SUMMARYAnthocyanins 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 PHOTOMORPH-OGENIC1/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.

In plants, the cryptochrome photoreceptors suppress the activity of the COP1/SPA ubiquitin ligase to initiate photomorphogenesis in blue light. Both CRY1 and CRY2 interact with the COP1/SPA complex in a blue light-dependent manner. The mechanisms underlying the inhibition of COP1 activity through direct interactions with photoactivated CRYs are not fully understood. Here we tested the hypothesis that CRY2 inhibits COP1 by displacing the degradation substrates from COP1. To this end, we analyzed the role of a conserved valine-proline (VP) motif in the C-terminal domain of CRY2 (CCT2), which resembles the core COP1-WD40–binding sequences present in the substrates of COP1. We show that the VP motif in CRY2 is essential for the interaction of CRY2 with COP1 in yeast two-hybrid assays andin planta. Mutations in the VP motif of CRY2 abolished the CRY2 activity in photomorphogenesis, indicating the importance of VP. The interaction between COP1 and its VP-containing substrate PAP2 was prevented in the presence of coexpressed CRY2, but not in the presence of CRY2 carrying a VP mutation. Thus, since both PAP2 and CRY2 engage VP motifs to bind to COP1, these results demonstrate that CRY2 outcompetes PAP2 for binding to COP1. We further found that the previously unknown interaction between SPA1-WD and CCT2 occurs via the VP motif in CRY2, suggesting structural similarities in the VP-binding pockets of COP1-WD40 and SPA1-WD40 domains. A VP motif present in CRY1 is also essential for binding to COP1. Thus, CRY1 and CRY2 might share this mechanism of COP1 inactivation.

The plant homologs of the archaeal DNA topoisomerase VI complex are required for the progression of endoreduplication cycles. Here, we describe the identification of MIDGET (MID) as a novel component of topoisomerase VI. We show that mid mutants show the same phenotype as rhl1, rhl2, and top6B mutants and that MID protein physically interacts with RHL1. The phenotypic analysis revealed new phenotypes, indicating that topoisomerase VI is involved in chromatin organization and transcriptional silencing. In addition, genetic evidence is provided suggesting that the ATR-dependent DNA damage repair checkpoint is activated in mid mutants, and CYCB1;1 is ectopically activated. Finally, we demonstrate that overexpression of CYCB1;2 can rescue the endoreduplication defects in mid mutants, suggesting that in mid mutants, a specific checkpoint is activated preventing further progression of endoreduplication cycles.