The Arabidopsis COP1/SPA complex is a key repressor of photomorphogenesis that suppresses light signaling in the dark. Both COP1 and SPA proteins are essential components of this complex. Although COP1 also exists in humans, SPA genes are specific to the green lineage.To elucidate the evolution of SPA genes we analyzed SPA functions in the moss Physcomitrella patens by characterizing knockout mutants in the two Physcomitrella SPA genes PpSPAa and PpSPAb.Light-grown PpspaAB double mutants exhibit smaller gametophores than the wild-type. In the dark, PpspaAB mutant gametophores show enhanced continuation of growth but etiolate normally. Gravitropism in the dark is reduced in PpspaAB mutant protonemata. The expression of light-regulated genes is mostly not constitutive in PpspaAB mutants. PpSPA and PpCOP1 interact; PpCOP1 also interacts with the transcription factor PpHY5 and, indeed, PpHY5 is destabilized in dark-grown Physcomitrella. Degradation of PpHY5 in darkness, however, does not require PpSPAa and PpSPAb.The data suggest that COP1/SPA-mediated light signaling is only partially conserved between Arabidopsis and Physcomitrella. Whereas COP1/SPA interaction and HY5 degradation in darkness is conserved, the role of SPA proteins appears to have diverged. PpSPA genes, unlike their Arabidopsis counterparts, are only required to suppress a subset of light responses in darkness.

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.

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