SO_2诱导拟南芥气孔运动与保卫细胞凋亡效应研究
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摘要
二氧化硫(SO_2)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。SO_2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;SO_2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SO_2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SO_2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SO_2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SO_2可诱导拟南芥气孔关闭,活性氧(ROS)和Ca~(2+)在气孔关闭过程中起重要作用;高浓度SO_2可诱导保卫细胞凋亡,ROS、Ca~(2+)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SO_2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Ca~(2+)螯合剂乙二醇四乙酸酯(EGTA)与150μmol·L~(-1)SO_2衍生物共同作用时,气孔开度显著增加。外源H_2O_2或CaCl_2可诱导气孔开度减小,EGTA能逆转H_2O_2诱导的气孔开度缩小。利用ROS荧光指示剂(DCFH-DA)及Ca~(2+)荧光指示剂(Fluo-3AM)检测发现,150μmol·L~(-1)SO_2衍生物处理可提高保卫细胞内ROS水平,增加胞质内Ca~(2+)([Ca~(2+)]cyt)浓度。上述结果表明,一定浓度的SO_2可诱导植株叶片气孔关闭,引起胞内ROS水平升高及Ca~(2+)浓度增大,推测SO_2胁迫通过诱导活性氧增加,使[Ca~(2+)]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SO_2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SO_2衍生物共同作用时,保卫细胞死亡率显著降低。Ca~(2+)螯合剂EGTA或Ca~(2+)通道抑制剂LaCl_3与SO_2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SO_2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~(-1)SO_2衍生物处理可提高保卫细胞内ROS水平,增加[Ca~(2+)]cyt浓度。研究表明,一定浓度的SO_2可诱导拟南芥保卫细胞凋亡,引起胞内ROS水平升高及[Ca~(2+)]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Ca~(2+)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SO_2能诱导气孔运动,开度减小,使进入植物体内的SO_2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SO_2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SO_2进入植物体内的量,这可能是植物受到伤害的原因。
Sulfur dioxide(SO_2) is a common global air pollutant.SO_2 enters into plant through the stomata and its effects related to absorbed dose.SO_2 could stimulate the stomata opening or closing abnormally,which induces physiological disorder and leaf necrosis.Exposure to high concentration of SO_2 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 SO_2 is not clear by now,and the reports concerning the death of guard cells induced by SO_2 in plants is rather scantly.
In this study,the effects of SO_2 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 SO_2 derivatives at concentrations of 7.5～150μmol·L~(-1).However,stomatal aperture increased significantly when epidermal strips exposed to 150μmol·L~(-1) SO_2 derivatives combined with 1mmol/L ascorbic acid(AsA) or EGTA(chelator of Ca~(2+)). Stomatal aperture decreased when epidermal strips exposed to exogenous H_2O_2(0.1～10μmol·L~(-1)) or CaCl_2(10～200μmol·L~(-1)),but stomatal aperture increased after exposed strips to 10μmol·L~(-1) H_2O_2 combined with 1 mmol.L~(-1) EGTA.Exposure to 150μmol·L~(-1) SO_2 derivatives caused an elevation in the ROS level,and an increase in the content of[Ca~(2+)]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 SO_2 derivatives.The results of our present study suggested that SO_2 derivatives regulate stomatal aperture through inducing reactive oxygen species(ROS) generation and activating calcium messenger system,resulting in increased plant tolerance to SO_2 stress.
SO_2 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 SO_2 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 SO_2 derivatives combined with EGTA(0.1,1 mmol·L~(-1)) or LaCl_3(Ca~(2+) channel blocker,0.1 mmol·L~(-1)).The cell death percentage also decreased after exposed the strips to SO_2 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~(-1)SO_2 derivatives caused an elevation in the ROS level,and an increase in the content of[Ca~(2+)]cyt which existed in guard cells.The results of the present study indicated that SO_2 causes cell apoptosis in A.thaliana via reactive oxygen species(ROS) production.ROS activates plasma membrane Ca~(2+) channels,leads to Ca~(2+) influx and an increase of[Ca~(2+)]cyt level,which activates Caspase-like proteases and induces finally cell death.
In summary,SO_2 induces stomatal movement at low concentration, decreases stomatal aperture and reduces the amount of SO_2 entering the plant, which strengthens the plant stress resistance to SO_2.However,SO_2 induces guard ceils dead at high concentration,resulting in the disturbance of stomatal regulation,which is responsible for the injury of plant.
本文以拟南芥叶片下表皮为材料,研究了SO_2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SO_2可诱导拟南芥气孔关闭,活性氧(ROS)和Ca~(2+)在气孔关闭过程中起重要作用;高浓度SO_2可诱导保卫细胞凋亡,ROS、Ca~(2+)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SO_2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Ca~(2+)螯合剂乙二醇四乙酸酯(EGTA)与150μmol·L~(-1)SO_2衍生物共同作用时,气孔开度显著增加。外源H_2O_2或CaCl_2可诱导气孔开度减小,EGTA能逆转H_2O_2诱导的气孔开度缩小。利用ROS荧光指示剂(DCFH-DA)及Ca~(2+)荧光指示剂(Fluo-3AM)检测发现,150μmol·L~(-1)SO_2衍生物处理可提高保卫细胞内ROS水平,增加胞质内Ca~(2+)([Ca~(2+)]cyt)浓度。上述结果表明,一定浓度的SO_2可诱导植株叶片气孔关闭,引起胞内ROS水平升高及Ca~(2+)浓度增大,推测SO_2胁迫通过诱导活性氧增加,使[Ca~(2+)]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SO_2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SO_2衍生物共同作用时,保卫细胞死亡率显著降低。Ca~(2+)螯合剂EGTA或Ca~(2+)通道抑制剂LaCl_3与SO_2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SO_2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~(-1)SO_2衍生物处理可提高保卫细胞内ROS水平,增加[Ca~(2+)]cyt浓度。研究表明,一定浓度的SO_2可诱导拟南芥保卫细胞凋亡,引起胞内ROS水平升高及[Ca~(2+)]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Ca~(2+)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SO_2能诱导气孔运动,开度减小,使进入植物体内的SO_2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SO_2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SO_2进入植物体内的量,这可能是植物受到伤害的原因。
Sulfur dioxide(SO_2) is a common global air pollutant.SO_2 enters into plant through the stomata and its effects related to absorbed dose.SO_2 could stimulate the stomata opening or closing abnormally,which induces physiological disorder and leaf necrosis.Exposure to high concentration of SO_2 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 SO_2 is not clear by now,and the reports concerning the death of guard cells induced by SO_2 in plants is rather scantly.
In this study,the effects of SO_2 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 SO_2 derivatives at concentrations of 7.5～150μmol·L~(-1).However,stomatal aperture increased significantly when epidermal strips exposed to 150μmol·L~(-1) SO_2 derivatives combined with 1mmol/L ascorbic acid(AsA) or EGTA(chelator of Ca~(2+)). Stomatal aperture decreased when epidermal strips exposed to exogenous H_2O_2(0.1～10μmol·L~(-1)) or CaCl_2(10～200μmol·L~(-1)),but stomatal aperture increased after exposed strips to 10μmol·L~(-1) H_2O_2 combined with 1 mmol.L~(-1) EGTA.Exposure to 150μmol·L~(-1) SO_2 derivatives caused an elevation in the ROS level,and an increase in the content of[Ca~(2+)]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 SO_2 derivatives.The results of our present study suggested that SO_2 derivatives regulate stomatal aperture through inducing reactive oxygen species(ROS) generation and activating calcium messenger system,resulting in increased plant tolerance to SO_2 stress.
SO_2 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 SO_2 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 SO_2 derivatives combined with EGTA(0.1,1 mmol·L~(-1)) or LaCl_3(Ca~(2+) channel blocker,0.1 mmol·L~(-1)).The cell death percentage also decreased after exposed the strips to SO_2 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~(-1)SO_2 derivatives caused an elevation in the ROS level,and an increase in the content of[Ca~(2+)]cyt which existed in guard cells.The results of the present study indicated that SO_2 causes cell apoptosis in A.thaliana via reactive oxygen species(ROS) production.ROS activates plasma membrane Ca~(2+) channels,leads to Ca~(2+) influx and an increase of[Ca~(2+)]cyt level,which activates Caspase-like proteases and induces finally cell death.
In summary,SO_2 induces stomatal movement at low concentration, decreases stomatal aperture and reduces the amount of SO_2 entering the plant, which strengthens the plant stress resistance to SO_2.However,SO_2 induces guard ceils dead at high concentration,resulting in the disturbance of stomatal regulation,which is responsible for the injury of plant.
引文
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