The blue light-induced interaction of cryptochrome 1 with COP1 requires SPA proteins during Arabidopsis light signaling

PLoS Genet. 2017 Oct 9;13(10):e1007044. doi: 10.1371/journal.pgen.1007044. eCollection 2017 Oct.

Abstract

Plants constantly adjust their growth, development and metabolism to the ambient light environment. Blue light is sensed by the Arabidopsis photoreceptors CRY1 and CRY2 which subsequently initiate light signal transduction by repressing the COP1/SPA E3 ubiquitin ligase. While the interaction between cryptochromes and SPA is blue light-dependent, it was proposed that CRY1 interacts with COP1 constitutively, i.e. also in darkness. Here, our in vivo co-immunoprecipitation experiments suggest that CRY1 and CRY2 form a complex with COP1 only after seedlings were exposed to blue light. No association between COP1 and CRY1 or CRY2 was observed in dark-grown seedlings. Thus, our results suggest that cryptochromes bind the COP1/SPA complex after photoactivation by blue light. In a spa quadruple mutant that is devoid of all four SPA proteins, CRY1 and COP1 did not interact in vivo, neither in dark-grown nor in blue light-grown seedlings. Hence, SPA proteins are required for the high-affinity interaction between CRY1 and COP1 in blue light. Yeast three-hybrid experiments also show that SPA1 enhances the CRY1-COP1 interaction. The coiled-coil domain of SPA1 which is responsible for COP1-binding was necessary to mediate a CRY1-SPA1 interaction in vivo, implying that-in turn-COP1 may be necessary for a CRY1-SPA1 complex formation. Hence, SPA1 and COP1 may act cooperatively in recognizing and binding photoactivated CRY1. In contrast, the blue light-induced association between CRY2 and COP1 was not dependent on SPA proteins in vivo. Similarly, ΔCC-SPA1 interacted with CRY2, though with a much lower affinity than wild-type SPA1. In total, our results demonstrate that CRY1 and CRY2 strongly differ in their blue light-induced interaction with the COP1/SPA complex.

MeSH terms

  • Arabidopsis / genetics*
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism*
  • Cryptochromes / genetics
  • Cryptochromes / metabolism*
  • Gene Deletion
  • Gene Expression Regulation, Plant
  • Immunoprecipitation
  • Light Signal Transduction / genetics*
  • Light*
  • Seedlings / genetics
  • Seedlings / growth & development
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism*

Substances

  • Arabidopsis Proteins
  • CRY1 protein, Arabidopsis
  • CRY2 protein, Arabidopsis
  • Cryptochromes
  • AT2G32950 protein, Arabidopsis
  • Ubiquitin-Protein Ligases

Grant support

This work was funded by grants from the Deutsche Forschungsgemeinschaft (HO 2793/3-1) and the Cluster of Excellence on Plant Sciences (CEPLAS) to UH and a grant from AFOSR (FA9550-14-0-0409) to MA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.