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Light signals recognized by phytochromes are transduced through interactions between down-stream signaling components. Phytochrome-interacting ankyrin repeat protein 2 (PIA2) was found to interact with phytochrome interacting factor 3 (PIF3), a wellknown repressor of plant photomorphogenesis in response to phytochrome-mediated light signalling. Both PIA2 and PIF3 are known to be positive regulators of anthocyanin accumulation in Arabidopsis seedlings under far-red conditions. Thus, we investigated the functional relationship between PIA2 and PIF3 in light signalling. We found that PIA2 suppressed PIF3 phosphorylation by phyA. To elucidate how PIA2 modulates phyA-mediated PIF3 phosphorylation, we generated non-phosphorylation mutants and N-terminal α-helix breaking mutants of PIA2. PIF3 phosphorylation by phyA was not suppressed by α-helix breaking PIA2 mutants. The α-helix breaking mutations also resulted in remarkably decreased interactions between PIA2 and PIF3. However, the non-phosphorylation mutants exhibited no effect on phyA-mediated PIF3 phosphorylation. In addition, decreased anthocyanin accumulation in pia2 knockout plant seedlings was not rescued by overexpression of the α-helix breaking mutant in transgenic plants under far-red conditions. These results suggest that PIA2 modulates phyAmediated PIF3 phosphorylation by physical interaction with PIF3 and that the secondary structure of the PIA2 N-terminus is important in this modulation. [ABSTRACT FROM AUTHOR]

Phytochrome A (phyA) is a light labile phytochrome that mediates plant development under red/far-red light condition. Degradation of phyA is initiated by red light-induced phyA-ubiquitin conjugation through the 26S proteasome pathway. The N-terminal of phyA is known to be important in phyA degradation. To determine the specific lysine residues in the N-terminal domain of phyA involved in light-induced ubiquitination and protein degradation, we aligned the amino acid sequence of the N-terminal domain of Arabidopsis phyA with those of phyA from other plant species. Based on the alignment results, phytochrome over-expressing Arabidopsis plants were generated. In particular, wild-type and mutant (substitutions of conserved lysines by arginines) phytochromes fused with GFP were expressed in phyA-211 Arabidopsis plants. Degradation kinetics of over-expressed phyA proteins revealed that degradation of the K206R phyA mutant protein was delayed. Delayed phyA degradation of the K206R phyA mutant protein resulted in reduction of red-light-induced phyA-ubiquitin conjugation. Furthermore, seedlings expressing the K206R phyA mutant protein showed an enhanced phyA response under far-red light, resulting in inhibition of hypocotyl elongation as well as cotyledon opening. Together, these results suggest that lysine 206 is the main lysine for rapid ubiquitination and protein degradation of Arabidopsis phytochrome A. [ABSTRACT FROM AUTHOR]