Molecular mechanisms of chilling injury and freezing injury

本研究取得的实验结果概括如下：1)冷害的分子机制。确定了以下两个因素在植物细胞冷害中起主要作用。首先，a)由于低温诱导磷脂酰甘油在脂双层中的饱和分子物种的相分离而导致的叶绿体功能障碍。在橡胶科和木本植物中，不饱和磷脂酰甘油的含量与植物的冷敏性之间都建立了相关性。采用微区延迟荧光法测定了低温下完整膜体系中饱和磷脂酰甘油的相分离。从冷敏感的南瓜子叶中分离纯化了控制饱和磷脂酰甘油合成的关键酶，并成功地克隆了cDNA。第二，b)Du…细胞质标志性环境的干扰更多的是由于低温引起的液泡膜质子转运功能障碍。在绿豆细胞中，液泡膜H^+-ATPase在体内较短的冷藏时间内受到损害，导致细胞质酸化。从绿豆幼苗中分离纯化了两种质子转运酶，即H^+-ATPase和H^+-PPase，并对其进行了鉴定。低温诱导的H^+-ATPase失活与57、68和32 kDa亚基的特异性释放有关。2)冻害机理。根据电子显微镜的研究，证实了冷冻引起的质膜超微结构的变化是细胞损伤的主要因素。细胞致死性冷冻后，细胞膜内颗粒聚集，形成无颗粒区，形成与冷害相关的六方相结构。然而，这些超微结构的变化似乎与植物种类和细胞类型有关，表明冻害机制存在多样性。在植物的驯化过程中，质膜的分子重排，包括质膜蛋白的质变和不饱和磷脂的积累，被认为对质膜的稳定性起着重要的作用，这些结果可能为今后植物冷害和冻害机理的分子生物学研究提供依据。较少

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