【作者】 吴婷；

【导师】 仪慧兰；

【作者基本信息】 山西大学 ， 植物学， 2009， 硕士

【摘要】 二氧化硫（SO₂）是一种常见的全球性大气污染物,它通过气孔进入植物体,其对植物的伤害程度取决于进入植物体内的量。SO₂能诱导气孔运动,刺激气孔不正常地开放或关闭,影响植物体正常的生理机能;SO₂还能阻止植物细胞进入分裂态,可导致根尖细胞核固缩,细胞死亡。但是SO₂通过什么样的机制调控气孔运动,诱导气孔开度减小,使植物适应逆境胁迫;高浓度的SO₂会不会影响保卫细胞存活率,破坏气孔正常的调控机能,未见报道。本文以拟南芥叶片下表皮为材料,研究了SO₂体内衍生物-亚硫酸钠和亚硫酸氢钠混合液(3:1,μmol·L^-1/μmol·L^-1)对拟南芥叶片气孔运动和保卫细胞凋亡的影响。发现低浓度SO₂可诱导拟南芥气孔关闭,活性氧（ROS）和Ca²⁺在气孔关闭过程中起重要作用;高浓度SO₂可诱导保卫细胞凋亡,ROS、Ca²⁺及类Caspase蛋白酶在植物细胞凋亡过程中起重要作用。研究结果表明:浓度为7.5～150μmol·L^-1的SO₂衍生物处理3 h后叶表皮气孔开度明显减小;用抗氧化剂抗坏血酸（AsA）或Ca²⁺螯合剂乙二醇四乙酸酯（EGTA）与150μmol·L^-1SO₂衍生物共同作用时,气孔开度显著增加。外源H₂O₂或CaCl₂可诱导气孔开度减小,EGTA能逆转H₂O₂诱导的气孔开度缩小。利用ROS荧光指示剂（DCFH-DA）及Ca²⁺荧光指示剂（Fluo-3AM）检测发现,150μmol·L^-1SO₂衍生物处理可提高保卫细胞内ROS水平,增加胞质内Ca²⁺([Ca²⁺]cyt)浓度。上述结果表明,一定浓度的SO₂可诱导植株叶片气孔关闭,引起胞内ROS水平升高及Ca²⁺浓度增大,推测SO₂胁迫通过诱导活性氧增加,使[Ca²⁺]cyt浓度升高,激活钙信号系统调节气孔开度,进而调节气体的进入,对胁迫条件下叶片和植株的生理进行保护。浓度为0.5～4.5 mmol·L^-1的SO₂衍生物处理表皮3 h可诱导保卫细胞凋亡,细胞死亡率呈浓度依赖性增高。AsA或过氧化氢酶（CAT）与SO₂衍生物共同作用时,保卫细胞死亡率显著降低。Ca²⁺螯合剂EGTA或Ca²⁺通道抑制剂LaCl₃与SO₂衍生物共同作用时,保卫细胞死亡率显著降低;天冬氨酸特异性半胱氨酸蛋白酶（CaSpase蛋白酶）抑制剂Z-Asp-CH2-DCB或丝氨酸/半胱氨酸蛋白酶抑制剂TPCK与SO₂衍生物共同作用时,保卫细胞死亡率亦显著降低。利用DCFH-DA和Fluo-3AM检测发现,3 mmol·L^-1SO₂衍生物处理可提高保卫细胞内ROS水平,增加[Ca²⁺]cyt浓度。研究表明,一定浓度的SO₂可诱导拟南芥保卫细胞凋亡,引起胞内ROS水平升高及[Ca²⁺]cyt浓度增大,保卫细胞中具有类Caspase蛋白酶活性,推测胁迫可能通过诱导ROS、激活质膜钙通道,造成胞外Ca²⁺内流,继而激活类Caspase蛋白酶活性引发细胞凋亡。总之,低浓度的SO₂能诱导气孔运动,开度减小,使进入植物体内的SO₂量减少,进而减轻其对植物的伤害,提高植物的抗逆能力;而高浓度的SO₂使气孔保卫细胞死亡,干扰气孔正常的生理调控,气孔关闭机能失调,增加了SO₂进入植物体内的量,这可能是植物受到伤害的原因。更多还原

【Abstract】 Sulfur dioxide（SO₂） is a common global air pollutant.SO₂ enters into plant through the stomata and its effects related to absorbed dose.SO₂ could stimulate the stomata opening or closing abnormally,which induces physiological disorder and leaf necrosis.Exposure to high concentration of SO₂ could prevent the plant root cell from going split and induce the cell pyknosis and cell death.But the regulation mechanism of somatal movement induced by SO₂ is not clear by now,and the reports concerning the death of guard cells induced by SO₂ in plants is rather scantly.In this study,the effects of SO₂ derivatives(mixture of sodium sulfite and sodium bisulfite-3:1,μmol·L^-1/μmol·L^-1) on stomatal movements and guard cells apoptosis were investigated in Arabidopsis thaliana.The signal transduction pathways on stomatal movement and apoptosis were studied, some regulation mechanisms were discussed.The results of our present study showed that stomatal aperture decreased significantly when epidermal strips exposed to SO₂ derivatives at concentrations of 7.5～150μmol·L^-1.However,stomatal aperture increased significantly when epidermal strips exposed to 150μmol·L^-1 SO₂ derivatives combined with 1mmol/L ascorbic acid（AsA） or EGTA(chelator of Ca²⁺). Stomatal aperture decreased when epidermal strips exposed to exogenous H₂O₂(0.1～10μmol·L^-1) or CaCl₂(10～200μmol·L^-1),but stomatal aperture increased after exposed strips to 10μmol·L^-1 H₂O₂ combined with 1 mmol.L^-1 EGTA.Exposure to 150μmol·L^-1 SO₂ derivatives caused an elevation in the ROS level,and an increase in the content of[Ca²⁺]cyt in guard cells.The experimental resuts indicated that the stimulation of calcium messenger system and accumulation of active oxygen free radical were closely related to stomatal movements induced by SO₂ derivatives.The results of our present study suggested that SO₂ derivatives regulate stomatal aperture through inducing reactive oxygen species（ROS） generation and activating calcium messenger system,resulting in increased plant tolerance to SO₂ stress.SO₂ derivatives significantly induced cell apoptosis at concentrations from 0.5 to 4.5 mmol·L^-1.However,the percentage of the cell death decreased when the strips exposed to SO₂ derivatives combined with AsA(0.1,1 mmol·L^-1) or CAT(200 U·mL^-1).The cell death percentage decreased after the strips exposure to SO₂ derivatives combined with EGTA(0.1,1 mmol·L^-1) or LaCl₃(Ca²⁺ channel blocker,0.1 mmol·L^-1).The cell death percentage also decreased after exposed the strips to SO₂ derivatives combined with Z-Asp-CH2-DCB(broad-range caspase inhibitor,0.1,0.5μmol·L^-1) or TPCK (serine/cysteine protease inhibitor,0.1μmol·L^-1).Exposure to 3 mmol·L^-1SO₂ derivatives caused an elevation in the ROS level,and an increase in the content of[Ca²⁺]cyt which existed in guard cells.The results of the present study indicated that SO₂ causes cell apoptosis in A.thaliana via reactive oxygen species（ROS） production.ROS activates plasma membrane Ca²⁺ channels,leads to Ca²⁺ influx and an increase of[Ca²⁺]cyt level,which activates Caspase-like proteases and induces finally cell death.In summary,SO₂ induces stomatal movement at low concentration, decreases stomatal aperture and reduces the amount of SO₂ entering the plant, which strengthens the plant stress resistance to SO₂.However,SO₂ induces guard ceils dead at high concentration,resulting in the disturbance of stomatal regulation,which is responsible for the injury of plant.更多还原