| Aluminum (Al) toxicity is the major limiting factor for plants growth on acidic soils, organic acids (OAs) plays an important role in alleviating Al toxicity. Recent studies have found that the expression of14-3-3and plasma membrane H+-ATPase isoform genes was induced under low phosphorus stress or Al stress. Meanwhile, the citrate secretion was correlation with plasma membrane H+-ATPase activity, plasma membrane H+-ATPase may be involved in the citrate secretion regulation through a citrate-proton transport system, and14-3-3proteins play an important role in regulating the activity of plasma membrane H+-ATPase. Adding FC (activitor) or AMP (inhibitor) treatment, the activity of plasma membrane H+-ATPase increase or decrease under Al stress, at the same time the citrate secretion was also up-or down-regulation. Therefore, the regulation of OAs secration by changing the plasma membrane H+-ATPase activity may be another important mechanism which is independent with the OAs secration channel protein. In this study, as aluminum-Resistant Black Soybean (RB) and aluminum-Sensitive black soybean (SB) experimental materials, it was investigated the expression and interaction of14-3-3proteins and plasma membrane H+-ATPase in response mechanism to Al stress at the molecular level. The following results:1. This effect of the transcription translation and interaction of the14-3-3protein and PM H+-ATPase were investigated in Al-Resistant (RB) and Al-Sensitive (SB) black soybeans, respectively, under50μM Al stress. The results showed that Al stress significantly induced the transcription of the14-3-3a gene in RB but not in SB roots. The Al-enhanced transcription of the PM H+-ATPase encoding gene (gha2) was higher in RB than in SB roots. The translation and phosphorylation of PM H+-ATPase, the interaction between the14-3-3protein and the phosphorylated PM H+-ATPase all showed an increasing pattern in RB but displayed a descending pattern in SB during the whole Al stress period. Consistently, the H+pump activity and citrate secretion were also higher in RB than in SB under Al stress. Furthermore, the citrate secretion was positively correlated with the PM H+-ATPase activity in RB and SB under Al stress. These results indicated that Al-enhanced expression of14-3-3protein and PM H+-ATPase and the interaction between the14-3-3protein and the phosphorylated PM H+-ATPase increased the activity of PM H+-ATPase and the H+pump, which thereby augmented citrate secretion in RB. This might be an important molecular mechanism underlying the higher Al-tolerance in RB than SB.2.FC (Fusicoccin) increased plasma membrane H+-ATPase activity through promoting interaction14-3-3proteins and the phosphorylated plasma membrane H+-ATPase; while AMP was an inhibitor of interaction14-3-3proteins and the phosphorylated plasma membrane H+-ATPase, and it had a role to inhibit the plasma membrane H+-ATPase activity. It was investigated that under Al stress whether the change of interaction14-3-3proteins and PM H+-ATPase by FC treatment for SB or AMP for RB was involved in the regulation of citrate secration and aluminum tolerance. The results shows that under50μM Al stress, adding1μM FC treatment for8h, the inhibition of SB root growth was significantly alleviated (RRG increase of36.4%), while add100μM AMP treatment for8h, the aluminum tolerance of RB significantly was reduced (RRG decrease of41.4%). Co-immunoprecipitation analysis showed that FC promoted the interaction14-3-3proteins and phosphorylated plasma membrane H+-ATPase, increased the activity of plasma membrane H+-ATPase (about1-fold), enhanced H+pump activity and H+efflux, and ascended citrate secration (about2-fold) in SB under Al stress. However, AMP reduced the interaction14-3-3proteins and phosphorylaed plasma membrane H+-ATPase, decreased the plasma membrane H+-ATPase activity (about1-fold)、H+pump and H+efflux, and decended citrate secretion (about1-fold) in RB under Al stress. These results confirmed that the interaction14-3-3proteins and plasma membrane H+-ATPase was involved in the regulation of citrate secretion in soybean under Al stress, and the change of interaction14-3-3proteins and plasma membrane H+-ATPase could affect the ability of Al-tolerance of Black Soybean.3. Because, tobacco was reproduced and transformed, so it was one of model plant in plant research field. In this study, In order to test the black soybean14-3-3 proteins and PM H+-ATPase in response mechanim to Al stress by transgenic tobacco, it was investigated that the14-3-3proteins and PM H+-ATPase in response mechanism to Al stress in wide-type tobacco (WT). The results indicated that WT was Al-sensistent, and the expression and interaction14-3-3proteins and PM H+-ATPase was decreased, and citrate secration and H+effult was reduced with increasing Al treatment time under50μM Al stress. And adding FC promoted increasing of PM H+-ATPase activity、citrate secretion and Al-tolerance in WT tobacco, but adding AMP was opposite results. These results indicated that the expression and interaction14-3-3proteins and plasma membrane H+-ATPase was correlatived with PM H+-ATPase activity and citrate secretion in WT tobacco under Al stress, and it confirmed that14-3-3proteins and plasma membrane H+-ATPase in response mechanism to Al stress was similar to Al-sensitent black soybean (SB).4. To further investigate the role of expression and interaction of Al-induced14-3-3proteins and plasma membrane H+-ATPase of RB in the regulation of citrate secretion and Al-Resistant. The SGF14a and without C-terminal self-suppression domain (△GHA2) were respectively overexpressed in tobacco, while the tobacco14-3-3e and plasma membrane H+-ATPase (pma4) were inhibited expression through RNAi (RNA interference). Further analysis results showed that in overexpression SGF14a transgene tobacco, amounts of14-3-3proteins could combine with phosphorylated plasma membrane H+-ATPase, and maintain the stability of the phosphorylation level of plasma membrane H+-ATPase, which not only can enhance the activity of plasma membrane H+-ATPase and H+secretion, but also antagonist to Al or inhibitor AMP-induced the down-regulation of its activity, and increase citrate secretion of in transgenic tobacco root tip, significantly reduce H2O2accumulation and oxidative stress under Al stress, so it enhanced the ability of Al-resistant and adaption to low phosphorus stress on red soil. While RNAi-inhibited the expression of14-3-3protein in tobacco, the phosphorylated plasma membrane H+-ATPase was lack of14-3-3proteins, thereby its phosphorylation state was instability to occur dephosphorylation, and reduced plasma membrane H+-ATPase activity and citrate secretion in transgenic, and reduced Al-resistant and the ability of adaption to low phosphorus stress on red soil. Overexpression AGHA2in tobacco has no effect the phosphorylation level of plasma membrane H+-ATPase and interaction with14-3-3proteins, but it significantly improved plasma membrane H+-ATPase activity and citrate secretion and its ability of Al-tolerance, and the growth of transgenic tobacco was better than WT on red soil. While inhibited expression of plasma membrane H+-ATPase (pma4) in tobacco was significantly reduced the combination14-3-3proteins with phosphorylated plasma membrane H+-ATPase, and marked droped the activity of plasma membrane H+-ATPase、H+secretion and citrate secretion, and which it increased H2O2accumulation under Al stress, reduced Al-tolerance and adaptation to low phosphorus stress. These results provide a new strategy and genetic resources for using genetic engineering and molecular biology methods to improve the ability of Al-resistant in plant. |
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