Phototropins are blue-light (BL) receptor serine (Ser)/threonine kinases, and contain two light, oxygen, and voltage (LOV) domains, and are members of the PAS domain superfamily. They mediate phototropism, chloroplast movement, leaf expansion, and stomatal opening of higher plants in response to BL. In stomatal guard cells, genetic analysis has revealed that phototropins mediate activation of the plasma membrane H ϩ -ATPase by phosphorylation and drive stomatal opening. However, biochemical evidence for the involvement of phototropins in the BL response of stomata is lacking. Using guard cell protoplasts, we showed that broad bean (Vicia faba) phototropins (Vfphots) were phosphorylated by BL, and that this phosphorylation of Vfphots reached to the maximum level earlier than that of the H ϩ -ATPase. Phosphorylation of both Vfphots and H ϩ -ATPase showed similar sensitivity to BL and were similarly suppressed by protein kinase and flavoprotein inhibitors. We found that a 14-3-3 protein was bound to Vfphots upon phosphorylation, and this binding occurred earlier than the H ϩ -ATPase phosphorylation. Vfphots (Vfphot1a and Vfphot1b) were expressed in Escherichia coli, and phosphorylation sites were determined to be Ser-358 for Vfphot1a and Ser-344 for Vfphot1b, which are localized between LOV1 and LOV2. We conclude that Vfphots act as BL receptors in guard cells and that phosphorylation of a Ser residue between LOV1 and LOV2 and subsequent 14-3-3 protein binding are likely to be key steps of BL response in stomata. The binding of a 14-3-3 protein to Vfphot was found in etiolated seedlings and leaves in response to BL, suggesting that this event was common to phototropin-mediated responses.Stomatal pores surrounded by a pair of guard cells in the epidermis regulate gas exchange between leaves and the atmosphere and allow CO 2 entry for both photosynthesis and the transpirational stream in higher plants (Zeiger, 1983; Assmann, 1993). Stomata open through the activation of a H ϩ pump in guard cells in response to blue light (BL; Assmann et al., 1985; Shimazaki et al., 1986). The BL-activated pump creates an inside-negative, electrical potential across the plasma membrane and drives K ϩ uptake through voltage-gated K ϩ channels (Hedrich and Schroeder, 1989; Assmann and Shimazaki, 1999; Schroeder et al., 2001). The H ϩ pump has been demonstrated to be the plasma membrane H ϩ -ATPase and is activated via phosphorylation of its C terminus with concomitant binding of the 14-3-3 protein (Kinoshita and Shimazaki, 1999; Emi et al., 2001; Palmgren, 2001). Although physiological responses downstream of BL perception have been elucidated extensively in guard cells, the mechanism of BL perception itself, an initial event in the BL response of stomata, had yet to be elucidated. Recently, genetic analysis of Arabidopsis mutants strongly suggests that phototropins (Atphot1 and Atphot2) function as BL receptors in a redundant manner and mediate BL-dependent stomatal opening (Kinoshita et al., 2001), although the biochemical eviden...

To elucidate the role of guard-cell chloroplasts (GCCs) in stomatal movement, we investigated the effects of oligomycin, an inhibitor of oxidative phosphorylation, and 3-

Blue light-dependent proton pumping in guard cell protoplasts and light-induced stomatal opening in the epidermis were inhibited by 1 mM verapamil, a Ca2+ channel blocker. Proton pumping and stomatal opening induced by fusicoccin, an activator of plasma membrane proton pump, were not inhibited by verapamil. These results suggest that verapamil inhibits blue light signaling in guard cells without inhibiting the pump.