• 关键词：蚕豆保卫细胞  SO₂衍生物  细胞死亡  活性氧  Ca²⁺

• 基金项目：国家自然科学基金项目(No.30870454,30470318)

• 尹晶晶
• 山西大学生命科学学院, 太原 030006

• 刘鑫
• 山西大学生命科学学院, 太原 030006

• 仪慧兰
• 山西大学生命科学学院, 太原 030006

• 杨民乐
• 山西大学生命科学学院, 太原 030006

• 摘要：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.

• Abstract：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 HSO₃^-and SO₃^2-in cytoplasm before turning into SO₄^2-.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,ROSand 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.Asynchronous increase in cell death rate and intercellular ROSand 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 ROSlevel 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 SO_2.LaCl₃ decreased the cell death caused by H₂O₂,which indicated that Ca²⁺ plays an important role in the process of ROSinduced cell death.These results clearly demonstrated that SO₂^-caused cell death was associated with the increases in intercellular ROSand Ca²⁺ levels.ROSgeneration,Ca²⁺ influx and intercellular Ca²⁺ level increase are involved in the mechanism of SO₂induced cell death.

• Key words：Vicia faba guard cells  SO₂ derivatives  cell death  ROS  Ca₂ +

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