Up-regulation and interaction of the plasma membrane H(+)-ATPase and the 14-3-3 protein are involved in the regulation of citrate exudation from the broad bean (Vicia faba L.) under Al stress

Abstract

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.

Keywords: 1,3-bis-[tris(hy- droxymethyl)-methylamino]-propane; 14-3-3 Protein; 5’-AMP; AD; Al; Al stress; BTP; Broad bean; Citrate exudation; DDT; FC; OA; PM; PMSF; Plasma membrane H(+)-ATPase; VHA2p; Vicia faba 14-3-3-like protein b; Vicia faba plasma membrane H(+)-ATPase; Vicia faba. L cv. Anhui Candou; Vicia faba. L cv. Yunnan Candou 8363; YD; adenosine 5′-monophosphate; aluminum; dithiothreitol; fusicoccin; organic acid; phenylmethylsulfonyl fluoride; phosphorylated VHA2 at Thr-951 in C terminus; plasma membrane; vf14-3-3b; vha2.