外源草酸对三七皂苷Rg_1自毒效应的缓解作用及机制研究
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摘要
【目的】明确外源添加草酸缓解皂苷Rg_1对三七根系自毒的缓解效应及作用机理,为缓解三七自毒危害提供依据。【方法】采用外源添加的方式,测定了1.0mg/L皂苷Rg_1溶液单独处理及外源添加质量浓度0.1,1.0,5.0mg/L草酸的混合处理对三七种苗根系萎蔫和鲜质量的影响,同时以体积分数0.1%甲醇为空白对照,分析外源添加草酸对Rg_1引起的三七种苗自毒效应的缓解作用。利用硝基四唑蓝氯化物(NBT)活性氧染色法,比较外源添加草酸后三七根尖活性氧的积累情况,测定抗氧化酶系统中与活性氧清除相关的过氧化物酶(POD)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)的活性,以及抗坏血酸-谷胱甘肽(ASC-GSH)循环中抗坏血酸过氧化物酶(APX)和脱氢抗坏血酸还原酶(DHAR)的活性及抗坏血酸(ASC)和脱氢抗坏血酸(DHA)含量,探讨外源添加草酸对Rg_1自毒作用的缓解机制。【结果】1.0mg/L Rg_1单独处理会导致三七植株出现明显萎蔫,萎蔫率达到46.70%,鲜质量减少率达18.10%。外源添加质量浓度为0.1,1.0,5.0mg/L草酸后,三七的萎蔫率显著降低,仅为0～2.50%,鲜质量减少率降低至3.64%～6.17%,明显缓解了由皂苷Rg_1引起的自毒效应。NBT染色结果显示,Rg_1能诱导三七根尖活性氧大量积累,外源添加草酸后根尖积累的活性氧显著减少。酶活性分析表明,Rg_1能降低抗坏血酸-谷胱甘肽(ASC-GSH)循环中的抗坏血酸过氧化物酶(APX)活性和抗坏血酸(ASC)含量,从而降低细胞对氧自由基的清除能力,导致细胞内活性氧的大量积累。外源添加草酸能通过增强过氧化物酶(POD)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)和脱氢抗坏血酸还原酶(DHAR)的活性及增加抗坏血酸(ASC)的含量来提高根系的抗氧化能力。【结论】外源添加草酸能提高ASC-GSH循环中APX和DHAR的活性及抗氧化剂ASC含量,进而增强三七根系的抗氧化能力,有效缓解皂苷Rg_1对三七生长的自毒活性。
【Objective】The aim of this study was to identify the effect and mechanism of exogenous oxalic acid on alleviating the autotoxicity of Rg_1on Panax notoginsengroot and provide evidence for alleviating P.notoginsengautotoxicity damage.【Method】Effect of 1.0mg/L Rg_1solution alone or 1.0mg/L Rg_(1 )with 0.1,1.0,and 5.0mg/L oxalic acid on wilt and fresh weight of P.notoginsengseedlings was tested using exogenously application method.The alleviating effect of oxalic acid on the autotoxicity of Rg_1was analyzed using 0.1%methanol as blank control.The reactive oxygen species(ROS)accumulation in root tip was observed by NBT reactive oxygen stain.The activities of ROS-related enzymes including superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT)and ascorbate-glutathione(ASC-GSH)cycle enzymes including ascorbate peroxidase(APX)and dehydrogenase ascorbate reductase(DHAR),as well as the contents of non-enzymatic antioxidants including ascorbic acid(ASC)and dehydrogenase ascorbic acid(DHA)were determined to explore the alleviating mechanism.【Result】Rg_(1 )with the concentration of 1.0mg/L caused significant wilt and fresh weight reduction of P.notoginsengseedlings.The wilt ratio and fresh weight reduction ratio were 46.70%and 18.10%,respectively.Exogenous oxalic acid with the concentrations of 0.1,1.0,5.0mg/L could significantly reduce the wilt ratio and fresh weight reduction ratio to 0-2.50%and 3.64%-6.17%,which clearly alleviated the toxic influence of Rg_1.NBT staining results showed that Rg_1could lead to accumulation of reactive oxygen in the root tip of P.notoginseng.After adding oxalic acid,the reactive oxygen accumulation in root tip decreased significantly.Enzyme activity analysis showed that Rg_1could inhibit APX in ASC-GSH and decrease the content of ASC,so as to reduce the ability of cells to scavenge oxygen free radicals and lead to accumulation of ROS in cell.Exogenous oxalic acid could help root cell scavenge over-accumulated ROS by promoting activities of SOD,POD,CAT,APX and DHAR and content of ASC.【Conclusion】Exogenous oxalic acid can increase activities of APX and DHAR as well as content of ASC in ASC-GSH cycle,and enhance the anti-oxidative ability of root to effectively relieve the toxicity of Rg_1on P.notoginsenggrowth.
【Objective】The aim of this study was to identify the effect and mechanism of exogenous oxalic acid on alleviating the autotoxicity of Rg_1on Panax notoginsengroot and provide evidence for alleviating P.notoginsengautotoxicity damage.【Method】Effect of 1.0mg/L Rg_1solution alone or 1.0mg/L Rg_(1 )with 0.1,1.0,and 5.0mg/L oxalic acid on wilt and fresh weight of P.notoginsengseedlings was tested using exogenously application method.The alleviating effect of oxalic acid on the autotoxicity of Rg_1was analyzed using 0.1%methanol as blank control.The reactive oxygen species(ROS)accumulation in root tip was observed by NBT reactive oxygen stain.The activities of ROS-related enzymes including superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT)and ascorbate-glutathione(ASC-GSH)cycle enzymes including ascorbate peroxidase(APX)and dehydrogenase ascorbate reductase(DHAR),as well as the contents of non-enzymatic antioxidants including ascorbic acid(ASC)and dehydrogenase ascorbic acid(DHA)were determined to explore the alleviating mechanism.【Result】Rg_(1 )with the concentration of 1.0mg/L caused significant wilt and fresh weight reduction of P.notoginsengseedlings.The wilt ratio and fresh weight reduction ratio were 46.70%and 18.10%,respectively.Exogenous oxalic acid with the concentrations of 0.1,1.0,5.0mg/L could significantly reduce the wilt ratio and fresh weight reduction ratio to 0-2.50%and 3.64%-6.17%,which clearly alleviated the toxic influence of Rg_1.NBT staining results showed that Rg_1could lead to accumulation of reactive oxygen in the root tip of P.notoginseng.After adding oxalic acid,the reactive oxygen accumulation in root tip decreased significantly.Enzyme activity analysis showed that Rg_1could inhibit APX in ASC-GSH and decrease the content of ASC,so as to reduce the ability of cells to scavenge oxygen free radicals and lead to accumulation of ROS in cell.Exogenous oxalic acid could help root cell scavenge over-accumulated ROS by promoting activities of SOD,POD,CAT,APX and DHAR and content of ASC.【Conclusion】Exogenous oxalic acid can increase activities of APX and DHAR as well as content of ASC in ASC-GSH cycle,and enhance the anti-oxidative ability of root to effectively relieve the toxicity of Rg_1on P.notoginsenggrowth.
引文
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[6]郑小林.外源草酸对水果的保鲜效应及其机制研究进展[J].果树学报,2010,27(4):605-610.Zheng X L.Effects of exogenous oxalic acid on fruit during postharvest storage and its mechanism[J].Journal of Fruit Science,2010,27(4):605-610.
[7]梁巧兰,魏列新,徐秉良,等.三种化学物质诱导观赏百合对黑斑病抗性的研究[J].植物保护,2011,37(2):36-40.Liang Q L,Wei L X,Xu B L,et al.Resistance of ornamental lily against Alternaria alternata induced by three chemicals[J].Plant Protection,2011,37(2):36-40.
[8]刘喜存,刘红彦,倪云霞,等.不同化学诱抗剂对金银花叶片防御酶系的影响[J].植物保护,2009,35(2):75-77.Liu X C,Liu H Y,Ni Y X,et al.Effects of different chemical inducers on the defense-related enzymes in honeysuckle[J].Plant Protection,2009,35(2):75-77.
[9]陈年来,朱振家,安翠香,等.诱抗处理对甜瓜叶片防卫酶活性的影响[J].西北植物学报,2008,28(7):1354-1358.Chen N L,Zhu Z J,An C X,et al.Effects of chemical elicitors on several defense-related enzyme activities in muskmelon leaves[J].Acta Botanica Boreali-Occidentalia Sinica,2008,28(7):1354-1358.
[10]陈晓亚,薛红卫.植物生理与分子生物学[M].4版.北京:高等教育出版社,2012:628-629.Chen X Y,Xue H W.Plant physiology and molecular biology[M].4th ed.Beijing:Higher Education Press,2012:628-629.
[11]Dutilleul C,Garmier M,Noctor G,et al.Leaf mitochondria modulate whole cell redox homeostasis,set antioxidant capacity,and determine stress resistance through altered signaling and diurnal regulation[J].Plant Cell,2003,15(5):1212-1226.
[12]Wu L B,Ueda Y,Lai S K,et al.Shoot tolerance mechanisms to iron toxicity in rice(Oryza sativa L.)[J].Plant Cell&Environment,2017,40(4):570-584.
[13]Chen L,Ying H,Hao J,et al.Nitrogen nutrient status induces sexual differences in responses to cadmium in Populus yunnanensis[J].Journal of Experimental Botany,2011,62(14):5037-5050.
[14]Yu J Q,Ye S F,Zhang M F,et al.Effects of root exudates and aqueous root extracts of cucumber(Cucumis sativus),and allelochemicals on photosynthesis and antioxidant enzymes in cucumber[J].Biochemical Systematics and Ecology,2003,31:129-139.
[15]Laranu1ez A,Romeroromero T,Ventura J L,et al.Allelochemical stress causes inhibition of growth and oxidative damage in Lycopersicon esculentum Mill[J].Plant Cell&Environment,2006,29(11):2009-2016.
[16]Kobayashi D,Kondo K,Uehara N,et al.Endogenous reactive oxygen species is an important mediator of miconazole antifungal effect[J].Antimicrobial Agents&Chemotherapy,2002,46(10):3113-3117.
[17]Wu B,Long H Q,Gao Y,et al.Comprehensive characterization of a time-course transcriptional response induced by autotoxins in Panax ginseng using RNA-Seq[J].BMC Genomics,2015,16(1):1010-1025.
[18]Chi W,Chen Y,Hsiung Y,et al.Autotoxicity mechanism of Oryza sativa:transcriptome response in rice roots exposed to ferulic acid[J].BMC Genomics,2013,14:1-18.
[19]Lin W X,Kim K U,Shin D H.Rice allelopathic potential and its modes of action on barnyardgrass(Echinochloa crusgalli)[J].Allelopathy Journal,2000,7(2):215-224.
[20]Zeng R S,Luo S M,Shi Y H,et al.Physiological and biochemical mechanism of allelopathy of secalonic acid F on higher plants[J].Agronomy Journal,2001,93(1):72-79.
[21]Baxter A,Mittler R,Suzuki N.ROS as key players in plant stress signalling[J].Journal of Experimental Botany,2014,65(5):1229-1240.
[22]Shi W G,Li H,Liu T X,et al.Exogenous abscisic acid alleviates zinc uptake and accumulation in Populus canescens exposed to excess zinc[J].Plant Cell&Environment,2015,38(1):207-223.
[23]Gill S S,Tuteja N.Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants[J].Plant Physiology&Biochemistry,2010,48(12):909-930.
[24]Green M A,Fry S C.Vitamin C degradation in plant cells via enzymatic hydrolysis of 4-O-oxalyl-l-threonate[J].Nature,2005,433(7021):83-87.
[25]Beyer R E.The role of ascorbate in antioxidant protection of biomembranes:interaction with vitamin E and coenzyme Q[J].Journal of Bioenergetics&Biomembranes,1994,26(4):349-358.
[26]Navari-Izzo F,Meneguzzo S,Loggini B,et al.The role of the glutathione system during dehydration of Boea hygroscopica[J].Physiologia Plantarum,1997,99(1):23-30.
[27]Jiang Y,Yang B,Harris N S,et al.Comparative proteomic analysis of NaCl stress-responsive proteins in Arabidopsis roots[J].Journal of Experimental Botany,2007,58(13):3591-3607.
[28]Bose J,Rodrigomoreno A,Shabala S.ROS homeostasis in halophytes in the context of salinity stress tolerance[J].Journal of Experimental Botany,2014,65(5):1241-1257.
[29]丁继军.外源ASC、GSH对土壤Cd胁迫下石竹幼苗抗性生理的影响及其机理[D].成都:四川农业大学,2013.Ding J J.Effects and mechanism of exogenous ascorbic acid,glutathiose on the resistance physiology of Dianthus chinensis seedlings exposed to cadmium in the soil[D].Chengdu:Sichuan Agricultural University,2013.
[30]陈梅,陈亚华,沈振国,等.外源有机酸对小麦幼苗铝毒的缓解作用[J].植物生理与分子生物学学报,2003,29(4):281-288.Chen M,Chen Y H,Shen Z G,et al.Alleviation of aluminum toxicity in wheat seedlings by exogenous organic acid[J].Journal of Plant Physiology and Molecular Biology,2003,29(4):281-288.
[31]刘拥海,俞乐,陈秀云,等.外源草酸对水稻幼苗铝毒害的缓解作用[J].湖南农业大学学报(自然科学版),2008,34(3):281-283,340.Liu Y H,Yu L,Chen X Y,et al.Alleviation of aluminum toxicity in rice seedlings by exogenous oxalic acid[J].Journal of Hunan Agricultural University(Natural Sciences Edition),2008,34(3):281-283,340.
[32]金婷婷,刘鹏,徐根娣,等.外源有机酸对铝毒胁迫下大豆根系形态的影响[J].中国油料作物学报,2006,28(3):302-308.Jin T T,Liu P,Xu G D,et al.Effect of exogenous organic acids on morphological characteristics of soybean roots under aluminum stress[J].Chinese Journal of Oil Crop Sciences,2006,28(3):302-308.
[2]刘莉,刘大会,金航,等.三七连作障碍的研究进展[J].山地农业生物学报,2011,30(1):70-75.Liu L,Liu D H,Jin H,et al.Overview on mechanisms and control methods of sequential cropping obstacle of Panax notoginseng F.H.Chen[J].Journal of Mountain Agriculture and Biology,2011,30(1):70-75.
[3]Yang M,Zhang X D,Xu Y G,et al.Autotoxic ginsenosides in the rhizosphere contribute to the replant failure of Panax notoginseng[J].PLoS One,2015,10(2):e0118555.
[4]杨敏.三七根系皂苷的自毒作用机制研究[D].昆明:云南农业大学,2015.Yang M.Investigation on autotoxic mechanism of ginsenosides from Panax notoginseng roots[D].Kunming:Yunnan Agricultural University,2015.
[5]刘高峰.NO和钙信使系统在草酸诱导黄瓜叶片抗霜霉病中的作用[J].西北植物学报,2012,32(5):969-974.Liu G F.Role of nitric oxide and calcium signal of cucumber leaves to Pseudoperonospora cubensis[J].Acta Botanica Boreali-Occidentalia Sinica,2012,32(5):969-974.
[6]郑小林.外源草酸对水果的保鲜效应及其机制研究进展[J].果树学报,2010,27(4):605-610.Zheng X L.Effects of exogenous oxalic acid on fruit during postharvest storage and its mechanism[J].Journal of Fruit Science,2010,27(4):605-610.
[7]梁巧兰,魏列新,徐秉良,等.三种化学物质诱导观赏百合对黑斑病抗性的研究[J].植物保护,2011,37(2):36-40.Liang Q L,Wei L X,Xu B L,et al.Resistance of ornamental lily against Alternaria alternata induced by three chemicals[J].Plant Protection,2011,37(2):36-40.
[8]刘喜存,刘红彦,倪云霞,等.不同化学诱抗剂对金银花叶片防御酶系的影响[J].植物保护,2009,35(2):75-77.Liu X C,Liu H Y,Ni Y X,et al.Effects of different chemical inducers on the defense-related enzymes in honeysuckle[J].Plant Protection,2009,35(2):75-77.
[9]陈年来,朱振家,安翠香,等.诱抗处理对甜瓜叶片防卫酶活性的影响[J].西北植物学报,2008,28(7):1354-1358.Chen N L,Zhu Z J,An C X,et al.Effects of chemical elicitors on several defense-related enzyme activities in muskmelon leaves[J].Acta Botanica Boreali-Occidentalia Sinica,2008,28(7):1354-1358.
[10]陈晓亚,薛红卫.植物生理与分子生物学[M].4版.北京:高等教育出版社,2012:628-629.Chen X Y,Xue H W.Plant physiology and molecular biology[M].4th ed.Beijing:Higher Education Press,2012:628-629.
[11]Dutilleul C,Garmier M,Noctor G,et al.Leaf mitochondria modulate whole cell redox homeostasis,set antioxidant capacity,and determine stress resistance through altered signaling and diurnal regulation[J].Plant Cell,2003,15(5):1212-1226.
[12]Wu L B,Ueda Y,Lai S K,et al.Shoot tolerance mechanisms to iron toxicity in rice(Oryza sativa L.)[J].Plant Cell&Environment,2017,40(4):570-584.
[13]Chen L,Ying H,Hao J,et al.Nitrogen nutrient status induces sexual differences in responses to cadmium in Populus yunnanensis[J].Journal of Experimental Botany,2011,62(14):5037-5050.
[14]Yu J Q,Ye S F,Zhang M F,et al.Effects of root exudates and aqueous root extracts of cucumber(Cucumis sativus),and allelochemicals on photosynthesis and antioxidant enzymes in cucumber[J].Biochemical Systematics and Ecology,2003,31:129-139.
[15]Laranu1ez A,Romeroromero T,Ventura J L,et al.Allelochemical stress causes inhibition of growth and oxidative damage in Lycopersicon esculentum Mill[J].Plant Cell&Environment,2006,29(11):2009-2016.
[16]Kobayashi D,Kondo K,Uehara N,et al.Endogenous reactive oxygen species is an important mediator of miconazole antifungal effect[J].Antimicrobial Agents&Chemotherapy,2002,46(10):3113-3117.
[17]Wu B,Long H Q,Gao Y,et al.Comprehensive characterization of a time-course transcriptional response induced by autotoxins in Panax ginseng using RNA-Seq[J].BMC Genomics,2015,16(1):1010-1025.
[18]Chi W,Chen Y,Hsiung Y,et al.Autotoxicity mechanism of Oryza sativa:transcriptome response in rice roots exposed to ferulic acid[J].BMC Genomics,2013,14:1-18.
[19]Lin W X,Kim K U,Shin D H.Rice allelopathic potential and its modes of action on barnyardgrass(Echinochloa crusgalli)[J].Allelopathy Journal,2000,7(2):215-224.
[20]Zeng R S,Luo S M,Shi Y H,et al.Physiological and biochemical mechanism of allelopathy of secalonic acid F on higher plants[J].Agronomy Journal,2001,93(1):72-79.
[21]Baxter A,Mittler R,Suzuki N.ROS as key players in plant stress signalling[J].Journal of Experimental Botany,2014,65(5):1229-1240.
[22]Shi W G,Li H,Liu T X,et al.Exogenous abscisic acid alleviates zinc uptake and accumulation in Populus canescens exposed to excess zinc[J].Plant Cell&Environment,2015,38(1):207-223.
[23]Gill S S,Tuteja N.Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants[J].Plant Physiology&Biochemistry,2010,48(12):909-930.
[24]Green M A,Fry S C.Vitamin C degradation in plant cells via enzymatic hydrolysis of 4-O-oxalyl-l-threonate[J].Nature,2005,433(7021):83-87.
[25]Beyer R E.The role of ascorbate in antioxidant protection of biomembranes:interaction with vitamin E and coenzyme Q[J].Journal of Bioenergetics&Biomembranes,1994,26(4):349-358.
[26]Navari-Izzo F,Meneguzzo S,Loggini B,et al.The role of the glutathione system during dehydration of Boea hygroscopica[J].Physiologia Plantarum,1997,99(1):23-30.
[27]Jiang Y,Yang B,Harris N S,et al.Comparative proteomic analysis of NaCl stress-responsive proteins in Arabidopsis roots[J].Journal of Experimental Botany,2007,58(13):3591-3607.
[28]Bose J,Rodrigomoreno A,Shabala S.ROS homeostasis in halophytes in the context of salinity stress tolerance[J].Journal of Experimental Botany,2014,65(5):1241-1257.
[29]丁继军.外源ASC、GSH对土壤Cd胁迫下石竹幼苗抗性生理的影响及其机理[D].成都:四川农业大学,2013.Ding J J.Effects and mechanism of exogenous ascorbic acid,glutathiose on the resistance physiology of Dianthus chinensis seedlings exposed to cadmium in the soil[D].Chengdu:Sichuan Agricultural University,2013.
[30]陈梅,陈亚华,沈振国,等.外源有机酸对小麦幼苗铝毒的缓解作用[J].植物生理与分子生物学学报,2003,29(4):281-288.Chen M,Chen Y H,Shen Z G,et al.Alleviation of aluminum toxicity in wheat seedlings by exogenous organic acid[J].Journal of Plant Physiology and Molecular Biology,2003,29(4):281-288.
[31]刘拥海,俞乐,陈秀云,等.外源草酸对水稻幼苗铝毒害的缓解作用[J].湖南农业大学学报(自然科学版),2008,34(3):281-283,340.Liu Y H,Yu L,Chen X Y,et al.Alleviation of aluminum toxicity in rice seedlings by exogenous oxalic acid[J].Journal of Hunan Agricultural University(Natural Sciences Edition),2008,34(3):281-283,340.
[32]金婷婷,刘鹏,徐根娣,等.外源有机酸对铝毒胁迫下大豆根系形态的影响[J].中国油料作物学报,2006,28(3):302-308.Jin T T,Liu P,Xu G D,et al.Effect of exogenous organic acids on morphological characteristics of soybean roots under aluminum stress[J].Chinese Journal of Oil Crop Sciences,2006,28(3):302-308.