Functional Characterization of Phytochrome Autophosphorylation in Plant Light Signaling

Abstract

Plant phytochromes, molecular light switches that regulate various aspects of plant growth and development, are phosphoproteins that are also known to be autophosphorylating serine/threonine kinases. Although a few protein phosphatases that directly interact with and dephosphorylate phytochromes have been identifi ed, no protein kinase that acts on phytochromes has been reported thus far, and the exact site of phytochrome autophosphorylation has not been identifi ed. In this study, we investigated the functional role of phytochrome autophosphorylation. We fi rst mapped precisely the autophosphorylation sites of oat phytochrome A (phyA), and identifi ed Ser8 and Ser18 in the 65 amino acid N-terminal extension (NTE) region as being the autophosphorylation sites. The in vivo functional roles of phytochrome autophosphorylation were examined by introducing autophosphorylation site mutants into phyAdefi cient Arabidopsis thaliana . We found that all the transgenic plants expressing the autophosphorylation site mutants exhibited hypersensitive light responses, indicating an increase in phyA activity. Further analysis showed that these phyA mutant proteins were degraded at a signifi cantly slower rate than wild-type phyA under light conditions, which suggests that the increased phyA activity of the mutants is related to their increased protein stability. In addition, protoplast transfection analyses with green fl uorescent protein (GFP)-fused phyA constructs showed that the autophosphorylation site mutants formed sequestered areas of phytochrome (SAPs) in the cytosol much more slowly than did wild-type phyA. These results suggest that the autophosphorylation of phyA plays an important role in the regulation of plant phytochrome signaling through the control of phyA protein stability.