alpha -Tubulin is rapidly phosphorylated in response to hyperosmotic stress in rice and Arabidopsis.

摘要

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.