Arabidopsis Phytochrome B Directly Regulates COP1and SPA1Nuclear Activities To Suppress The Photomorphogenesis Under Far-Red Light

As sessile organisms, plants have evolved a high degree of developmental plasticity to optimizetheir growth and reproduction in response to their changing environments. Light is one of the importantfactors modulating many developmental processes of plants, from seed germination to seedlingflowering. There are a group of photoreceptors for plants to monitor light quality, quantity, direction,duration and periodicity. Prominent amongst these are the red/far–red reversible photoreceptors thephytochromes. The five distinct phytochromes in Arabidopsis, designated phyA to phyE, have unique aswell as partially redundant or antagonistic roles in different photomorphogenic responses. PhyA is theprimary photoreceptor mediating de–etiolation under far red (FR) light, whereas phyB predominantlyregulates the light responses under red light.The suppressor of phyA–105(SPA1) is one of the most important repressing factors in lightsignaling pathway. SPA1forms the E3complex with constitutive photomorphogenic1(COP1), whichis responsible for the degradation of various photomorphogenesis–promoting factors, resulting indesensitisation to light signaling. Previous studies have reported that overexpression of PHYB promotesseedling etiolation under FR light. However, the role of phyB in FR–light signaling and the regulatorypathway from light–activated phytochromes to the COP1–SPA1E3complex are largely unknown.This report describes the genetic, molecular and biochemical mechanisms responsible for thenegative effects of phyB on plant photomorphogenesis in response to continuous FR light.1. PhyB represses photomorphogeneseis under far–red light. Seedlings overexpressing PHYB–GFP have drastic etiolation phenotypes, with elongated hypocotyl and reduced anthoc yanin and nuclearHY5protein accumulations under FR light. Conversely, the PHYB–deficient phyB–9mutant displaysde–etiolation phenotypes in FR light conditions. These results suggest that phyB plays a repressive rolein continuous FR light.2. PhyB rapidly responds to far–red light treatment. When shifting from dark to FR light,PHYB transcript abundance gradually increases seven times in wild type (Col–0) seedlings. In responseto FR light, phyB–GFP forms dim, small NBs and effectively accumulates into nuclei. The nuclearactivities of phyB remain unchanged in a phyA–211mutant background. The R/FR transition stillpromotes phyB–GFP nuclear kinetics and increases nuclear import, with large nuclear bodiesconverting into small ones and dispersing throughout the nucleoplasm.3. PhyB represses seedling etiolation in response to FR light in a phyA–independent manner.Overexpression of PHYB–GFP causes25.1%or11.4%greater hypocotyl elongation compared withwild type (Col–0) or the phyA–211single mutant, respectively. Promotion of hypocotyl elongation,cotyledon folding and chlorophyll accumulation by PHYB–GFP in both the presence and absence ofphyA suggests that phyB represses seedling photomorphogenesis under FR light, independently ofphyA 4. PhyB acts upstream of SPA1in FR–light signaling. Under FR light, spa1–100completelysuppresses the de–etiolation and etiolation phenotypes caused by phyB–9and PHYB–GFP, suggestingnot only that phyB acts upstream of SPA1, but also that under FR light its functions is dependent onSPA1.5. PhyB physically interacts with SPA1, and they both form a complex in planta. Theseobservations of yeast two–hybrid assays suggest that the carboxy–terminal domain of SPA1, includingboth coiled–coil and WD40domains, is required for the phyB–SPA1interaction. Byimmunofluorescence assay, phyB–GFP and Myc–SPA1co–localised into the nucleus under FR lightcondition. In vivo co–IP assays indicate that phyB and SPA1co–localise in the same protein complexunder FR light, and the interaction between phyB and SPA1is important for their activity.6. PhyB antagonizes phyA in terms of SPA1stability under far–red light. This reportdemonstrates that the Myc–SPA1protein levels in phyA–211or phyB–9mutant backgrounds are70%higher and40%lower than those in the wild–type background under FR light, respectively. The Myc–SPA1protein levels in the phyA–211phyB–9double–mutant background are exactly between the phyA–211and phyB–9background levels. This suggests that the effects of phyB and phyA on SPA1proteinaccumulation are antagonised in an au pair manner in FR light.7. PhyB enhances nuclear transport of SPA1under FR light. During the transition from dark toFR light, phyB is rapidly imported into the nucleus and facilitates nuclear SPA1accumulation. Thenuclear accumulation of Myc–SPA1is related to the strength of its binding to phyB–GFP during the Dkto FR transition.8. SPA1is required for proper COP1nuclear accumulation under FR light. The results ofnuclear–enriched staining and immunoblot analysis show that the nuclear GUS–COP1in No–0wildtype background is dramatically higher than in the spa1–3mutant background under FR light. Theresults imply that both COP1and SPA1are functionally interdependent, and SPA1enhances COP1activation under FR–light condition.These findings support the notion that phyB plays a role in repressing FR–light signaling. Theregulation of SPA1nuclear accumulation by light–activated phytochromes is a pivotal regulatory step inlight signaling.