Functional Analysis of the SPA Gene Family in Arabidopsis thaliana

Ambient light conditions affect development throughout the plant life cycle, including seed germination, seedling development and the induction of flowering. In the model plant Arabidopsis, the COP1-SPA ubiquitin ligase complex plays a central role in suppressing light signaling in darkness. The COP1-SPA complex targets positively acting factors like HY5, a protein necessary for normal seedling development in the light, several photoreceptors and the flowering time regulator CONSTANS for degradation via the 26S proteasome. Therefore, one of the major functions of the light signal transduction pathways is the inactivation of the COP1-SPA complex. While COP1 is a single copy gene, the SPA proteins are encoded by four different loci (SPA1-SPA4). All SPA proteins have redundant, but also distinct functions in regulating plant development. SPA1 and SPA2 are the key regulators that suppress photomorphogenesis in dark-grown seedlings. Over-stimulation in light-grown seedlings is primarily prevented by SPA1, and to a minor extent, also by SPA3 and SPA4. SPA2, in contrast has only negligible function in the light. SPA1 is sufficient for repressing flowering under non-inductive short-day conditions. Here, I show that distinct functions of the SPA genes partially correlate with their distinct gene expression patterns. RNA gel blot-analysis revealed that the expression of SPA1, SPA3 and SPA4, but not that of SPA2, is positively influenced by light of different wavelengths. All main photoreceptors contribute to the up-regulation of these SPA transcripts, implying that photoreceptors initiate a negative feedback regulation, which might protect plants from over-stimulation by light. GUS reporter gene experiments show that SPA genes exhibit somewhat distinct tissue-specific expression patterns, which might be important for tissue specific regulation of COP1-SPA targets. However, differences in SPA gene expression cannot account for all distinct SPA gene functions. Promoter-swap experiments with SPA1, SPA2 and SPA4 show that all SPA proteins are potent repressors in dark-grown seedlings. SPA1 and SPA4 also act as repressor in the light. SPA2, however, can never act as a repressor in the light, not even when it is expressed from the strong light-induced SPA1 promoter. These results show that SPA proteins themselves feature properties that determine characteristic SPA protein functions. All SPA proteins feature a characteristic domain structure with a C-terminal WD-repeat, a central coiled-coil domain and a less well-conserved N-terminus that includes a kinase-like motif. The WD-repeat domain and the coiled-coil domain are essential for formation of the COP1-SPA complex as well as interactions with various ubiquitination targets. In contrast, the function of the N-terminal domain is unknown. Aiming to determine the importance of the N-terminal domain of SPA1, I conducted a structure-function analysis. While the N-terminal domain of SPA1 is dispensable for SPA1 function in the seedling stage, this domain is required for SPA1-mediated repression of flowering in non-inductive short-day conditions. These results indicate, that the SPA1 N-terminal domain can full-fill an essential function

The SPA Quartet: A Family of WD-Repeat Proteins with a Central Role in Suppression of Photomorphogenesis in Arabidopsis

The Arabidopsis thaliana proteins suppressor of phytochrome A-105 1 (SPA1), SPA3, and SPA4 of the four-member SPA1 protein family have been shown to repress photomorphogenesis in light-grown seedlings. Here, we demonstrate that spa quadruple mutant seedlings with defects in SPA1, SPA2, SPA3, and SPA4 undergo strong constitutive photomorphogenesis in the dark. Consistent with this finding, adult spa quadruple mutants are extremely small and dwarfed. These extreme phenotypes are only observed when all SPA genes are mutated, indicating functional redundancy among SPA genes. Differential contributions of individual SPA genes were revealed by analysis of spa double and triple mutant genotypes. SPA1 and SPA2 predominate in dark-grown seedlings, whereas SPA3 and SPA4 prevalently regulate the elongation growth in adult plants. Further analysis of SPA2 function indicated that SPA2 is a potent repressor of photomorphogenesis only in the dark but not in the light. The SPA2 protein is constitutively nuclear localized in planta and can physically interact with the repressor COP1. Epistasis analysis between spa2 and cop1 mutations provides strong genetic support for a biological significance of a COP1–SPA2 interaction in the plant. Taken together, our results have identified a new family of proteins that is essential for suppression of photomorphogenesis in darkness

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

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 DN 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

Light exposure of arabidopsis seedlings causes rapid de-stabilization as well as selective post-translational inactivation of the repressor of photomorphogenesis SPA2

Summary The COP1/SPA complex acts as an E3 ubiquitin ligase to repress photomorphogenesis by targeting activators of the light response for degradation. Genetic analysis has shown that the four members of the SPA gene family (SPA1-SPA4) have overlapping but distinct functions. In particular, SPA1 and SPA2 differ in that SPA1 encodes a potent repressor in light- and dark-grown seedlings, but SPA2 fully loses its function when seedlings are exposed to light, indicating that SPA2 function is hyper-inactivated by light. Here, we have used chimeric SPA1/SPA2 constructs to show that the distinct functions of SPA1 and SPA2 genes in light-grown seedlings are due to the SPA protein sequences and independent of the SPA promoter sequences. Biochemical analysis of SPA1 and SPA2 protein levels shows that light exposure leads to rapid proteasomal degradation of SPA2, and, more weakly, of SPA1, but not of COP1. This suggests that light inactivates the COP1/SPA complex partly by reducing SPA protein levels. Although SPA2 was more strongly degraded than SPA1, this was not the sole reason for the lack of SPA2 function in the light. We found that the SPA2 protein is inherently incapable of repressing photomorphogenesis in light-grown seedlings. The data therefore indicate that light inactivates the function of SPA2 through a post-translational mechanism that eliminates the activity of the remaining SPA2 protein in the cell