RLF1亚硝基化介导生长素调控水稻侧根发育的分子机制

Auxin and NO have been recognized to act as crucial signaling molecules in regulating lateral root (LR) development. However, the regularoty signaling mechanisms of LR development by these two signaling molecules is still largely unknown. By analyzing the expression profiles of rice roots treated with IAA, we have screened an IAA-responsive gene and named it as RLF1 (reduced lateral root formation 1) since lateral root number was significantly reduced in the rlf1 mutant compared with wild-type plants. Interestingly, RLF1 was found to be highly expressed in LR primordium and emerged LR, implying that RLF1 may be a key regulator of auxin–dependent LR development. It is known that IAA treatment induces NO accumulation in roots. By performing biotin switch assay, we found that RLF1 could be S-nitrosylated by nitric oxide (NO) in vitro. To dissect the possible molecular mechanisms of RLF1 in regulating auxin-dependent LR development of rice, we will perform the biochemical analysis to investigate the effect of NO on S-nitrosylation of RLF1. LC-MS/MS will be employed to identify the candidate S-nytrosylated residue of RLF1. Furthermore, ChIP-Seq could be used to identify the RLF1 downstream-genes and the generated transgenic lines will be taken to investigate its role in auxin-mediated rice root development, especially in LR development. Our findings will gain a deep insight into the mechanism involved in NO-dependent S-nitrosylation of RLF1 in rice LR development, and provide a strategy to improve crop tolerance to limited soil resources by genetic control of root development.

生长素和一氧化氮（NO）是调控侧根发育的重要信号分子，但是目前在水稻中有关其调控侧根发育的机理还有很多待阐明的方面。通过水稻根系生长素（IAA）响应基因的表达谱分析及相应候选基因的突变体观察，我们发现了一个侧根数目显著减少的突变体rlf1（reduced lateral root formation 1）。RLF1编码一个具有DNA结合结构域的转录调控因子，RLF1基因的表达受IAA的快速诱导，并且在水稻侧根原基及突出的侧根部位呈现高水平表达，表明RLF1参与水稻侧根发育过程。此外，NO可以在体外亚硝基化RLF1蛋白。为了更深入地研究RLF1调控水稻侧根发育的机理，将进一步分析并鉴定出RLF1的亚硝基化修饰位点，利用ChIP-Seq筛选出RLF1亚硝基化调控的参与水稻侧根发育的下游靶基因，并结合ChIP-PCR、EMSA等生化验证及遗传分析阐明蛋白亚硝基化修饰调控侧根发育的机理。

生长素和一氧化氮（NO）是调控侧根发育的重要信号分子。NO除了作为信使分子，还可以通过对一些蛋白的直接修饰作用即亚硝基化（S-nitrosylation）来调节蛋白功能进而影响生理过程。但是目前在水稻中还没见有关蛋白亚硝基化修饰调控侧根发育的相关报道。通过分析响应生长素（IAA）基因的芯片结果，我们发现了一个侧根数目显著减少的突变体rlf1（reduced lateral root formation 1）。RLF1 编码一个ARF（Auxin responsive factor）类转录因子，RLF1 基因的表达受生长素的快速诱导，并且在水稻侧根部位呈现高水平表达，表明RLF1 参与水稻侧根发育调节过程。生长素能诱导NO在根部的积累，并且生化证据表明NO可以亚硝基化RLF1蛋白；通过LC-MS/MS分析鉴定出两个亚硝基化修饰位点：37位半胱氨酸（C37）和155位半胱氨酸（C155）。此外，我们进一步确定了RLF1蛋白调控的下游靶基因：我们证实RLF1可以专一性地结合OsCOG1启动子区域的Auxin Responsive Element (ARE)，而亚硝基化修饰过程可以显著提高RLF1 蛋白结合OsCOG1启动子的活性。OsCOG1编码一个推定的细胞分裂素糖基转移酶，通过糖基化修饰的形式调控体内活性细胞分裂素水平。过量表达OsCOG1 可促进水稻侧根发生，而抑制该基因的表达则会显著减少侧根发生。以上研究结果阐明了生长素通过下游NO信号亚硝基化修饰RLF1转录因子，从而调控细胞分裂素内源糖基化修饰影响侧根发育的分子机理。项目研究结果阐明了生长素通过下游NO亚硝基化修饰RLF1介导水稻侧根发育的分子机理和信号转导过程。我们的研究首次从NO依赖的亚硝基化蛋白修饰这一新的视角深入揭示生长素诱导水稻侧根发生的分子机理和信号转导过程。