Cryptochrome 2 competes with COP1 substrates to repress COP1 ubiquitin ligase activity during Arabidopsis photomorphogenesis

Abstract

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.

Chimeric Activators and Repressors Define HY5 Activity and Reveal a Light-Regulated Feedback Mechanism

“…We propose a model, based on our results and the existing literature, in which HY5 is regulated at the transcript level by a number of factors including itself. At the protein level, HY5 is controlled by COP1/SPA, multiple photoreceptors, and by the availability of other light-dependent HY5-interacting protein(s) ( Figure 6) (Ang et al, 1998;Lau and Deng, 2012;Zheng et al, 2013;Abbas et al, 2014;Binkert et al, 2014;Lu et al, 2015;Yang et al, 2018;Lau et al, 2019;Ponnu et al, 2019;Xu, 2019). In the dark, HY5 and its known partners are degraded, consistent with the observation that overexpression of HY5 alone cannot promote de-etiolation.…”

CONSTITUTIVELY PHOTOMORPHOGENIC1 promotes ABA‐mediated inhibition of post‐germination seedling establishment

“…promoting its exclusion from the nucleus, disrupting the COP1-SPA interaction or degradation of the SPA proteins (Podolec and Ulm, 2018). Light-activated phyA, phyB, CRY1 and CRY2 inhibit COP1 activity by one or more of these mechanisms (Lu et al, 2015;Sheerin et al, 2015;Ponnu et al, 2019). As our observations showed that COP1 promotes ABA-mediated post-germination growth arrest, we asked if photoreceptors regulate this process in light by modulating COP1 activity.…”

“…Both CRY1 and CRY2 interact with SPA1 to suppress COP1 activity (Liu et al, 2011;Zuo et al, 2011). CRY1 and CRY2 also interact with COP1 via their VP motifs and thus prevent COP1 from binding to its substrates inhibiting its function (Lau et al, 2019;Ponnu et al, 2019). The hypersensitivity of cry1cry2 seedlings in ABA could be due to enhanced activity of COP1 in the absence of the cryptochromes (Figure 2a,b).…”

Transcriptional Regulation of Photomorphogenesis in Brassica Napus Under Different Light Quality

“…On the contrary, COP1 is regarded as a central negative factor on plant photomorphogenesis [22,23]. It has been reported that COP1-SPAs complex not only target positive regulators (HY5, HFR1, etc) for ubiquitination and degradation, but also promote the accumulation of negative factors such as PIFs [17,24,25].…”