Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan.
Research Fellow of the Japan Society for the Promotion of Science, Tiyoda-ku, Tokyo 102-8471, Japan.
Plant J. 2010 Jul;63(2):269-282. doi: 10.1111/j.1365-313X.2010.04239.x. Epub 2010 Apr 28.
Nitrate is a major nitrogen source for land plants and also acts as a signaling molecule that induces changes in growth and gene expression. To identify the cis-acting DNA element involved in nitrate-responsive gene expression, we analyzed the promoter of the Arabidopsis gene encoding nitrite reductase (NIR1). A region from positions -188 to -1, relative to the translation start site, was found to contain at least one cis-element necessary for the nitrate-dependent activation of the promoter, in which the activity of nitrate transporter NRT2.1 and/or NRT2.2 plays a critical role. To define this nitrate-responsive cis-element (NRE), we compared the sequences of several nitrite reductase gene promoters from various higher plants and identified a conserved sequence motif as the putative NRE. A synthetic promoter in which the four copies of a 43-bp sequence containing the motif were fused to the 35S minimal promoter was found to direct nitrate-responsive transcription. Furthermore, mutations within this conserved motif in the native NIR1 promoter markedly reduced the nitrate-responsive activity of the promoter, indicating that the 43-bp sequence is an NRE that is both necessary and sufficient for nitrate-responsive transcription. We also show that both the native NIR1 promoter and the synthetic promoter display a similar level of sensitivity to nitrate, but respond differentially to exogenously supplied glutamine, indicating independent modulation of NIR1 expression by NRE-mediated nitrate induction and feedback repression mediated by other cis-element(s). These findings thus define the presence of multiple cis-elements involved in the nitrogen response in Arabidopsis.
硝酸盐是陆地植物的主要氮源，也是一种信号分子，可诱导生长和基因表达的变化。为了鉴定参与硝酸盐响应基因表达的顺式作用 DNA 元件，我们分析了拟南芥基因编码亚硝酸盐还原酶（NIR1）的启动子。发现相对于翻译起始位点，-188 到-1 位置的区域至少包含一个顺式元件，对于启动子的硝酸盐依赖激活是必需的，其中硝酸盐转运蛋白 NRT2.1 和/或 NRT2.2 的活性起着关键作用。为了定义这个硝酸盐响应的顺式元件（NRE），我们比较了来自各种高等植物的几个亚硝酸盐还原酶基因启动子的序列，并鉴定了一个保守的序列基序作为可能的 NRE。发现含有该基序的 43 个碱基对序列的四个拷贝融合到 35S 最小启动子的合成启动子可指导硝酸盐响应的转录。此外，在天然 NIR1 启动子中该保守基序内的突变显著降低了启动子的硝酸盐响应活性，表明 43 个碱基对序列是 NRE，对于硝酸盐响应的转录是必要和充分的。我们还表明，天然 NIR1 启动子和合成启动子对硝酸盐表现出相似的敏感性，但对外源谷氨酰胺的反应不同，表明 NIR1 表达的独立调节通过 NRE 介导的硝酸盐诱导和由其他顺式元件（多个）介导的反馈抑制。这些发现因此定义了拟南芥中参与氮响应的多个顺式元件的存在。
