【作者】 杨野；

【导师】 赵竹青；

【作者基本信息】 华中农业大学 ， 植物营养学， 2011， 博士

【摘要】 酸性土壤中的铝毒害抑制植物生长,降低作物品质和产量。因此对植物耐铝胁迫机理的研究具有重要意义。本文以不同耐铝型小麦品种ET8(耐铝型)和ES8(铝敏感型)为试验材料,对两品种耐铝差异进行了比较研究。通过对铝胁迫下不同耐铝型小麦品种根尖细胞影响细胞壁结构的物质代谢的研究,阐明了不同耐铝型小麦品种根生长差异机理。通过对铝胁迫下不同耐铝型小麦品种活性氧代谢系统的研究,阐明了不同耐铝型小麦品种耐铝胁迫差异机理。通过对铝胁迫下不同耐铝型小麦品种根尖细胞质膜H+ -ATPase的研究,阐明了不同耐铝型小麦品种改变根际pH值能力差异机理。此外,通过对植物激素在铝胁迫下不同耐铝型小麦品种苹果酸分泌中作用的研究,揭示了铝激活苹果酸分泌机理。最后,通过对铝胁迫下不同耐铝型小麦品种根尖细胞有机酸代谢的研究,阐明了不同耐铝型小麦品种苹果酸分泌差异机理。结果概述如下：1.铝胁迫下不同耐铝型小麦品种根生长差异机理随着铝处理浓度的升高及处理时间的延长,ET8和ES8根相对伸长率及根尖细胞相对长度均显著降低,两者呈极显著正相关(R2=0.866**)。50μmol/L铝胁迫24h后,ET8和ES8根尖伸长区皮层细胞变扁平,细胞间隙变小,细胞壁褶皱,并呈齿轮状交合,ET8细胞核解体,高尔基体、线粒体依然完整,但是线粒体出现肿胀,且初生壁内出现多层次生壁,ES8细胞内含物消失,次生壁生长量更大,细胞壁明显变厚,ES8细胞受伤害程度较ET8显著。铝胁迫下ET8和ES8根尖细胞木质素、H202及胼胝质含量上升,纤维素含量下降,苯丙氨酸解氨酶(PAL)、肉桂醇脱氢酶(CAD)和过氧化物酶(POD)活性升高,胼胝质酶和纤维素酶活性降低,相同处理条件下ES8变化显著大于ET8。根相对伸长率与胼胝质、H202和纤维素含量间均具有极显著的相关性(R2=0.958**,R2=0.8806**,R2=0.9182**)。综上所述,铝胁迫下ES8根尖细胞结构受破坏程度较ET8显著,且由于ES8根尖细胞影响细胞壁结构的物质代谢酶活性的变化显著大于ET8,并造成这些物质变化也显著大于ET8,从而导致细胞壁结构更加不稳定,降低了细胞壁延展性,进而导致细胞伸长显著小于ET8,最终导致铝胁迫下两不同耐型小麦品种根生长差异。2.铝胁迫下不同耐铝型小麦品种活性氧代谢差异及与小麦耐铝差异关系随着铝处理浓度的升高,ET8和ES8根尖O2·-产生速率和H202含量随之显著升高,根尖活性氧代谢酶,如超氧化物歧化酶(SOD)、过氧化物酶(POD)、抗坏血酸过氧化物酶(APX)、过氧化氢酶(CAT)及谷胱甘肽还原酶(GR)等酶活性也均显著升高,ET8根尖活性氧累积量显著低于ES8,且抗氧化酶效率显著高于ES8。对O2·-和H2O2的组织化学定位研究表明,两者主要累积于根尖分生区及伸长区,而且ET8根尖活性氧累积量显著低于ES8。ET8和ES8根尖细胞丙二醛(MDA)含量显著升高,质膜透性随着MDA含量的升高而增加。对质膜完整性的组织化学染色表明,质膜受破坏的细胞在根尖的部位及受破坏程度与2·-和H202累积部位、累积量相吻合。综上所述,铝胁迫下不同耐铝型小麦品种活性氧清除酶系统效率显著差异是耐铝差异机理之一。3.铝胁迫下不同耐铝型小麦品种根尖细胞质膜H+-ATPase调节的根际pH值差异与耐铝差异关系ET8和ES8根际pH值随培养时间的延长及铝浓度的降低而显著升高,相同处理条件下ET8根际pH值显著高于ES8,根际pH值与根尖铝含量呈极显著负相关(R2=0.9321**)关系,与根相对伸长率呈极显著正相关(R2=0.9209**)关系。H+-ATPase专一性抑制剂DCCD(25μmol/L)处理显著抑制根际pH值的上升。根尖细胞质膜H+-ATPase活性随铝处理浓度的升高而显著降低,100μmol/L Al处理24h ET8和ES8根尖细胞质膜H+-ATPase活性分别为各自无铝处理的69.8%和60.0%,相同处理条件下,ET8根尖细胞质膜H+-ATPase相对活性显著高于ES8。细胞质膜H+-ATPase相对活性与根际pH值呈极显著正相关关系(R2=0.8319**)。综上所述,由于铝胁迫下耐铝型小麦品种ET8根尖细胞能够吸收根际H+的质膜H+-ATPase活性显著高于铝敏感型小麦品种ES8,因此其根际pH值也显著高于ES8,从而降低了根际活性铝含量,减少了铝在根尖细胞的累积,减轻了铝的毒害作用,铝胁迫下不同耐铝型小麦品种显著的质膜H+-ATPase活性差异导致的根际pH值差异也是不同耐铝型小麦品种耐铝差异机理之一。4.植物激素对铝胁迫下不同耐铝型小麦品种苹果酸分泌的影响1)与对照处理相比,无铝条件下,不同浓度的ABA、GA和IAA处理对小麦4d幼苗及20 d大苗的苹果酸分泌均无显著影响；与铝单独处理相比,铝存在条件下,ABA或GA处理对小麦根苹果酸的分泌也无显著影响,但IAA(50,100μmol/L)处理,ET8苹果酸分泌速率显著升高,ES8苹果酸分泌速率无显著变化,说明外源ABA和GA对铝胁迫下ET8和ES8苹果酸分泌无显著影响,但IAA可以增加ET8苹果酸的分泌；2)当ET8和ES8经铝预处理3h后,无铝条件下,再经不同浓度IAA处理,ET8苹果酸分泌速率随IAA浓度的升高而显著升高,ES8苹果酸分泌速率却无显著变化；ET8和ES8经0或50IAA处理3h后,无IAA条件下,再经铝处理,经IAA预处理过的ET8根苹果酸分泌速率显著高于无IAA预处理根的苹果酸分泌速率,但ES8根经IAA预处理与否,苹果酸分泌速率均无显著变化,进一步说明外源IAA可增加铝胁迫下ET8苹果酸的分泌；3)铝胁迫显著促进ET8和ES8根尖内源ABA及IAA含量的升高,但降低GA含量；IAA分解代谢酶,IAAoxidase相对活性则随铝处理浓度的升高而显著降低；相同处理条件下ET8根内源IAA含量显著高于ES8,IAA oxidase活性显著低于ES8,说明IAA oxidase活性差异与内源IAA含量差异有关；根苹果酸分泌速率同内源IAA含量间显著相关性(R2=0.8532**)表明IAA参与调节铝胁迫下小麦根苹果酸的分泌；4)分根处理条件下,ET8植株两侧根系(part A和part B)分别经0和50μmol/L铝处理后,0μmol/L铝处理一侧未检测到转运自铝处理一侧的铝,但该侧根苹果酸分泌速率及内源IAA含量均显著高于植株两侧均为0μmol/L铝处理(part A,0μmol/L和part B,0μmol/L)条件下的根苹果酸分泌速率及内源IAA含量,表明铝不必直接与根接触即可诱导苹果酸分泌速率和内源IAA含量同时升高,说明IAA参与苹果酸分泌；ET8植株两侧分别经part A (0μmol/L)和part B (50μmol/L Al+50μmol/L IAA)处理后,part A根系苹果酸分泌速率显著高于part A (0μmol/L)和part B (50μmol/L Al)处理中partA的苹果酸分泌速率,该结果更加说明IAA参与调节铝胁迫下小麦根苹果酸的分泌；5)同铝单独处理相比,IAA和铝共处理可显著减少铝在ET8根尖的累积,但对ES8无显著影响；6)IAA极性运输抑制剂NPA或TIBA可以显著抑制铝胁迫下ET8和ES8根苹果酸的分泌,有力说明IAA参与调节铝胁迫下小麦根苹果酸的分泌；7)外源IAA处理可以显著抑制阴离子通道抑制剂对铝胁迫下ET8和ES8根苹果酸分泌的抑制作用,表明IAA可能通过调节阴离子通道参与铝胁迫下小麦根苹果酸的分泌；8)IAA和铝共处理,ET8根尖细胞ALMT1表达量显著升高。综上所述,虽然外源ABA和GA对小麦根苹果酸的分泌无显著影响,但IAA在Al存在条件下,能够通过调节阴离子通道参与铝胁迫下小麦分泌苹果酸,IAA对不同耐铝型小麦品种苹果酸分泌调节作用的差异也是不同耐铝型小麦品种苹果酸分泌差异机理之一。5.铝胁迫下不同耐铝型小麦品种有机酸代谢与苹果酸分泌差异的关系不同浓度铝处理下ET8和ES8根尖细胞内源苹果酸含量均无显著变化,相同处理条件下ET8和ES8间内源苹果酸含量亦无显著差异,分别为0.48,0.46,0.57,0.52nmol root apex-1和0.45,0.51,0.51,0.54 nmol root apex-1;同无铝处理相比,50或100μmol/L铝处理显著促进ET8和ES8根尖细胞磷酸烯醇式丙酮酸酶(PEPC)活性升高,但各铝浓度处理对柠檬酸合成酶(CS)和苹果酸脱氢酶(MDH)活性均无显著影响；相同处理条件下ET8和ES8根尖细胞PEPC, CS和MDH三种酶活性均无显著差异。综上所述,铝胁迫下根尖细胞内源苹果酸含量及有机酸代谢关键酶活性对不同耐铝型小麦品种苹果酸分泌差异无显著影响。更多还原

【Abstract】 Aluminum (A1) is believed to be one of the major factors limiting the growth of roots and the overall development of plants in acid soils. So the researches on A1 resistant mechanism are of great importance. The Al resistance mechanism differences of two wheat(Triticum aestivum L.) cultivars ET8 and ES8 that differing in A1 tolerances were studied in this research. Root growth differences of wheat differing in A1 tolerance were reveled by studies of cell wall chemical components metabolism system. A1 resistant mechanisms of wheat differing in Al tolerance were reveled by studies of reactive oxygen species. Rhizosphere pH changing capacities of wheat differing in Al tolerance were reveled by studies of plasma membrane H+-ATPase. Mechanisms of Al-induced efflux of malic acid were revealed by studies of effects of phytohormone on Al-induced malic acid efflux. Malic acid efflux differences of wheat differing in A1 tolerance were revealed by studies of organic acid metabolism system in root apex cell. The main results obtained were summarized as follows:1. Mechanisms of root growth differences of wheat differing in Al toleranceRelative root elongation (RRE) and relative root cell length (RCL) of ET8 and ES8 decreased with the increasing Al concentrations and time prolonging. A good correlation was obtained between RRE and RCL (R2=0.866**). After 50μmol/L A1 treatment for 24 h, longitudinal sections of ET8 and ES8 both reveal that the cells in the elongation zone became flat, the cell wall folded and ragged, intercellular space was decreased and occluded like gears; in ET8 the nucleolus was degradated, the srtucture of golgi and mitochondria kept integrety, but mitochondria swelled, multilayer secondry cell wall occured within primary cell wall; in ES8 cell contents disappeared, large amount of secondary cell wall occurred. Damages to cell structure of ES8 were more serious than that of ET8. Activities of phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD) increased; activities of callase, cellulase decrease; contents of lignin, H2O2 and callose increased; contents of cellulose decreased. Changes of enzyme activities and cell wall chemical components were significant of both lines, but more prominent in the ES8 line. There were a good correlation among RRE and contents of callose, H2O2 and cellulose (R2=0.958**, R2=0.8806**, R2=0.9182**). Analysis indicated that damages to cell structure of ES8 was more serious than that of ET8, and the A1 induced changes of root apex cell wall chemical components regulated by the key enzyme in cell wall chemical components metabolism were significant of ES8 than that of ET8, which caused rigidity of cell wall and followed by significant inhibition of cell elongation. 2. Active oxygen metabolism differences of wheat differing in Al tolerance and the relation to Al tolerance differencesWith the increasing of Al concentrations, contents of O2·- and H2O2, and activates of antioxidant enzymes in root apexes e.g., supper oxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR) all increased significantly in both ET8 and ES8. The accumulation of ROS in ET8 was less than that of ES8, and ET8 showed a higher efficiency of active oxygen metabolism systems than that of ES8. Contents of malondialdehyde (MDA) in ET8 and ES8 were increased. Membrane permeability increased with the increasing contents of MDA. Histochemical localization experiments showed that plasma membrane (PM) integrity decreased, consistent with the increasing MDA contents. Location of cells whose PM were destroyed, and the destruction degree of those cells were consistent with the accumulation location and extent of O2·- and H2O2. In conclusion, active oxygen metabolism systems efficiency difference of ET8 and ES8 was one of the mechanisms of Al tolerance difference.3. Relation ship between rhizosphere pH differences regulated by PM H+-ATPase and Al tolerant difference of wheat differing in Al toleranceRhizosphere pH of ET8 and ES8 increased with treatment time prolonging, but decreased with increasing Al concentrations. It was much higher of ET8 than that of ES8 under same treatment. Significant correlations were obtained among rhizosphere pH and relative root elongation (R2=0.9209**), or Al content in root apexes (R2=0.9321**). The elevation of rhizosphere pH was inhibited by H+-ATPase specific inhibitor DCCD (dicylcohexylcarbodiimide,25μmol/L). PM (plasma membrane) H+-ATPase activities decreased with increasing Al concentrations. It was 69.8% and 60.0% of ET8 and ES8 respectively, under the treatment of 100μmol/L Al for 24 h. Relative PM H+-ATPase activity of ET8 was significantly higher than that of ES8 under the same treatment. Significant correlation between relative PM H+-ATPase activity and rhizosphere pH (R2=0.8319**) were obtained. Taken together, PM H+-ATPase activities of ET8 were significantly higher than that of ES8, which induced higher rhizosphere pH of ET8 under Al stress. Less Al was accumulated in ET8 than that in ES8, and the Al toxic effects to ET8 was lighter than that of ES8. The Al-tolerant line showed a stronger capacity of up-regulating rhizosphere pH by PM H+-ATPase than the Al-sensitive line, which may explain the observed differences in Al tolerance between the two wheat cultivars.4. Effect of phytohormone on Al-induced malic acid efflux from wheat differing in Al tolerance1) ABA, GA or IAA treatments alone did not affect the efflux of mailc acid from 4 or 20 days old wheat of ET8 and ES8; the efflux of malic acid also could not be induced by the co-treatments of ABA or GA and Al, but could be induced by IAA (50,100μmol/L) in ET8, not in ES8 (25,50μmol/L); results above indicated that exogenous ABA or GA treatment did not affect the efflux of malic acid, but exogenous IAA could enhance the efflux of malic acid from ET8 under Al stress; 2) after a pretreatment with Al for 3 h, different concentrations IAA was applied at the Al free condition for 24 h, the efflux of malic acid increased significantly with the increasing concentrations of IAA in the ET8 but not ES8; after a pretreatment with 0 or 50μmol/L IAA for 3 h, the efflux of malic acid of the roots that were pretreated with IAA under Al stress was significantly higher than that without IAA pretreatment in ET8, the pretreatment of IAA did not affect the efflux of malic acid in ES8; results above further confirm that exogenous IAA could enhance the efflux of malic acid from ET8 under A1 stress; 3) Al stress induced the accumulation of ABA and IAA in root apex of ET8 and ES8, but decreased the content of GA; activities of IAA oxidase decreased with increasing A1 concentrations; endogenous IAA contents of ET8 were significantly higher than that of ES8 under the same treatment, which indicated that endogenous IAA content differences related to IAA oxidase activities differences; the highly positive correlation between malic acid efflux rate and endogenous IAA content indicated that IAA was involved in the regulation of A1 induced efflux of malic acid; 4) in split-root Al stress experiments (part A,0μmol/L and part B,50μmol/L Al), Al could not be transported from Al treatment portion (part B) to the control treatment portion (part A) of ET8 and ES8, but the malic acid efflux rate and endogenous IAA content in part A (CK) of ET8 were higher than that both side were treated without Al (part A,0μmol/L and part B,0μmol/L) in ET8 not ES8, which indicated that A1 could induce the efflux of malic acid without interact with root directly. The simultaneously increase of malic acid and endogenous IAA content indicated that IAA was involved in the Al induced efflux of malic acid; when part A and part B were treated with control and A1+IAA (part A, CK and part B, Al+IAA) respectively, the malic acid efflux rate in part A of ET8 was enhanced by the application of IAA in part B compared with the part A in the group that the other part was treated with Al (part A, CK and part B Al); results above proved that IAA was involved in regulating mailc acid efflux much more; 5) compared to Al treatment alone, root apex A1 content of ET8 decreased by the application of IAA, but not ES8; 6) compared to A1 treatments, the efflux rate of malic acid in ET8 and ES8 decreased under the co-treatments of IAA transport inhibitors NAP (or TIBA) and Al, which suggested that IAA was involved in the Al-induced malic acid efflux powerfully; 7) in addition, anion channel inhibitor treatment experiment showed that IAA (50μM) relieved the inhibiting effect of 5μM A9C (or NIF) on malic acid efflux induced by Al (50μM), compared to the co-treatment of A1 (50μM) and 5μM A9C (or NIF) it was thus speculated that the anion channel might have been activated when IAA was involved in malic acid efflux; 8) the expression of ALMT1 was induced significantly under the co-treatments of IAA and Al. In conclusion, although ABA and GA did not affect the efflux of malic acid of ET8 and ES8 under Al stress, IAA could be involved in regulating Al-induced malic acid efflux of wheat ET8 and ES8 via anion channel. The different regulating effects of IAA on the efflux of malic acid of wheat differing in Al tolerance may one of the mechanisms related to malic acid efflux differences.5. Relationship between malic acid secretion differences and organic acid metabolism of wheat differing in Al toleranceEndogenous malic acid contents of ET8 and ES8 were not affected by Al, and there were no differences between ET8 and ES8 when they received same treatment. Endogenous malic acid contents were 0.48,0.46,0.57,0.52 nmol root apex-1 and 0.45,0.51,0.51,0.54 nmol root apex-1, respectively of ET8 and ES8. Compared with Al free treatment, activities of phosphoenolpyruvate carboxylase (PEPC) were increased significantly under Al treatment, but not citrate synthase (CS) or malate dehydrogenase (MDH). The activities of PEPC (or CS and MDH) of ET8 and ES8 were close to each other under the same treatment. In conclusion, the malic acid secretion differences of different Al-tolerant wheat were independent on the endogenous malic acid content and the organic acid metabolism.更多还原