIGT1（硝酸盐诱导的 GARP 型转录抑制剂 1）。DNA 结合位点选择实验和电泳迁移率变动分析的结果表明，NIGT1 结合到含有两个共识序列中的任一个的 DNA 上，GAATC 或 GAATATTC。在瞬时报告基因测定中，发现 NIGT1 在体内抑制包含鉴定的 NIGT1 结合序列的启动子的转录。此外，NIGT1 抑制其自身启动子的活性，表明存在自抑制机制。一致地，发现硝酸盐诱导的 NIGT1 表达在硝酸盐处理期间短暂峰值后被下调，并且该基因在转基因水稻植物中被组成型过表达时，硝酸盐诱导的 NIGT1 表达减少。此外，在以硝酸盐培养基而不是以铵盐培养基生长的水稻 NIGT1 过表达体中，可作为氮利用标志物的叶绿素含量降低。因此，我们提出 NIGT1 作为一种硝酸盐诱导和自抑制的转录抑制剂，可能在水稻的氮响应中发挥作用。与 NIGT1 结合位点在拟南芥 NIGT1 同源物的启动子序列中保守的事实相结合，我们的研究结果表明，存在一个依赖时间的硝酸盐响应转录调节的复杂系统，该系统在单子叶植物和双子叶植物中都保守。
