H_2O_2预处理及高温胁迫下杜鹃叶片活性氧及抗氧化酶亚细胞定位分析
|
摘要
为明确H_2O_2在杜鹃耐热性形成中的作用机制以及高温胁迫下杜鹃活性氧清除系统的亚细胞分布,该研究以3个耐热性不同的杜鹃品种为实验材料,进行H_2O_2预处理后再进行高温胁迫,分析叶绿体、线粒体和细胞溶质等亚细胞组分中活性氧产生和清除系统的变化。结果表明:(1)‘胭脂蜜’、‘红月’、‘红珊瑚’分别为耐热、较耐热和热敏感品种。(2)高温胁迫下,3个杜鹃品种H_2O_2含量的亚细胞分布为细胞溶质>叶绿体>线粒体,各亚细胞组分中超氧阴离子自由基■产生速率差异不显著;3个杜鹃品种均以细胞溶质中的丙二醛(MDA)含量最高,且热敏感杜鹃‘红月’和‘红珊瑚’中MDA亚细胞分布与H_2O_2相一致,但‘胭脂蜜’线粒体中MDA含量高于叶绿体。(3)高温胁迫下,‘胭脂蜜’和‘红珊瑚’亚细胞组分超氧化物歧化酶(SOD)和过氧化物酶(POD)活性排序为细胞溶质>叶绿体>线粒体,‘红月’相应排序为细胞溶质>线粒体>叶绿体;过氧化氢酶(CAT)活性在‘胭脂蜜’和‘红月’亚细胞组分中的排序相同,线粒体中CAT活性高于细胞溶质和叶绿体,而在‘红珊瑚’中叶绿体CAT活性最高。(4)H_2O_2预处理可以通过增强耐热杜鹃的抗氧化防御能力,减轻杜鹃幼苗的过氧化损伤,尤其是对耐热性较差的杜鹃品种效果显著。研究发现,杜鹃叶片MDA、活性氧、抗氧化酶活性的亚细胞分布在高温胁迫下存在品种差异,MDA亚细胞分布与活性氧的分布并不完全一致,热敏感杜鹃‘红月’和‘红珊瑚’叶绿体所受的过氧化损伤程度高于耐热品种‘胭脂蜜’;高温胁迫下,H_2O_2预处理对不同杜鹃品种各亚细胞器的活性氧和抗氧化酶的影响程度也表现出品种间差异。
In order to make clear the response mechanism of antioxidant defense to heat stress at the subcellular level and hydrogen peroxide-induced heat tolerance of rhododendrons, we used three Rhododendron cultivars with different heat tolerance as experimental materials to analyze the effects of heat stress and H_2O_2 pre-treatment on the level of reactive oxygen species(ROS) and the activities of antioxidant system in the chloroplasts, mitochondria and cytosol of Rhododendron leaves. The results showed that:(1) the order of the three Rhododendron cultivars according to their heat resistance were R. obtusum ‘Yanzhimi', R. ‘Hongyue' and R. ‘Hongshanhu', respectively.(2) Under heat stress, the subcellular distribution of H_2O_2: cytosol > chloroplast > mitochondria. There was no significant difference in the production rate of superoxide radical ■ in three subcellular organelles. The MDA content in cytosol was the highest in three Rhododendron cultivars, the subcellular distribution of MDA in R. ‘Hongyue' and R. ‘Hongshanhu' was the same with H_2O_2 distribution, but MDA in the mitochondria of R. obtusum ‘Yanzhimi' was higher than that in the chloroplast.(3) Under heat stress, the order of superoxide dismutase(SOD) and peroxidase(POD) activities in R. obtusum ‘Yanzhimi' and R. ‘Hongshanhu': cytosol > chloroplast > mitochondria, however in R. ‘Hongyue', the order was cytosol > mitochondria > chloroplast. The subcellular distribution of catalase(CAT) in R. obtusum ‘Yanzhimi' was the same with R. ‘Hongyue', their CAT activity in the mitochondria was higher than that in the cytosols and chloroplasts, but in R. ‘Hongshanhu' the activity of CAT in the chloroplasts was the highest.(4) H_2O_2 pretreatment could improve the heat tolerance of rhododendron seedlings by enhancing the antioxidant defense ability of reducing the peroxidative damage under heat stress especially for the seedlings with poor heat tolerance. The results indicated that the subcellular distributions of ROS, MDA and antioxidant enzymes were different among three cultivars. The distribution of MDA was not completely consistent with that of ROS, and the chloroplasts of heat-sensitive cultivars R. ‘Hongyue' and R. ‘Hongshanhu' were more susceptible to oxidative damage than those of R. obtusum ‘Yanzhimi'. The effects of H_2O_2 pretreatment on the level of ROS and the activities of antioxidant enzymes in subcellular organelles appeared to be different among the three cultivars.
In order to make clear the response mechanism of antioxidant defense to heat stress at the subcellular level and hydrogen peroxide-induced heat tolerance of rhododendrons, we used three Rhododendron cultivars with different heat tolerance as experimental materials to analyze the effects of heat stress and H_2O_2 pre-treatment on the level of reactive oxygen species(ROS) and the activities of antioxidant system in the chloroplasts, mitochondria and cytosol of Rhododendron leaves. The results showed that:(1) the order of the three Rhododendron cultivars according to their heat resistance were R. obtusum ‘Yanzhimi', R. ‘Hongyue' and R. ‘Hongshanhu', respectively.(2) Under heat stress, the subcellular distribution of H_2O_2: cytosol > chloroplast > mitochondria. There was no significant difference in the production rate of superoxide radical ■ in three subcellular organelles. The MDA content in cytosol was the highest in three Rhododendron cultivars, the subcellular distribution of MDA in R. ‘Hongyue' and R. ‘Hongshanhu' was the same with H_2O_2 distribution, but MDA in the mitochondria of R. obtusum ‘Yanzhimi' was higher than that in the chloroplast.(3) Under heat stress, the order of superoxide dismutase(SOD) and peroxidase(POD) activities in R. obtusum ‘Yanzhimi' and R. ‘Hongshanhu': cytosol > chloroplast > mitochondria, however in R. ‘Hongyue', the order was cytosol > mitochondria > chloroplast. The subcellular distribution of catalase(CAT) in R. obtusum ‘Yanzhimi' was the same with R. ‘Hongyue', their CAT activity in the mitochondria was higher than that in the cytosols and chloroplasts, but in R. ‘Hongshanhu' the activity of CAT in the chloroplasts was the highest.(4) H_2O_2 pretreatment could improve the heat tolerance of rhododendron seedlings by enhancing the antioxidant defense ability of reducing the peroxidative damage under heat stress especially for the seedlings with poor heat tolerance. The results indicated that the subcellular distributions of ROS, MDA and antioxidant enzymes were different among three cultivars. The distribution of MDA was not completely consistent with that of ROS, and the chloroplasts of heat-sensitive cultivars R. ‘Hongyue' and R. ‘Hongshanhu' were more susceptible to oxidative damage than those of R. obtusum ‘Yanzhimi'. The effects of H_2O_2 pretreatment on the level of ROS and the activities of antioxidant enzymes in subcellular organelles appeared to be different among the three cultivars.
引文
[1] 耿玉英.中国杜鹃花属植物[M].上海:上海科学技术出版社,2014:612.
[2] 耿兴敏,胡才民,杨秋玉,等.杜鹃花对各种非生物逆境胁迫的抗性研究进展[J].中国野生植物资源,2014,33(3):18-21.GENG X M,HU C M,YANG Q Y,et al.Research advance of resistance to abiotic stresses of Rhododendrons[J].Chinese Wild Plant Resources,2014,33(3):18-21.
[3] 张乐华,孙宝腾,周广,等.高温胁迫下五种杜鹃花属植物的生理变化及其耐热性比较[J].广西植物,2011,31(5):651-658.ZHANG L H,SUN B T,ZHOU G,et al.Physiological changed and heat tolerance comparison of five Rhododendrons species under high-temperature stress[J].Guihaia,2011,31(5):651-658.
[4] 张乐华,周广,孙宝腾,等.高温胁迫对两种常绿杜鹃亚属植物幼苗生理生化特性的影响[J].植物科学学报,2011,29(3):362-369.ZHANG L H,ZHOU G,SUN B T,et al.Physiological and biochemical effects of high temperature stress on the seedlings of two Rhododendron species of subgenus hymenanthes[J].Plant Science Journal,2011,29(3):362-369.
[5] SHEN H F,ZHAO B,XU J J,et al.Effects of heat stress on changes in physiology and anatomy in two cultivars of Rhododendron[J].South African Journal of Botany,2017,112:338-345.
[6] 李小玲,雒玲玲,华智锐.高温胁迫下高山杜鹃的生理生化响应[J].西北农业学报,2018,27(2):1-6.LI X L,LUO L L,HUA Z R.Physiological and biochemical responses of alpine Rhododendrons under high temperature stress [J].Acta Agriculturae Boreali-Occidentalis Sinica,2018,27(2):1-6.
[7] SLESAK I,LIBIK M,KARPINSKA B,et al.The role of hydrogen peroxide in regulation of plant metabolism and cellular signaling in response to environmental stresses[J].Acta Biochim Pol,2007,54(1):39-50.
[8] 于飞,陈银萍,杨宗娟,等.低温胁迫对两种圆柏属植物亚细胞抗氧化酶活性的影响[J].广西植物,2014,34(5):686-693.YU F,CHEN Y P,YANG Z J,et al.Effect of low temperature stress on antioxidant enzymes activities in the subcellular of two Sabina species[J].Guihaia,2014,34(5):686-693.
[9] GU K,GENG X M,YUE Y,et al.Contribution of keeping more stable anatomical structure under high temperature to heat resistance of Rhododendron seedlings[J].Journal of Faculty of Agriculture,Kyushu University,2016,61(2):273-279.
[10] DAS K,ROYCHOUDHURY A.Reactive oxygen species (ROS) and response of antioxidants as ROS-scavengers during environmental stress in plants [J].Frontiers in Environmental Science,2014,2(53):1-13.
[11] PEREZ B I,BROWN P.The role of ROS signaling in cross-tolerance:from model to crop[J].Frontiers in Plant Science,2014,5:1-6.
[12] 余土元,冯颖竹.低温对香蕉幼苗的伤害及外源H2O2、CaCl2对香蕉抗寒性的影响[J].中国农学通报,2011,27(25):219-223.YU S Y,FENG Y Z.Low-temperature injury of banana seedlings and effects of exogenous H2O2 and CaCl2 on cold resistance in banana[J].Chinese Agricultural Science Bulletin,2011,27(25):219-223.
[13] GONG M,CHEN B,LI Z G,et al.Heat-shock-induced cross adaptation to heat,chilling,drought and salt stress in maize seedlings and involvement of H2O2[J].Journal of Plant Physiology,2001,158(9):1 125-1 130.
[14] LUNA C M,PASTORI G M,DRISCOLL S,et al.Drought controls on H2O2 accumulation,catalase (CAT) activity and CAT gene expression in wheat[J].Journal of Experimental Botany,2004,56(411):417-423.
[15] LI H,LIU S S,YI C Y,et al.Hydrogen peroxide mediates abscisic acid-induced HSP70 accumulation and heat tolerance in grafted cucumber plants[J].Plant,Cell and Environment,2014,37(12):2 768-2 780.
[16] ZANDALINAS S I,BALFAGON D,AROBONA V,et al.Modulation of antioxidant defense system is associated with combined drought and heat stress tolerance in Citrus[J].Frontiers in Plant Science,2017,8(953):1-10.
[17] MITTLER R,VANDERAUWERA S,GOLLERY M,et al.Reactive oxygen gene network of plant[J].Trends in Plant Science,2004,9(10):490-498.
[18] 李建坤,汪宗立.涝渍逆境下玉米叶片各细胞器超氧物歧化酶活性的变化及6-BA的保护作用[J].江苏农业学报,1992,8(3):20-24.LI J K,WANG Z L.Changes in the activity of superoxide dismutase in various organelles of maize leaves under waterlogging stress and the protective effect of 6-BA[J].Jiangsu Journal of Agricultural Sciences,1992,8(3):20-24.
[19] 陈一舞,邵桂花,常汝镇.盐胁迫对大豆幼苗子叶各细胞器超氧化物歧化酶(SOD)的影响[J].作物学报,1997,23(2):214-219.CHEN Y W,SHAO G H,CHANG R Z.Effect of salt stress on superoxide dismutase (SOD) in cotyledon of soybean seedling[J].Acta Agronomica Sinica,1997,23(2):214-219.
[20] BOWLER C,MONTAGU M,INZE D.Superoxide dismutase and stress tolerance[J].Annual Review of Plant Physiology and Plant Molecular Biology,1992,43:83-116.
[21] 韩娜,冯汉青,李燕鸿,等.外源H2O2对水稻幼苗抗寒性的调节作用[J].西北植物学报,2009,29(6):1 214-1 219.HAN N,FENG H Q,LI Y H,et al.Regulation of exogenous H2O2 on rice seedlings chilling tolerance[J].Acta Botanica Boreali-Occidentalia Sinica,2009,29(6):1 214-1 219.
[22] 徐芬芬,徐鹏,胡志涛,等.外源过氧化氢对盐胁迫下水稻幼苗根系生长和抗氧化系统的影响[J].杂交水稻,2017,32(1):74-77.XU F F,XU P,HU Z T,et al.Effects of exogenous hydrogen peroxide on root growth and antioxidant system of rice seedling under salt stress[J].Hybrid Rice,2017,32(1):74-77.
[23] GU W Y,MO P H,YANG J S,et al.Exogenous nitric oxide and hydrogen peroxide regulate the acclimation of chicory (Cichorium intybus) to salt stress[J].Chinese Journal of Ecology,2014,33(1):89-97.
[24] 刘忠静,郭延奎,林少航,等.外源过氧化氢对干旱胁迫下温室黄瓜叶绿体超微结构和抗氧化酶的影响[J].园艺学报,2009,36(8):1 140-1 146.LIU Z J,GONG Y K,LIN S H,et al.Effects of exogenous hydrogen peroxide on ultrastructure of chloroplast and activities of antioxidant enzyme in greenhouse cucumber under drought stress[J].Acta Horticulturae Sinica,2009,36(8):1 140-1 146.
[25] ZHOU J,XIA X J,ZHOU Y H,et al.RBOH1-dependent H2O2 production and subsequent activation of MPK1/2 play an important role in acclimation-induced cross-tolerance in tomato[J].Journal of Experimental Botany,2014,65(2):595-607.
[26] 王瑛,杨海荣,张莉,等.H2O2和ABA对干旱高温复合胁迫诱导的玉米叶片抗氧化防护基因表达的影响[J].河南农业大学学报,2011,45(6):634-639.WANG Y,YANG H R,ZHANG L,et al.Influence of H2O2 and ABA on antioxidant defense gene expression of maize seedlings leaves under the combination of drought and heat stress[J].Journal of Henan Agricultural University,2011,45(6):634-639.
[2] 耿兴敏,胡才民,杨秋玉,等.杜鹃花对各种非生物逆境胁迫的抗性研究进展[J].中国野生植物资源,2014,33(3):18-21.GENG X M,HU C M,YANG Q Y,et al.Research advance of resistance to abiotic stresses of Rhododendrons[J].Chinese Wild Plant Resources,2014,33(3):18-21.
[3] 张乐华,孙宝腾,周广,等.高温胁迫下五种杜鹃花属植物的生理变化及其耐热性比较[J].广西植物,2011,31(5):651-658.ZHANG L H,SUN B T,ZHOU G,et al.Physiological changed and heat tolerance comparison of five Rhododendrons species under high-temperature stress[J].Guihaia,2011,31(5):651-658.
[4] 张乐华,周广,孙宝腾,等.高温胁迫对两种常绿杜鹃亚属植物幼苗生理生化特性的影响[J].植物科学学报,2011,29(3):362-369.ZHANG L H,ZHOU G,SUN B T,et al.Physiological and biochemical effects of high temperature stress on the seedlings of two Rhododendron species of subgenus hymenanthes[J].Plant Science Journal,2011,29(3):362-369.
[5] SHEN H F,ZHAO B,XU J J,et al.Effects of heat stress on changes in physiology and anatomy in two cultivars of Rhododendron[J].South African Journal of Botany,2017,112:338-345.
[6] 李小玲,雒玲玲,华智锐.高温胁迫下高山杜鹃的生理生化响应[J].西北农业学报,2018,27(2):1-6.LI X L,LUO L L,HUA Z R.Physiological and biochemical responses of alpine Rhododendrons under high temperature stress [J].Acta Agriculturae Boreali-Occidentalis Sinica,2018,27(2):1-6.
[7] SLESAK I,LIBIK M,KARPINSKA B,et al.The role of hydrogen peroxide in regulation of plant metabolism and cellular signaling in response to environmental stresses[J].Acta Biochim Pol,2007,54(1):39-50.
[8] 于飞,陈银萍,杨宗娟,等.低温胁迫对两种圆柏属植物亚细胞抗氧化酶活性的影响[J].广西植物,2014,34(5):686-693.YU F,CHEN Y P,YANG Z J,et al.Effect of low temperature stress on antioxidant enzymes activities in the subcellular of two Sabina species[J].Guihaia,2014,34(5):686-693.
[9] GU K,GENG X M,YUE Y,et al.Contribution of keeping more stable anatomical structure under high temperature to heat resistance of Rhododendron seedlings[J].Journal of Faculty of Agriculture,Kyushu University,2016,61(2):273-279.
[10] DAS K,ROYCHOUDHURY A.Reactive oxygen species (ROS) and response of antioxidants as ROS-scavengers during environmental stress in plants [J].Frontiers in Environmental Science,2014,2(53):1-13.
[11] PEREZ B I,BROWN P.The role of ROS signaling in cross-tolerance:from model to crop[J].Frontiers in Plant Science,2014,5:1-6.
[12] 余土元,冯颖竹.低温对香蕉幼苗的伤害及外源H2O2、CaCl2对香蕉抗寒性的影响[J].中国农学通报,2011,27(25):219-223.YU S Y,FENG Y Z.Low-temperature injury of banana seedlings and effects of exogenous H2O2 and CaCl2 on cold resistance in banana[J].Chinese Agricultural Science Bulletin,2011,27(25):219-223.
[13] GONG M,CHEN B,LI Z G,et al.Heat-shock-induced cross adaptation to heat,chilling,drought and salt stress in maize seedlings and involvement of H2O2[J].Journal of Plant Physiology,2001,158(9):1 125-1 130.
[14] LUNA C M,PASTORI G M,DRISCOLL S,et al.Drought controls on H2O2 accumulation,catalase (CAT) activity and CAT gene expression in wheat[J].Journal of Experimental Botany,2004,56(411):417-423.
[15] LI H,LIU S S,YI C Y,et al.Hydrogen peroxide mediates abscisic acid-induced HSP70 accumulation and heat tolerance in grafted cucumber plants[J].Plant,Cell and Environment,2014,37(12):2 768-2 780.
[16] ZANDALINAS S I,BALFAGON D,AROBONA V,et al.Modulation of antioxidant defense system is associated with combined drought and heat stress tolerance in Citrus[J].Frontiers in Plant Science,2017,8(953):1-10.
[17] MITTLER R,VANDERAUWERA S,GOLLERY M,et al.Reactive oxygen gene network of plant[J].Trends in Plant Science,2004,9(10):490-498.
[18] 李建坤,汪宗立.涝渍逆境下玉米叶片各细胞器超氧物歧化酶活性的变化及6-BA的保护作用[J].江苏农业学报,1992,8(3):20-24.LI J K,WANG Z L.Changes in the activity of superoxide dismutase in various organelles of maize leaves under waterlogging stress and the protective effect of 6-BA[J].Jiangsu Journal of Agricultural Sciences,1992,8(3):20-24.
[19] 陈一舞,邵桂花,常汝镇.盐胁迫对大豆幼苗子叶各细胞器超氧化物歧化酶(SOD)的影响[J].作物学报,1997,23(2):214-219.CHEN Y W,SHAO G H,CHANG R Z.Effect of salt stress on superoxide dismutase (SOD) in cotyledon of soybean seedling[J].Acta Agronomica Sinica,1997,23(2):214-219.
[20] BOWLER C,MONTAGU M,INZE D.Superoxide dismutase and stress tolerance[J].Annual Review of Plant Physiology and Plant Molecular Biology,1992,43:83-116.
[21] 韩娜,冯汉青,李燕鸿,等.外源H2O2对水稻幼苗抗寒性的调节作用[J].西北植物学报,2009,29(6):1 214-1 219.HAN N,FENG H Q,LI Y H,et al.Regulation of exogenous H2O2 on rice seedlings chilling tolerance[J].Acta Botanica Boreali-Occidentalia Sinica,2009,29(6):1 214-1 219.
[22] 徐芬芬,徐鹏,胡志涛,等.外源过氧化氢对盐胁迫下水稻幼苗根系生长和抗氧化系统的影响[J].杂交水稻,2017,32(1):74-77.XU F F,XU P,HU Z T,et al.Effects of exogenous hydrogen peroxide on root growth and antioxidant system of rice seedling under salt stress[J].Hybrid Rice,2017,32(1):74-77.
[23] GU W Y,MO P H,YANG J S,et al.Exogenous nitric oxide and hydrogen peroxide regulate the acclimation of chicory (Cichorium intybus) to salt stress[J].Chinese Journal of Ecology,2014,33(1):89-97.
[24] 刘忠静,郭延奎,林少航,等.外源过氧化氢对干旱胁迫下温室黄瓜叶绿体超微结构和抗氧化酶的影响[J].园艺学报,2009,36(8):1 140-1 146.LIU Z J,GONG Y K,LIN S H,et al.Effects of exogenous hydrogen peroxide on ultrastructure of chloroplast and activities of antioxidant enzyme in greenhouse cucumber under drought stress[J].Acta Horticulturae Sinica,2009,36(8):1 140-1 146.
[25] ZHOU J,XIA X J,ZHOU Y H,et al.RBOH1-dependent H2O2 production and subsequent activation of MPK1/2 play an important role in acclimation-induced cross-tolerance in tomato[J].Journal of Experimental Botany,2014,65(2):595-607.
[26] 王瑛,杨海荣,张莉,等.H2O2和ABA对干旱高温复合胁迫诱导的玉米叶片抗氧化防护基因表达的影响[J].河南农业大学学报,2011,45(6):634-639.WANG Y,YANG H R,ZHANG L,et al.Influence of H2O2 and ABA on antioxidant defense gene expression of maize seedlings leaves under the combination of drought and heat stress[J].Journal of Henan Agricultural University,2011,45(6):634-639.