NO和冷信号交互作用对桃果实细胞膜鞘脂响应贮藏冷害的调控作用

The decrease in fluidity of cell membrane is a key early-response of plant to cold stress. As an important component of membrane lipid, sphingolipid involves in regulating the response of cell membrane of plant to chilling injury. This project investigates the regulation by the crosstalk between nitric oxide (NO) and cold signal on the responses of sphingolipid of cell membrane in peach fruit to chilling injury during storage with modern analytical instruments and methods of physical chemistry and molecular biology. (1) By using surface plasmon resonance (SPR), proteomics and other technologies, the posttranslational modification of cold-responsive proteins and the key enzymes in process of sphingolipid metabolism by NO and cold stress are detected, and the effects of the posttranslational modification of these proteins on the fluidity of cell membrane are studied. (2) The changes in the contents of reactive oxygen species, activities of antioxidant enzymes, the components and the peroxidation of membrane lipids including sphingolipid are also assayed. (3) By using molecular biology and other technologies, the regulations by NO and cold on the expressions of cold-responsive genes CBF family and genes encoding the key enzymes in process of sphingolipid metabolism are explored. (4) By using modern analytical instruments and methods of physical chemistry, physiology and biochemistry, the roles of NO and cold in solid-liquid phase change process and the phase transformation behavior of sphingolipid under treatments with are measured, and the changes in sphingolipid homeostasis and membrane fluidity are discussed. With methods of molecular biology and physical chemistry, this project studies the roles of NO and cold in regulating the responses of sphingolipid to chilling injury of peach fruit during storage. The results of this project provide evidences to shed light on the regulation mechanism of NO on alleviating the chilling injury and improving the quality of peach fruit during cold storage.

细胞膜流动性降低是植物感受冷信号的关键早期反应之一，鞘脂作为重要的膜脂组分参与调控细胞膜对低温冷害的响应。本项目结合现代仪器分析方法和物理化学、分子生物学等技术，在明确桃果实中一氧化氮（NO）和冷信号交互作用的基础上，研究NO和冷信号对细胞膜鞘脂响应贮藏冷害的调控作用。（1）利用表面等离子体共振和蛋白质组学等方法研究NO和冷信号对细胞膜冷响应蛋白和鞘脂代谢关键酶的翻译后修饰及蛋白质翻译后修饰对细胞膜流动性的影响；（2）研究NO和冷信号对鞘脂等膜脂组分变化和膜脂过氧化作用的影响；（3）采用分子生物学手段研究NO对冷响应基因CBF家族和鞘脂代谢关键酶基因的表达调控作用；（4）采用物理化学、现代仪器分析手段研究NO和冷胁迫对鞘脂相变过程和相变行为、鞘脂稳态变化的影响。本项目从生物学和物理化学等方面研究NO和冷信号对鞘脂响应贮藏冷害的调控作用，为缓解桃果实贮藏冷害、提高桃果实贮藏品质提供理论依据。

适宜的细胞膜流动性是细胞维持正常生理功能的必要条件。膜流动性降低是植物感受冷信号的关键早期反应之一。鞘脂组分的相对稳态平衡对保持膜脂流动性具有重要的作用，且鞘脂能参与调控植物对低温冷害的响应。目前已知NO与鞘脂的相互作用存在于植物信号途径中，但是冷胁迫中NO调控膜脂功能的作用机制却仍然未知。本项目将肥城桃果实分别用5、15、30 μmol/L NO和5 μmol/L c-PTIO溶液浸泡处理后分别于0 ℃和25 ℃贮藏。结果发现，15 μmol/L NO 处理能够有效减轻桃果实的冷害损伤程度，而c-PTIO处理桃果实冷害情况加重。外源NO处理能够提高桃果实中能量合成酶的活性，通过维持桃果实线粒体内能量合成酶的活性进而保证ATP、ADT等能量物质的合成，保持了桃果实较高的能量和能荷水平，降低果实内氧化产物的含量，延缓果实冷害的发生。而在冷胁迫发生时，NO处理能够通过提高细胞膜脂肪酸代谢关键酶的活性进而提高总脂肪酸含量和亚麻酸和亚油酸占比，提高桃果实内激素水平，引起果实细胞气孔关闭，进而保护细胞膜完整性，降低膜脂相变温度从而使细胞膜保持较好的流动性，减少膜通透性，使细胞膜尽可能具有正常结构和生理功能。iTRAQ技术鉴定到4776种蛋白，涉及135个代谢过程，iTRAQ检测到的与膜脂代谢相关的101种蛋白主要参与了脂肪酸的降解、生物合成、延伸及多种脂肪酸代谢等生命过程，而NO处理可以有效上调这些脂类代谢的关键蛋白质，减缓温度对膜脂代谢相关蛋白的影响。而在细胞膜鞘脂代谢过程中，iTRAQ检测到31种蛋白质，其中9个差异蛋白出现在鞘脂合成初期以及下游代谢过程。低温能够提高磷脂酶Dα（PLDα）基因表达水平，而NO能够降低PLDα基因表达水平。低温降低了桃果实中鞘脂含量、鞘脂代谢关键酶活性，而外源NO处理显著延缓了这种降低，表明外源NO能缓解桃果实的低温冷害作用。冷藏桃果实中存在的NO与冷信号的交互作用可以调控桃果实中鞘脂等细胞膜脂组分和代谢，进而影响膜的流动性等生物学功能并表现出对贮藏冷害的响应。