Evaluation Of Hypoxia Tolerance For Malus Rootstock Seedlings And Study On Physiological Metabolism Of Adaptation To Hypoxia Stress

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.