【作者】 李翠英；

【导师】 马锋旺；

【作者基本信息】 西北农林科技大学 ， 果树学， 2008， 博士

【摘要】 苹果是世界上重要的栽培果树之一，是我国北方落叶果树中栽培面积最大的树种。在苹果生产中，时常因水淹、排灌不利、土壤质地紧实等原因造成苹果树根际缺氧。而关于低氧胁迫对苹果树的影响，国内外研究较少。苹果树的抗逆性主要取决于砧木，因此了解苹果属砧木资源的耐低氧能力，筛选耐低氧砧木，对充分利用砧木资源、发展苹果生产、合理选择和定向培育耐涝果树砧木都具有重要意义。深入研究低氧胁迫对苹果砧木伤害的生理原因，探明苹果砧木对低氧逆境的适应机制，有利于丰富果树抗逆生理理论，并且能为解决果树生产中土壤低氧胁迫问题提供一定的理论依据。本研究以中国12个苹果属砧木资源为试材，采用营养液培养的方法，结合淹水试验，利用湿害指数（WI）、抗逆系数（ARC）、模糊数学中隶属函数值和聚类分析等多种方法分析不同苹果属砧木资源幼苗耐低氧性的差异，对12个供试砧木资源进行耐低氧性综合评价。以筛选出的耐低氧能力具有显著差异的两个苹果砧木为材料，进一步采用营养液培养的方法，通过分析低氧胁迫下植株体内活性氧（ROS）积累和膜脂过氧化作用的特点，以及抗氧化酶和呼吸酶活性、抗氧化剂的适应性变化，研究低氧胁迫对苹果砧木幼苗部分生理特性、ROS代谢和根系呼吸代谢的影响，探究苹果砧木幼苗低氧胁迫的生理伤害机理以及抗氧化系统和根系呼吸代谢对低氧胁迫的调节机制，并分析这些生理生化变化与苹果砧木幼苗耐低氧性的关系。主要研究结果如下：1．低氧胁迫15 d时，苹果属砧木资源幼苗新叶数、根系长、最长新根长、株高和生物量明显降低，根系中超氧阴离子（O₂^-）产生速率和丙二醛（MDA）含量增加，可溶性蛋白含量下降，相对膜透性（RMP）增大，植株受到伤害；同时根系中脯氨酸（Pro）含量增加，超氧化物歧化酶（SOD）和过氧化物酶（POD）活性升高，而根系活力和过氧化氢酶（CAT）活性因砧木资源不同而表现不一。淹水胁迫30 d时，苹果属砧木资源幼苗叶绿素含量降低并且不同叶绿素之间的比例发生改变，植株生物量明显减少。2．不同砧木资源对低氧逆境的敏感性存在较大差异。采用WI、ARC、隶属函数值等多种方法结合聚类分析的综合评价结果显示，供试的12个苹果属砧木资源可以分为4大类：（1）耐低氧性强的砧木资源，包括‘平邑甜茶’（Malus hupehensis（Pamp）Rehd．）、‘平顶海棠’（M．robusta Rehd．）和‘卢氏红果’（M．sieboldii（Regel）Rehd．）；（2）耐低氧性中等的砧木资源，包括‘毛山荆子’（M．manshurica（Maxim）Komarov．）、‘楸子’（M．prunifolia （Willd）Borkh．）、‘西府海棠’（M．micromalus Hemsl．）、‘八棱海棠’（M．robusta Rehd．）和‘东北黄海棠’（M．prunifolia Borkh．）；（3）耐低氧性较弱的砧木资源，包括‘红三叶海棠’（M．sieboldii（Regel）Rehd．）和‘大果红三叶海棠’（M．sieboldii（Regel）Rehd．）；（4）耐低氧性弱的砧木资源，包括‘新疆野苹果’（M．sieverii（Ledeb）Roem．）和‘变叶海棠’（M．toringoides（Rehd）Hughes．）。3．‘平邑甜茶’和‘变叶海棠’通气对照的幼苗在整个处理过程中光合色素含量及其比值、根系活力、可溶性蛋白和Pro含量变化不大。而低氧胁迫下两种苹果砧木幼苗叶绿素a（Chl a）、叶绿素b（Chl b）、总叶绿素（Chl）和类胡萝卜素（Car）含量都降低，并引起不同色素间比例的改变；根系活力先升后降并在胁迫中后期低于对照；叶片和根系中可溶性蛋白质含量持续下降；脯氨酸（Pro）含量逐渐增加，且叶片的变化幅度大于根系。耐低氧性强的‘平邑甜茶’除根系活力和Pro含量外，其余指标的变化幅度均小于耐性弱的‘变叶海棠’。4．正常通气条件下，‘平邑甜茶’和‘变叶海棠’两种砧木幼苗体内ROS代谢均无明显变化。但在低氧胁迫过程中，苹果砧木幼苗叶片和根系中O₂^-产生速率和H₂O₂含量增加，MDA含量上升，且随时间延长呈快慢快的变化趋势，RMP随之增加；同时叶片和根系中SOD、POD、CAT的活性增加，抗坏血酸过氧化物酶（APX）和谷胱甘肽还原酶（GR）的活性前期有所提高而后期降低并低于对照，抗坏血酸（AsA）和还原型谷胱甘肽（GSH）含量在胁迫早期增加而后逐渐减少，中期和后期低于对照，脱氢抗坏血酸（DHA）和氧化型谷胱甘肽（GSSG）含量略增，并由此引起总抗坏血酸（AsA+DHA）和总谷胱甘肽（GSH+GSSG）水平的变化与AsA和GSH含量基本一致，而表示抗坏血酸和谷胱甘肽氧化还原势的AsA／DHA和GSH／GSSG比值在胁迫前期增加而后下降，在胁迫后期显著低于对照。说明低氧胁迫下苹果砧木幼苗叶片和根系中ROS积累，细胞膜脂质过氧化加重，植株叶片和根系的抗氧化防御系统能在一定程度上减轻低氧胁迫所造成的膜伤害，但当胁迫持续加重时，植株将受到更大的氧化伤害。‘变叶海棠’叶片和根系细胞膜受到的氧化伤害大于‘平邑甜茶’，其抗氧化系统的防御能力弱于后者；而根系受到的氧化伤害大于叶片。5．正常通气条件下，‘平邑甜茶’和‘变叶海棠’幼苗根系的呼吸代谢未发生明显变化。低氧胁迫使苹果砧木幼苗根系中异柠檬酸脱氢酶（IDH）、琥珀酸脱氢酶（SDH）、苹果酸脱氢酶（MDH）和细胞色素C氧化酶（CCO）活性显著降低，丙酮酸大量积累；同时丙酮酸脱羧酶（PDC）、乙醇脱氢酶（ADH）和乳酸脱氢酶（LDH）活性先升后降，并在胁迫中期酶活性达到最高；乙醛、乙醇和乳酸含量呈先增后减的趋势。说明低氧胁迫下，苹果砧木幼苗根系三羧酸循环（TCAC）受阻，电子呼吸链的末端氧化能力下降，根系有氧呼吸代谢受到抑制；同时根系加强了乙醇发酵和乳酸发酵，导致根系中乙醇、乙醛和乳酸积累。整体来看，耐低氧性强的‘平邑甜茶’幼苗根系中有氧呼吸酶活性下降幅度较小而无氧呼吸酶活性的上升幅度较大，乙醇和乙醛积累较少。更多还原

【Abstract】 Apple is one kind of the world’s major fruit trees of cultivated species and the mostwidely cultivated deciduous fruit trees in North of China.In apple production,Some factorssuch as flooding,unfavorable irrigation,tight soil texture cause the occurrence of hypoxiaaround apple tree’s root-zone.There are few reports about the hypoxia stress on apple trees inthe world.The stress resistance of apple trees mainly depends on rootstocks,therefore tostudy the resistant capacity of Malus rootstocks to hypoxia,and select hypoxia-resistantrootstocks is of great significance to fully utilize the resources of Malus rootstocks,developapple’s production,reasonably choose and orientately breed fruit trees varieties with tolerantto waterlogging.Through deeply studying on physiological damage to Malus rootstocks andproving the adaptive mechanism of hypoxia stress,it can provide some physiological stresstheory and certain theoretical basis to solve the soil stress of hypoxia in production of fruittrees.In this research,12 Malus rootstocks in China were used as materials.In order toanalyze the difference of tolerance to hypoxia among different Malus rootstock seedlings andcomprehensively evaluate their hypoxia tolerance,many means by waterlogging index（WI）,adversity resistance coefficient（ARC）,subordinate function value in fuzzy math and clusteranalysis were used,combining with nutrient solution culture with waterlogging trail.TwoMalus rootstocks with significant difference in hypoxia tolerance were selected for furtherstudy.Through analyzing the changes of accumulation of reactive oxygen species（ROS）andmembrane lipid oxidation,the activities of antioxidant enzymes and enzymes related to root’srespiratory and the contents of antioxidants,the results revealed the physiological mechanismof damage and the regulation metabolism of antioxidant system and root’s respiratorymetabolism in Malus rootstock seedlings under hypoxia stress,and further analyzing therelationship between these physiological changes and hypoxia tolerance of Malus rootstockseedlings. The main results were as follows:1.After 15 d under hypoxia stress,some indicators showed significant decreases amongin Malus rootstock seedlings,such as the number of new leaf,root length and maximumlength of new root,plant height and biomass,however the generation rate of superoxideanion radical（O₂^-）,content of malonaldehyde（MDA）and relative membrane permeability（RMP）in root increased,and content of the soluble protein decreased,which indicated thatthe seedlings under hypoxia stress were damaged.Meanwhile,in root of Malus rootstockseedlings under hypoxia stress,the content of the proline（Pro）increased,and the activities ofsuperoxide dismutase（SOD）and peroxidase（POD）raised,while the root vitality and activityof catalase（CAT）showed different changes among different Malus rootstocks.After 30 d ofwaterlogging stress,in all rootstock seedlings the contents of chlorophyll reduced and theratios of different chlorophyll types changed,and the plant biomass decreased significantly.2.More significant differences in hypoxia sensitivity among different Malus rootstocksexisted.Comprehensive evaluation with WI,ARC,subordinate function value in fuzzy mathand cluster analysis results showed these rootstocks could be divided into 4 categories.（1）high hypoxia tolerance rootstocks,including ’Pingyi begonia’（Malus hupehensis（Pamp）Rehd.）,’Pinding begonia’（M.robusta Rehd.）and ’Lushi red fruit’（M.sieboldii（Regel）Rehd.）;（2）medium hypoxia tolerance rootstocks,including ’Maoshang jingzi’（M.manshurica（Maxim）Komarov.）,’Qiuzi’（M.prunifolia（Willd）Borkh.）,’Xifu begonia’（M.micromalus Hemsl.）,’Baleng begonia’（M.robusta Rehd.）and’Northeast yellow begonia’（M.prunifolia Borkh.）;（3）low hypoxia tolerance rootstocks,including’Red tri-leaf begonia’（M.sieboldii（Regel）Rehd.）and ’Dig fruit red tri-leaf begonia’（M.sieboldii（Regel）Rehd.）;（4）week hypoxia tolerance rootstocks,including ’Xinjiang wild apple’（M.sieverii（Ledeb）Roem.）and ’Bianye begonia’（M.toringoides（Rehd）Hughes.）.3.During the normal ventilation treatment,in ’M.hupehensis’ and ’M.toringoides’seedlings,there were little changes in the contents and ratios of photosynthetic pigments,rootvitality,contents of soluble protein and Pro in leaf and root.While in the seedlings underhypoxia stress,the contents of chlorophyll a（Chl a）,chlorophyll b（Chl b）,total chlorophyll（Chl）and carotinase（Car）decreased,and the ratios of different photosynthetic pigmentschanged.At the same time,the root vitality rose first and decreased afterwards,and waslower than that of control at middle-late-term under hypoxia stress;and the contents ofsoluble protein continually declined in leaf and root.But the content of Pro increasedgradually,and the variation range in leaf was larger than that in root.There were different in two rootstocks under hypoxia stress.Except the root vitality and the content of Pro,therewere larger variation ranges of other indicators in ’M.hupehensis’ seedlings with highhypoxia tolerance than that of’M.toringoides’ seedlings with week hypoxia tolerance.4.There were no significant changes in ROS metabolism in ’M.hupehensis’ and ’M.toringoides’ seedlings under normal ventilated conditions.However,many indicatorsincreased in leaf and root of Malus rootstock seedlings during hypoxia stress,such as thegeneration rate of O₂^-,content of hydrogen peroxide（H₂O₂）and MDA and RMP,showedthe similar trend of’fast-slow-fast’ with the time passing.At the same time,the activities ofSOD,POD and CAT increased;activities of ascorbate peroxidase（APX）and glutathionereductase（GR）increased first and decreased afterwards,and were lower than that of controlat later-term under hypoxia stress.Moreover,contents of ascorbic acid（AsA）and reducedglutathione（GSH）increased in the early and reduced gradually in leaf and root of Malusrootstock seedlings under hypoxia stress,and were lower than that of control at middle-term;there were slight increases in contents of dehydroascorbate（DHA）and oxidized glutathione（GSSG）,which lead to that the contents of total ascorbic acid（AsA + DHA）and totalglutathione（GSH + GSSG）showed basically in accordance with AsA and GSH.As a result,ratios of AsA/DHA and GSH/GSSG indicating redox potential of ascorbate and glutathioneincreased at earlier term and then decreased gradually,and were significantly lower than thatof control at later term of hypoxia stress.All these suggested that hypoxia stress in Malusrootstock seedlings lead to the accumulation of ROS and more serious membrane lipidperoxidation,however plants could soften the damage from oxidative stress;but theantioxidant defense system including antioxidantive enzymes and antioxidants could reducethese damages to some extent,but when the stress continued over the limit,the plants wouldexpose to greater oxidative damage.The damages to leaf and root of ’M.toringoides’seedlings were greater than that of’M.hupehensis’,and the capacity of antioxidant defensesystem were lower than that of the latter,and the damage of oxidative stress to root wasgreater than that of leaf.5.There were no significant changes in respiratory metabolism of root in ’M.hupehensis’ and ’M.toringoides’ seedlings under normal ventilated conditions.Underhypoxia stress,the activities of some enzymes related to respiration decreased significantly inroot of Malus rootstock seedlings,such as isocitrate dehydrogenase（IDH）,succinatedehydrogenase（SDH）,malate dehydrogenase（MDH）and cytochrome C oxidase（CCO）,andpyruvate accumulated.At the same time,the activities of pyruvate decarboxylase（PDC）, alcohol dehydrogenase（ADH）and lactate dehydrogenase（LDH）rose first and decreasedafterwards,and reached the highest in the middle-term under hypoxia stress.There were sametrends of increasing-decreasing for acetaldehyde,alcohol and lactate.All these indicated thatthe tricarboxylic acid cycle（TCAC）was inhibited in root of Malus rootstock seedlings underhypoxia stress,the terminal oxidation capacity of electronic respiratory chain decreased,andthe aerobic respiration in root was inhibited.At the same time,the anaerobic respirationincluding alcohol fermentation and lactate fermentation was strengthen in root of Malusrootstock seedlings,which caused the accumulations of alcohol,acetaldehyde and lactate.Overall,the decreasing ranges of the activities of enzymes related to aerobic respiration wereless in root of ’M.hupehensis’ seedlings with high hypoxia tolerance,while the activities ofenzymes related to anaerobic respiratory increased larger,and showed less accumulations ofalcohol and acetaldehyde in root.更多还原