泛素连接酶DWL调控水稻干旱反应的机制解析

Cuticular waxes are the outmost surface of plant, which play important roles in decreasing plant water loss by restricting non-stomatal transpiration under drought stress. Altered epicuticular wax structure can significantly affect tolerance to water deficit in plant. In our previous study, a rice RING finger domain containing transcription factor, DWL (Decreased wax and lethal), was identified. Dwl functions as an active ubiquitin E3 ligase. DWL over expressing lines show dramatic drought sensitive phenotype, some even lethal. But dwl mutant shows enhanced tolerance to water limiting conditions. Further, it was found that, compared with wild type, cuticular wax contents decreased significantly in DWL overexpression lines, whereas increased in dwl mutant. Cuticular wax in DWL overexpression lines were also seriously defects. Moreover, we found that DWL can interact with transcription factor DIP (DWL interaction protein), and promote degradation of DIP via the 26S proteasome pathway, which suggests that DWL regulate biosysthesis of wax by controlling the degradation of DIP, and thus modify the response to water dificit of rice. In this proposal, we seek to dissect the biological funtion of DWL and DIP gene, and explore the molecular mechanism of DWL and DIP in regulating the wax biosynthesis. To this end, several part of work will be performed. First, genetic relationship between DWL and DIP will be explored. Second, it will be studied if DWL controls wax biosynthsis by mediating the degradation of DIP. Finally, we will elucidate how the interaction of DWL and DIP functions in regulating wax biosynthsis and in response to drought stess.

表皮蜡质是植物最外面的疏水保护层，对干旱胁迫条件下减少植物非气孔途径的水分流失具有重要作用。表皮蜡质结构的改变，会严重影响植物对干旱胁迫的反应。前期工作中，我们发现一个水稻RING finger基因Dwl，该基因过量表达的水稻对干旱极其敏感；dwl突变体抗旱能力显著提高。进一步研究表明，DWL是通过调控水稻表皮蜡质合成，来调节干旱反应。生化试验表明，DWL具有泛素连接酶活性，能够与水稻转录因子DIP互作，促进DIP通过26S蛋白酶体降解。由此推测DWL可能通过泛素化修饰DIP来调节表皮蜡质发育，进而调控水稻对干旱胁迫的反应。本申请拟从遗传学角度研究DWL和DIP在调控蜡质合成、干旱胁迫反应中的生物学功能及二者遗传学关系；利用生化手段研究 DWL是否对DIP进行泛素化修饰。从而深入解析DWL和DIP共同介导水稻表皮蜡质合成的调控网络，以及该调控网络在水稻干旱胁迫反应中的作用机制。

植物的表皮蜡质层对植物防御逆境胁迫具有重要作用，尤其在干旱胁迫条件下对减少植物通过非气孔途径水分蒸腾起着重要作用。目前已鉴定到许多参与蜡质合成关键酶或转录因子，但是对于蜡质合成的转录后调节途径尚不清楚。本项目研究发现一个水稻蜡质合成关键调节因子Decreased wax and lethal (Dwl)，Dwl 过量表达植株表皮蜡质严重缺陷，而dwl突变体表皮蜡质相对于野生型显著增加，从而导致Dwl 过量表达植株对干旱超敏感，而dwl突变体则具有耐旱表型。Dwl编码RING-figer 类蛋白，具有E3连接酶活性，能够与HD-ZIP Ⅳ家族的转录因子DIP (DWL interaction protein)相互作用。通过创制Dip过量表达和dip突变体水稻，研究其表皮蜡质结构和干旱胁迫反应，发现Dip正调控表皮蜡质合成，从而正调控干旱胁迫反应。植物体内DIP蛋白具有泛素化形式，并且通过26S蛋白酶体途径降解，DWL能够促进DIP的降解。DIP作用于DWL的遗传学下游，而BDGL是DWL-DIP途径的直接作用底物，DIP可以直接作用于BDGL的启动子，并激活BDGL的表达。综合以上结果，揭示了DWL通过促进DIP降解从而负调控表皮蜡质合成的分子机制，DWL-DIP调控的水稻蜡质合成网络在水稻干旱胁迫反应中的具有重要作用，对于提高水稻抗旱性育种研究具有重要的应用价值。