Mutations in the N‐terminal kinase‐like domain of the repressor of photomorphogenesis SPA1 severely impair SPA1 function but not light responsiveness in Arabidopsis

Abstract

The COP1/SPA complex is an E3 ubiquitin ligase that acts as a key repressor of photomorphogenesis in dark-grown plants. While both COP1 and the four SPA proteins contain coiled-coil and WD-repeat domains, SPA proteins differ from COP1 in carrying an N-terminal kinase-like domain that is not present in COP1. Here, we have analyzed the effects of deletions and missense mutations in the N-terminus of SPA1 when expressed in a spa quadruple mutant background devoid of any other SPA proteins. Deletion of the large N-terminus of SPA1 severely impaired SPA1 activity in transgenic plants with respect to seedling etiolation, leaf expansion and flowering time. This ΔN SPA1 protein showed a strongly reduced affinity for COP1 in vitro and in vivo, indicating that the N-terminus contributes to COP1/SPA complex formation. Deletion of only the highly conserved 95 amino acids of the kinase-like domain did not severely affect SPA1 function nor interactions with COP1 or cryptochromes. In contrast, missense mutations in this part of the kinase-like domain severely abrogated SPA1 function, suggesting an overriding negative effect of these mutations on SPA1 activity. We therefore hypothesize that the sequence of the kinase-like domain has been conserved during evolution because it carries structural information important for the activity of SPA1 in darkness. The N-terminus of SPA1 was not essential for light responsiveness of seedlings, suggesting that photoreceptors can inhibit the COP1/SPA complex in the absence of the SPA1 N-terminal domain. Together, these results uncover an important, but complex role of the SPA1 N-terminus in the suppression of photomorphogenesis.

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

“…We found that COP1 preferentially interacts with the phosphorylated, higher-mobility isoforms of CRY1 and CRY2 that are formed upon B-irradiation. A similar observation was made for the B-dependent association of SPA1 and SPA2 with the higher-mobility isoforms of cryptochromes [ 26 , 37 ]. The B-induced phosphorylation of cryptochromes is a key step in cryptochrome activation and function [ 5 ].…”

“…These experiments were performed as described in [ 37 ]. Briefly, seedlings were homogenized in extraction buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.1% Triton X-100, 5 mM DTT, 1% protease inhibitor cocktail (Sigma-Aldrich), 10 μM MG132, phosSTOP phosphatase inhibitor cocktail (Roche, 1 tablet/5 ml buffer)).…”

The Transcription Factor COL12 Is a Substrate of the COP1/SPA E3 Ligase and Regulates Flowering Time and Plant Architecture

“…LUMIER protein-protein interaction assays in mammalian HEK293 cell culture was performed as described in (Holtkotte et al, 2016).…”

Direct phosphorylation of HY5 by SPA1 kinase to regulate photomorphogenesis in Arabidopsis

“…It has been suggested that the kinase domain of SPA1 may provide structural information critical for SPA1 function (Holtkotte et al, 2016). SPA1 R517E mutant shows a defect in PIF1 degradation and seed germination in response to light (Paik et al, 2019).…”

“…SPA1 kinase domain is conserved in SPA1 sequences from multiple plants (Paik et al, 2019). The R517 residue in SPA1 is part of a conserved Glu-Arg salt bridge that defines eukaryotic protein kinases (Yang et al, 2012) and has recently been shown to be critical for its biological function in the dark (Holtkotte et al, 2016). To investigate the importance of SPA1-Ser/Thr kinase activity for HY5 phosphorylation, we used the point mutant version of SPA1 (mSPA1) that has the R517E mutation in the kinase domain.…”