Functional characterization of phytochrome autophosphorylation in plant light signaling.
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 identified, no protein kinase that acts on phytochromes has been reported thus far, and the exact site of phytochrome autophosphorylation has not been identified. In this study, we investigated the functional role of phytochrome autophosphorylation. We first mapped precisely the autophosphorylation sites of oat phytochrome A (phyA), and identified 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 phyA-deficient 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 significantly 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 fluorescent 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.
Han
Yun-Jeong
YJ
Department of Biotechnology and Kumho Life Science Laboratory, Chonnam National University, Gwangju 500-757, Korea.
Kim
Hwan-Sik
HS
Kim
Yong-Min
YM
Shin
Ah-Young
AY
Lee
Si-Seok
SS
Bhoo
Seong Hee
SH
Song
Pill-Soon
PS
Kim
Jeong-Il
JI
D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D015153:Blotting, Western; D018506:Gene Expression Regulation, Plant / Q000235:genetics / Q000528:radiation effects; D008027:Light; D010766:Phosphorylation / Q000528:radiation effects; D051942:Phytochrome A / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D055550:Protein Stability; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics / Q000528:radiation effects*
D029681:Arabidopsis Proteins; C497973:PHYA protein, Arabidopsis; D051942:Phytochrome A