低温和机械损伤双因子胁迫协同诱导茶(Camellia sinensis)叶重要香气成分吲哚蓄积机制的研究

Plants can synthesize many aroma compounds under stresses. Numerous studies have validated that single or independent stress can affect the formation of plant aroma compounds, while less attention has been paid to the effect of multiple or co-occurring stress factors on the regulation of plant aromas formation. Tea (Camellia sinensis) aroma is an important factor, affecting tea quality. Many stresses are involved in formation of tea aromas during the tea growth and tea manufacturing processes. Our preliminary experiments proposed that the combination of low temperature stress and mechanical damage had a synergistic effect on formation of indole (a key odorant responsible for tea floral aroma) and its synthetic gene CsTSB2 expression level. Furthermore, jasmonic acid content and its biosynthesis-related key transcript factor MYC2 expression level were also significantly enhanced by the dual stresses. In addition, we obtained the preliminary evidences of involvement of jasmonic acid in biosynthesis of indole in response to the dual stresses. This research proposal will further elucidate regulation mechanism of CsTSB2 by MYC2, and investigate effect of interaction of ICE (a low temperature-related key transcript factor) and JAZ (a mechanical damage -related key transcript factor) on MYC2 based on transient transactivation assay, yeast two-hybrid assay, BiFC analysis, pull-down, and Co-IP. This research proposal will provide evidences of occurrences of transcript factors regulating biosynthesis of indole in plants for the first time, and advance our understanding of synergic regulation mechanism of formation of plant aroma compounds in response to dual stresses. The results obtained within these studies also can be essential for the future utilization of stress-responses of tea secondary metabolism for improvement of quality components of tea.

胁迫可诱导植物合成许多香气物质，目前大多集中于单一胁迫对植物香气的影响研究，然而多个胁迫的影响报道极少。香气是茶叶核心品质成分。在茶叶生长与加工过程中，多个胁迫参与了茶叶香气形成。我们前期研究发现，与单一胁迫相比，低温和机械损伤双胁迫共同作用可显著提高茶叶重要香气吲哚的含量及其合成关键基因CsTSB2表达量。此外，茉莉酸及其调控途径的关键转录因子MYC2也被显著上调，获得了茉莉酸可能参与调控吲哚的初步证据。本项目拟采用瞬时转录激活、凝胶迁移、酵母双杂、双荧光互补、蛋白质体外结合等技术，进一步明确MYC2对CsTSB2的调控作用，同时解析低温相关的ICE转录因子和机械损伤相关的JAZ转录因子互作对MYC2的调控机制。本项目有望首次揭示植物中尚未知的调控吲哚合成的转录因子，同时提高人们对多个胁迫协同诱导植物香气合成机制的认知，为今后利用茶叶对逆境胁迫的代谢应答研发茶叶安全提香技术提供理论基础。

香气物质是茶叶的核心品质成分之一。在茶树生长与茶叶加工过程中，多个胁迫参与了茶叶香气形成。本项目主要阐明了低温和机械损伤双因子胁迫协同诱导茶叶香气成分吲哚蓄积机制，取得的主要研究结果包括： .（1）茶叶中吲哚合成关键酶CsTSA和CsTSB2定位在质体上，且存在互作；低温（15oC）和损伤双胁迫可通过激活茶叶CsTSB2的表达进而诱导吲哚含量的蓄积；双因子胁迫促进了茉莉酸（JA）和茉莉酮基异亮氨酸（JA-Ile）的生物合成，转录因子CsMYC2a在JA信号中调控了吲哚的生物合成；大多数CsJAZs的表达水平在双因子胁迫下显著上调，CsICEs的表达水平则不被显著影响，CsICE2和CsJAZ2的互作释放了CsMYC2a，进而激活了CsTSB2表达，从而促进了双因子胁迫条件下吲哚的生物合成。.（2）连续损伤处理后，茶叶CsTSB2启动子的DNA甲基化水平和组蛋白H3K9me2水平呈现显著性下降趋势，CsTSB2启动子上较低的DNA甲基化水平使得转录因子CsMYC2a在CsTSB2启动子区域的结合能力增强，进而激活CsTSB2表达；此外，结合于CsTSB2启动子的CsHDA8含量减少，使得其H3乙酰化水平增加，染色质变得松散，进而促进CsTSB2的转录表达，最终促使吲哚的形成。.本研究结果一方面有助于人们深入理解茶叶香气物质吲哚生物合成的分子调控机制，另一方面为今后利用茶叶对逆境胁迫的代谢应答机制研发茶叶安全提香技术提供理论基础。.项目实施期间，已在相关领域的英文期刊发表论文6篇（均已标注本项目资助）；获授权发明专利1件。