Blue-Light- and Phosphorylation-Dependent Binding of a 14-3-3 Protein to Phototropins in Stomatal Guard Cells of Broad Bean

Abstract

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...

Roles of ion channels and transporters in guard cell signal transduction

“…When inactive, the C terminus of the H + -ATPase acts as an autoinhibitory domain. Upon blue light illumination, a type 1 protein phosphatase is activated [8] which, via an unknown mechanism, activates a protein kinase that phosphorylates the C terminus of the H + -ATPase [9][10][11]. Consequent H + -ATPase binding with 14-3-3 protein results in displacement of the auto-inhibitory domain and activation of the H + -ATPase [11] (Fig.…”

Phosphorylation of BLUS1 kinase by phototropins is a primary step in stomatal opening

“…4d), with a re-phosphorylation of BLUS1 following a second pulse. The time course of BLUS1 phosphorylation revealed a more rapid response than described for H þ -ATPase phosphorylation 12,26 and coincided with the timing of phot1 autophosphorylation 9,26 . Thus, it appeared conceivable that phototropins directly phosphorylate BLUS1.…”

The Arabidopsis rcn1-1 Mutation Impairs Dephosphorylation of Phot2, Resulting in Enhanced Blue Light Responses

“…A single mutation in the ATP binding site (D806N in Arabidopsis thaliana phot1) inactivates the autophosphorylation (Christie et al, 2002). Phosphorylation of a Ser between the LOV1 and LOV2 domains of the broad bean phototropins (Vicia faba Phot1a and Phot1b) has been shown to form the phosphoserine recognized by a 14-3-3 protein, and binding of the 14-3-3 protein is required for stomatal opening induced by blue light (Kinoshita et al, 2003). Phosphorylation of Arabidopsis phot1 ser851 in the kinase activation loop is also necessary for phot1 physiological activity (Inoue et al, 2008a).…”