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

Abstract

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 microM Al (9.1 microM Al3+ 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.

Figure 1.

Effect of Al concentration (A and B) and duration of Al treatment (C and D) on citrate secretion (A and C) and the activity of plasma membrane H⁺-ATPase (B and D). Soybean seedlings were incubated in nutrient solution for 8 d, then treated with Al in the nutrient solution at pH 4.5. In A and B, the duration of Al treatment was 4 h, and in C and D, the concentration of Al was 30 μm. After treatment, root exudates were analyzed for citrate by HPLC, and the root apices (1 cm) were used for analysis of the activity of plasma membrane H⁺-ATPase. See details in “Materials and Methods.” Each value is the mean ± se (n = 5). Different letters in the same column or cultivar indicate that the values are significantly different at the 0.05 level, according to Duncan's Multiple Range Test.

Figure 2.

Effects of FC and VA on the Al-induced citrate secretion (A and C) and the activity of plasma membrane H⁺-ATPase (B and D) in soybean cultivars. Soybean seedlings were incubated in nutrient solution for 8 d, then treated with 30 μm Al together with 5 μm FC or 100 μm VA. After a 4-h treatment, exudates from soybean roots were analyzed for citrate by HPLC. The root apices (1 cm) of soybean were analyzed for the activity of plasma membrane H⁺-ATPase. See details in “Materials and Methods.” Each value is the mean ± se (n = 5). Different letters in the same column or cultivar indicate that the values are significantly different at the 0.05 level, according to Duncan's Multiple Range Test.

Figure 3.

Effect of Al concentration on the secretion (A) of citrate and the activity (B) of plasma membrane H⁺-ATPase in Arabidopsis roots. Arabidopsis seedlings were incubated in one-sixth Murashige and Skoog solution for 14 d, then exposed to 15 or 30 μm Al (9.1 μm Al³⁺ activity) in one-sixth Murashige and Skoog solution (pH 4.5) for 2 h. After treatment, the incubation solution was analyzed for citrate by an enzymatic method. The whole roots of Arabidopsis were analyzed for the activity of plasma membrane H⁺-ATPase. See details in “Materials and Methods.” Each value is the mean ± se (n = 5). Different letters in the same column or plant indicate that the values are significantly different at the 0.05 level, according to Duncan's Multiple Range Test.

Figure 4.

Quantitative real-time RT-PCR analysis of the transcriptional level of the plasma membrane H⁺-ATPase gene. After incubation in nutrient solution for 8 d, the seedling roots were treated in the nutrient solution (pH 4.5) as follows. A, Duration of Al treatment was 2 h. B, Concentration of Al was 30 μm. C, Concentration of Al and FC was 30 and 5 μm, respectively, and the duration of treatment was 2 h. D, Concentration of Al and VA was 30 and 100 μm, respectively, and the duration of treatment was 2 h. After the treatment, the root apices (1 cm) were excised and mRNA was isolated. See details in “Materials and Methods.” Each value is the mean ± se (n = 5). Different letters in the same column or cultivar indicate that the values are significantly different at the 0.05 level, according to Duncan's Multiple Range Test.

Figure 5.

Western-blot analysis (A–D) and immunolocalization (E–G) of plasma membrane H⁺-ATPase. After an 8-d incubation in nutrient solution, soybean roots of Al-resistant (Al-R) and Al-sensitive (Al-S) cultivars were treated in the nutrient solution (pH 4.5) as follows. A, Marker. B, Control and 30 μm Al for 2 h. C and D, Relative induction of protein expression of plasma membrane H⁺-ATPase after setting the expression level of each cultivar at control or 0 h as 1. Treatment time was 2 h in C, and concentration of Al was 30 μm Al in D; E, Al-R, control. F, Al-R, 30 μm Al. G, Al-R, negative control. Bar in G = 250 μm. Treatment time in E to G was 2 h. After treatment, root apices were used for preparation of plasma membrane H⁺-ATPase proteins or for immunolocalization. See details in “Materials and Methods.” The results shown in Figure 5, C and D, are representative of three independent experiments. Different letters in the same column or cultivar indicate that the values are significantly different at the 0.05 level, according to Duncan's Multiple Range Test.

Figure 6.

Phosphorylated H⁺-ATPase on plasma membrane. After an 8-d incubation in nutrient solution, Al-resistant soybean roots were treated in nutrient solution (pH 4.5) as follows. A, Duration of Al treatment was 2 h. B, Concentration of Al was 30 μm. After the treatment, the root apices (1 cm) were excised and used for preparation of plasma membrane H⁺-ATPase proteins. See details in “Materials and Methods.”