Molecular mechanisms of chilling injury and freezing injury

本课题实验结果总结如下：1)低温伤害的分子机制。以下两个因素被确定为对植物细胞冷害的主要影响因素。首先，a)由于低温诱导脂质双层中磷脂酰甘油不饱和分子的相分离而导致叶绿体功能障碍。在重质植物和木本植物中均建立了不饱和磷脂酰甘油含量与植物冷敏感性的相关关系。用延时荧光法测定了低温下完整膜体系中不饱和磷脂酰甘油的相分离。从冷敏南瓜子叶中纯化了控制磷脂酰甘油不饱和分子种合成的关键酶，并成功克隆了CDNA。其次，b)细胞质标志性环境的干扰，更可能是低温诱导的细胞质质子运输功能障碍。在绿豆细胞中，细胞质H^+- atp酶在体内相对较短的冷却时间内受损，导致细胞质酸化。从绿豆幼苗中纯化出两种质子转运酶，即H^+- atp酶和H^+-PPase，并对其进行了表征。低温诱导的H^+- atp酶失活与57,68和32 kDa亚基的特异性释放有关。2)冻伤机制。电镜研究证实，冷冻诱导的质膜超微结构变化是细胞损伤的主要因素。细胞在致死性冷冻后，膜内颗粒聚集，无颗粒区形成，六角形THY相结构形成，这与冷损伤有关。然而，这些超微结构变化似乎依赖于植物种类和细胞类型，表明冻害的机制存在多样性。在植物的低温驯化过程中，质膜的分子重排，包括膜蛋白的质变和不饱和磷脂的富集，被认为在稳定质膜抵御冰冻胁迫方面起着重要作用。这些结果为今后从分子生物学角度研究植物冷害和冻害机理提供了基础。少

Experimental results obtained through this Research Project are summarized as follows: 1) Molecular mechanisms of chilling injury. Following two factors were determined to be primary important for chilling injury in plant cells. First, a) a dysfunction of chloroplasts due to a low temperature-induced phase separation of disaturated molecular species of phosphatidylglycerol in the lipid bilayers. The correlation between the content of disaturated phosphatidylglycerol and the chilling sensitivity of plants was established both in hevaceous and woody plant species. The phase separation of disaturated phosphatoidylglycerol in intact membrane systems under low temperatures was determined by an analysis of msec delayed fluorescence.The key enzyme which controls the synthesis of the disaturated molecular species of phosphatidylglycerol was purified from chilling sensitive squash cotyledons and the CDNA was successfully cloned. Second, b) a disturbance of an iconic environment of cytoplasms du … More e to a low temperature-induced dysfunction of tonoplast proton transports. In mung bean cells, the tonoplast H^+-ATPase is impaired by a relatively short time of chilling in vivo, giving rise to an acidification of the cytoplasms. Two types of the proton transporting enzymes, i.e., H^+-ATPase and H^+-PPase, were purified from mung bean seedlings and characterized. The low temperature-induced inactivation of the H^+-ATPase is found to be related to a specific release of 57, 68 and 32 kDa subunits. 2) Mechanisms of freezing injury. Based on electronmicroscopic studies, it was confirmed that a freeze-induced ultrastructural changes in plasma membranes is primary important factor for cell injury. Upon a lethal freezing of cells, an aggegation of intramebranous particles and a formation of particle-free area, and a formation of Hexagonal THY phase structure were observed in plasma membranes specifically related to chill injury. However, these ultrastructural changes seemed to be dependent on plant species and the cell types, suggesting a diversity in the mechanisms of freezing injury. In sold acclimation of plants, a molecular rearrangement of plasma membranes, which includes qualitative changes in membrane proteins and enrichment of unsaturated phospholipids, is considered to play an important role in stabilization of plasma membranes against freezing stress.These results may provide a basis for molecularbiological approaches to the mechanisms of chilling injury and freezing injury in plants in future. Less