Changes in Tubulin Protein Expression in Guard Cells of Vicia faba L. Accompanied with Dynamic Organization of Microtubules during the Diurnal Cycle

We previously reported that the organization of microtubules (MTs) in guard cells of Vicia faba L. shows dynamic diurnal changes [Fukuda et al. (1998) Plant Cell Physiol. 39: 80]. Here, we report a method to directly extract total proteins from guard cells to investigate the biochemical changes in guard cells of Vicia faba L. during the diurnal cycle. Electrophoretic profiles of total proteins of guard cells showed distinct patterns with the time of extraction. Immunoblot analysis also demonstrated changes in alpha-tubulin and beta-tubulin contents with the diurnal cycle. Both tubulins were abundant at 6:00 h and 12:00 h but were almost undetectable at 24:00 h. Although treatment with either actinomycin D or cycloheximide at 18:00 h inhibited neither radial organization of cortical MTs nor stomatal opening, that at 6:00 h inhibited both. These results suggest that the dynamic diurnal changes in the organization of MTs in guard cells and stomatal movement of Vicia faba L. may be, at least partly, regulated by de novo synthesis and decomposition of tubulin molecules in guard cells.