二氧化硫诱导蚕豆气孔保卫细胞死亡效应研究

Sulfur dioxide induces guard cell death in Vicia faba

采用蚕豆叶面气孔保卫细胞,研究SO2衍生物(Na2SO3与NaHSO3混合液,3:1,mmol·L-1)对细胞的致死效应.结果表明,在浓度1～4mmol·L-1内,SO2衍生物暴露3h可使表皮保卫细胞活性降低,部分细胞死亡,并致胞内活性氧和Ca2+水平升高;随着处理浓度的提高细胞死亡率增高.一定浓度的抗坏血酸(AsA)或过氧化氢酶(CAT)与SO2衍生物共同作用时,胞内活性氧水平降低,细胞死亡率下降.Ca2+螯合剂乙二醇双四乙酸(EGTA)或Ca2+通道抑制剂LaCl3与SO2衍生物共同作用时,胞内钙水平与细胞死亡率降低.LaCl3能降低H2O2诱导的细胞死亡率.研究结果表明,SO2致蚕豆保卫细胞死亡与胞内活性氧水平增高有关,活性氧能激活质膜Ca2+通道,使胞外Ca2+内流,造成胞内Ca2+浓度升高,介导细胞死亡;胁迫组气孔保卫细胞活性降低或死亡,将导致气孔运动失调.

Sulfur dioxide（SO2） is a major air pollutant,with harmful effects to plants,animals and human beings.Previous studies have shown that SO2 can cause chlorosis and necrosis,injure the photosynthetic process and influence the activities of enzymes for scavenging reactive oxygen species（ROS） in plant cells.SO2enters into plants mainly through the stomata,and exists as HSO 3-and SO23-in cytoplasm before turning into SO24-.The toxicity of SO2 depends on the amount of SO2 hydrates in the cells.Stomata movement regulates gas exchange including SO2,and the viability of guard cells plays an important role in gas exchange.In this study,the effect of SO2 derivatives（a mixture of sodium sulfite and sodium bisulfite,3：1 mmol·L-1） on guard cell viability was investigated in Vicia faba leaves.Dry seeds of Vicia faba were soaked for 24 h in tap water.When germinated,they were grown in soil in a greenhouse.Young,fully expanded leaves from 4-week-old plants were used in the test.Epidermal strips were obtained by peeling off the lower epidermis and incubated in 2-（Nmorpholino） ethanesulfonic（MES） buffer containing SO2 derivatives for 3 h in white light at 23℃.After treatment,the strips were stained with fluorescence reagent fluorescein diacetate（FDA）,2＇,7＇-dichlorofluorescein diacetate（DCFH-DA） and fluo-3 acetomethoxyester（Fluo-3 AM）,respectively,specific for cell viability assay,ROS and Ca2 ＋ detection,and observed under a fluorescence microscope.The results of the present study show that SO2 exposure significantly decreased cell viability and induced guard cell death at concentrations of 1,2,3 and 4 mmol·L-1.A synchronous increase in cell death rate and intercellular ROS and Ca2 ＋ levels occurred in Vicia faba guard cells exposed to SO2 derivatives for 3 h.The antioxidant substances ascorbic acid（AsA,0.1 mmol·L-1 and 1.0 mmol·L-1） and catalase（CAT,200U·mL-1） significantly inhibited SO2-induced cell death and also decreased the intercellular ROS level in SO2-treated guard cells.Both the Ca2 ＋ chelating agent ethylene glycol bis（2-aminoethyl） tetraacetic acid（EGTA,0.1 mmol·L-1 and 1.0 mmol·L-1） and the Ca2 ＋ channel blocker LaCl3（0.1 mmol·L-1） significantly decreased the cell death rate and the intercellular Ca2 ＋ level caused by SO2.LaCl3 decreased the cell death caused by H2O2,which indicated that Ca2 ＋ plays an important role in the process of ROS induced cell death.These results clearly demonstrated that SO2-caused cell death was associated with the increases in intercellular ROS and Ca2 ＋ levels.ROS generation,Ca2 ＋ influx and intercellular Ca2 ＋ level increase are involved in the mechanism of SO2induced cell death.