我们先前的研究表明,柠檬酸盐的排泄以及伴随的质子释放与蚕豆的铝(Al)抗性有关。此外,编码质膜(PM)H()-ATPase(vha2)和14-3-3蛋白(vf14-3-3b)的基因在抗Al(YD)蚕豆根中被Al上调。在这项研究中,进一步研究了抗Al(YD)和Al敏感(AD)蚕豆品种中PMH()-ATPase(E.C.3.3.6.3.6.6)和14-3-3蛋白在柠檬酸盐分泌调节中的作用在Al胁迫下。结果表明,与AD相比,YD根中的柠檬酸渗出量较大与PMH()-ATPase活性较高呈正相关。实时RT-PCR分析显示,vha2在YD中明显被Al上调,而在AD根中未被Al上调,而vf14-3-3b的转录水平在YD和AD根中以时间依赖性方式升高。免疫沉淀和Western分析表明,随着Al处理时间的增加,YD根中VHA2的磷酸化和与vf14-3-3b蛋白的相互作用得到增强,而AD根中则没有。镰刀霉素或腺苷5'-单磷酸增加或减少磷酸化VHA2和vf14-3-3b蛋白之间的相互作用,在Al胁迫条件下,两个品种的PMH()-ATPase活性和柠檬酸盐渗出均增强或降低,分别。一起来看,这些结果表明Al增强了PMH()-ATPase与14-3-3蛋白的表达和相互作用,从而导致PMH()-ATPase的活性更高,并且从YD植物中渗出更多的柠檬酸盐。
Our previous study showed that citrate excretion coupled with a concomitant release of protons was involved in aluminum (Al) resistance in the broad bean. Furthermore, genes encoding plasma membrane (PM) H(+)-ATPase (vha2) and the 14-3-3 protein (vf14-3-3b) were up-regulated by Al in Al-resistant (
YD) broad bean roots. In this study, the roles of PM H(+)-ATPase (E.C. 3.6.3.6) and the 14-3-3 protein in the regulation of citrate secretion were further investigated in Al-resistant (
YD) and Al-sensitive (AD) broad bean cultivars under Al stress. The results showed that greater citrate exudation was positively correlated with higher activities of PM H(+)-ATPase in roots of YD than AD. Real-time RT-PCR analysis revealed that vha2 was clearly up-regulated by Al in
YD but not in AD roots, whereas the transcription levels of vf14-3-3b were elevated in a time-dependent manner in both
YD and AD roots. Immunoprecipitation and Western analysis suggested that phosphorylation and interaction with the vf14-3-3b protein of the VHA2 were enhanced in YD roots but not in AD roots with increasing Al treatment time. Fusicoccin or adenosine 5\'-monophosphate increased or decreased the interaction between the phosphorylated VHA2 and the vf14-3-3b protein, followed by an enhancement or reduction of the PM H(+)-ATPase activity and citrate exudation in both cultivars under Al stress conditions, respectively. Taken together, these results suggested that Al enhanced the expression and interaction of the PM H(+)-ATPase and the 14-3-3 protein, which thereby led to higher activity of the PM H(+)-ATPase and more citrate exudation from
YD plants.