α-Tubulin is Rapidly Phosphorylated in Response to Hyperosmotic Stress in Rice and Arabidopsis

By using high-resolution two-dimensional PAGE followed by phosphoprotein-specific staining and peptide mass fingerprint analysis along with other assays, we found that alpha-tubulin is phosphorylated in response to hyperosmotic stress in rice and Arabidopsis. The onset of the phosphorylation response was as early as 2 min after hyperosmotic stress treatment, and a major proportion of alpha-tubulin was phosphorylated after 60 min in root tissues. However, the phosphorylated form of alpha-tubulin was readily dephosphorylated upon stress removal. The phosphorylation site was identified as Thr349 by comprehensive mutagenesis of serine/threonine residues in a rice alpha-tubulin isoform followed by evaluation in cultured cell protoplasts. This residue is located at the surface for the interaction with beta-tubulin in polymerized alpha-beta tubulin dimers and has been proposed to be directly involved in this interaction. Thus, alpha-tubulin phosphorylation was considered to occur on free tubulin dimers in response to hyperosmotic stress. The incorporation of green fluorescent protein (GFP)-alpha-tubulin into cortical microtubules was completely inhibited in transgenic Arabidopsis when Thr349 was substituted with glutamate or aspartate. Using transgenic Arabidopsis plants expressing GFP-alpha-tubulin, we found that hyperosmotic stress causes extensive cortical microtubule depolymerization. Microtubule-destabilizing treatments such as propyzamide or oryzalin and temperature stresses resulted in alpha-tubulin phosphorylation, whereas hyperosmotic stress-induced alpha-tubulin phosphorylation was partially inhibited by taxol, which stabilizes microtubules. These results and the three-dimensional location of the phosphorylation site suggested that microtubules are depolymerized in response to hyperosmotic stress via alpha-tubulin phosphorylation. Together, the results of the present study reveal a novel mechanism that globally regulates the microtubule polymerization.