Identification of 14-3-3 protein binding site in phototropins from Vicia guard cells.

Abstract

Blue light (BL), absorbed by phototropins, activates the plasma membrane H⁺-ATPase in guard cells, and drives stomatal opening. However, there is no biochemical evidence for early events of phototropins in stomatal guard cells. In vivo phosphorylation analysis using ³²P-labeled guard-cell protoplasts from Vicia faba revealed that Vfphots were reversibly phosphorylated in response to BL, and that phosphorylation of vfphots was faster than that of the H⁺-ATPase. We found that the 14-3-3 protein was bound to Vfphots upon phosphorylation. Amino acid substitution analysis using recombinant Vfphots (Vfphot1a and Vfphot1b) expressed Escherichia coli showed that binding of 14-3-3 protein requires phosphorylations of Ser358 residue for Vfphot1a (RRKS₃₅₈) and Ser344 residue for Vfphot1b (RRKS₃₄₄), which are localized between LOV1 and LOV2, respectively. We conclude that phosphorylation of a Ser residue between LOV1 and LOV2 and subsequent 14-3-3 binding are likely to be key step of BL responses in stomata.