Physiological And Molecular Mechanisms Of Soybean (Glycine Max.) Adaptation To Manganese Stress

Soybean [Glycine max (L.) Merr] is one of the important legume crops containing high protein and fat, which is one of main protein resources for human. Manganese (Mn) toxicity is one of the main factors limiting soybean growth on acid soils. However, physiological and molecular mechanisms of soybean tolerance to manganese toxicity remain unclear. In this study, three soybean varieties contrasting in Mn tolerance were used, including Bendi2 (BD2), Huachun2 (HC2) and Huachun3 (HC3). Effects of Mn availability on acquisition and transportation of Mn, iron (Fe) and phosphorus (P) were evaluated in plants grown in nutrient solution. In order to elucidate the molecular mechanisms of Mn tolerance in soybean, proteomic analysis platform of soybean roots was constructed and optimized. Subsequently, protein expression profiles in soybean roots were compared in Mn tolerant cultivar BD2 and Mn sensitive cultivar HC2 under both normal and Mn toxic conditions. A group of proteins responsive to Mn toxicity were isolated and identified, which partially elucidated molecular mechanisms of soybean tolerance to Mn toxicity. The main results were shown as follows:1. Brown spots were observed in the old leaves, and soybean growth was inhibited at 50μmol·L-1 Mn level. With the increase of Mn application, amount of the brown spots and the degree of inhibited growth of plants were increased. At 400μmol·L-1 Mn level, the amount of brown spots was 15 times higher than that at 50μmol·L-1 Mn, and biomass was separately decreased by 36% in shoots and 26% in roots, compared with plants grown under normal conditions. Furthermore, increase of Mn application in the medium resulted in increase of Mn and Mn/Fe in plants. However, Fe concentration in plants was decreased, P concentration in plants was not affected by the increase of Mn availability in the medium. Interestingly, Mn toxicity resulted in more Mn accumulation in old leaves and more Fe accumulation in young leaves, indicating that Mn toxicity could affect Mn and Fe distribution in leaves.2. Manganese toxicity tolerance was compared among the three soybean cultivars, including BD2, HC2 and HC3. Among them, BD2 seemed the most Mn tolerant cultivar because the amount of brown spots in old leaves, the ratio of Mn/Fe and the reduced growth were the least in BD2 at 50μmol·L-1 Mn level. Superior capability of Mn toxicity tolerance in BD2 might be partially caused by the highest Fe concentration and the lowest Mn/Fe in BD2 under Mn toxic conditions.3. In order to construct and optimize proteomic analysis platform of soybean roots, three protein-extracting methods (direct extracted by lysate, extracted by 0.5M Tris-HCl, extracted by 40mM Tris-HCl) and effects of different immobilized pH gradient (IPG) strips were evaluated using the two dimensional electrophoresis. Our results showed that extraction of protein using 0.5M Tris-HCl buffer combined with using 24 cm IPG (pH3-10) strips could result in the best separation of root proteins. A total of 849 protein spots were observed in the gel. Furthermore, separation of different protein spots was confirmed through MALDI-TOF-TOF/MS analysis of one randomly selected protein spot from the gel.4. Totally,20 and 17 protein spots responsive to Mn toxicity were separately isolated in BD2 and HC2 through the two dimensional electrophoresis analysis. Subsequently, MALDI-TOF-TOF/MS analysis results showed that the identified proteins were involved in several biological processes, including stress responses, metabolism, translation, transcription. Furthermore, the expression levels of several proteins, including PR-1, PR-10 and isocitrate dehydrogenase were higher in BD2 than those in HC2, suggesting that these accumulated proteins might be involved in the molecular mechanisms underlying Mn tolerance in BD2.