Summary: Anthocyanins are natural pigments that accumulate only in light‐grown and not in dark‐grown Arabidopsis plants. Repression of anthocyanin accumulation in darkness requires the CONSTITUTIVELY PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA‐105 (COP1/SPA) ubiquitin ligase, as cop1 and spa mutants produce anthocyanins also in the dark. Here, we show that COP1 and SPA proteins interact with the myeloblastosis (MYB) transcription factors PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP)1 and PAP2, two members of a small protein family that is required for anthocyanin accumulation and for the expression of structural genes in the anthocyanin biosynthesis pathway. The increased anthocyanin levels in cop1 mutants requires the PAP1 gene family, indicating that COP1 functions upstream of the PAP1 gene family. PAP1 and PAP2 proteins are degraded in the dark and this degradation is dependent on the proteasome and on COP1. Hence, the light requirement for anthocyanin biosynthesis results, at least in part, from the light‐mediated stabilization of PAP1 and PAP2. Consistent with this conclusion, moderate overexpression of PAP1 leads to an increase in anthocyanin levels only in the light and not in darkness. Here we show that SPA genes are also required for reducing PAP1 and PAP2 transcript levels in dark‐grown seedlings. Taken together, these results indicate that the COP1/SPA complex affects PAP1 and PAP2 both transcriptionally and post‐translationally. Thus, our findings have identified mechanisms via which the COP1/SPA complex controls anthocyanin levels in Arabidopsis that may be useful for applications in biotechnology directed towards increasing anthocyanin content in plants.

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.

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.

Significance: Plants adapt their growth and development to ambient light conditions by modulating the activity of the COP1/SPA ubiquitin ligase. In Arabidopsis , photoactivated cryptochromes interact directly with COP1/SPA to suppress the activity of COP1 in blue light. The molecular mechanisms of CRY-mediated inhibition of COP1 are not fully understood. Here we show that CRY2 inhibits COP1 by displacing degradation substrates from COP1. This mechanism requires a valine-proline (VP) motif present in the C-terminal domain of CRY2. These amino acids are also present in interactors of Arabidopsis and human COP1 and have been previously shown to directly bind the WD repeat of Arabidopsis and human COP1. Thus, the evolution of the plant-specific, VP-containing C-terminal domain of CRY2 might have allowed for light control of COP1 activity.

CONSTITUTIVE PHOTOMORPHOGENIC 1 functions as an E3 ubiquitin ligase in plants and animals. Discovered originally in Arabidopsis thaliana, COP1 acts in a complex with SPA proteins as a central repressor of light-mediated responses in plants. By ubiquitinating and promoting the degradation of several substrates, COP1/SPA regulates many aspects of plant growth, development and metabolism. In contrast to plants, human COP1 acts as a crucial regulator of tumorigenesis. In this review, we discuss the recent important findings in COP1/SPA research including a brief comparison between COP1 activity in plants and humans.