铜胁迫对蚕豆根尖细胞凋亡及线粒体功能的影响
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摘要
为了揭示重金属铜对植物的伤害机制,对铜胁迫下蚕豆根尖细胞凋亡与线粒体功能关系进行了分析.用1.0mmol·L-1硫酸铜处理新萌发的蚕豆(Vicia faba)根尖4h,根系生长抑制率达60%,根尖细胞活力明显受到抑制.经荧光染料丫啶橙(AO)和溴化乙锭(EB)双染后,可见细胞凋亡特征,凋亡率达61.7%.同时,线粒体膜通透性明显增大,线粒体膜电位和线粒体内Cyt c/a吸光度比值下降,说明在铜胁迫下线粒体膜受到损伤.二氨基联苯胺(3,3′-diaminobenzidine,DAB)染色实验证明硫酸铜可以诱导蚕豆根尖活性氧的积累,根尖中H2O2的含量比对照组提高2.5倍.利用过氧化氢酶(catalase,CAT)预处理分解铜胁迫诱导的活性氧,可以降低细胞凋亡率和线粒体膜损伤.实验结果证明,活性氧可能介导铜胁迫造成的根尖生长抑制,是植物在遭受重金属铜胁迫时细胞凋亡和线粒体膜损伤的重要生理信号.
To reveal the mechanism of heavy metal copper(Cu)toxicity on plants,the effects of Cu on cell apoptosis and mitochondrial functions were investigated in root tips of Vicia faba L.We found that CuSO4 at 1.0mmol·L-1stunted root growth by 60% and depressed the root viability seriously.Cu treatment also induced the apoptosis characterized by AO/EB dye,while the apoptosis rate reached 61.7%after 4htreatment of CuSO4.Simultaneously,the mitochondrial permeability was increased by Cu treatment,while mitochondrial membrane potential decreased significantly.In addition,the ratio of Cyt c/a was decreased by Cu treatment,suggesting a mitochondrial dysfunction induced by Cu.The dye of 3,3′-diaminobenzidine(DAB)demonstrated an accumulation of reactive oxygen species in root tips.The H2O2 production was enhanced by 2.5-fold under Cu stress.The cell apoptosis and the mitochondrial injury induced by Cu were mitigated while using pretreatment of catalase(CAT),indicating a role of ROS in theCu-induced physiological responses.Experimental results demonstrated that ROS could mediate Cu-induced growth inhibition and is important plant signal for cell apoptosis and mitochondrial dysfunction under Cu stress.
To reveal the mechanism of heavy metal copper(Cu)toxicity on plants,the effects of Cu on cell apoptosis and mitochondrial functions were investigated in root tips of Vicia faba L.We found that CuSO4 at 1.0mmol·L-1stunted root growth by 60% and depressed the root viability seriously.Cu treatment also induced the apoptosis characterized by AO/EB dye,while the apoptosis rate reached 61.7%after 4htreatment of CuSO4.Simultaneously,the mitochondrial permeability was increased by Cu treatment,while mitochondrial membrane potential decreased significantly.In addition,the ratio of Cyt c/a was decreased by Cu treatment,suggesting a mitochondrial dysfunction induced by Cu.The dye of 3,3′-diaminobenzidine(DAB)demonstrated an accumulation of reactive oxygen species in root tips.The H2O2 production was enhanced by 2.5-fold under Cu stress.The cell apoptosis and the mitochondrial injury induced by Cu were mitigated while using pretreatment of catalase(CAT),indicating a role of ROS in theCu-induced physiological responses.Experimental results demonstrated that ROS could mediate Cu-induced growth inhibition and is important plant signal for cell apoptosis and mitochondrial dysfunction under Cu stress.
引文
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[17]Tonshin A A,Saprunova V B,Solodovnikova I M,et al.Functional activity and ultrastructure of mitochondria isolated from myocardial apoptotic tissue.Biochemistry,2003,68(8):875-881.
[18]Kerr J F,Wyllie A H,Currie A R.Apoptosis:a basic biological phenomenon with wide-ranging implications in tissue kinetics.British Journal of Cancer,1972,26(4):239-257.
[19]McConkey D J,Hartzell P,Nicotera P,et al.Stimulation of endogenous endonuclease activity in hepatocytes exposed to oxidative stress.Toxicology Letters,1988,42(2):123-130.
[20]Hautegem T V,Waters A J,Goodrich J,et al.Only in dying,life:programmed cell death during plant development.Trends in Plant Science,2015,20(2):102-113.
[21]Delledonne M,Zeier J,Marocco A,et al.Signal interactions between nitric oxide and reactive oxygen intermediates in the plant hypersensitive diseaseresistanceresponse. Proceedingof National Academy of Sciences USA,2001,98(23):13454-13459.
[22]Desikan R,Reynolds A,Hancock J T,et al.Harpin and hydrogen peroxide both initiate programmed cell death but have differential effects on defence gene expression in Arabidopsis suspension cultures.Biochemistry Journal,1998,330:115-120.
[23]Tiwari B S,Belenghi B,Levine A.Oxidative stress increased respiration and generation of reactive oxygen species,resulting in depletion,opening of mitochondrial permeability transition,and programmed cell death.Plant Physiology,2002,128(4):1271-1281.
[2] Fernandes J C,Henrique F S.Biochemical,physiological and structural effects of excess copper in plants.Botanical Review,1991,57:246-273.
[3] Wainwright S J, Woolhouse H W. Some physiological aspects of copper and zinc tolerance in Agrostis tenuis Sibth.:cell elongation and membrane damage.Journal of Experimental Botany,1977,28:1029-1036.
[4] Mittler R,Vanderauwera S,Gollery M,et al.Abiotic stress series.Reactive oxygen gene network of plants.Trends of Plant Science,2004,9(10):490-498.
[5] Khatun S,Hahn E J,Paek K Y.Copper toxicity in Withania somnifera:Growth and antioxidant enzyme responses of in vitro grown plants.Environmental and Experimental Botany,2008,64:279-285.
[6] Hung W C,Huang D D,Chien P S,et al.Protein tyrosine dephosphorylation during copper-induced cell death in rice roots.Chemosphere,2007,69:55-62.
[7] Houot V,Etienne P,Suty L.Hydrogen peroxide induces programmed cell death features in cultured tobacco BY-2cells,in a dose-dependent manner.Journal of Experimental Botany,2001,52:1721-1730.
[8] Zandalinas S I,Mittler R.ROS-induced ROS release in plant and animal cells.Free Radical Biology and Medicine,2018,122:21-27.
[9] Wang Y,Lin J S,Wang G X.Calcium-mediated mitochondrial permeability transition involved in hydrogen peroxide-induced apoptosis in tobacco protoplasts.Journal of Integrative Plant Biology,2006,48(1):1-5.
[10]Pan J W,Zhu M Y,Chen H.Aluminum-induced celldeathinroot-tipcellsofbarley.Environmental Experimental Botany,2001,46:71-79.
[11]Baker C J,Mock N M.An improved method for monitoring cell death in cell suspension and leaf disc assays using evans blue.Plant Cell,Tissue Organ Culture,1994,39(1):7-12.
[12]Tewari R K,Kumar P,Sharma P N.Antioxidant responses to enhanced generation of superoxide anion radical and hydrogen peroxide in the copper-stressed mulberry plants.Planta,2006,223:1145-1153.
[13]Sergiev I,Alexieva V,Karanov E.Effect of spermine,atrazine and combination between them on some endogenous protective systems and stress markers in plant.Comptes Rendus de l Academie Bulgare des Sciences,1997,51:121-124.
[14]Petrussa E,Bertolini A,Krajnakova J.Isolation of mitochondria from embryogenic cultures of Picea abies(L.)Karst.and Abies cephalonica Loud.:characterization of a K+ATPchannel.Plant Cell Reports,2008,27:137-146.
[15]金超芳,沈生荣,赵保路.EGCG对线粒体PT孔开放及Ca2+转运的影响.茶叶科学,2002,22(1):14-18.(Jin C R,Shen S R,Zhao B L.Influences of EGCG on mitochondrial PTP opening and Ca2+transportation.Journal of Tea Science,22(1):14-18.)
[16]Braidot E,Petrussa E,Macri F,et al.Plant mitochondrial electrical potential monitored by fluorescence quenchingofrhodamine123.Biologia Plantarum,1998,2:193-201.
[17]Tonshin A A,Saprunova V B,Solodovnikova I M,et al.Functional activity and ultrastructure of mitochondria isolated from myocardial apoptotic tissue.Biochemistry,2003,68(8):875-881.
[18]Kerr J F,Wyllie A H,Currie A R.Apoptosis:a basic biological phenomenon with wide-ranging implications in tissue kinetics.British Journal of Cancer,1972,26(4):239-257.
[19]McConkey D J,Hartzell P,Nicotera P,et al.Stimulation of endogenous endonuclease activity in hepatocytes exposed to oxidative stress.Toxicology Letters,1988,42(2):123-130.
[20]Hautegem T V,Waters A J,Goodrich J,et al.Only in dying,life:programmed cell death during plant development.Trends in Plant Science,2015,20(2):102-113.
[21]Delledonne M,Zeier J,Marocco A,et al.Signal interactions between nitric oxide and reactive oxygen intermediates in the plant hypersensitive diseaseresistanceresponse. Proceedingof National Academy of Sciences USA,2001,98(23):13454-13459.
[22]Desikan R,Reynolds A,Hancock J T,et al.Harpin and hydrogen peroxide both initiate programmed cell death but have differential effects on defence gene expression in Arabidopsis suspension cultures.Biochemistry Journal,1998,330:115-120.
[23]Tiwari B S,Belenghi B,Levine A.Oxidative stress increased respiration and generation of reactive oxygen species,resulting in depletion,opening of mitochondrial permeability transition,and programmed cell death.Plant Physiology,2002,128(4):1271-1281.