盐及干旱胁迫对油菜抗氧化系统和RbohC、RbohF基因表达的影响
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摘要
以白菜型油菜‘陇油6号’和‘天油2号’为试验材料,经MAPK抑制剂U0126、H_2O_2清除剂DMTU、NADPH氧化酶抑制剂DPI和IMD预处理后再分别进行盐胁迫、PEG-6000模拟干旱胁迫,研究其对两种油菜幼苗活性氧、抗氧化酶活性和RbohC、RbohF基因表达的影响.结果表明:盐胁迫和PEG-6000模拟干旱胁迫下,两种白菜型油菜中H_2O_2积累量上升,O_2~(-·)积累量下降,抗氧化酶(超氧化物歧化酶SOD、过氧化氢酶CAT、抗坏血酸过氧化物酶APX和谷胱甘肽还原酶GR)活性和RbohC、RbohF基因表达均升高.与单独胁迫处理相比,两种油菜O_2~(-·)积累、抗氧化酶活性和RbohC、RbohF基因的表达量均明显降低,经DMTU、DPI和IMD预处理后再分别进行盐和干旱胁迫,H_2O_2积累量下降,但U0126预处理后再进行胁迫处理,H_2O_2积累量上升.说明NADPH氧化酶、MAP激酶级联途径、H_2O_2参与了盐、干旱胁迫下活性氧产生、抗氧化酶活性变化和RbohC、RbohF基因表达的调控.
To investigate the effects of salt stress and PEG-6000 simulating drought stress on the active oxygen and antioxidant enzyme activities, as well as the expression level of RbohC and RbohF genes, the seedlings of two Brassica campestris, Longyou 6 and Tianyou 2, were treated with U0126(a MAPKK inhibitor), DMTU(a H_2O_2 scavenger), as well as DPI and IMD(NADPH oxidase inhibitors). The results showed that under both stresses, H_2O_2 accumulation as well as antioxidant enzyme(SOD, CAT, APX, GR) activities and the expression of RbohC and RbohF genes increased, while O_2~(-·) accumulation decreased. The O_2~(-·) accumulation, antioxidant enzyme activity and RbohC and RbohF genes expression in both varieties all significantly decreased. Compared to seedlings with on pretreatment before salt and PEG-6000 simulating drought stress, the accumulation of H_2O_2 decreased in seedlings pretreated with DMTU, DPI and IMD. However, the accumulation of H_2O_2 increased in those pretreated with U0126. Those results indicated that the NADPH oxidase, MAP kinase cascade and H_2O_2 were involved in the regulation of active oxygen production and antioxidant enzyme activity, as well as the expression of RbohC and RbohF under salt stress and drought stress.
To investigate the effects of salt stress and PEG-6000 simulating drought stress on the active oxygen and antioxidant enzyme activities, as well as the expression level of RbohC and RbohF genes, the seedlings of two Brassica campestris, Longyou 6 and Tianyou 2, were treated with U0126(a MAPKK inhibitor), DMTU(a H_2O_2 scavenger), as well as DPI and IMD(NADPH oxidase inhibitors). The results showed that under both stresses, H_2O_2 accumulation as well as antioxidant enzyme(SOD, CAT, APX, GR) activities and the expression of RbohC and RbohF genes increased, while O_2~(-·) accumulation decreased. The O_2~(-·) accumulation, antioxidant enzyme activity and RbohC and RbohF genes expression in both varieties all significantly decreased. Compared to seedlings with on pretreatment before salt and PEG-6000 simulating drought stress, the accumulation of H_2O_2 decreased in seedlings pretreated with DMTU, DPI and IMD. However, the accumulation of H_2O_2 increased in those pretreated with U0126. Those results indicated that the NADPH oxidase, MAP kinase cascade and H_2O_2 were involved in the regulation of active oxygen production and antioxidant enzyme activity, as well as the expression of RbohC and RbohF under salt stress and drought stress.
引文
[1] Asada K. Production and scavenging of reactive oxygen species in chloroplasts and their functions. Plant Physio-logy, 2006, 141: 391-396
[2] Parida AK, Das AB, Mohanty P. Defense potentials to NaCl in a mangrove, Bruguiera parviflora: Differential changes of isoforms of some antioxidative enzymes. Journal of Plant Physiology, 2004, 161: 531-542
[3] Pandey S, Nelson DC, Assmann SM. Two novel GPCR-type G proteins are abscisic acid receptors in Arabidopsis. Cell, 2009, 136: 136-148
[4] Miller G, Shulaev V, Mittler R. Reactive oxygen signaling and abiotic stress. Physiologia Plantarum, 2010, 133: 481-489
[5] Herve C, Tonon T, Collen J, et al. NADPH oxidases in Eukaryotes: Red algae provide new hints! Current Genetics, 2006, 49: 190-204
[6] BaileySerres J, Mittler R. The roles of reactive oxygen species in plant cells. Plant Physiology, 2006, 141: 311, doi: https://doi.org/10.1104/pp.104.900191
[7] Leshem Y, Seri L, Levine A. Induction of phosphatidylinositol 3-kinase-mediated endocytosis by salt stress leads to intracellular production of reactive oxygen species and salt tolerance. Plant Journal, 2010, 51: 185-197
[8] Sakamoto H, Matsuda O, Iba K. ITN1, a novel gene encoding an ankyrin-repeat protein that affects the ABA-mediated production of reactive oxygen species and is involved in salt-stress tolerance in Arabidopsis thaliana. Plant Journal, 2010, 56: 411-422
[9] Nühse TS, Bottrill AR, Jones AM, et al. Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses. Plant Journal, 2010, 51: 931-940
[10] Jiang M, Zhang J. Cross-talk between calcium and reactive oxygen species originated from NADPH oxidase in abscisic acid-induced antioxidant defence in leaves of maize seedlings. Plant, Cell and Environment, 2010, 26: 929-939
[11] Aziz A, Heyraud A, Lambert B. Oligogalacturonide signal transduction, induction of defense-related responses and protection of grapevine against Botrytis cinerea. Planta, 2004, 218: 767-774
[12] Zhang T-G (张腾国), Nie T-T (聂婷婷), Sun W-C (孙万仓), et al. Effects of diverse stresses on gene expression and enzyme activity of glutathione reductase in Brassica campestris. Chinese Journal of Applied Ecology (应用生态学报), 2018, 29(1): 213-222 (in Chinese)
[13] Sun W-C (孙万仓), Ma W-G (马卫国), Lei J-M (雷建民), et al. Study on adaptation and introduction possibility of winter rapeseed to dry and cold areas in Northwest China. Scientia Agricultura Sinica (中国农业科学), 2007, 40(12): 2716-2726 (in Chinese)
[14] Scarpeci TE, Zanor MI, Carrillo N, et al. Generation of superoxide anion in chloroplasts of Arabidopsis thaliana during active photosynthesis: A focus on rapidly induced genes. Plant Molecular Biology, 2008, 66: 361-378
[15] Gao X, Chen X, Lin W, et al. Bifurcation of Arabidopsis NLR immune signaling via Ca2+-dependent protein kinases. PLoS Pathogens, 2013, 9: e1003127
[16] Sergiev I, Alexieva V, Karanov E. Effect of spermine, atrazine and combination between them on some endogenous protective systems and stress markers in plants. Comptes Rendus de l’Academie Bulgare des Sciences, 1997, 51: 121-124
[17] Ries SK. In vitro production of superoxide radical from paraquat and its interactions with monuron and diuron. Weed Science, 1977, 25: 298-303
[18] Aebi H. Catalase in vitro. Methods in Enzymology, 1984, 105: 121-126
[19] Esterbauer H, Grill D. Seasonal variation of glutathione and glutathione reductase in needles of Picea abies. Plant Physiology, 1978, 61: 119-121
[20] Nakano Y, Asada K. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant and Cell Physiology, 1981, 22: 867-880
[21] Mittler R, Vanderauwera S, Gollery M, et al. Reactive oxygen gene network of plants. Trends in Plant Science, 2004, 9: 490-498
[22] Hu X, Jiang M, Zhang J, et al. Calcium-calmodulin is required for abscisic acid-induced antioxidant defense and functions both upstream and downstream of H2O2 production in leaves of maize (Zea mays) plants. New Phytologist, 2007, 173: 27-38
[23] Li G-Q (李国旗), An S-Q (安树青), Zhang J-L (张纪林), et al. Impact of salt stress on peroxidase activity in Populus deltoides cambium and its consequence. Chinese Journal of Applied Ecology (应用生态学报), 2003, 14(6): 871-874 (in Chinese)
[24] Chen J (陈珺), Lai Q-X (赖齐贤), He B-L (何宝龙), et al. Production of activated oxygen and antioxidant enzyme activity in ornamental species under drought stress. Acta Botanica Boreali-Occidentalia Sinica (西北植物学报), 2014, 34(7): 1390-1396 (in Chinese)
[25] Vaidyanathan H, Sivakumar P, Chakrabarty R, et al. Scavenging of reactive oxygen species in NaCl-stressed rice (Oryza sativa L.): Differential response in salt-tolerant and sensitive varieties. Plant Science, 2003, 165: 1411-1418
[26] Schopfer P, Plachy C, Frahry G. Release of reactive oxygen intermediates (superoxide radicals, hydrogen peroxide, and hydroxyl radicals) and peroxidase in germinating radish seeds controlled by light, gibberellin, and abscisic acid. Plant Physiology, 2001, 125: 1591-1602
[27] Leshem Y, Seri L, Levine A. Induction of phosphatidylinositol 3-kinase-mediated endocytosis by salt stress leads to intracellular production of reactive oxygen species and salt tolerance. Plant Journal, 2010, 51: 185-197
[28] Gupta DK, Inouhe M, Rodríguez-Serrano M, et al. Oxidative stress and arsenic toxicity: Role of NADPH oxidases. Chemosphere, 2013, 90: 1987-1996
[29] Pitzschke A, Hirt H. Disentangling the complexity of mitogen-activated protein kinases and reactive oxygen species signaling. Plant Physiology, 2009, 149: 606-615
[30] Zhang A, Jiang M, Zhang J, et al. Mitogen-activated protein kinase is involved in abscisic acid-induced antioxidant defense and acts downstream of reactive oxygen species production in leaves of maize plants. Plant Physiology, 2006, 141: 475-487
[31] Zhang A, Jiang M, Zhang J, et al. Nitric oxide induced by hydrogen peroxide mediates abscisic acid-induced activation of the mitogen-activated protein kinase cascade involved in antioxidant defense in maize leaves. New Phytologist, 2007, 175: 36-50
[32] Mittler R, Vanderauwera S, Gollery M, et al. Reactive oxygen gene network of plants. Trends in Plant Science, 2004, 9: 490-498
[33] Dat JF, Pellinen R, Beeckman T, et al. Changes in hydrogen peroxide homeostasis trigger an active cell death process in tobacco. The Plant Journal, 2010, 33: 621-632
[34] Yoshioka H, Numata N, Nakajima K, et al. Nicotiana benthamiana gp91 phox homologs NbrbohA and NbrbohB participate in H2O2 accumulation and resistance to Phytophthora infestans. Plant Cell, 2003, 15: 706-718
[35] Zhang H, Liu Y, Feng W, et al. A novel rice C2H2-type zinc finger protein, ZFP36, is a key player involved in abscisic acid-induced antioxidant defence and oxidative stress tolerance in rice. Journal of Experimental Botany, 2014, 65: 5795-5809
[36] Kwak JM, Mori IC, Pei ZM, et al. NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis. EMBO Journal, 2014, 22: 2623-2633
[2] Parida AK, Das AB, Mohanty P. Defense potentials to NaCl in a mangrove, Bruguiera parviflora: Differential changes of isoforms of some antioxidative enzymes. Journal of Plant Physiology, 2004, 161: 531-542
[3] Pandey S, Nelson DC, Assmann SM. Two novel GPCR-type G proteins are abscisic acid receptors in Arabidopsis. Cell, 2009, 136: 136-148
[4] Miller G, Shulaev V, Mittler R. Reactive oxygen signaling and abiotic stress. Physiologia Plantarum, 2010, 133: 481-489
[5] Herve C, Tonon T, Collen J, et al. NADPH oxidases in Eukaryotes: Red algae provide new hints! Current Genetics, 2006, 49: 190-204
[6] BaileySerres J, Mittler R. The roles of reactive oxygen species in plant cells. Plant Physiology, 2006, 141: 311, doi: https://doi.org/10.1104/pp.104.900191
[7] Leshem Y, Seri L, Levine A. Induction of phosphatidylinositol 3-kinase-mediated endocytosis by salt stress leads to intracellular production of reactive oxygen species and salt tolerance. Plant Journal, 2010, 51: 185-197
[8] Sakamoto H, Matsuda O, Iba K. ITN1, a novel gene encoding an ankyrin-repeat protein that affects the ABA-mediated production of reactive oxygen species and is involved in salt-stress tolerance in Arabidopsis thaliana. Plant Journal, 2010, 56: 411-422
[9] Nühse TS, Bottrill AR, Jones AM, et al. Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses. Plant Journal, 2010, 51: 931-940
[10] Jiang M, Zhang J. Cross-talk between calcium and reactive oxygen species originated from NADPH oxidase in abscisic acid-induced antioxidant defence in leaves of maize seedlings. Plant, Cell and Environment, 2010, 26: 929-939
[11] Aziz A, Heyraud A, Lambert B. Oligogalacturonide signal transduction, induction of defense-related responses and protection of grapevine against Botrytis cinerea. Planta, 2004, 218: 767-774
[12] Zhang T-G (张腾国), Nie T-T (聂婷婷), Sun W-C (孙万仓), et al. Effects of diverse stresses on gene expression and enzyme activity of glutathione reductase in Brassica campestris. Chinese Journal of Applied Ecology (应用生态学报), 2018, 29(1): 213-222 (in Chinese)
[13] Sun W-C (孙万仓), Ma W-G (马卫国), Lei J-M (雷建民), et al. Study on adaptation and introduction possibility of winter rapeseed to dry and cold areas in Northwest China. Scientia Agricultura Sinica (中国农业科学), 2007, 40(12): 2716-2726 (in Chinese)
[14] Scarpeci TE, Zanor MI, Carrillo N, et al. Generation of superoxide anion in chloroplasts of Arabidopsis thaliana during active photosynthesis: A focus on rapidly induced genes. Plant Molecular Biology, 2008, 66: 361-378
[15] Gao X, Chen X, Lin W, et al. Bifurcation of Arabidopsis NLR immune signaling via Ca2+-dependent protein kinases. PLoS Pathogens, 2013, 9: e1003127
[16] Sergiev I, Alexieva V, Karanov E. Effect of spermine, atrazine and combination between them on some endogenous protective systems and stress markers in plants. Comptes Rendus de l’Academie Bulgare des Sciences, 1997, 51: 121-124
[17] Ries SK. In vitro production of superoxide radical from paraquat and its interactions with monuron and diuron. Weed Science, 1977, 25: 298-303
[18] Aebi H. Catalase in vitro. Methods in Enzymology, 1984, 105: 121-126
[19] Esterbauer H, Grill D. Seasonal variation of glutathione and glutathione reductase in needles of Picea abies. Plant Physiology, 1978, 61: 119-121
[20] Nakano Y, Asada K. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant and Cell Physiology, 1981, 22: 867-880
[21] Mittler R, Vanderauwera S, Gollery M, et al. Reactive oxygen gene network of plants. Trends in Plant Science, 2004, 9: 490-498
[22] Hu X, Jiang M, Zhang J, et al. Calcium-calmodulin is required for abscisic acid-induced antioxidant defense and functions both upstream and downstream of H2O2 production in leaves of maize (Zea mays) plants. New Phytologist, 2007, 173: 27-38
[23] Li G-Q (李国旗), An S-Q (安树青), Zhang J-L (张纪林), et al. Impact of salt stress on peroxidase activity in Populus deltoides cambium and its consequence. Chinese Journal of Applied Ecology (应用生态学报), 2003, 14(6): 871-874 (in Chinese)
[24] Chen J (陈珺), Lai Q-X (赖齐贤), He B-L (何宝龙), et al. Production of activated oxygen and antioxidant enzyme activity in ornamental species under drought stress. Acta Botanica Boreali-Occidentalia Sinica (西北植物学报), 2014, 34(7): 1390-1396 (in Chinese)
[25] Vaidyanathan H, Sivakumar P, Chakrabarty R, et al. Scavenging of reactive oxygen species in NaCl-stressed rice (Oryza sativa L.): Differential response in salt-tolerant and sensitive varieties. Plant Science, 2003, 165: 1411-1418
[26] Schopfer P, Plachy C, Frahry G. Release of reactive oxygen intermediates (superoxide radicals, hydrogen peroxide, and hydroxyl radicals) and peroxidase in germinating radish seeds controlled by light, gibberellin, and abscisic acid. Plant Physiology, 2001, 125: 1591-1602
[27] Leshem Y, Seri L, Levine A. Induction of phosphatidylinositol 3-kinase-mediated endocytosis by salt stress leads to intracellular production of reactive oxygen species and salt tolerance. Plant Journal, 2010, 51: 185-197
[28] Gupta DK, Inouhe M, Rodríguez-Serrano M, et al. Oxidative stress and arsenic toxicity: Role of NADPH oxidases. Chemosphere, 2013, 90: 1987-1996
[29] Pitzschke A, Hirt H. Disentangling the complexity of mitogen-activated protein kinases and reactive oxygen species signaling. Plant Physiology, 2009, 149: 606-615
[30] Zhang A, Jiang M, Zhang J, et al. Mitogen-activated protein kinase is involved in abscisic acid-induced antioxidant defense and acts downstream of reactive oxygen species production in leaves of maize plants. Plant Physiology, 2006, 141: 475-487
[31] Zhang A, Jiang M, Zhang J, et al. Nitric oxide induced by hydrogen peroxide mediates abscisic acid-induced activation of the mitogen-activated protein kinase cascade involved in antioxidant defense in maize leaves. New Phytologist, 2007, 175: 36-50
[32] Mittler R, Vanderauwera S, Gollery M, et al. Reactive oxygen gene network of plants. Trends in Plant Science, 2004, 9: 490-498
[33] Dat JF, Pellinen R, Beeckman T, et al. Changes in hydrogen peroxide homeostasis trigger an active cell death process in tobacco. The Plant Journal, 2010, 33: 621-632
[34] Yoshioka H, Numata N, Nakajima K, et al. Nicotiana benthamiana gp91 phox homologs NbrbohA and NbrbohB participate in H2O2 accumulation and resistance to Phytophthora infestans. Plant Cell, 2003, 15: 706-718
[35] Zhang H, Liu Y, Feng W, et al. A novel rice C2H2-type zinc finger protein, ZFP36, is a key player involved in abscisic acid-induced antioxidant defence and oxidative stress tolerance in rice. Journal of Experimental Botany, 2014, 65: 5795-5809
[36] Kwak JM, Mori IC, Pei ZM, et al. NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis. EMBO Journal, 2014, 22: 2623-2633
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