Abstract
Arabidopsis Long Hypocotyl in Far-Red Light 1 (HFR1), a bHLH transcription factor, plays a critical role in promoting seedling photomorphogenesis and in balancing the shade-avoidance response under canopy shade conditions. Previous studies have established that HFR1 protein is degraded in darkness and is stabilized under light conditions to promote light signaling. How light regulates HFR1 stability is not well understood. In this study, we show that Arabidopsis HFR1 can be phosphorylated by recombinant casein kinase II (CKII) and plant extract in vitro and that phosphorylation of HFR1 can be effectively reduced by treatments with two CKII-specific inhibitors, 5,6-dichloro-1-beta-d-ribofuranosyl-benzimidazole (DRB) and heparin. We demonstrate that HFR1 physically interacts with the CKB1 and CKB2 regulatory subunits of CKII. Mutagenesis studies indicate that HFR1 is phosphorylated at multiple serine (Ser) residues in the N-terminal regulatory domain of HFR1. We also show that phosphorylation of HFR1 is promoted by light and that a predicted CKII site, Ser(122), represents a major phosphorylation site of HFR1 under both dark and light conditions. Comparison of wild-type, phosphorylation-deficient, and phosphorylation-mimic mutant proteins suggests that phosphorylation acts to reduce the degradation rate of HFR1. Together, our results suggest that CKII-mediated phosphorylation represents an important post-translational modification influencing the stability and signaling activity of Arabidopsis HFR1.
Highlights
750 nm) sensing phytochromes, and the blue (B)/UV-A (320 –500 nm) absorbing cryptochromes and phototropins [1, 2]
We demonstrate that Hypocotyl in Far-Red Light 1 (HFR1) is phosphorylated at multiple serine (Ser) residues in the N-terminal regulatory domain of HFR1 and that a predicted casein kinase II (CKII) site, Ser122, is a major phosphorylation site of HFR1 under both dark and light conditions
HFR1 Can Be Phosphorylated by CKII and Plant Extracts in Vitro—A recent study reported that HFR1 is a phosphoprotein in vivo [13]; the phosphorylation site(s) is unknown, and the responsible kinase(s) remains to be identified
Summary
750 nm) sensing phytochromes (phy), and the blue (B)/UV-A (320 –500 nm) absorbing cryptochromes (cry) and phototropins [1, 2]. Phytochromes have been well characterized for their roles in regulating seedling de-etiolation and mediating shade-avoidance response. Light is depleted of red but not far-red wavelengths, lowering the R/FR ratio By sensing this change in light quality, plants initiate the shade-avoidance response by increasing the elongation growth of petioles and stems, the length-to-width ratio of leaves, and accelerating flowering [5]. Genetic studies revealed that Arabidopsis HFR1 acts as a positive regulator of phyA-mediated FR and cry1-mediated B light signaling in regulating seedling de-etiolation, and as a general negative regulator of shade-avoidance response (6 –9). The observed rapid accumulation of HFR1 protein under light conditions (detected in less than 30 min of light exposure; Ref. 13) suggests that there must be additional mechanisms regulating HFR1 stability. Using an in vitro degradation assay, we gathered evidence to support the proposition that phosphorylation acts to reduce the degradation rate of HFR1, contributing to the regulation of HFR1 stability and signaling activity
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