CCR2调控的细胞壁木质素形成在氢气介导的苜蓿耐镉性中的作用及其机理

Hydrogen gas (H2) is a novel gaseous signaling molecule. Our recent study found that the release of endogenous hydrogen in roots of alfalfa increased under cadmium stress, and the exogenous hydrogen treatment could mimic the physiological function of endogenous hydrogen, such as regulating the tolerance to cadmium, mercury and other heavy metals in alfalfa. According to proteomic analysis of hydrogen-rich water treatment under cadmium stress, hydrogen-induced cellular processes and molecular mechanisms of tolerating cadmium stress were found. These results showed that hydrogen-induced cadmium tolerance in alfalfa might be related to the formation of cell wall. Furthermore, we found the cell wall lignin synthesis, which was regulated by cinnamoyl-CoA reductase 2 (CCR2), might be involved in hydrogen-induced alfalfa cadmium tolerance. This project will focus on CCR2, which is the key gene in the pathway of the cell wall lignin synthesis, combined with CCR2 genetic material in Medicago truncatula, to study the role of lignin synthesis regulated by CCR2 in hydrogen-induced cadmium resistance. Meanwhile, we will combine transcriptomics and proteomics analysis to dig the key targets in the upstream and downstream, as well as corresponding regulatory pathways. We will further construct the genetic materials of key genes to verify their roles in hydrogen-induced alfalfa cadmium tolerance which is mediated by CCR2. Finally, we will use molecular biology and pharmacological experiments to explore the relationship between hydrogen-induced cell wall formation, which is mediated by CCR2, and reactive oxygen metabolism. The results will further enrich the molecular mechanism of hydrogen regulation of heavy metal tolerance in alfalfa and provide ideas for the production practices and quality improvement of alfalfa.

氢气（H2）是新发现的气体信号，我们最近的研究发现镉胁迫下苜蓿内源氢气的释放增加，利用外源氢气处理能模拟内源氢气的生理功能，如调节苜蓿对镉、汞等重金属的耐性。我们通过蛋白组学分析，发现了氢气诱导的耐镉性的细胞进程和分子机制，暗示氢气诱导的耐镉性可能与细胞壁形成有关；进一步的实验发现，肉桂酰辅酶A还原酶2（CCR2）调控的细胞壁木质素合成可能参与了这一过程。本课题将以木质素合成关键基因CCR2为重点，结合蒺藜苜蓿CCR2遗传学材料，研究其调控的木质素合成在氢气诱导的耐镉性中的作用；通过转录组学和蛋白组学分析，挖掘CCR2上下游的关键靶点及调控通路；进一步构建挖掘到的关键基因遗传学材料，验证其在CCR2介导的氢气诱导苜蓿耐镉性中的作用；利用分子生物学和药理学实验，探查CCR2调控细胞壁形成过程与活性氧代谢的关系。研究结果将丰富苜蓿对重金属耐性的分子机制，也将为苜蓿生产实践及品种改良提供思路。

早期的研究结果表明，氢气可能作为信号分子介导苜蓿的耐镉性，富氢水外源处理能模拟内源性氢气对镉积累的抑制以及对细胞氧化伤害的缓解作用，但是具体的分子机制并不清楚。本课题前期的预实验发现，氢气降低植物对镉的吸收和积累、增强对镉的耐性的同时，植物细胞的细胞壁木质化增加。肉桂酰辅酶A还原酶（CCR）是植物木质素合成的关键酶，在植物次生细胞壁发育和环境胁迫防御中发挥重要作用。紫花苜蓿是一种优良的豆科优势饲料，但问题土壤严重影响其品质，且木质素含量对饲料消化率有负面影响。因此，本课题从CCR调控的木质素合成的角度出发，通过遗传学、分子生物学、组织化学以及生物信息学等手段，探查了CCR表达和木质素合成在氢气介导的，耐镉性形成中的作用及通路中上下游关键组分。. 项目首先进行了紫花苜蓿CCR基因家族基因特征分析、表达分析和生化特性分析，理清了紫花苜蓿中CCR基因组成及各成员在木质素合成中的作用。其次，通过转录组和蛋白组分析找到了响应氢气信号的关键通路如碳代谢、氧化还原代谢等，以及参与调控的关键蛋白，如HB8、LRR-RLK等。随后的分子生物学实验利用酵母单杂交获得了与苜蓿CCR2基因启动子互作的蛋白，筛选出关键蛋白GAPA并通过ChIP-qPCR进行了验证，亚细胞定位分析显示其在镉及氢气处理下从叶绿体向细胞核转移来行使兼职功能。同时，利用遗传学材料结合分子互作实验证实了miR166/HB8参与氢气信号，并通过下游SND2来调控木质素合成途径中的CAD1和4CL5基因表达；也发现了LRR-RLK感知镉胁迫并通过依赖RBOHD的活性氧信号激活了内源氢气的释放。最后，初步实验结果还显示可变剪接、独脚金内酯信号也可能参与了氢气的信号通路。. 总之，这些结果不仅丰富了植物-环境互作过程中信号转导的模式，填补了氢气发挥生物学效应的相关通路机制，同时也对豆科牧草品质改良和生物燃料工业工程的应用具有潜在的价值。