Ogo Yuko, Kobayashi Takanori, Nakanishi Itai Reiko, Nakanishi Hiromi, Kakei Yusuke, Takahashi Michiko, Toki Seiichi, Mori Satoshi, Nishizawa Naoko K
Graduate School of Agricultural and Life Sciences, the University of Tokyo, 1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
J Biol Chem. 2008 May 9;283(19):13407-17. doi: 10.1074/jbc.M708732200. Epub 2008 Feb 28.
Iron is essential for most living organisms, and thus iron deficiency poses a major abiotic stress in crop production. Plants induce iron utilization systems under conditions of low iron availability, but the molecular mechanisms of gene regulation under iron deficiency remain largely unknown. We identified a novel transcription factor of rice and barley, IDEF2, which specifically binds to the iron deficiency-responsive cis-acting element 2 (IDE2) by yeast one-hybrid screening. IDEF2 belongs to an uncharacterized branch of the NAC transcription factor family and exhibits novel properties of sequence recognition. An electrophoretic mobility shift assay and cyclic amplification and selection of targets experiment revealed that IDEF2 predominantly recognized CA(A/C)G(T/C)(T/C/A)(T/C/A) within IDE2 as the core-binding site. IDEF2 transcripts are constitutively present in rice roots and leaves. Repression of the function of IDEF2 by the RNA interference (RNAi) technique and chimeric repressor gene-silencing technology (CRES-T) caused aberrant iron homeostasis in rice. Several genes up-regulated by iron deficiency, including the Fe(II)-nicotianamine transporter gene OsYSL2, were less induced by iron deficiency in the RNAi rice of IDEF2, suggesting that IDEF2 is involved in the regulation of these genes. Many genes with repressed expression in IDEF2 RNAi rice possessed the IDEF2-binding core sites in their promoters, and the flanking sequences were also highly homologous to IDE2. IDEF2 bound to OsYSL2 promoter region containing the binding core site, suggesting direct regulation of OsYSL2 expression. These results reveal novel cis-element/trans-factor interactions functionally associated with iron homeostasis.
铁对大多数生物来说至关重要，因此缺铁是作物生产中的一种主要非生物胁迫。植物在铁有效性较低的条件下会诱导铁利用系统，但缺铁情况下基因调控的分子机制仍 largely 未知。我们通过酵母单杂交筛选鉴定出一种水稻和大麦的新型转录因子 IDEF2，它能特异性结合缺铁响应顺式作用元件 2（IDE2）。IDEF2 属于 NAC 转录因子家族中一个未被表征的分支，具有序列识别的新特性。电泳迁移率变动分析和靶标的循环扩增与选择实验表明，IDEF2 主要识别 IDE2 内的 CA(A/C)G(T/C)(T/C/A)(T/C/A)作为核心结合位点。IDEF2 转录本在水稻根和叶中组成性存在。通过 RNA 干扰（RNAi）技术和嵌合阻遏物基因沉默技术（CRES-T）抑制 IDEF2 的功能导致水稻中铁稳态异常。包括 Fe(II)-烟酰胺转运蛋白基因 OsYSL2 在内的几个受缺铁上调的基因，在 IDEF2 的 RNAi 水稻中受缺铁诱导程度较低，这表明 IDEF2 参与了这些基因的调控。许多在 IDEF2 RNAi 水稻中表达受抑制的基因在其启动子中具有 IDEF2 结合核心位点，且侧翼序列也与 IDE2 高度同源。IDEF2 与含有结合核心位点的 OsYSL2 启动子区域结合，表明对 OsYSL2 表达有直接调控作用。这些结果揭示了与铁稳态功能相关的新型顺式元件/反式因子相互作用。
