NO和H2S交互调控采后桃果实细胞壁结构的协同分子机制

Nitric oxide (NO) and hydrogen sulfide (H2S) are two important gaseous signaling molecules and there is important crosstalk between the signaling pathways of NO and H2S on the regulation of ripening of fruits. This project investigates molecular mechanism of synergistic regulation of cell wall architecture of peaches by crosstalk between NO and H2S with modern analytical instruments and methods of glycobiology and molecular biology. (1) The crosstalk between NO and H2S and the regulations of cell wall architecture of peach fruits are investigated using carbohydrate gel electrophoresis, oligosaccharide fingerprinting, Fourier transform infrared (FTIR), Raman spectroscopies and atomic force microscopy (AFM). (2) Effects of NO and H2S on the activities and gene expressions of the key enzymes- plygalacturonase(PG), pectin methylesterase (PE), endo-1,4-β-glucanases (EGase), pectate lyase (PEL), xyloglucan endotransglucosylase-hydrolases (XTH), α-L-Arabinofuranosidase（a-L-Af), 3-glucuronidase (3-Gal) in process of degradation of cell walls. (3) By using UV chromatography, fluorescence chromatography, circular dichroism chromatography (CD) and X-ray structure analysis, the combination mode between NO, H2S and the key enzymes and the changes of the spatial configuration of complex are studied. (4) By using biotin switch assay and modified biotin switch (S-sulfhydration) assay and MS, the interactions between NO and special Cys residues of the key enzymes are studied to examine the effects of S-nitrosylation and S-sulfhydration for the regulation of softening of peach fruits. The results of this project will provide evidences to improve the quality of peach fruits during storage.

NO和H2S是调控果实成熟衰老的重要的内源气体信号，两者之间存在重要的相互作用。本项目利用现代仪器分析、糖生物学和分子生物学技术研究NO和H2S交互调控采后桃果实细胞壁结构的协同分子机制。（1）利用凝胶色谱、寡糖指纹图谱、红外色谱、拉曼色谱和原子力显微镜研究NO和H2S对桃果实细胞壁结构调控及交互作用。（2）研究NO和H2S对桃果实细胞壁代谢过程中的关键酶-PG、PE、EGase、PEL、XTH、a-L-Af、3-Gal活性及基因表达的影响。（3）利用紫外、荧光、圆二色谱、X-射线等技术研究NO和H2S与细胞壁代谢关键酶的结合方式和形成复合物空间构型变化。（4）生物素标签法及改良的生物素标签法结合质谱研究NO和H2S与细胞壁代谢关键酶特定Cys残基的作用，揭示酶蛋白S-亚硝基和S-巯基修饰在调控桃果实软化中的作用。本项目的顺利开展可为改善采后桃果实品质提供理论依据。

NO和H2S是调控果实成熟衰老的重要的内源气体信号，两者之间存在重要的相互作用。本项目利用电子投射电镜、组学技术和分子生物学技术研究NO和H2S交互调控采后桃果实细胞壁结构的协同分子机制。主要研究结果如下：（1）研究NO、H2S、NO协同H2S处理对桃果实硬度、可溶性固形物、细胞壁结构和细胞壁代谢酶活性的影响，H2S和NO协同处理的果实比单独处理效果好，能更好的抑制桃果实硬度的下降和可溶性固形物含量的提高。电子投射电镜结果表明，协同处理更好的保持细胞壁的结构。协同处理果实显著抑制了exo-PG和endo-PG活性，果胶甲酯酶活性（PE）和葡聚糖酶活性（Egase）。（2）利用转录组、蛋白质组学技术研究NO和H2S协同处理交互作用的分子基础，明确响应NO、H2S和协同处理主要的蛋白为果胶甲酯酶、多聚半乳糖醛酸酶、木葡聚糖内转葡糖基化酶/水解酶蛋白和纤维素合成酶。（3）体外重组多聚半乳糖醛酸酶，果胶甲酯酶和木葡聚糖内转葡糖基化酶/水解酶蛋白，利用紫外、荧光等技术研究NO和H2S与细胞壁代谢关键酶的结合方式。NO和H2S及协同处理的对三个酶蛋白都有荧光淬灭作用，H2S、NO和蛋白分别形成1:2、1:1复合物。（4）利用转录组学技术研究了NO和H2S调控猕猴桃果实软化的的关键基因，响应NO调控猕猴桃果实的基因主要是ß-淀粉酶、果胶裂解酶、ß-半乳糖苷酶、纤维素合酶及乙烯转导相关基因，响应H2S调控猕猴桃果实的基因主要β-glu,β-gal and PME、PMEI及乙烯转导相关基因。本项目NO和H2S协同调控桃果实软化的分子机制，为NO和H2S应用到果蔬保鲜提供理论依据。