胞外ATP对AlCl_3诱导的细胞死亡过程中胞内H_2O_2和Ca~(2+)水平的影响及其生理机制分析
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摘要
该实验以烟草悬浮细胞BY-2为材料,在烟草悬浮细胞中分别加入0.05、0.10、0.15、0.20 mmol·L~(-1)AlCl_3,以等体积去离子水处理的悬浮细胞液为对照,并依据前述实验结果选择0.15 mmol·L~(-1) AlCl_3,分别添加5 mmol·L~(-1) DMTU(H_2O_2抑制剂)、20μmol·L~(-1)CaCl_2、15μmol·L~(-1) LaCl_3(Ca~(2+)通道抑制剂)和50μmol·L~(-1) ATP设计多项处理,分析胞外ATP(eATP)对铝离子(Al~(3+))胁迫引起的植物细胞死亡及其胞内H_2O_2、Ca~(2+)的影响,以揭示Al~(3+)胁迫下植物调节细胞死亡的可能机制,进一步扩展对eATP功能的认知。结果显示:(1)随着AlCl_3胁迫浓度的提高,细胞死亡水平和胞内H_2O_2水平上升,而胞内Ca~(2+)和eATP水平则逐渐降低。(2)外援施加H_2O_2抑制剂DMTU(二甲基硫脲)和Ca~(2+)能够有效缓解AlCl_3诱导的细胞死亡水平的上升;而Ca~(2+)通道抑制剂LaCl_3(三氯化镧)则加剧了AlCl_3胁迫下的细胞死亡。(3)在AlCl_3胁迫下对细胞添加外源ATP,能够缓解AlCl_3胁迫下胞内H_2O_2水平上升和Ca~(2+)水平下降的同时,并显著降低AlCl_3胁迫导致的细胞死亡。研究表明, Al~(3+)以剂量依赖的模式提升细胞死亡和细胞内H_2O_2的水平并降低胞内Ca~(2+)和eATP水平,AlCl_3诱导的细胞死亡受到H_2O_2和Ca~(2+)水平变化的调节,eATP可以通过调节H_2O_2与Ca~(2+)水平缓解AlCl_3诱导的细胞死亡。推测Al~(3+)胁迫可能通过抑制钙离子通道而破坏了细胞内H_2O_2和Ca~(2+)之间的协同关系,外源ATP对Al~(3+)诱导H_2O_2上升的缓解作用可能是由于其提升了细胞的抗氧化能力。
In this experiment, tobacco suspension cell BY-2 was used as the material, and 0.05, 0.10, 0.15, 0.20 mmol·L~(-1) AlCl_3 was added to the tobacco suspension cells, and the suspension cell solution treatment with equal volume of deionization water was used as a control. And, according to the above experimental results, 0.15 mmol·L~(-1) AlCl_3 was selected, 5 mmol·L~(-1) DMTU(H_2O_2 inhibitor), 20 μmol·L~(-1) CaCl_2, 15 μmol·L~(-1) LaCl_3(Ca~(2+) channel inhibitor) and 50 μmol·L~(-1) ATP was used to analyze the effects of extra-cellular ATP(eATP) on plant cell death and intra-cellular H_2O_2 and Ca~(2+) induced by aluminum ion(Al~(3+)) stress and to reveal plant the possible mechanism of eATP in regulating cell death under Al~(3+) stress, and further extend the perception of eATP function. The results showed that:(1) with the increase of AlCl_3 concentration, the levels of cell death and intracellular H_2O_2 were increased, while the levels of intracellular Ca~(2+)and eATP were decreased;(2) Application of DMTU(dimethylthiourea, a scavenger of H_2O_2) and exogenous Ca~(2+) effectively attenuated the AlCl_3-induced cell death, while barium trichloride(LaCl_3, the Ca~(2+) channel inhibitor) aggravated the AlCl_3-induced cell death, indicating that AlCl3-induced cell death is regulated by H_2O_2 and Ca~(2+);(3) The addition of exogenous ATP effectively impeded the increase of intracellular H_2O_2 production and decrease of intracellular Ca~(2+) under AlCl_3 stress, and the cell death induced by AlCl_3 were also alleviated by exogenous ATP. It can be seen that aluminum-induced cell death is regulated by changes in H_2O_2 and Ca~(2+) levels, and extracellular ATP affects the aluminum-induced cell death by regulating H_2O_2 and Ca~(2+) levels.Theses observations indicated that the level of cell death and intracellular H_2O_2 were increased, while the level of intracellular Ca~(2+) and eATP were decreased in a dose-dependent manner under Al~(3+) stress. It can be seen that aluminum-induced cell death is regulated by changes in H_2O_2 and Ca~(2+) levels, and extracellular ATP affects the aluminum-induced cell death by regulating H_2O_2 and Ca~(2+) levels. We predicated that Al~(3+) stress may destroy the synergistic relationship between H_2O_2 and Ca~(2+) in cells by inhibiting calcium channel, and the alleviation effect of exogenous ATP on Al~(3+) induced H_2O_2 elevation may be due to its enhanced antioxidant capacity.
In this experiment, tobacco suspension cell BY-2 was used as the material, and 0.05, 0.10, 0.15, 0.20 mmol·L~(-1) AlCl_3 was added to the tobacco suspension cells, and the suspension cell solution treatment with equal volume of deionization water was used as a control. And, according to the above experimental results, 0.15 mmol·L~(-1) AlCl_3 was selected, 5 mmol·L~(-1) DMTU(H_2O_2 inhibitor), 20 μmol·L~(-1) CaCl_2, 15 μmol·L~(-1) LaCl_3(Ca~(2+) channel inhibitor) and 50 μmol·L~(-1) ATP was used to analyze the effects of extra-cellular ATP(eATP) on plant cell death and intra-cellular H_2O_2 and Ca~(2+) induced by aluminum ion(Al~(3+)) stress and to reveal plant the possible mechanism of eATP in regulating cell death under Al~(3+) stress, and further extend the perception of eATP function. The results showed that:(1) with the increase of AlCl_3 concentration, the levels of cell death and intracellular H_2O_2 were increased, while the levels of intracellular Ca~(2+)and eATP were decreased;(2) Application of DMTU(dimethylthiourea, a scavenger of H_2O_2) and exogenous Ca~(2+) effectively attenuated the AlCl_3-induced cell death, while barium trichloride(LaCl_3, the Ca~(2+) channel inhibitor) aggravated the AlCl_3-induced cell death, indicating that AlCl3-induced cell death is regulated by H_2O_2 and Ca~(2+);(3) The addition of exogenous ATP effectively impeded the increase of intracellular H_2O_2 production and decrease of intracellular Ca~(2+) under AlCl_3 stress, and the cell death induced by AlCl_3 were also alleviated by exogenous ATP. It can be seen that aluminum-induced cell death is regulated by changes in H_2O_2 and Ca~(2+) levels, and extracellular ATP affects the aluminum-induced cell death by regulating H_2O_2 and Ca~(2+) levels.Theses observations indicated that the level of cell death and intracellular H_2O_2 were increased, while the level of intracellular Ca~(2+) and eATP were decreased in a dose-dependent manner under Al~(3+) stress. It can be seen that aluminum-induced cell death is regulated by changes in H_2O_2 and Ca~(2+) levels, and extracellular ATP affects the aluminum-induced cell death by regulating H_2O_2 and Ca~(2+) levels. We predicated that Al~(3+) stress may destroy the synergistic relationship between H_2O_2 and Ca~(2+) in cells by inhibiting calcium channel, and the alleviation effect of exogenous ATP on Al~(3+) induced H_2O_2 elevation may be due to its enhanced antioxidant capacity.
引文
[1]沈宏,严小龙.铝对植物的毒害和植物抗铝毒机理及其影响因素[J].土壤通报,2001,(6):281-285.SHEN H,YAN X L.Aluminum toxicity to plants and plant resistance to aluminum toxicity and its influencing factors[J].Soil Bulletin,2001,(6):281-285.
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[5]LADISLAV TAMAS,JANA HUTTOVA,IGOR MISTRIK.Inhibition of Al-induced root elongation and enhancement of Al-induced peroxidase activity in Al-sensitive and Al-resistant barley cultivars are positively correlated[J].Plant and Soil,2003,250(2):193-200.
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[8]CHIVASA S,NDIMBA B K,SIMON W J,et al.Extracellular ATP functions as an endogenous external metabolite regulating plant cell viability[J].The Plant Cell,2005,17(11):3 019-3 034.
[9]CHIVASA S,TOME D F A,HAMILTON J M,et al.Proteomic analysis of extracellular ATP-regulated proteins identifies ATP synthaseβ-subunit as a novel plant cell death regulator[J].Molecular&Cellular Proteomics,2011,10(3):M110.003905.
[10]SUN J,ZHANG C L A N,DENG S H U R,et al.An ATPsignalling pathway in plant cells:extracellular ATP triggers programmed cell death in Populus euphratica[J].Plant,Cell&Environment,2012,35(5):893-916.
[11]YANG S W,KIM S K,KIM W T.Perturbation of NgTRF1expression induces apoptosis-like cell death in tobacco BY-2cells and implicates NgTRF1in the control of telomere length and stability[J].The Plant Cell,2004,16(12):3 370-3 385.
[12]JONES K,KIM D W,PARK J S,et al.Live-cell fluorescence imaging to investigate the dynamics of plant cell death during infection by the rice blast fungus Magnaporthe oryzae[J].BMC Plant Biology,2016,16(1):69.
[13]KAUR N,SHARMA I,KIRAT K,et al.Detection of reactive oxygen species in Oryza sativa L.(Rice)[J].Bio-protocol,2016,6(24):e2061.doi:1021769/Bioprotoc.2061.
[14]XU X,ZHANG Y,LING Y.The dynamic changes and origin analysis of reactive oxygen species during isolation and culture of Citrus reticulata blanco protoplasts[J].Botanical Research,2016,5(3),67-73.
[15]TANAKA K,GILORY S,JONES A M,et al.Extracellular ATP signaling in plants[J].Trends in Cell Biology,2010,20(10):601-608.
[16]IKEGAWA H,YAMAMOTO Y,MATSUMOTO H.Cell death caused by a combination of aluminum and iron in cultured tobacco cells[J].Physiologia Plantarum,1998,104(3):474-478.
[17]IKEGAWA H,YAMAMOTO Y,MATSUMOTO H.Responsesto aluminiumof suspension-cultured tobacco cells in a simple calcium solution[J].Soil Science and Plant Nutrition,2000,46(2):503-514.
[18]窦伟.硫化氢对盐和铝胁迫下小麦种子萌发及氧化损伤的缓解效应[D].合肥:合肥工业大学,2010.DOU W.Alleviation effect of hydrogen sulfide on wheat seed germination and oxidative damage under salt and aluminum stress[D].Hefei:Hefei University of Technology,2010.
[19]PEJCHAR P,MARTINEC J.Aluminum ions alter the function of non-specific phospholipase C through the changes in plasma membrane physical properties[J].Plant Signaling&Behavior,2015,10(6):e1031938.
[20]YANG D,CHEN D,WANG P,et al.Aluminium-inhibited NO3-uptake is related to Al-increased H2O2content and Aldecreased plasma membrane ATPase activity in the root tips of Al-sensitive black soybean[J].Functional Plant Biology,2017,44(2):198-207.
[21]YU Y,ZHOU W,ZHOU K,et al.Polyamines modulate aluminum-induced oxidative stress differently by inducing or reducing H2O2production in wheat[J].Chemosphere,2018,212:645-653.
[22]RENTEL M C,KNIGHT M R.Oxidative stress-induced calcium signaling in Arabidopsis[J].Plant Physiology,2004,135(3):1 471-1 479.
[23]TAKEDA S,GAPPER C,KAYA H,et al.Local positive feedback regulation determines cell shape in root hair cells[J].Science,2008,319(5 867):1 241-1 244.
[24]KAWANO T,KADNON T,FUMOTO K,et al.Aluminum as a specific inhibitor of plant TPC1Ca2+channels[J].Biochemical and Biophysical Research Communications,2004,324(1):40-45.
[25]PRASAD T K,ANDERSON M D,STEWART C R.Acclimation,hydrogen peroxide,and abscisic acid protect mitochondria against irreversible chilling injury in maize seedlings[J].Plant Physiology,1994,105(2):619-627.
[26]DEMIDCHIK V,SHANG Z,SHIN R,et al.Plant extracellular ATP signalling by plasma membrane NADPH oxidase and Ca2+channels[J].The Plant Journal,2009,58(6):903-913
[27]SUN J,ZHANG X,DENG S,et al.Extracellular ATP signaling is mediated by H2O2and cytosolic Ca2+in the salt response of Populus euphratica cells[J].PLoS One,2012b,7(12):e53136.
[28]CLARK G,ROUX S.Role of Ca2+in mediating plant responses to extracellular ATP and ADP[J].International Journal of Molecular Sciences,2018,19(11):3 590.
[29]ZIPFEL C.Plant pattern-recognition receptors[J].Trends in Immunology,2014,35(7):345-351.
[2]王庆文,冯汉青,戎富虎,等.交替氧化酶对AlCl3胁迫下烟草BY-2悬浮细胞死亡发生的影响[J].兰州大学学报(自然科学版),2015,51(4):564-568.WANG Q W,FENG H Q,RONG F H,et al.Effect of alternating oxidase on death of BY-2suspension cells in tobacco under AlCl3stress[J].Journal of Lanzhou University(Natural Sciences),2015,51(4):564-568.
[3]VON UEXKLL HR,MUTERT E Global extent,developmentand economic impact of acid soils[J].Plant Soil,1995,171:1-15.
[4]NOGUEIROL RC,MONTERIRO FA,GRATAO PL,et al.Tropical soils with high aluminum concentrations cause oxidative stress in two tomato genotypes[J].Environ Monit Assess,2015,187:73-89.
[5]LADISLAV TAMAS,JANA HUTTOVA,IGOR MISTRIK.Inhibition of Al-induced root elongation and enhancement of Al-induced peroxidase activity in Al-sensitive and Al-resistant barley cultivars are positively correlated[J].Plant and Soil,2003,250(2):193-200.
[6]霍垲,陆巍,李霞,等.植物细胞外信号分子eATP研究进展[J].植物学报,2014,49(5):618-625.HUO K,LU W,LI X,CHEN P B.Research progress on plant extracellular signal molecule eATP[J].Plant Journal,2014,49(5):618-625.
[7]冯汉青,白晶月,管冬冬,等.胞外ATP通过刺激NADPH氧化酶缓解水杨酸诱导的细胞死亡[J].植物生理学报,2014,50(11):1 639-1 644.FENG H Q,BAI J Y,GUAN D D,JIA L Y,SUN K.Extracellular ATP relieves salicylic acid-induced cell death by stimulating NADPH oxidase[J].Journal of Plant Physiology.2014,50(11):1 639-1 644.
[8]CHIVASA S,NDIMBA B K,SIMON W J,et al.Extracellular ATP functions as an endogenous external metabolite regulating plant cell viability[J].The Plant Cell,2005,17(11):3 019-3 034.
[9]CHIVASA S,TOME D F A,HAMILTON J M,et al.Proteomic analysis of extracellular ATP-regulated proteins identifies ATP synthaseβ-subunit as a novel plant cell death regulator[J].Molecular&Cellular Proteomics,2011,10(3):M110.003905.
[10]SUN J,ZHANG C L A N,DENG S H U R,et al.An ATPsignalling pathway in plant cells:extracellular ATP triggers programmed cell death in Populus euphratica[J].Plant,Cell&Environment,2012,35(5):893-916.
[11]YANG S W,KIM S K,KIM W T.Perturbation of NgTRF1expression induces apoptosis-like cell death in tobacco BY-2cells and implicates NgTRF1in the control of telomere length and stability[J].The Plant Cell,2004,16(12):3 370-3 385.
[12]JONES K,KIM D W,PARK J S,et al.Live-cell fluorescence imaging to investigate the dynamics of plant cell death during infection by the rice blast fungus Magnaporthe oryzae[J].BMC Plant Biology,2016,16(1):69.
[13]KAUR N,SHARMA I,KIRAT K,et al.Detection of reactive oxygen species in Oryza sativa L.(Rice)[J].Bio-protocol,2016,6(24):e2061.doi:1021769/Bioprotoc.2061.
[14]XU X,ZHANG Y,LING Y.The dynamic changes and origin analysis of reactive oxygen species during isolation and culture of Citrus reticulata blanco protoplasts[J].Botanical Research,2016,5(3),67-73.
[15]TANAKA K,GILORY S,JONES A M,et al.Extracellular ATP signaling in plants[J].Trends in Cell Biology,2010,20(10):601-608.
[16]IKEGAWA H,YAMAMOTO Y,MATSUMOTO H.Cell death caused by a combination of aluminum and iron in cultured tobacco cells[J].Physiologia Plantarum,1998,104(3):474-478.
[17]IKEGAWA H,YAMAMOTO Y,MATSUMOTO H.Responsesto aluminiumof suspension-cultured tobacco cells in a simple calcium solution[J].Soil Science and Plant Nutrition,2000,46(2):503-514.
[18]窦伟.硫化氢对盐和铝胁迫下小麦种子萌发及氧化损伤的缓解效应[D].合肥:合肥工业大学,2010.DOU W.Alleviation effect of hydrogen sulfide on wheat seed germination and oxidative damage under salt and aluminum stress[D].Hefei:Hefei University of Technology,2010.
[19]PEJCHAR P,MARTINEC J.Aluminum ions alter the function of non-specific phospholipase C through the changes in plasma membrane physical properties[J].Plant Signaling&Behavior,2015,10(6):e1031938.
[20]YANG D,CHEN D,WANG P,et al.Aluminium-inhibited NO3-uptake is related to Al-increased H2O2content and Aldecreased plasma membrane ATPase activity in the root tips of Al-sensitive black soybean[J].Functional Plant Biology,2017,44(2):198-207.
[21]YU Y,ZHOU W,ZHOU K,et al.Polyamines modulate aluminum-induced oxidative stress differently by inducing or reducing H2O2production in wheat[J].Chemosphere,2018,212:645-653.
[22]RENTEL M C,KNIGHT M R.Oxidative stress-induced calcium signaling in Arabidopsis[J].Plant Physiology,2004,135(3):1 471-1 479.
[23]TAKEDA S,GAPPER C,KAYA H,et al.Local positive feedback regulation determines cell shape in root hair cells[J].Science,2008,319(5 867):1 241-1 244.
[24]KAWANO T,KADNON T,FUMOTO K,et al.Aluminum as a specific inhibitor of plant TPC1Ca2+channels[J].Biochemical and Biophysical Research Communications,2004,324(1):40-45.
[25]PRASAD T K,ANDERSON M D,STEWART C R.Acclimation,hydrogen peroxide,and abscisic acid protect mitochondria against irreversible chilling injury in maize seedlings[J].Plant Physiology,1994,105(2):619-627.
[26]DEMIDCHIK V,SHANG Z,SHIN R,et al.Plant extracellular ATP signalling by plasma membrane NADPH oxidase and Ca2+channels[J].The Plant Journal,2009,58(6):903-913
[27]SUN J,ZHANG X,DENG S,et al.Extracellular ATP signaling is mediated by H2O2and cytosolic Ca2+in the salt response of Populus euphratica cells[J].PLoS One,2012b,7(12):e53136.
[28]CLARK G,ROUX S.Role of Ca2+in mediating plant responses to extracellular ATP and ADP[J].International Journal of Molecular Sciences,2018,19(11):3 590.
[29]ZIPFEL C.Plant pattern-recognition receptors[J].Trends in Immunology,2014,35(7):345-351.