拟南芥两个BLH转录因子调控种皮粘液质果胶去甲酯化的分子机制

Pectins are synthesized in Golgi and transported to cell wall in a highly methyesterified state, and then they are de-methylesterified by pectin methylesterases (PMEs). However, until now knowledge about the regulation mechanism of pectin de-methylesterification is limited. Arabidopsis seed coat mucilage is a specialized cell wall, the main component of which are pectins, thus has been used as a model system for studing the metablism and structure modification of pecins in recent years. In our previous work, we found that the seed coat mucilage of the Arabidopsis double mutant of two BEL1-like homeodomain genes BLH2 and BLH4 shows incompact structure, more easily detached from seed surface, and lighter staining compared to the wild type. Immunolabeling assay indicated that the degree of methylesterification of pectin in blh2 blh4 seed coat mucilage is dramatically increased. Further analysis suggesting that BLH2 and BLH4 may function in promoting the pectin de-methylesterification process through regulating the expression of PME genes. Based on these results, this project aim to reveal the functions of BLH2 and BLH4 in regulating pectin de-methylesterification of seed coat mucilage, identify and functionally analyze the key direct target genes and interacting proteins of BLH2 and BLH4, elucidating the underlying molecular mechanism of BLH2 and BLH4 regulating pectin de-methylesterification in Arabidopsis seed coat mucilage. This work will lay foundation for the understanding of the regulation mechanism of pectin de-methylesterification.

果胶在高尔基体内合成后是以高甲酯化的状态存在的，被分泌到细胞壁后需要在果胶甲酯酶的作用下发生适当的去甲酯化才能发挥作用。然而，目前关于果胶去甲酯化的调控机制还不清楚。拟南芥种皮粘液质是一种特质化的细胞壁，主要成分是果胶，近年来已成为研究果胶代谢和结构修饰的模式体系。我们前期研究发现，拟南芥BEL1-like同源异型盒转录因子BLH2与BLH4的双突变体呈现种皮粘液质内层结构松散、易脱落、染色浅的表型，免疫组化分析表明该表型是由于突变体中果胶甲酯化程度升高所致。进一步分析发现BLH2/4可能通过调控果胶甲酯酶基因的表达来促进果胶去甲酯化。在此基础上，本项目将深入解析BLH2/4在调控种皮粘液质果胶去甲酯化中的功能，鉴定其下游靶基因和互作蛋白，并对关键靶基因和互作蛋白进行功能分析，揭示BLH2/4调控种皮粘液质果胶去甲酯化的分子机制，为深入理解果胶去甲酯化调控机制奠定基础。

果胶是一种重要的细胞壁多糖，在植物生长发育、信号传导、免疫反应等过程中发挥重要功能。果胶在高尔基体内合成后是以高甲酯化的状态存在的，被分泌到细胞壁后需要在果胶甲酯酶（PME）的作用下发生适当的去甲酯化才能发挥作用。果胶甲酯化修饰程度对于果胶结构与功能起着至关重要的作用，然而目前关于果胶去甲酯化的调控机制还不清楚。拟南芥种皮粘液质是一种特质化的细胞壁，主要成分是果胶，近年来已成为研究果胶代谢和结构修饰的模式体系。本研究利用这一体系鉴定了两个在果胶去甲酯化过程中起正调控作用的功能冗余的转录因子BEL1-Like homeodomain 2 (BLH2)与BLH4。研究发现blh2 blh4突变体粘液结构松散、易脱落，通过单糖测定、免疫组化分析、及甲酯化度测定等表明该表型是由于突变体中果胶甲酯化程度升高所致。与之相对应的是，blh2 blh4突变体种皮PME活性降低。基因表达分析发现，blh2 blh4突变体中PME58基因表达显著降低，进一步通过EMSA、ChIP-qPCR、转录激活等分析，证实了BLH2/4可以直接结合PME58基因的启动子并激活其表达。此外， pme58突变体呈现与blh2 blh4突变体相似的粘液质表型，且blh2 blh4 pme58 三突变体粘液质表型也与blh2 blh4表型相当，其粘液质甲酯化度与种皮PME活性也呈现与blh2 blh4呈现相似程度的变化，这表明blh2 blh4突变体的粘液质表型主要是由于PME58基因下降表达造成的。我们还创建了在blh2 blh4突变体中过量表达PME58的转基因回补材料（PME58-OE blh2 blh4），钌红染色、免疫组化及PME活性分析结果显示，回补系的粘液质表型、甲酯化度、以及种皮PME活性均恢复到野生型水平。这些结果在遗传上证明了BLH2与BLH4通过调控PME58基因的表达调控粘液质果胶去甲酯化。本研究揭示了果胶去甲酯化的分子机制，为果胶代谢调控网络的研究奠定了基础。