Microtubules Are Essential for Guard-Cell Function in Vicia and Arabidopsis

Radially arranged cortical microtubules are a prominent feature of guard cells. Guard cells expressing GFP- tubulin showed consistent changes in the appearance of microtubules when stomata opened or closed. Guard cells showed fewer microtubule structures as stomata closed, whether induced by transfer to darkness, ABA, hydrogen per- oxide, or sodium hydrogen carbonate. Guard cells kept in the dark （closed stomata） showed increases in microtubule struc- tures and stomatal aperture on light treatment. GFP-EB1, marking microtubule growing plus ends, showed no change in number of plus ends or velocity of assembly on stomatal closure. Since the number of growing plus ends and the rate of plus-end growth did not change when microtubule structure numbers declined, microtubule instability and/or rearrange- ment must be responsible for the apparent loss of microtubules. Guard cells with closed stomata showed more cytosolic GFP-fluorescence than those with open stomata as cortical microtubules became disassembled, although with a large net loss in total fluorescence. Microtubule-targeted drugs blocked guard-cell function in Vicia and Arabidopsis. Oryzalin dis- rupted guard-cell microtubules and prevented stomatal opening and taxol stabilized guard-cell microtubules and delayed stomatal closure. Gas exchange measurements indicated that the transgenes for fluorescent-labeled proteins did not dis- rupt normal stomatal function. These dynamic changes in guard-cell microtubules combined with our inhibitor studies provide evidence for an active role of microtubules in guard-cell function.

The Subcellular Localization and Blue-Light- Induced Movement of Phototropin 1-GFP in Etiolated Seedlings of Arabidopsis thalianaw

Phototropin 1 （photl） is a photoreceptor for phototropism, chloroplast movement, stomatal opening, leaf expansion, and solar tracking in response to blue light. Following earlier work with PHOTI：：GFP （Sakamoto and Briggs, 2002）, we investigated the pattern of cellular and subcellular localization of photl in 3-4 d old etiolated seedlings of Arabidopsis thalinana. As expressed from native upstream sequences, the PHOTT：GFP fusion protein is expressed strongly in the abaxial tissues of the cotyledons and in the elongating regions of the hypocotyl. It is moderately expressed in the shoot/root transition zone and in cells near the root apex. A fluorescence signal is undetectable in the root epidermis, root cap, and root apical meristem itself. The plasma membranes of mesophyll cells near the cotyledon margin appear labeled uniformly but cross-walls created by recent cell divisions are more strongly labeled. The pattern of labeling of individual cell types varies with cell type and developmental stage. Blue-light treatment causes PHOTI：：GFP, initially relatively evenly distributed at the plasma membrane, to become reorganized into a distinct mosaic with strongly labeled punctate areas and other areas completely devoid of fluorescenco a phenomenon best observed in cortical cells in the hypocotyl elongation region. Concomitant with or following this reorganization, PHOTT：GFP moves into the cytoplasm in all cell types investigated except for guard cells. It disappears from the cytoplasm by an unidentified mechanism after several hours in darkness. Neither its appearance in the cytoplasm nor its eventual disappearance in darkness is prevented by the translation inhibitor cycloheximide, although the latter process is retarded. We hypothesize that blue-light-induced photl relocalization modulates blue-light-activated signal transduction.