| 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. |
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