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<sup>+</sup>-ATPase<sup><a href="#fn1" rid="fn1" class=" fn">1</a></sup>
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 μm Al (9.1 μm Al 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.
Aluminum (Al) toxicity in plants is one of the major limitations to crops grown on acid soils (Horst, 1995; Kochian, 1995; Taylor, 1995; Matsumoto, 2000; Ryan et al., 2001). Exudation of organic acids is an important mechanism for plants to resist Al toxicity. Many plants were found to secrete organic acids in response to Al stress. For instance, citrate is released from the roots of soybean (Glycine max; Yang et al., 2001) and maize (Zea mays; Piñeros et al., 2002), oxalate from buckwheat (Fagopyrum esculentum; Ma et al., 1997), and malate is released from the roots of Al-tolerant genotypes of wheat (Triticum aestivum; Delhaize et al., 1993; Ryan et al., 1995). However, it remains unclear how Al activates organic acid secretion and how this process is regulated (Ryan et al., 2001). Recent studies indicated that the Al-dependent secretion of organic acids in soybean, wheat, and rye is poorly associated with changes in internal organic acid concentrations or with the activities of enzymes that synthesize these acids in the root cells (Ryan et al., 1995; Yang et al., 2001; Hayes and Ma, 2003). The Al-dependent secretion of malate from wheat roots and citrate from maize roots occurs via anion channels on the plasma membrane of the root cells (Ryan et al., 1997; Zhang et al., 2001; Piñeros et al., 2002; Sasaki et al., 2004). The activity of these channels depends on some direct or indirect interaction between Al and the channel protein, although the nature of these is unclear. Indirect interactions with Al might include kinase or phosphatase enzymes, or the activation of secondary messenger pathways that involve changes in cytoplasmic calcium or hormones (Haug et al., 1994; Ryan et al., 1995; Osawa and Matsumoto, 2001; Shen et al., 2004).
The plasma membrane H-ATPase is the most abundant protein on the plasma membrane and is involved in multiple stress responses. This protein activates a series of secondary transporters since the transmembrane movement of many solutes, assimilates, or metabolites is dependent on proton motive force (Serrano, 1989; Sussman, 1994). Recent studies indicated that plasma membrane H-ATPase is involved in the regulation of responses to a variety of environmental stimuli, such as Al stress (Ahn et al., 2004), NaCl stress (Niu et al., 1993; 1996), phosphorus deficiency (Yan et al., 2002), and ammonium stress (Jernejc and Legisa, 2001). Plasma membrane H-ATPase is also involved in auxin-mediated cell elongation during wheat embryo development (Rober-Kleber et al., 2003). Transcript levels of plasma membrane H-ATPase increase more in the roots of halophytes treated with NaCl than glycophytes (Niu et al., 1993), suggesting that the capacity of the plasma membrane H-ATPase gene to respond to NaCl may contribute to the greater salt tolerance of halophytes. Furthermore, in tomato, modulation of plasma membrane H-ATPase activity differentially activates wound and pathogen defense responses (Schaller and Oecking, 1999).
Since proton exudation contributes to charge balance during the secretion of organic acid anions from lupin roots (Yan et al., 2002), we hypothesized that plasma membrane H-ATPase would also be involved in the Al-dependent secretion of citrate from soybean roots. In this study, we investigated whether plasma membrane H-ATPase is involved in the Al-induced secretion of citrate from soybean roots and, if so, how it modulates this response.
Acknowledgments
We thank Michiyo Ariyoshi for culturing Arabidopsis seedlings, Sanae Rikiishi for experimental assistance in the laboratory (Research Institute for Bioresources, Okayama University, Japan), and Dr. Peter R. Ryan (Commonwealth Scientific and Industrial Research Organization Plant Industry, Australia) for his critical review of the manuscript.
Notes
This work was supported by the Program for the Promotion of Basic Research Activities in Innovative Biosciences and a Grant-in-Aid for General Research from the Ministry of Education, Sports, Culture, Science and Technology of Japan (grant no. 14206008 to H.M.), by the International Foundation for Science (grant no. C/3042–2), by the National Natural Scientific Foundation of China (grant no. 30471040/30230220), by the Guangdong Natural Scientific Foundation (grant no. 000642), and by the Japan Society for the Promotion of Science (postdoctoral fellowships to H. Shen).
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.058065.