麝香草酚靶向HDAs激活水稻免疫耐镉的分子机制

Cadmium (Cd) contamination in rice paddies has been drawing great attention. Exogenous regulation of rice tolerance to Cd is relatively easy to go. Our previous study suggested that phytochemical thymol (a self-developed agricultural bio-agent) efficiently conferred rice tolerance to Cd. By using the theory of drug-protein interaction in medical study to make calculation and prediction, we found that thymol may trigger rice immune response by directly targeting histone deacetylase (HDA). To understand its molecular mechanism, this proposal intends to finish the following studies. (1) The physiological and biochemical effects of thymol on the regulation of rice tolerance to Cd will be analyzed extensively. (2) The interactive target HDAs of thymol will be identified by screening their potential of interaction in vitro. (3) The interaction mode between thymol and target HDAs will be positioned preciously by using site-directed mutation both in vivo and in vitro. (4) Both HDAs-overexpressing and HADs-RNAi transgenic lines will be obtained. The transcriptome profile of these transgenic lines under thymol or Cd treatment will be analyzed to investigate the downstream immune network regulated by target HDAs. Based on the above research, we would identify the directly interactive target of thymol from rice, and understand the role of thymol-HAD interaction in triggering rice immune response against Cd. These studies not only provide scientific basis for the exogenous-regulation of crop tolerance to heavy metals, but also shed new light on the identification of the immune targets of natural small molecules during the regulation of plant responses to abiotic stress.

水稻产地环境面临镉污染，外源调控水稻耐镉是相对简单易行的栽培技术。我们前期发现，自主研发的农用植物源生物制剂麝香草酚调控水稻高效耐镉。借鉴医学药物-蛋白互作理论，计算并预测出麝香草酚可能直接靶向组蛋白脱乙酰化酶（HDA）激活水稻免疫，为了进一步解析其分子机理，本项目拟开展以下工作：（1）全面分析麝香草酚调控水稻耐镉的生理生化效应；（2）体外互作筛选麝香草酚的靶标水稻HDAs；（3）体内、体外定点突变HDAs，精确定位麝香草酚-HDAs互作模式；（4）构建靶标HDAs超表达和RNAi转基因水稻，分析比较其响应麝香草酚和镉胁迫的表达谱，明确麝香草酚-HDAs互作调控的下游免疫基因网络。通过上述研究，挖掘出麝香草酚在水稻中的直接互作靶标HDAs，揭示这种互作激活水稻免疫高效耐镉的分子机制。不但为外源调控作物抗重金属提供理论基础，亦为天然小分子化合物在调控植物抗非生物逆境中的靶标挖掘提供新思路。

本项目前期原创性发现天然小分子化合物麝香草酚（Thymol）能够高效调控水稻耐镉，本项目挖掘到了水稻体内Thymol直接在的作用靶标组蛋白脱乙酰化酶HDA704，解析了两者的互作模式。体外表达和定点突变结果表表明：Thymol能够与HDA704三个活性位点（Pro328、His331、Asp458）结合，阻碍了HDA704活性口袋与底物的结合，进而抑制其活性。通过比较比较野生型（WT）水稻和功能缺失型突变体hda704响应镉胁迫的转录组分析，获得了HDA704调控水稻耐镉的下游基因，包括植物螯合肽合成酶基因、抗氧化酶基因、Cd转运蛋白基因等。另外，通过原位标记和药剂学试验，发现Ca2+可能作为HDA的上游和下游信号组分，在Thymol-HDA704调控水稻耐镉的过程中发挥重要作用。本研究结果初步阐明了Thymol调控水稻耐镉的分子机制，即：Thymol 可直接与 HDA704 结合互作抑制其活性，导致组蛋白乙酰化程度增加，进而松弛染色体结构，激活下游耐镉基因的表达。后续将进一步研究HDA704调控染色体组蛋白的乙酰化位点，并挖掘其调控下游基因表达可能所需的转录因子，解析Thymol-HDA704调控耐镉基因表达的分子模块，以期为Thymol应用于镉污染地区的水稻安全生产提供理论依据。