外源H_2O_2对盐胁迫下黄瓜幼苗氧化胁迫及抗氧化系统的影响
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摘要
为阐明外源H_2O_2缓解盐胁迫的生理机制,以水培法培养的黄瓜‘春夏秋王’幼苗为试材,采用0、1、5、10μmol·L~(-1) H_2O_2处理200 mmol·L~(-1)NaCl胁迫的黄瓜幼苗。通过测定黄瓜幼苗的叶面积、株高、根长、叶鲜质量、根鲜质量及根干质量等指标,结果显示外源H_2O_2缓解了NaCl胁迫对植株生长的抑制,其中5μmol·L~(-1)H_2O_2对盐胁迫下黄瓜幼苗的缓解效应最有效。H_2O_2不仅降低盐胁迫下黄瓜幼苗叶片和根系O■、H_2O_2和丙二醛质量摩尔浓度及相对电导率,也提高了还原型谷胱甘肽质量分数、抗坏血酸质量摩尔浓度和过氧化物酶、谷胱甘肽还原酶活性。此外,不同浓度H_2O_2处理引起盐胁迫下黄瓜叶片甲基转移酶PMT5和铁氧还原蛋白的谷氨酸合成酶上调-下调-上调的变化模式,而在根中网格蛋白重链1表现出逐渐下调和热休克蛋白70样蛋白15逐渐上调的模式。以上结果表明,外源H_2O_2通过增强黄瓜的抗氧化系统来降低盐胁迫引起的氧化胁迫,同时调控相关蛋白质的表达,从而缓解NaCl胁迫造成的伤害。
In order to elucidate the physiological mechanism of exogenous H_2O_2, so as to alleviate salt stress, seedlings of Cucumis sativus L.‘Chunxiaqiuwang' grown in hydroponic culture were used as test materials. 0, 1, 5, 10 μmol·L~(-1) H_2O_2 were chosen to treat the seedlings of cucumber in response to 200 mmol·L~(-1) NaCl stress. The results showed that exogenous H_2O_2 treatment alleviated the growth inhibition of cucumber under NaCl stress, which was reflected by the indexes of leaf area, shoot height, root length, shoot fresh mass, root fresh mass, root dry mass and so on. 5 μmol·L~(-1) H_2O_2 was the most effective in relieving the cucumber seedlings under salt stress. Application of H_2O_2 not only reduced the molality of endogenous O■, H_2O_2 and malondialdehyde as well as relative conductivity in both leaves and roots of cucumber seedlings under salt stress, but also increased the content of reduced glutathione and ascorbic acid and the activities of peroxidase and glutathione reductase. Additionally, different concentrations of H_2O_2 treatment resulted in a up-down-up expression model of the probable methyltransferase PMT5 and ferredoxin-dependent glutamatesynthase(chloroplastic isoform X2) in the cucumber leaves, while clathrin heavy chain 1 showed a down-regulation pattern and heat shock protein 70-like protein 15 a showed a up-regulation in the roots of cucumber seedlings with the H_2O_2 treatments. The results indicated that exogenous H_2O_2 alleviated the damage caused by NaCl stress, enhancing the antioxidant system could reduce the oxidative stress and regulate the expression of some proteins in cucumber.
In order to elucidate the physiological mechanism of exogenous H_2O_2, so as to alleviate salt stress, seedlings of Cucumis sativus L.‘Chunxiaqiuwang' grown in hydroponic culture were used as test materials. 0, 1, 5, 10 μmol·L~(-1) H_2O_2 were chosen to treat the seedlings of cucumber in response to 200 mmol·L~(-1) NaCl stress. The results showed that exogenous H_2O_2 treatment alleviated the growth inhibition of cucumber under NaCl stress, which was reflected by the indexes of leaf area, shoot height, root length, shoot fresh mass, root fresh mass, root dry mass and so on. 5 μmol·L~(-1) H_2O_2 was the most effective in relieving the cucumber seedlings under salt stress. Application of H_2O_2 not only reduced the molality of endogenous O■, H_2O_2 and malondialdehyde as well as relative conductivity in both leaves and roots of cucumber seedlings under salt stress, but also increased the content of reduced glutathione and ascorbic acid and the activities of peroxidase and glutathione reductase. Additionally, different concentrations of H_2O_2 treatment resulted in a up-down-up expression model of the probable methyltransferase PMT5 and ferredoxin-dependent glutamatesynthase(chloroplastic isoform X2) in the cucumber leaves, while clathrin heavy chain 1 showed a down-regulation pattern and heat shock protein 70-like protein 15 a showed a up-regulation in the roots of cucumber seedlings with the H_2O_2 treatments. The results indicated that exogenous H_2O_2 alleviated the damage caused by NaCl stress, enhancing the antioxidant system could reduce the oxidative stress and regulate the expression of some proteins in cucumber.
引文
[1] LIANG W J,MA X L,WAN P,et al.Plant salt-tolerance mechanism:a review.[J].Biochemical & Biophysical Research Communications,2018,495(1):286-291.
[2] YANG Y Q,GUO Y.Unraveling salt stress signaling in plants[J].Journal of Integrative Plant Biology,2018,60(9):796-804.
[3] KALEEM F,SHABIR G,ASLAM K,et al.An overview of the genetics of plant response to salt stress:present status and the way forward[J].Applied Biochemistry and Biotechnology,2018,186(2):306-334.
[4] 单长卷,杨天佑.谷胱甘肽对盐胁迫下玉米幼苗抗氧化特性和光合性能的影响[J].西北农业学报,2017,26(2):185-191.SHAN CH J,YANG T Y.Effects of glutathione on antioxidant and photosynthetic properties of maize seedlings under salt stress[J].Acta Agriculturae Boreali-occidentalis Sinica,2017,26(2):185-191.
[5] ZHOU Y L,HUO S F,WANG L T,et al.Exogenous 24-epibrassinolide alleviates oxidative damage from copper stress in grape(Vitis vinifera l.) cuttings[J].Plant Physiology and Biochemistry,2018,130,555-565.
[6] LI J T,QIU Z B,ZHANG X W,et al.Exogenous hydrogen peroxide can enhance tolerance of wheat seedlings to salt stress[J].Acta Physiologiae Plantarum,2011,33(3):835-842.
[7] WU J Q,SHU S,LI C C,et al.Spermidine-mediated hydrogen peroxide signaling enhances the antioxidant capacity of salt-stressed cucumber roots[J].Plant Physiology and Biochemistry,2018,128:152-162.
[8] 马风勇,石晓霞,许兴,等.拟南芥SOS基因家族与植物耐盐性研究进展[J].中国农学通报,2013,29(21):121-125.MA F Y,SHI X X,XU X,et al.Research development of SOS gene family underlying salt tolerance in plant[J].Chinese Agricultural Science Bulletin,2013,29(21):121-125.
[9] 蔡丹,郑易之,兰英.大豆LEA蛋白Em的表达可提高大肠杆菌和烟草耐盐性[J].深圳大学学报理工版,2006,23(3):230-236.CAI D,ZHENG Y ZH,LAN Y.Expression of Em gene(LEA1) from soybean mimature seeds confers salt tolerance to Escherichia coli and tobacco plants[J].Journal of Shenzhen University Science and Engineering,2006,23(3):230-236.
[10] 赫卫,刘林,关荣霞,等.大豆耐盐相关基因GmNcl1的序列单倍型及表达分析[J].中国农业科学,2014,47(3):411-421.HE W,LIU L,GUAN R X,et al.Haplotypes and expression analysis of salt-tolerant gene GmNcl1 in soybean[J].Scientia Agricultura Sinica,2014,47(3):411-421.
[11] 蒋景龙,沈季雪,徐卫平,等.外源H2O2对低温胁迫下大红柑生长及叶片生理指标的影响[J].浙江农业学报,2016,28(7):1164-1170.JIANG J L,SHEN J X,XU W P,et al.Effects of exogenous hydrogen peroxide on growth and physiological index of Citrus reticulata Blanco cv.Dahonggan leaves under low temperature stress[J].Acta Agriculturae Zhejiangensis,2016,28(7):1164-1170.
[12] LIU Z J,GUO Y K,BAI J G.Exogenous hydrogen peroxide changes antioxidant enzyme activity and protects ultrastructure in leaves of two cucumber ecotypes under osmotic stress[J].Journal of Plant Growth Regulation,2010,29(2):171-183.
[13] GAO Y,GUO Y K,LIN S H,et al.Hydrogen peroxide pretreatment alters the activity of antioxidant enzymes and protects chloroplast ultrastructure in heat-stressed cucumber leaves[J].Scientia Horticulturae,2010,126(1):20-26.
[14] ZHANG X L,JIA X F,YU B,et al.Exogenous hydrogen peroxide influences antioxidant enzyme activity and,lipid peroxidation in cucumber leaves at low light[J].Scientia Horticulturae,2011,129(4):656-662.
[15] SUN Y,WANG H,LIU S,et al.Exogenous application of hydrogen peroxide alleviates drought stress in cucumber seedlings[J].South African Journal of Botany,2016,106:23-28.
[16] 龚建华,向军.黄瓜群体叶面积无破坏性速测方法研究[J].中国蔬菜,2001(4):7-9.GONG J H,XIANG J.Studies on a quick intact measurement to cucumber colony’s leaf area[J].China Vegetables,2001(4):7-9.
[17] 蒋景龙.H2O2胁迫对山黧豆生理生化指标的影响[J].西北农业学报,2016,25(2):243-248.JIANG J L.Effects of H2O2 stress on physiological and biochemical indexes of grass pea[J].Acta Agriculturae Boreali-occidentalis Sinica,2016,25(2):243-248.
[18] 田雲,蒋景龙,沈季雪,等.低温胁迫对5个栽培品种黄瓜生长及生理指标的影响[J].分子植物育种,2017,15(5):352-358.TIAN Y,JIANG J L,SHEN J X,et al.Effect of low temperature stress on growth and physiological indexes of five cultivars of cucumber[J].Molecular Plant Breeding,2017,15(5):352-358.
[19] JIANG J L,SU M ,WANG L Y,et al.Exogenous hydrogen peroxide reversibly inhibits root gravitropism and induces horizontal curvature of primary root during grass pea germination[J].Plant Physiology & Biochemistry Ppb,2012,53:84-93.
[20] WU Q F,LI C,KE L,et al.A high-efficiency,two-dimensional gel electrophoresis platform for mature leaves of grass pea(Lathyrus sativus L.)[J].Acta Physiologiae Plantarum,2011,33(6):2387-2397.
[21] 李金亭,赵萍萍,邱宗波,等.外源H2O2对盐胁迫下小麦幼苗生理指标的影响[J].西北植物学报,2012,32(9):1796-1801.LI J T,ZHAO P P,QIU Z B,et al.Effects of exogenous hydrogen peroxide on the physiological index of wheat seedlings under salt stress[J].Acta Botanica Boreali-Occidentalia Sinica,2012,32(9):1796-1801.
[22] 石玉,潘媛媛,张毅,等.光碳核肥对盐胁迫下黄瓜幼苗生长抑制的缓解效应[J].西北农业学报,2017,26(5):752-758.SHI Y,PAN Y Y,ZHANG Y,et al.Mitigative effect of carbon dioxide trapping agent on inhibiting growth of cucumber seedings under nacl stress[J].Acta Agriculturae Boreali-occidentalis Sinica,2017,26(5):752-758.
[23] FEDINA I S,NEDEVA D,CICEK N.Pre-treatment with H2O2 induces salt tolerance in barley seedlings[J].Biologia Plantarum,2009,53(2):321-324.
[24] 孙德智,韩晓日,彭靖,等.外源NO和SA对盐胁迫下番茄幼苗叶片膜脂过氧化及AsA-GSH循环的影响[J].植物科学学报,2018,36(4):612-622.SUN D ZH,HAN X R,PENG J,et al.Effects of exogenous nitric oxide and salicylic acid on membrane peroxidation and the ascorbate-glutathione cycle in leaves of Lycopersicon esculentum seedlings under NaCl stress[J].Plant Science Journal,2018,36(4):612-622.
[25] 曹齐卫,李利斌,孔素萍,等.不同黄瓜品种幼苗对等渗 Mg(NO3)2和NaCl胁迫的生理响应[J].应用生态学报,2015,26(4):1171-1178.CAO Q W,LI L B,KONG S P,et al.Physiological responses of different cucumber cultivars seedlings to iso-osmotic Mg(NO3)2 and NaCl stress[J].Chinese Journal of Applied Ecology,2015,26(4):1171-1178.
[26] 杨建军,张国斌,郁继华,等.盐胁迫下内源NO对黄瓜幼苗活性氧代谢和光合特性的影响[J].中国农业科学,2017,50(19):3778-3788.YANG J J,ZHANG G B,YU J H,et al.Effects of endogenous NO on reactive oxygen metabolism and photosynthetic characteristics of cucumber seedlings under salt stress[J].Scientia Agricultura Sinica,2017,50(19):3778-3788.
[27] ZHANG Z,ZHANG S,ZHANG Y,et al.Arabidopsis floral initiator SKB1 confers high salt tolerance by regulating transcription and pre-mRNA splicing through altering histone H4R3 and small nuclear ribonucleoprotein LSM4 methylation[J].Plant Cell,2011,23(1):396-411.
[28] 侯毅枫,邓娴,陆天聪,等.蛋白质精氨酸甲基化参与基因转录后调控的研究进展[J].生命科学,2015,27(3):351-362.HOU Y F,DENG X,LU T C,et al.Research progress of protein arginine methylation in post-transcriptional regulation[J].Chinese Bulletin of Life Sciences,2015,27(3):351-362.
[29] POPOVA O V,ISMAILOV S F,POPOVA T N,et al.Salt-induced expression of NADP-dependent isocitrate dehydrogenase and ferredoxin-dependent glutamate synthase in Mesembryanthemum crystallinum[J].Planta,2002,215(6):906-913.
[30] 王超,潘建伟.高等动植物网格蛋白介导的内吞[J].浙江师范大学学报(自然科学版),2012,35(4):453-458.WANG CH,PAN J W.Clathrin-mediated endocytosis in higher animals and plants[J].Journal of Zhejiang Normal University(Natural Sciences),2012,35(4):453-458.
[31] 杨小妮,王俊花,杨剑锋,等.干旱胁迫下平邑甜茶叶片差异表达蛋白分析[J].西北农林科技大学学报(自然科学版),2018,46(11):70-80.YANG X N,WANG J H,YANG J F,et al.Proteomic analysis of apple leaf under water stress[J].Journal of Northwest A&F University(Natural Science Edition),2018,46(11):70-80.
[2] YANG Y Q,GUO Y.Unraveling salt stress signaling in plants[J].Journal of Integrative Plant Biology,2018,60(9):796-804.
[3] KALEEM F,SHABIR G,ASLAM K,et al.An overview of the genetics of plant response to salt stress:present status and the way forward[J].Applied Biochemistry and Biotechnology,2018,186(2):306-334.
[4] 单长卷,杨天佑.谷胱甘肽对盐胁迫下玉米幼苗抗氧化特性和光合性能的影响[J].西北农业学报,2017,26(2):185-191.SHAN CH J,YANG T Y.Effects of glutathione on antioxidant and photosynthetic properties of maize seedlings under salt stress[J].Acta Agriculturae Boreali-occidentalis Sinica,2017,26(2):185-191.
[5] ZHOU Y L,HUO S F,WANG L T,et al.Exogenous 24-epibrassinolide alleviates oxidative damage from copper stress in grape(Vitis vinifera l.) cuttings[J].Plant Physiology and Biochemistry,2018,130,555-565.
[6] LI J T,QIU Z B,ZHANG X W,et al.Exogenous hydrogen peroxide can enhance tolerance of wheat seedlings to salt stress[J].Acta Physiologiae Plantarum,2011,33(3):835-842.
[7] WU J Q,SHU S,LI C C,et al.Spermidine-mediated hydrogen peroxide signaling enhances the antioxidant capacity of salt-stressed cucumber roots[J].Plant Physiology and Biochemistry,2018,128:152-162.
[8] 马风勇,石晓霞,许兴,等.拟南芥SOS基因家族与植物耐盐性研究进展[J].中国农学通报,2013,29(21):121-125.MA F Y,SHI X X,XU X,et al.Research development of SOS gene family underlying salt tolerance in plant[J].Chinese Agricultural Science Bulletin,2013,29(21):121-125.
[9] 蔡丹,郑易之,兰英.大豆LEA蛋白Em的表达可提高大肠杆菌和烟草耐盐性[J].深圳大学学报理工版,2006,23(3):230-236.CAI D,ZHENG Y ZH,LAN Y.Expression of Em gene(LEA1) from soybean mimature seeds confers salt tolerance to Escherichia coli and tobacco plants[J].Journal of Shenzhen University Science and Engineering,2006,23(3):230-236.
[10] 赫卫,刘林,关荣霞,等.大豆耐盐相关基因GmNcl1的序列单倍型及表达分析[J].中国农业科学,2014,47(3):411-421.HE W,LIU L,GUAN R X,et al.Haplotypes and expression analysis of salt-tolerant gene GmNcl1 in soybean[J].Scientia Agricultura Sinica,2014,47(3):411-421.
[11] 蒋景龙,沈季雪,徐卫平,等.外源H2O2对低温胁迫下大红柑生长及叶片生理指标的影响[J].浙江农业学报,2016,28(7):1164-1170.JIANG J L,SHEN J X,XU W P,et al.Effects of exogenous hydrogen peroxide on growth and physiological index of Citrus reticulata Blanco cv.Dahonggan leaves under low temperature stress[J].Acta Agriculturae Zhejiangensis,2016,28(7):1164-1170.
[12] LIU Z J,GUO Y K,BAI J G.Exogenous hydrogen peroxide changes antioxidant enzyme activity and protects ultrastructure in leaves of two cucumber ecotypes under osmotic stress[J].Journal of Plant Growth Regulation,2010,29(2):171-183.
[13] GAO Y,GUO Y K,LIN S H,et al.Hydrogen peroxide pretreatment alters the activity of antioxidant enzymes and protects chloroplast ultrastructure in heat-stressed cucumber leaves[J].Scientia Horticulturae,2010,126(1):20-26.
[14] ZHANG X L,JIA X F,YU B,et al.Exogenous hydrogen peroxide influences antioxidant enzyme activity and,lipid peroxidation in cucumber leaves at low light[J].Scientia Horticulturae,2011,129(4):656-662.
[15] SUN Y,WANG H,LIU S,et al.Exogenous application of hydrogen peroxide alleviates drought stress in cucumber seedlings[J].South African Journal of Botany,2016,106:23-28.
[16] 龚建华,向军.黄瓜群体叶面积无破坏性速测方法研究[J].中国蔬菜,2001(4):7-9.GONG J H,XIANG J.Studies on a quick intact measurement to cucumber colony’s leaf area[J].China Vegetables,2001(4):7-9.
[17] 蒋景龙.H2O2胁迫对山黧豆生理生化指标的影响[J].西北农业学报,2016,25(2):243-248.JIANG J L.Effects of H2O2 stress on physiological and biochemical indexes of grass pea[J].Acta Agriculturae Boreali-occidentalis Sinica,2016,25(2):243-248.
[18] 田雲,蒋景龙,沈季雪,等.低温胁迫对5个栽培品种黄瓜生长及生理指标的影响[J].分子植物育种,2017,15(5):352-358.TIAN Y,JIANG J L,SHEN J X,et al.Effect of low temperature stress on growth and physiological indexes of five cultivars of cucumber[J].Molecular Plant Breeding,2017,15(5):352-358.
[19] JIANG J L,SU M ,WANG L Y,et al.Exogenous hydrogen peroxide reversibly inhibits root gravitropism and induces horizontal curvature of primary root during grass pea germination[J].Plant Physiology & Biochemistry Ppb,2012,53:84-93.
[20] WU Q F,LI C,KE L,et al.A high-efficiency,two-dimensional gel electrophoresis platform for mature leaves of grass pea(Lathyrus sativus L.)[J].Acta Physiologiae Plantarum,2011,33(6):2387-2397.
[21] 李金亭,赵萍萍,邱宗波,等.外源H2O2对盐胁迫下小麦幼苗生理指标的影响[J].西北植物学报,2012,32(9):1796-1801.LI J T,ZHAO P P,QIU Z B,et al.Effects of exogenous hydrogen peroxide on the physiological index of wheat seedlings under salt stress[J].Acta Botanica Boreali-Occidentalia Sinica,2012,32(9):1796-1801.
[22] 石玉,潘媛媛,张毅,等.光碳核肥对盐胁迫下黄瓜幼苗生长抑制的缓解效应[J].西北农业学报,2017,26(5):752-758.SHI Y,PAN Y Y,ZHANG Y,et al.Mitigative effect of carbon dioxide trapping agent on inhibiting growth of cucumber seedings under nacl stress[J].Acta Agriculturae Boreali-occidentalis Sinica,2017,26(5):752-758.
[23] FEDINA I S,NEDEVA D,CICEK N.Pre-treatment with H2O2 induces salt tolerance in barley seedlings[J].Biologia Plantarum,2009,53(2):321-324.
[24] 孙德智,韩晓日,彭靖,等.外源NO和SA对盐胁迫下番茄幼苗叶片膜脂过氧化及AsA-GSH循环的影响[J].植物科学学报,2018,36(4):612-622.SUN D ZH,HAN X R,PENG J,et al.Effects of exogenous nitric oxide and salicylic acid on membrane peroxidation and the ascorbate-glutathione cycle in leaves of Lycopersicon esculentum seedlings under NaCl stress[J].Plant Science Journal,2018,36(4):612-622.
[25] 曹齐卫,李利斌,孔素萍,等.不同黄瓜品种幼苗对等渗 Mg(NO3)2和NaCl胁迫的生理响应[J].应用生态学报,2015,26(4):1171-1178.CAO Q W,LI L B,KONG S P,et al.Physiological responses of different cucumber cultivars seedlings to iso-osmotic Mg(NO3)2 and NaCl stress[J].Chinese Journal of Applied Ecology,2015,26(4):1171-1178.
[26] 杨建军,张国斌,郁继华,等.盐胁迫下内源NO对黄瓜幼苗活性氧代谢和光合特性的影响[J].中国农业科学,2017,50(19):3778-3788.YANG J J,ZHANG G B,YU J H,et al.Effects of endogenous NO on reactive oxygen metabolism and photosynthetic characteristics of cucumber seedlings under salt stress[J].Scientia Agricultura Sinica,2017,50(19):3778-3788.
[27] ZHANG Z,ZHANG S,ZHANG Y,et al.Arabidopsis floral initiator SKB1 confers high salt tolerance by regulating transcription and pre-mRNA splicing through altering histone H4R3 and small nuclear ribonucleoprotein LSM4 methylation[J].Plant Cell,2011,23(1):396-411.
[28] 侯毅枫,邓娴,陆天聪,等.蛋白质精氨酸甲基化参与基因转录后调控的研究进展[J].生命科学,2015,27(3):351-362.HOU Y F,DENG X,LU T C,et al.Research progress of protein arginine methylation in post-transcriptional regulation[J].Chinese Bulletin of Life Sciences,2015,27(3):351-362.
[29] POPOVA O V,ISMAILOV S F,POPOVA T N,et al.Salt-induced expression of NADP-dependent isocitrate dehydrogenase and ferredoxin-dependent glutamate synthase in Mesembryanthemum crystallinum[J].Planta,2002,215(6):906-913.
[30] 王超,潘建伟.高等动植物网格蛋白介导的内吞[J].浙江师范大学学报(自然科学版),2012,35(4):453-458.WANG CH,PAN J W.Clathrin-mediated endocytosis in higher animals and plants[J].Journal of Zhejiang Normal University(Natural Sciences),2012,35(4):453-458.
[31] 杨小妮,王俊花,杨剑锋,等.干旱胁迫下平邑甜茶叶片差异表达蛋白分析[J].西北农林科技大学学报(自然科学版),2018,46(11):70-80.YANG X N,WANG J H,YANG J F,et al.Proteomic analysis of apple leaf under water stress[J].Journal of Northwest A&F University(Natural Science Edition),2018,46(11):70-80.