Poster: Stomatal Physiology

Abs # 389: Blue light-induced phosphorylation of phototropins in stomatal guard cells

Presenter:	Kinoshita, Toshinori , toshircb@mbox.nc.kyushu-u.ac.jp
Authors	Kinoshita, Toshinori  (A)   Emi, Takashi  (A)   Tominaga, Misumi  (A)   Shimazaki, Ken-ichiro  (A)
Affiliations:	(A): Department of Biology, Fuculty of Science, Kyushu University

Phototropins (phot1 and phot2) act as blue light (BL)-receptors and mediate physiological responses including, phototropism, chloroplast relocation, and stomatal opening in higher plants. In stomatal guard cells, molecular genetic analysis using Arabidopsis revealed that phots mediate BL-induced stomatal opening through the activation of plasma membrane H⁺-ATPase by phosphorylation. However, biochemical evidence for participation of phots in this BL response remains lacking. Here we show the biochemical evidence for phots as BL receptors in stomatal guard cells of Vicia faba L. We isolated cDNAs encoding phot, named vfphot1a and vfphot1b, from guard cells of Vicia faba L. In vivo phosphorylation analysis using ³²P-labeled guard cell protoplasts revealed that vfphots were reversibly phosphorylated in response to BL, and that phosphorylation of vfphots was earlier than that of the H⁺-ATPase. BL-induced phosphorylations of vfphots and H⁺-ATPase showed similar fluence dependency. Half-saturated BL was around 225 umol/m². Staurosporin, an inhibitor of serine/threonine protein kinase, and diphenyleneiodonium chloride (DPI), an inhibitor of flavoprotein, inhibited BL-dependent phosphorylations of vfphot and H⁺-ATPase with similar concentration-dependency. From these results we conclude that vfphots act as BL-receptors mediating stomatal opening. We also found that the 14-3-3 protein bound to vfphots upon phosphorylation, and the binding was faster than phosphorylation of the H⁺-ATPase in response to BL. The binding of 14-3-3 protein to vfphot was found in etiolated seedlings and leaves in response to BL, suggesting that binding of the 14-3-3 protein was an early event common to phot-mediated responses.