Takashi Emi

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

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.

Isolation of a Protein Interacting with Vfphot1a in Guard Cells of Vicia faba

A recent study has demonstrated that phototropins act as blue light receptors in stomatal guard cells. However, the downstream components responsible for phototropin signaling are largely unknown. In this study, using a yeast two-hybrid system, we isolated a Vicia faba protein that has a high similarity to dynein light chain in the C terminus, which interacts with Vicia faba phototropin 1a (Vfphot1a). Protein-blot and two-hybrid analyses revealed that Vfphot1a interacting protein (VfPIP) bound to the C-terminal region of Vfphot1a but did not bind to Vfphot1b. The interaction between VfPIP and Vfphot was indicated by a pull-down assay. Northern analysis revealed that the transcription level of VfPIP gene was more abundant in guard cells than in other tissues or cell types. The transiently expressed fusion protein of VfPIP-green fluorescent protein was localized on cortical microtubules in Vicia guard cells. Microtubule-depolymerizing herbicides partially inhibited both blue light-dependent H+ pumping in Vicia guard cell protoplasts and stomatal opening in the Vicia epidermis. From these results, we conclude that VfPIP may act as a downstream component of phototropin (Vfphot1a) in blue light signaling in guard cells. The possible role of VfPIP in blue light signaling of guard cells is discussed.

Effects of High Temperatures on Photosynthetic Systems: Fluorescence Fo Increases in Cyanobacteria

Oxygen evolution is one of the most heat-sensitive sites in photosynthetic organisms. To investigate effects of heat stress on photosynthesis, high-temperature (HT) -induced fluorescence Fo (minimum fluorescence) increases and Fm (maximum fluorescence) decreases had been studied by many workers (1). In higher plants, the Fo increase was attributed to irreversible detachment of light-harvesting chlorophyll a/b protein complexes from the reaction center (RC) complexes of Photosystem (PS) II, to partly reversible inactivation of PS 11 (2, 3), and to dark reduction of QA (4). The Fm decrease is related to inhibition of oxygen evolution (5, 6). However, Fm levels in the presence of 3- (3,4-dichlorophenyl) -1,1-dimethylurea and NH2OH, which cancelled the inhibitory effect of heat stress on oxygen evolution, were also decreased by HT treatments, showing that RCs of PS II were inactivated by high temperature-stress and that the inactivated PS II RC complexes become less fluorescent (6). In cyanobacteria, however, little is known about the effects of HTs on photosynthetic systems and on these fluorescence parameters. In this report, we show that the Fo increase in cyanobacteria is due to partly reversible release of phycobilisomes from the RC complexes of PS II and to partly reversible inactivation of PS II RC.