H2O2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

Sub-cellular Localization of ROS-scavenging System in Rhododendron Leaves under Heat Stress and H2O2 Pretreatment

为明确H2O2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H2O2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H2O2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(O2-·)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H2O2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)H2O2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,H2O2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 H2O2 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 H2O2: cytosol > chloroplast > mitochondria. There was no significant difference in the production rate of superoxide radical (O2-·) 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 H2O2 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) H2O2 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 H2O2 pretreatment on the level of ROS and the activities of antioxidant enzymes in subcellular organelles appeared to be different among the three cultivars.