Organization of protein complexes under photomorphogenic UV-B in Arabidopsis

Low-fluence and long-wavelength UV-B light promotes photomorphogenic development in Arabidopsis. CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) is a positive regulator in this pathway while it is a negative regulator of the traditional photomorphogenesis triggered by far-red and visible light. We have recently reported the mechanism by which the switch of COP1 function is accomplished in distinct light contexts. In response to photomorphogenic UV-B, the photoactivated UV RESISTANCE LOCUS 8 (UVR8) associates with the COP1- SUPRESSOR OF PHYA (SPA) core complexes, resulting in the physical and functional disassociation of COP1-SPA from the CULLIN4-DAMAGED DNA BINDING PROTEIN 1 (CUL4-DDB1) E3 scaffold. These UV-B dependent UVR8-COP1-SPA complexes promote the stability and activity of ELONGATED HYPOCOTYL 5 (HY5), and eventually cause COP1 to switch from repressing to promoting photomorphogenesis. In addition, it is possible that CUL4-DDB1 might simultaneously recruit alternative DDB1 BINDING WD40 (DWD) proteins to repress this UV-B-specific signaling. Further investigation is required, however, to verify this hypothesis. Overall, the coordinated organization of various protein complexes facilitates an efficient and balanced UV-B signaling.     

Targeting Proteins for Degradation by Arabidopsis COP1： Teamwork Is What Matters

Arsbidopsis COP1 （Constitutive Photomorphogenic 1） defines a key repressor of photomorphogenesis in darkness by acting as an E3 ubiquitin Iigase in the nucleus, and is responsible for the targeted degradation of a number of photomorphogenesis-promoting factors, including phyA, HY5, LAF1, and HFR1. Light activation of multiple classes of photoreceptors （including both phytochromes and cryptochromes） inactivates COP1 and reduces its nuclear abundance, allowing the accumulation of these positively acting light signaling intermediates to promote photomorphogenic development. Recent studies suggest that Arabidopsis COP1 teams up with a family of SPA proteins （SPA1-SPA4） to form the physiologically active COP1-SPA E3 ubiquitin ligase complexes. These COP1-SPA complexes play overlapping and distinct functions in regulating seedling photomorphogenesis under different light conditions and adult plant growth. Further, the COP1-SPA complexes act In concert at a biochemical level with the CDD （COP10, DET1, and DDB1） complex and COP9 signalosome （CSN） to orchestrate the repression of photomorphogenesis.     

HFR1, a phytochrome A-signalling component,acts in a separate pathway from HY5, downstream of COP1 in Arabidopsis thaliana

HFR1, a basic helix-loop-helix protein, is known to be required for a subset of phytochrome A (phyA)-dependent photoresponses. To investigate the role of HFR1 in light signalling, we have examined the genetic interaction between HFR1 and HY5, a positive regulator of light signalling, and COP1, a repressor of photomorphogenesis. Double mutant analysis suggests that HFR1 mediates phyA-dependent inhibition of hypocotyl elongation independently of HY5. HFR1 was shown to be necessary for a subset of cop1-triggered photomorphogenic phenotypes in the dark, including inhibition of hypocotyl elongation, gravitropic hypocotyl growth, and expression of the light-inducible genes CAB and RBCS. Phenotypic analysis of the triple mutant cop1hy5hfr1 indicated that both HFR1 and HY5 are required for cop1-mediated photomorphogenic seedling development in darkness, consistent with their additive roles in phyA-dependent signalling. Taken together, these results suggest that HFR1 might act downstream of COP1, in a separate pathway from HY5, to mediate photomorphogenesis in Arabidopsis.     

Biochemical evidence for ubiquitin ligase activity of the Arabidopsis COP1 interacting protein 8 (CIP8)

Arabidopsis COP1 is a negative regulator of photomorphogenesis, which targets HY5, a positive regulator of photomorphogenesis, for degradation via the proteasome pathway in the absence of light. COP1 and its interactive partner CIP8 both possess RING finger motifs, characteristic of some E3 ubiquitin ligases. Here we show that CIP8 promotes ubiquitin attachment to HY5 in E2-dependent fashion in vitro. CIP8 exhibits a strong interaction with the E2 enzyme AtUBC8 through its N-terminal domain. Phosphorylation of HY5 by casein kinase II requires the beta subunit 2, but does not affect HY5's susceptibility to ubiquitination. The RING domain of CIP8 is required but is not sufficient for ubiquitin ligase activity. Although the RING domain of CIP8 interacts with the RING domain of COP1, addition of recombinant COP1 fails to affect CIP8's ubiquitin ligase activity towards HY5 in vitro. However, recombinant COP1 can pull-down native CIP8 from the extract of dark-grown seedlings, but not from the extract of light-grown seedlings in a column-binding assay, implying a requirement for light-regulated modification in vivo. Our data suggest that CIP8 can form a minimal ubiquitin ligase in co-operation with the E2 enzyme AtUBC8. It is possible that the AtUBC8-CIP8 module might interact with COP1 in vivo, thereby participating in proteasome-mediated degradation of HY5.     

Arabidopsis phytochromes A and B synergistically repress SPA1 under blue light

In Arabidopsis, although studies have demonstrated that phytochrome A(phyA) and phyB are involved in blue light signaling, how blue light-activated phytochromes modulate the activity of the CONSTITUTIVELY PHOTOMORPHOGENIC1(COP1)-SUPPRESSOR OF PHYA-105(SPA1) E3 complex remains largely unknown. Here, we show that phyA responds to early and weak blue light, whereas phyB responds to sustainable and strong blue light. Activation of both phyA and phyB by blue light inhibits SPA1 activity.Specifically,...     

The COP/DET/FUS proteins-regulators of eukaryotic growth and development

Eleven recessive mutant loci define the class of cop / det / fus mutants of Arabidopsis. The cop / det / fus mutants mimic the phenotype of light-grown seedlings when grown in the dark. At least four cop / det / fus mutants carry mutations in subunits of the COP9 signalosome, a multiprotein complex paralogous to the 'lid' subcomplex of the 26S proteasome. COP1, another COP/DET/FUS protein, is itself not a subunit of the COP9 signalosome. In the dark, COP1 accumulates in the nucleus where it is required for the degradation of the HY5 protein, a positive regulator of photomorphogenesis. In the light, COP1 is excluded from the nucleus and the constitutively nuclear HY5 protein can accumulate. Nuclear accumulation of COP1 and degradation of HY5 are impaired in the cop / det / fus mutants that carry mutations in subunits of the COP9 signalosome. Although the cellular function of the COP/DET/FUS proteins is not yet well understood, taken together the current findings suggest that the COP/DET/FUS proteins repress photomorphogenesis in the dark by mediating specific protein degradation.     

Light-induced degradation of SPA2 via its N-terminal kinase domain is required for photomorphogenesis

Arabidopsis (Arabidopsis thaliana) CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) and members of the SUPPRESSOR OF PHYTOCHROMEA-105 (SPA) protein family form an E3 ubiquitin ligase that suppresses light signaling in darkness by polyubiquitinating positive regulators of the light response. COP1/SPA is inactivated by light to allow photomorphogenesis to proceed. Mechanisms of inactivation include light-induced degradation of SPA1 and, in particular, SPA2, corresponding to a particularly efficient inactivation of COP1/SPA2 by light. Here, we show that SPA3 and SPA4 proteins are stable in the light, indicating that light-induced destabilization is specific to SPA1 and SPA2, possibly related to the predominant function of SPA1 and SPA2 in dark-grown etiolating seedlings. SPA2 degradation involves cullin and the COP10-DEETIOLATED-DAMAGED-DNA BINDING PROTEIN (DDB1) CDD complex, besides COP1. Consistent with this finding, light-induced SPA2 degradation required the DDB1-interacting Trp-Asp (WD)-repeat domain of SPA2. Deletion of the N-terminus of SPA2 containing the kinase domain led to strong stabilization of SPA2 in darkness and fully abolished light-induced degradation of SPA2. This prevented seedling de-etiolation even in very strong far-red and blue light and reduced de-etiolation in red light, indicating destabilization of SPA2 through its N-terminal domain is essential for light response. SPA2 is exclusively destabilized by phytochrome A in far-red and blue light. However, deletion of the N-terminal domain of SPA2 did not abolish SPA2-phytochrome A interaction in yeast nor in vivo. Our domain mapping suggests there are two SPA2-phytochrome A interacting domains, the N-terminal domain and the WD-repeat domain. Conferring a light-induced SPA2-phyA interaction only via the WD-repeat domain may thus not lead to COP1/SPA2 inactivation.

Light inactivates the COP1/SPA2 repressor of photomorphogenesis through cullin- and CDD-mediated degradation of SPA2, whereas the family members SPA3 and SPA4 are stable in the light.