Poster: Photomorphogenesis

Abs # 382: Blue light-dependent binding of a 14-3-3 protein to phot1 in Arabidopsis thaliana.

Presenter:	Inoue, Shin-ichiro , shinrcb@mbox.nc.kyushu-u.ac.jp
Authors	Inoue, Shin-ichiro  (A)   Kinoshita, Toshinori  (A)   Shimazaki, Ken-ichiro  (A)
Affiliations:	(A): Department of Biology, Faculty of Science, Kyushu University

Phototropins (phot1 and phot2) are blue light (BL) receptor serine/threonine kinases that mediate many BL responses such as phototropism, chloroplast relocation, leaf expansion, and stomatal opening in Arabidopsis thaliana. Previous studies demonstrated that phototoropins are autophosphorylated in response to BL and that this phosphorylation is an early event in phototropin-mediated responses. A more recent investigation has shown that a 14-3-3 protein binds to phototropins upon phosphorylation in response to BL in guard cells from Vicia faba. However, there is no such evidence in Arabidopsis plants and the precise site of binding has yet to be determined. In this study, we provide biochemical evidence for binding of 14-3-3 protein to phot1 in Arabidopsis plants in BL- and phosphorylation-dependent manners. Far Western blot analysis using a recombinant 14-3-3 protein as a probe showed that a 14-3-3 protein binds to phot1 in etiolated seedlings, leaves, and guard cells within 1 min after the start of BL pulse. A half-saturating intensity of BL was 7.2 μmol m^-2 s^-1 when the illumination was provided for 30 sec. Interestingly, a 14-3-3 proein did not bind to phot2 in any fluence rate of BL. Furthermore, phot1 was co-precipitated with a 14-3-3 protein in etiolated seedlings only when the seedlings were illuminated with BL, suggesting that the 14-3-3 protein bound to phot1 in vivo via autophosphorylation. We determined the site of 14-3-3 protein binding using recombinant phot1 fragments, and indicated that the sites are phosphorylated Ser-350 (RALS₃₅₀) and/or Ser-376 (RRMS₃₇₆), which are localized in the hinge region between LOV1 and LOV2. These results suggest that binding of a 14-3-3 protein to phot1 is likely to be key step of phot1-mediated responses.