First published online November 6, 2003; 10.1104/pp.103.029629
Plant Physiology 133:1453-1463 (2003)
© 2003 American Society of Plant Biologists
Blue-Light- and Phosphorylation-Dependent Binding of a 14-3-3 Protein to Phototropins in Stomatal Guard Cells of Broad Bean1
Toshinori Kinoshita,
Takashi Emi,
Misumi Tominaga,
Koji Sakamoto,
Ayako Shigenaga,
Michio Doi and
Ken-ichiro Shimazaki*
Department of Biology, Faculty of Science, Kyushu University, Ropponmatsu, Fukuoka, Japan 810-8560
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+-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+-ATPase. Phosphorylation of both Vfphots and H+-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+-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.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.029629.
1 This work was supported by the Ministry of Education, Science, Sports, and Culture of Japan (grant nos. 14704003 to T.K. and 10170224 to K.S.).
* Corresponding author; e-mail kenrcb{at}mbox.nc.kyushu-u.ac.jp; fax 81-92-726-4758.
Received July 3, 2003;
returned for revision August 5, 2003;
accepted September 16, 2003.
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