Citrate Secretion Coupled with the Modulation of Soybean Root Tip under Aluminum Stress. Up-Regulation of Transcription, Translation, and Threonine-Oriented Phosphorylation of Plasma Membrane H+-ATPase

The aluminum (Al)-induced secretion of citrate has been regarded as an important mechanism for Al resistance in soybean ( Glycine max ). However, the mechanism of how Al induces citrate secretion remains unclear. In this study, we investigated the regulatory role of plasma membrane H + -ATPase on the Al-induced secretion of citrate from soybean roots. Experiments performed with plants grown in full nutrient solution showed that Al-induced activity of plasma membrane H + -ATPase paralleled secretion of citrate. Vanadate and fusicoccin, an inhibitor and an activator, respectively, of plasma membrane H + -ATPase, exerted inhibitory and stimulatory effects on the Al-induced secretion of citrate. Higher activity of plasma membrane H + -ATPase coincided with more citrate secretion in Al-resistant than Al-sensitive soybean cultivars. These results suggested that the effects of Al stress on citrate secretion were mediated via modulation of the activity of plasma membrane H + -ATPase. The relationship between the Al-induced secretion of citrate and the activity of plasma membrane H + -ATPase was further demonstrated by analysis of plasma membrane H + -ATPase transgenic Arabidopsis ( Arabidopsis thaliana ). When plants were grown on Murashige and Skoog medium containing 30 μ m Al (9.1 μ m Al 3+ activity), transgenic plants exuded more citrate compared with wild-type Arabidopsis. Results from real-time reverse transcription-PCR and immunodetection analysis indicated that the increase of plasma membrane H + -ATPase activity by Al is caused by transcriptional and translational regulation. Furthermore, plasma membrane H + -ATPase activity and expression were higher in an Al-resistant cultivar than in an Al-sensitive cultivar. Al activated the threonine-oriented phosphorylation of plasma membrane H + -ATPase in a dose- and time-dependent manner. Taken together, our results demonstrated that up-regulation of plasma membrane H + -ATPase activity was associated with the secretion of citrate from soybean roots.