The Arabidopsis CONSTITUTIVELY PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA-105 (COP1/SPA) complex is a key repressor of light signaling that inhibits light responses in darkness. It acts as an E3 ubiquitin ligase, which ubiquitinates positively acting light-signaling intermediates, mainly transcription factors, thereby targeting them for proteolytic degradation by the 26S proteasome. In the light, photoreceptors directly interact with the COP1/SPA complex, leading to its inactivation, which subsequently allows the target transcription factors to accumulate and initiate vast reprogramming of gene expression (Huang et al., 2014Huang X. Ouyang X. Deng X.W. Beyond repression of photomorphogenesis: role switching of COP/DET/FUS in light signaling.Curr. Opin. Plant Biol. 2014; 21C: 96-103Crossref Scopus (129) Google Scholar). Genetic and biochemical studies indicate that COP1 and SPA proteins act in concert to repress photomorphogenesis, i.e. as members of the COP1/SPA complex(es) (Laubinger et al., 2004Laubinger S. Fittinghoff K. Hoecker U. The SPA quartet: a family of WD-repeat proteins with a central role in suppression of photomorphogenesis in Arabidopsis.Plant Cell. 2004; 16: 2293-2306Crossref PubMed Scopus (177) Google Scholar, Yang and Wang, 2006Yang J. Wang H. The central coiled-coil domain and carboxyl-terminal WD-repeat domain of Arabidopsis SPA1 are responsible for mediating repression of light signaling.Plant J. 2006; 47: 564-576Crossref PubMed Scopus (43) Google Scholar, Zhu et al., 2008Zhu D. Maier A. Lee J.H. Laubinger S. Saijo Y. Wang H. Qu L.J. Hoecker U. Deng X.W. Biochemical characterization of Arabidopsis complexes containing CONSTITUTIVELY PHOTOMORPHOGENIC1 and SUPPRESSOR OF PHYA proteins in light control of plant development.Plant Cell. 2008; 20: 2307-2323Crossref PubMed Scopus (168) Google Scholar). However, a spa cop1 null mutant lacking the whole COP1/SPA complex has not been described so far. Moreover, the phenotypes of cop1 null mutants and spa quadruple mutants with mutations in all four SPA genes (SPA1-SPA4) are not identical, although this would be expected for a required co-action of COP1 and SPA proteins. cop1 null mutants arrest growth at the seedling stage, whereas a spa quadruple mutant proceeds through development and produces seed, despite being very dwarfed (McNellis et al., 1994McNellis T.W. Von Arnim A.G. Araki T. Komeda Y. Miséra S. Deng X.-W. Genetic and molecular analysis of an allelic series of cop1 mutants suggests functional roles for the multiple protein domains.Plant Cell. 1994; 6: 487-500Crossref PubMed Scopus (304) Google Scholar, Laubinger et al., 2004Laubinger S. Fittinghoff K. Hoecker U. The SPA quartet: a family of WD-repeat proteins with a central role in suppression of photomorphogenesis in Arabidopsis.Plant Cell. 2004; 16: 2293-2306Crossref PubMed Scopus (177) Google Scholar). However, the interpretation of SPA function in these spa mutants was hindered by the lack of null alleles. The spa quadruple mutant analyzed so far is not null for SPA2 since the spa2-1 allele produces and accumulates a truncated SPA2 protein lacking the C-terminal ∼100 amino acids (Laubinger et al., 2004Laubinger S. Fittinghoff K. Hoecker U. The SPA quartet: a family of WD-repeat proteins with a central role in suppression of photomorphogenesis in Arabidopsis.Plant Cell. 2004; 16: 2293-2306Crossref PubMed Scopus (177) Google Scholar, Zhu et al., 2008Zhu D. Maier A. Lee J.H. Laubinger S. Saijo Y. Wang H. Qu L.J. Hoecker U. Deng X.W. Biochemical characterization of Arabidopsis complexes containing CONSTITUTIVELY PHOTOMORPHOGENIC1 and SUPPRESSOR OF PHYA proteins in light control of plant development.Plant Cell. 2008; 20: 2307-2323Crossref PubMed Scopus (168) Google Scholar). Also, spa1-7 and spa4-1 carry T-DNA insertions at the proximity of the 3′ end of the respective coding sequence, so that there is a possibility that truncated SPA1 and SPA4 proteins are produced. Hence, it cannot be excluded that the viability of this spa quadruple mutant is due to residual production of partially functional SPA proteins. Here, we have isolated spa null mutant alleles and generated a spa quadruple null mutant and two different types of cop1 spa quintuple mutants to address the following questions with respect to the degree of COP1/SPA co-action. (1) Are Arabidopsis plants which fail to produce any SPA proteins viable, i.e. does COP1 indeed have residual activity in the absence of SPAs? (2) Are COP1 and SPAs necessary for embryogenesis, i.e. do SPA proteins have residual activity in the absence of COP1 during embryogenesis? (3) Is the C-terminal WD-repeat domain truly essential for COP1/SPA function and can the SPA WD-repeat domains partially replace the functions of the WD-repeat domain of COP1? By screening the MPIPZ T-DNA insertion collection, we identified genuine null alleles in SPA2 and SPA4 (spa2-2, spa4-3; Supplemental Figure 1). We crossed the new null alleles with the previously identified spa1-100 and spa3-1 null alleles to generate higher-order spa mutants. The null spa2-2 and spa4-3 single mutants and derived double and triple spa null mutants exhibited seedling and adult phenotypes that were indistinguishable from those of the previously characterized multiple mutant allele combinations (Supplemental Figures 2 and 3). The null spa quadruple mutant lacking all four SPA proteins, hereafter referred to as spaQn, undergoes constitutive photomorphogenesis, and is viable, fertile, and able to complete its life cycle (Figure 1A and 1B), as was reported previously for the spaQ mutant which is not null for all four SPAs (Laubinger et al., 2004Laubinger S. Fittinghoff K. Hoecker U. The SPA quartet: a family of WD-repeat proteins with a central role in suppression of photomorphogenesis in Arabidopsis.Plant Cell. 2004; 16: 2293-2306Crossref PubMed Scopus (177) Google Scholar). This result confirms that plants lacking all SPA proteins are indeed viable, which is in contrast to the seedling growth arrest observed in cop1 null mutants (McNellis et al., 1994McNellis T.W. Von Arnim A.G. Araki T. Komeda Y. Miséra S. Deng X.-W. Genetic and molecular analysis of an allelic series of cop1 mutants suggests functional roles for the multiple protein domains.Plant Cell. 1994; 6: 487-500Crossref PubMed Scopus (304) Google Scholar). Hence, we can now unambiguously conclude that COP1 alone, i.e. in the absence of SPA proteins, has residual activity that allows the plant to complete its life cycle. COP1 activity is nevertheless strongly enhanced by SPA proteins. spaQn mutants differed from spaQ mutants in that seedlings and plants appeared darker, suggesting higher anthocyanin content in spaQn than in spaQ plants (Figure 1A and 1B). Indeed, spaQn seedlings accumulated higher levels of anthocyanin than spaQ seedlings (Supplemental Figure 4A). Hence, the spaQ mutant has residual SPA activity, possibly due to the truncated SPA2-1 protein it produces. Besides controlling seedling deetiolation and leaf expansion, the COP1/SPA complex is required to suppress flowering under non-inductive short-day conditions (McNellis et al., 1994McNellis T.W. Von Arnim A.G. Araki T. Komeda Y. Miséra S. Deng X.-W. Genetic and molecular analysis of an allelic series of cop1 mutants suggests functional roles for the multiple protein domains.Plant Cell. 1994; 6: 487-500Crossref PubMed Scopus (304) Google Scholar, Laubinger et al., 2006Laubinger S. Marchal V. Gentilhomme J. Wenkel S. Adrian J. Jang S. Kulajta C. Braun H. Coupland G. Hoecker U. Arabidopsis SPA proteins regulate photoperiodic flowering and interact with the floral inducer CONSTANS to regulate its stability.Development. 2006; 133: 3213-3222Crossref PubMed Scopus (239) Google Scholar). Previous results indicated overlapping but also distinct functions of the four SPA genes in seedling growth and leaf expansion (Laubinger et al., 2004Laubinger S. Fittinghoff K. Hoecker U. The SPA quartet: a family of WD-repeat proteins with a central role in suppression of photomorphogenesis in Arabidopsis.Plant Cell. 2004; 16: 2293-2306Crossref PubMed Scopus (177) Google Scholar, Balcerowicz et al., 2011Balcerowicz M. Fittinghoff K. Wirthmueller L. Maier A. Fackendahl P. Fiene G. Koncz C. Hoecker U. Light exposure of Arabidopsis seedlings causes rapid de-stabilization as well as selective post-translational inactivation of the repressor of photomorphogenesis SPA2.Plant J. 2011; 65: 712-723Crossref PubMed Scopus (44) Google Scholar). The regulation of flowering time, however, has not yet been analyzed in this regard. Figure 1C shows that SPA1 and SPA4 are sufficient to strongly repress flowering in short days. Hence, SPA1 and SPA4 are the primary SPA genes responsible for photoperiodic flowering, while SPA2 and SPA3 provide only minor contributions in regulating the transition from vegetative to reproductive growth. spaQn mutants flowered very early in short days and long days and were, thus, fully insensitive to day length (Figure 1D). COP1 function is thought to be specific to light signal transduction. On the other hand, cop1 null mutants arrest growth at the seedling stage (McNellis et al., 1994McNellis T.W. Von Arnim A.G. Araki T. Komeda Y. Miséra S. Deng X.-W. Genetic and molecular analysis of an allelic series of cop1 mutants suggests functional roles for the multiple protein domains.Plant Cell. 1994; 6: 487-500Crossref PubMed Scopus (304) Google Scholar), suggesting fundamental defects that may not solely be related to light signaling. Consistent with this idea, cop1 mutants exhibit increased DNA damage, although this DNA damage can apparently be repaired prior to cell division (Dohmann et al., 2008Dohmann E.M. Levesque M.P. De Veylder L. Reichardt I. Jurgens G. Schmid M. Schwechheimer C. The Arabidopsis COP9 signalosome is essential for G2 phase progression and genomic stability.Development. 2008; 135: 2013-2022Crossref PubMed Scopus (78) Google Scholar). Human COP1 is also involved in DNA damage-induced cell cycle block by controlling the stability of p53 (Dornan et al., 2004Dornan D. Wertz I. Shimizu H. Arnott D. Frantz G.D. Dowd P. O'Rourke K. Koeppen H. Dixit V.M. The ubiquitin ligase COP1 is a critical negative regulator of p53.Nature. 2004; 429: 86-92Crossref PubMed Scopus (588) Google Scholar). However, Arabidopsis cop1 null mutants proceed through embryogenesis, a process with complex and well-defined cell division patterns, suggesting that cell division is not fundamentally impaired in the absence of COP1. We therefore asked whether SPA proteins are at least partially active during embryogenesis and thus allow seed formation in the absence of COP1. To this end, we aimed to generate a homozygous spaQn cop1-5 quintuple mutant which is fully devoid of both COP1 and SPAs. Indeed, homozygous spaQn cop1-5 quintuple mutant seeds were identified in progeny of a selfed spa123 (−/−) spa4-3 (+/−) cop1-5 (+/−) plant. Hence, embryogenesis clearly does not require COP1/SPA function. In conclusion, fundamental cellular processes can proceed in the absence of COP1/SPA activity. Interestingly, light is required for growth of the shoot apex and leaf organ initiation. Hence, major disturbances specifically in meristem function of cop1 and cop1 spa null mutants are likely responsible for the growth arrest at the seedling stage (Yoshida et al., 2011Yoshida S. Mandel T. Kuhlemeier C. Stem cell activation by light guides plant organogenesis.Genes Dev. 2011; 25: 1439-1450Crossref PubMed Scopus (117) Google Scholar). spaQn cop1-5 quintuple mutant seedlings had a shape very similar to that of cop1-5 single mutants in both darkness and light (Figure 1E). Both the quintuple mutant and the cop1-5 mutant failed to develop beyond the seedling stage. In total, these results show that SPA proteins have no activity in the absence of COP1. The only detectable difference between cop1-5 and the spaQn cop1-5 quintuple mutant was a higher anthocyanin content in the quintuple mutant when compared with cop1-5 or spaQn (Figure 1E and Supplemental Figure 4B). This suggests a possible COP1-independent function of SPA proteins in anthocyanin accumulation. However, since the cop1-5 and the spaQn alleles were derived from different Arabidopsis accessions, we cannot exclude the possibility that these differences are due to the mixed genetic background in the quintuple null mutant. In their C-termini, both COP1 and SPA carry a WD-repeat domain which mediates direct interactions with substrates and with DDB1 in the higher-order CUL4-DDB1COP1/SPA E3 ubiquitin ligase (Chen et al., 2010Chen H. Huang X. Gusmaroli G. Terzaghi W. Lau O.S. Yanagawa Y. Zhang Y. Li J. Lee J.H. Zhu D. et al.Arabidopsis CULLIN4-damaged DNA binding protein 1 interacts with CONSTITUTIVELY PHOTOMORPHOGENIC1-SUPPRESSOR OF PHYA complexes to regulate photomorphogenesis and flowering time.Plant Cell. 2010; 22: 108-123Crossref PubMed Scopus (168) Google Scholar, Huang et al., 2014Huang X. Ouyang X. Deng X.W. Beyond repression of photomorphogenesis: role switching of COP/DET/FUS in light signaling.Curr. Opin. Plant Biol. 2014; 21C: 96-103Crossref Scopus (129) Google Scholar). In general, mutations in the respective WD-repeat domain abolish COP1 and SPA1 function. Nevertheless, the cop1-4 mutant, which carries a premature STOP codon and therefore accumulates a truncated COP1 lacking all WD repeats, has only a partial loss-of-function phenotype (McNellis et al., 1994McNellis T.W. Von Arnim A.G. Araki T. Komeda Y. Miséra S. Deng X.-W. Genetic and molecular analysis of an allelic series of cop1 mutants suggests functional roles for the multiple protein domains.Plant Cell. 1994; 6: 487-500Crossref PubMed Scopus (304) Google Scholar). This mutant is viable and has a plant size intermediate between those of the spa quadruple mutant and the wild-type. Hence, the COP1-4 protein is partially functional despite the missing the WD-repeat domain. To investigate whether the SPA proteins are responsible for the observed residual COP1-4 activity, we generated cop1-4 spaQn quintuple mutants. Figure 1F shows that this quintuple mutant had a “fusca” phenotype that was more severe than those of the cop1-4 and spaQn mutants. The cop1-4 spaQn quintuple mutant exhibited a seedling phenotype very similar to that of the cop1-5 null mutant (Figure 1F) and, like cop1-5, failed to develop beyond the seedling stage (Supplemental Figure 5). This result indicates that the COP1-4 protein does not retain any activity in the absence of SPA proteins. We therefore conclude that the WD-repeat domains provided by the SPA proteins can at least partially substitute for the lack of the COP1 WD-repeat domain in the COP1-4 protein. The severe phenotype of the cop1-4 spaQn quintuple mutant further confirms that the WD repeats are essential for signaling activity of the COP1/SPA complex, i.e. a COP1-4 protein per se has no apparent activity. This work was supported by grants from the Deutsche Forschungsgemeinschaft DFG (SFB 635 TPC2 to U.H. and KO1438/16-1 to C.K.). X.Y. was a recipient of a graduate fellowship from the International Max Planck Research School of Molecular Plant Development which is co-funded by the Max Planck Institute for Plant Breeding Research and the University of Cologne.
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