The phytochrome A-specific signaling intermediate SPA1 interacts directly with COP1, a constitutive repressor of light signaling in Arabidopsis.

SPA1 is a phytochrome A (phyA)-specific signaling intermediate that acts as a light-dependent repressor of photomorphogenesis in Arabidopsis seedlings. It contains a WD-repeat domain that shows high sequence similarity to the WD-repeat region of the constitutive repressor of light signaling, COP1. Here, using yeast two-hybrid and in vitro interaction assays, we show that SPA1 strongly and selectively binds to COP1. Domain mapping studies indicate that the putative coiled-coil domain of SPA1 is necessary and sufficient for binding to COP1. Conversely, similar deletion analyses of the COP1 protein suggest that SPA1 interacts with the presumed coiled-coil domain of COP1. To further investigate SPA1 function in the phyA signaling pathway, we tested whether SPA1, like COP1, mediates changes in gene expression in response to light. We show that spa1 mutations increase the photoresponsiveness of certain light-regulated genes within 2 h of light treatment. Taken together, the results suggest that SPA1 may function to link the phytochrome A-specific branch of the light signaling pathway to COP1. Hence, our data provide molecular support for the hypothesis that COP1 is a convergence point for upstream signaling pathways dedicated to individual photoreceptors.     

The SPA1-like proteins SPA3 and SPA4 repress photomorphogenesis in the light

Suppressor of phyA-105 (SPA1) is a phytochrome A-specific signaling intermediate that acts as a light-dependent repressor of photomorphogenesis in Arabidopsis seedlings. SPA1 is part of a small gene family comprising three genes: SPA1-related 2 (SPA2), SPA1-related 3 (SPA3), and SPA1-related 4 (SPA4). Here, we investigate the functions of SPA3 and SPA4, two very closely related genes coding for proteins with 74% identical amino acids. Seedlings with mutations in SPA3 or SPA4 exhibit enhanced photomorphogenesis in the light, but show no phenotype in darkness. While there are small differences between the effects of spa3 and spa4 mutations, it is apparent that SPA3 and SPA4 function to inhibit light responses in continuous far-red, red, and blue light. Phytochrome A is necessary for all aspects of the spa4 mutant phenotype, suggesting that SPA4, like SPA1, acts specifically in phytochrome A signaling. Enhanced photoresponsiveness of spa3 mutants is also fully dependent on phytochrome A in far-red and blue light, but not in red light. Hence, SPA3 function in red light may be dependent on other phytochromes in addition to phytochrome A. Using yeast two-hybrid and in vitro interaction assays, we further show that SPA3 as well as SPA4 can physically interact with the constitutive repressor of light signaling COP1. Deletion analyses suggest that SPA3 and SPA4, like SPA1, bind to the coiled-coil domain of COP1. Taken together, our results have identified two new loci coding for negative regulators that may be involved in fine tuning of light responses by interacting with COP1.     

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.     

The SPA quartet: a family of WD-repeat proteins with a central role in suppression of photomorphogenesis in arabidopsis

The Arabidopsis thaliana proteins suppressor of phytochrome A-105 1 (SPA1), SPA3, and SPA4 of the four-member SPA1 protein family have been shown to repress photomorphogenesis in light-grown seedlings. Here, we demonstrate that spa quadruple mutant seedlings with defects in SPA1, SPA2, SPA3, and SPA4 undergo strong constitutive photomorphogenesis in the dark. Consistent with this finding, adult spa quadruple mutants are extremely small and dwarfed. These extreme phenotypes are only observed when all SPA genes are mutated, indicating functional redundancy among SPA genes. Differential contributions of individual SPA genes were revealed by analysis of spa double and triple mutant genotypes. SPA1 and SPA2 predominate in dark-grown seedlings, whereas SPA3 and SPA4 prevalently regulate the elongation growth in adult plants. Further analysis of SPA2 function indicated that SPA2 is a potent repressor of photomorphogenesis only in the dark but not in the light. The SPA2 protein is constitutively nuclear localized in planta and can physically interact with the repressor COP1. Epistasis analysis between spa2 and cop1 mutations provides strong genetic support for a biological significance of a COP1-SPA2 interaction in the plant. Taken together, our results have identified a new family of proteins that is essential for suppression of photomorphogenesis in darkness.     

The Signaling Mechanism of Arabidopsis CRY1 Involves Direct Interaction with COP1

Dark-grown transgenic Arabidopsis seedlings expressing the C-terminal domains (CCT) of the cryptochrome (CRY) blue light photoreceptors exhibit features that are normally associated only with light-grown seedlings, indicating that the signaling mechanism of Arabidopsis CRY is mediated through CCT. The phenotypic properties mediated by CCT are remarkably similar to those of the constitutive photomorphogenic1 (cop1) mutants. Here we show that Arabidopsis cryptochrome 1 (CRY1) and its C-terminal domain (CCT1) interacted strongly with the COP1 protein. Coimmunoprecipitation studies showed that CRY1 was bound to COP1 in extracts from both dark- and light-grown Arabidopsis. An interaction also was observed between the C-terminal domain of Arabidopsis phytochrome B and COP1, suggesting that phytochrome signaling also proceeds, at least in part, through direct interaction with COP1. These findings give new insight into the initial step in light signaling in Arabidopsis, providing a molecular link between the blue light receptor, CRY1, and COP1, a negative regulator of photomorphogenesis.     

Light exposure of Arabidopsis seedlings causes rapid de-stabilization as well as selective post-translational inactivation of the repressor of photomorphogenesis SPA2

The COP1/SPA complex acts as an E3 ubiquitin ligase to repress photomorphogenesis by targeting activators of the light response for degradation. Genetic analysis has shown that the four members of the SPA gene family (SPA1-SPA4) have overlapping but distinct functions. In particular, SPA1 and SPA2 differ in that SPA1 encodes a potent repressor in light- and dark-grown seedlings, but SPA2 fully loses its function when seedlings are exposed to light, indicating that SPA2 function is hyper-inactivated by light. Here, we have used chimeric SPA1/SPA2 constructs to show that the distinct functions of SPA1 and SPA2 genes in light-grown seedlings are due to the SPA protein sequences and independent of the SPA promoter sequences. Biochemical analysis of SPA1 and SPA2 protein levels shows that light exposure leads to rapid proteasomal degradation of SPA2, and, more weakly, of SPA1, but not of COP1. This suggests that light inactivates the COP1/SPA complex partly by reducing SPA protein levels. Although SPA2 was more strongly degraded than SPA1, this was not the sole reason for the lack of SPA2 function in the light. We found that the SPA2 protein is inherently incapable of repressing photomorphogenesis in light-grown seedlings. The data therefore indicate that light inactivates the function of SPA2 through a post-translational mechanism that eliminates the activity of the remaining SPA2 protein in the cell.     

Degradation of Arabidopsis CRY2 is regulated by SPA proteins and phytochrome A

The UV-A/blue light photoreceptor crytochrome2 (cry2) plays a fundamental role in the transition from the vegetative to the reproductive phase in the facultative long-day plant Arabidopsis thaliana. The cry2 protein level strongly decreases when etiolated seedlings are exposed to blue light; cry2 is first phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome. COP1 is involved in cry2 degradation, but several cop1 mutants show only reduced but not abolished cry2 degradation. SUPPRESSOR OF PHYA-105 (SPA) proteins are known to work in concert with COP1, and recently direct physical interaction between cry2 and SPA1 was demonstrated. Thus, we hypothesized that SPA proteins could also play a role in cry2 degradation. To this end, we analyzed cry2 protein levels in spa mutants. In all spa mutants analyzed, cry2 degradation under continuous blue light was alleviated in a fluence rate-dependent manner. Consistent with a role of SPA proteins in phytochrome A (phyA) signaling, a phyA mutant had enhanced cry2 levels, particularly under low fluence rate blue light. Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy studies showed a robust physical interaction of cry2 with SPA1 in nuclei of living cells. Our results suggest that cry2 stability is controlled by SPA and phyA, thus providing more information on the molecular mechanisms of interaction between cryptochrome and phytochrome photoreceptors.     

Blue light-dependent interaction of CRY2 with SPA1 regulates COP1 activity and floral initiation in Arabidopsis

Cryptochromes are blue light receptors that mediate light regulation of gene expression in all major evolution lineages, but the molecular mechanism underlying cryptochrome signal transduction remains not fully understood. It has been reported that cryptochromes suppress activity of the multifunctional E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) to regulate gene expression in response to blue light. But how plant cryptochromes mediate light suppression of COP1 activity remains unclear. We report here that Arabidopsis CRY2 (cryptochrome 2) undergoes blue light-dependent interaction with the COP1-interacting protein SUPPRESSOR OF PHYTOCHROME A 1 (SPA1). We demonstrate that SPA1 acts genetically downstream from CRY2 to mediate blue light suppression of the COP1-dependent proteolysis of the flowering-time regulator CONSTANS (CO). We further show that blue light-dependent CRY2-SPA1 interaction stimulates CRY2-COP1 interaction. These results reveal for the first time a wavelength-specific mechanism by which a cryptochrome photoreceptor mediates light regulation of protein degradation to modulate developmental timing in Arabidopsis.     

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.