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• W1516409823 abstract "SPA1 is a phytochrome A (phyA)-specific signaling intermediate that acts as a light-dependent repressor of photomorphogenesis in Arabidopsis seedlings. It contains a WD-repeat domain that shows high sequence similarity to the WD-repeat region of the constitutive repressor of light signaling, COP1. Here, using yeast two-hybrid and in vitro interaction assays, we show that SPA1 strongly and selectively binds to COP1. Domain mapping studies indicate that the putative coiled-coil domain of SPA1 is necessary and sufficient for binding to COP1. Conversely, similar deletion analyses of the COP1 protein suggest that SPA1 interacts with the presumed coiled-coil domain of COP1. To further investigate SPA1 function in the phyA signaling pathway, we tested whether SPA1, like COP1, mediates changes in gene expression in response to light. We show that spa1 mutations increase the photoresponsiveness of certain light-regulated genes within 2 h of light treatment. Taken together, the results suggest that SPA1 may function to link the phytochrome A-specific branch of the light signaling pathway to COP1. Hence, our data provide molecular support for the hypothesis that COP1 is a convergence point for upstream signaling pathways dedicated to individual photoreceptors. SPA1 is a phytochrome A (phyA)-specific signaling intermediate that acts as a light-dependent repressor of photomorphogenesis in Arabidopsis seedlings. It contains a WD-repeat domain that shows high sequence similarity to the WD-repeat region of the constitutive repressor of light signaling, COP1. Here, using yeast two-hybrid and in vitro interaction assays, we show that SPA1 strongly and selectively binds to COP1. Domain mapping studies indicate that the putative coiled-coil domain of SPA1 is necessary and sufficient for binding to COP1. Conversely, similar deletion analyses of the COP1 protein suggest that SPA1 interacts with the presumed coiled-coil domain of COP1. To further investigate SPA1 function in the phyA signaling pathway, we tested whether SPA1, like COP1, mediates changes in gene expression in response to light. We show that spa1 mutations increase the photoresponsiveness of certain light-regulated genes within 2 h of light treatment. Taken together, the results suggest that SPA1 may function to link the phytochrome A-specific branch of the light signaling pathway to COP1. Hence, our data provide molecular support for the hypothesis that COP1 is a convergence point for upstream signaling pathways dedicated to individual photoreceptors. phytochrome red light continuous red light far-red light continuous far-red light Gal4 activation domain polymerase chain reaction open reading frame polyacrylamide gel electrophoresis Plants, as sessile organisms, have evolved flexible differentiation programs that allow an adaptation of growth and development to ambient environmental conditions. Sunlight, the primary source of energy for plants, is one of the most important environmental factors and is perceived by plants through the actions of several photoreceptors (1Kendrick R.E. Kronenberg G.H.M. Photomorphogenesis in Plants. 2nd Ed. Kluwer Academic Publishers, Dordrecht, The Netherlands1994Crossref Google Scholar). The members of the phytochrome photoreceptor family monitor the red (660 nm) and far-red (730 nm) region of the electromagnetic spectrum and control light responses such as seed germination, deetiolation, shade avoidance, and the induction of flowering. There is evidence for differential activities among members, with phytochrome A (phyA)1sensing mainly continuous far-red light (FRc) signals and phytochrome B (phyB) controlling responses to continuous red light (Rc) (1Kendrick R.E. Kronenberg G.H.M. Photomorphogenesis in Plants. 2nd Ed. Kluwer Academic Publishers, Dordrecht, The Netherlands1994Crossref Google Scholar, 2Whitelam G.C. Devlin P.F. Plant Cell Environ. 1997; 20: 752-758Crossref Scopus (183) Google Scholar, 3Smith H. Nature. 2000; 407: 585-591Crossref PubMed Scopus (716) Google Scholar). Efforts to identify components involved in phytochrome signal transduction have included genetic screens for signaling defective mutants and screens for phytochrome-interacting factors using the yeast two-hybrid system (3Smith H. Nature. 2000; 407: 585-591Crossref PubMed Scopus (716) Google Scholar, 4Deng X.-W. Quail P.H. Semin. Cell Dev. Biol. 1999; 10: 121-129Crossref PubMed Scopus (172) Google Scholar, 5Neff M.M. Fankhauser C. Chory J. Genes Dev. 2000; 14: 257-271Crossref PubMed Google Scholar, 6Quail P.H. Semin. Cell Dev. Biol. 2000; 11: 457-466Crossref PubMed Scopus (69) Google Scholar). These screens identified two types of signaling proteins, those that appear dedicated to a single phytochrome photoreceptor, phyA or phyB, and those that function in a transduction pathway segment common to several photoreceptors, suggesting that phytochrome signaling involves an interplay of highly selective and less specific partners. A number of signaling components that apparently play a selective role in transducing the phyA-perceived light signal have been identified through genetic screens (EID1, FAR1, FHY1, FHY3, FIN2, FIN219, HFR1 (REP1, RSF1), PAT1, SPA1; Refs. 7Dieterle M. Zhou Y.-C. Schäfer E. Funk M. Kretsch T. Genes Dev. 2001; 15: 939-944Crossref PubMed Scopus (170) Google Scholar, 8Hudson M. Ringli C. Boylan M.T. Quail P.H. Genes Dev. 1999; 13: 2017-2027Crossref PubMed Scopus (171) Google Scholar, 9Whitelam G.C. Johnson E. Peng J. Carol P. Anderson M.L. Cowl J.S. Harberd N.P. Plant Cell. 1993; 5: 757-768Crossref PubMed Scopus (483) Google Scholar, 10Soh M.S. Hong S.H. Hanzawa H. Furuya M. Nam H.G. Plant J. 1998; 16: 411-419Crossref PubMed Google Scholar, 11Hsieh H.L. Okamoto H. Wang M. Ang L.H. Matsui M. Goodman H. Deng X.-W. Genes Dev. 2000; 14: 1958-1970PubMed Google Scholar, 12Fairchild C.D. Schumaker M.A. Quail P.H. Genes Dev. 2000; 14: 2377-2391PubMed Google Scholar, 13Soh M.-S. Kim Y.-M. Han S.-J. Song P.-S. Plant Cell. 2000; 12: 2061-2074Crossref PubMed Scopus (106) Google Scholar, 14Spiegelman J.I. Mindrinos M.N. Fankhauser C. Richards D. Lutes J. Chory J. Oefner P.J. Plant Cell. 2000; 12: 2485-2498Crossref PubMed Scopus (50) Google Scholar, 15Bolle C. Koncz C. Chua N.H. Genes Dev. 2000; 14: 1269-1278PubMed Google Scholar, 16Hoecker U. Tepperman J.M. Quail P.H. Science. 1999; 284: 496-499Crossref PubMed Scopus (226) Google Scholar). The SPA1 locus was identified in a screen for extragenic mutations capable of suppressing the phenotype of a weak phyA mutant in a light-dependent fashion. Recessive spa1 mutants are hypersensitive specifically to the phyA-perceived light signal, suggesting that SPA1 functions as a phyA-dependent negative regulator of light signaling activity (17Hoecker U. Xu Y. Quail P.H. Plant Cell. 1998; 10: 19-33PubMed Google Scholar). SPA1 encodes a nuclear-localized 114-kDa protein with several features of potential functional importance (16Hoecker U. Tepperman J.M. Quail P.H. Science. 1999; 284: 496-499Crossref PubMed Scopus (226) Google Scholar). In the C-terminal portion, it displays four WD-repeats, a structural motif that is common to many eukaryotic proteins with diverse functions and is thought to mediate protein-protein interactions (18Smith T.F. Gaitatzes C. Saxena K. Neer E.J. Trends Biochem. Sci. 1999; 24: 181-185Abstract Full Text Full Text PDF PubMed Scopus (1017) Google Scholar). N-terminal to the WD-repeats, SPA1 contains a putative coiled-coil structure and two likely nuclear localization sequences. The N terminus of SPA1 shares weak sequence homology with Ser/Thr/Tyr protein kinases. A downstream convergence of several photoreceptor-specific branches of the light signaling pathway is supported by the isolation of various constitutively photomorphogenic mutants exhibiting features of light-grown seedlings in complete darkness (4Deng X.-W. Quail P.H. Semin. Cell Dev. Biol. 1999; 10: 121-129Crossref PubMed Scopus (172) Google Scholar, 19Deng X.-W. Symp. Soc. Exp. Biol. 1998; 51: 93-96PubMed Google Scholar, 20McNellis T.W. Deng X.-W. Plant Cell. 1995; 7: 1749-1761Crossref PubMed Scopus (192) Google Scholar). The corresponding loci code for negative regulators of photomorphogenesis that are active in dark-grown Arabidopsis and are thought to be inactivated by light through the functions of several phytochromes and other types of photoreceptors (4Deng X.-W. Quail P.H. Semin. Cell Dev. Biol. 1999; 10: 121-129Crossref PubMed Scopus (172) Google Scholar, 19Deng X.-W. Symp. Soc. Exp. Biol. 1998; 51: 93-96PubMed Google Scholar, 20McNellis T.W. Deng X.-W. Plant Cell. 1995; 7: 1749-1761Crossref PubMed Scopus (192) Google Scholar). Among these repressors, the RING finger-containing WD-repeat protein COP1 appears to be a key regulatory player that is nuclear-localized in the dark and excluded from the nucleus in the light (21von Arnim A.G. Deng X.-W. Cell. 1994; 79: 1035-1045Abstract Full Text PDF PubMed Scopus (364) Google Scholar). Through its WD-repeat domain COP1 physically interacts with HY5, a bZIP-type transcription factor that functions as a positive regulator of photomorphogenesis (22Ang L.H. Chattopadhyay S. Wei N. Oyama T. Okada K. Batschauer A. Deng X.-W. Mol. Cell. 1998; 1: 213-222Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar). It is hypothesized that COP1 inactivates HY5 in the dark by targeting it for degradation in the nucleus (23Osterlund M.T. Hardtke C.S. Wei N. Deng X.-W. Nature. 2000; 405: 462-466Crossref PubMed Scopus (969) Google Scholar). While genetic data are consistent with the postulated function of COP1 as a convergence point for upstream photoreceptor-specific signaling pathways, there is thus far no direct molecular evidence supporting this hypothesis. Three principal pieces of evidence suggested to us that SPA1 might be a phyA-specific signaling intermediate acting in concert with the COP1 protein. First, SPA1, like COP1, appears to function in repressing photomorphogenesis, although, in contrast to COP1, it requires light for activity. Second, SPA1 and COP1 are related by sequence in that they contain highly similar WD-repeat domains in their C-terminal regions. We considered this sequence similarity to be particularly significant, because the COP1 WD-repeat domain had been identified as an autonomous repressor module (24Torii K.U. McNellis T.W. Deng X.-W. EMBO J. 1998; 17: 5577-5587Crossref PubMed Scopus (113) Google Scholar). Third, both SPA1 and COP1 are nuclear localized in etiolated seedlings. In this study we therefore investigated the possibility that SPA1 and COP1 are physically interacting proteins. For yeast two-hybrid interaction experiments, GAD-SPA1 and GAD-SPA1-NT696 were constructed by PCR-amplifying the full-length SPA1 open reading frame (ORF) or the N-terminal part of SPA1 encoding the first 696 amino acids using primers with restriction sites and ligation of the digested PCR products into the NcoI-XhoI sites of pACT2 (CLONTECH). The full-length COP1 ORF was ligated into the EcoRI site of pGBKT7 (CLONTECH) to produce GBT-COP1. For expression of proteins in vitro, all constructs were made by ligating a PCR-amplified ORF into theNcoI-XhoI sites of the vector pET15b (Novagen). The construct SPA1 expresses the complete ORF of SPA1, SPA1-NT696 the amino acids 1–696, SPA1-NT545 the amino acids 1–545, and SPA1-cc the amino acids 521–696. COP1 expresses the complete ORF of COP1, COP1-Δcc the amino acids 1–119 and 221–675, and COP1-NT268 the amino acids 1–268. GAD-SPA1 and GAD-COP1 were constructed by ligating a PCR-amplified fragment that contains the sequences coding for GAD and the hemagglutinin epitope tag from pACT2 into the NcoI site of the constructs SPA1 and COP1, respectively. The construct GAD was made by PCR-amplifying GAD-hemagglutinin from pACT2 and ligating it into the NcoI-XhoI sites of pET15b. All constructs made using PCR-amplified fragments were sequenced to confirm the accuracy of the sequence. Cultures of the yeast strain SFY526 co-transformed with bait and prey plasmids were grown to an A600 of approximately 0.7. Cells were washed once in Z-buffer (60 mmNa2HPO4, 40 mmNaH2PO4, 10 mm KCl, 1 mm MgSO4, pH 7.0), resuspended in Z-buffer, and frozen in liquid N2. After thawing, β-mercaptoethanol ando-nitrophenyl-β-d-galactopyranoside were added to final concentrations of 0.19% and 0.64 mg/ml, respectively, and extracts were incubated at 37 °C for 30 min. Reactions were stopped by adding Na2CO3 to a final concentration of 0.3 m. Absorbance at 420 nm was determined spectrophotometrically, and β-galactosidase activity was determined in Miller units. All proteins were synthesized in the reticulocyte TnT in vitro transcription/translation system (Promega) according to the manufacturer's instructions. Prey proteins were produced in the presence of 35S-labeled methionine, while bait proteins were produced in the presence of either unlabeled methionine (domain mapping experiments) or a mixture of35S-labeled and unlabeled methionine. Ten μl each of TnT-produced bait and prey proteins were added to 0.1 ml of binding buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm dithiothreitol, 0.1% Tween 20) with protease inhibitors (Complete, EDTA-free, Roche Molecular Biochemicals) and incubated on a rotating platform at 4 °C for 2–3 h. Following addition of 1 μg of monoclonal antibody against GAD (Santa Cruz Biotechnology) incubation continued for one more hour. Subsequently, 20 μl of protein A-coated magnetic beads (Dynal, Oslo, Norway) was added. After another hour of incubation, beads were washed three times with 1 ml of binding buffer. Pellet and supernatant fractions were resolved on SDS-PAGE gels with 6–15% acrylamide and visualized using a phosphorimager (Fudji). Wild-type and mutant Arabidopsis lines used were described previously (17Hoecker U. Xu Y. Quail P.H. Plant Cell. 1998; 10: 19-33PubMed Google Scholar). Seeds were plated on growth medium and induced to germinate as described previously (17Hoecker U. Xu Y. Quail P.H. Plant Cell. 1998; 10: 19-33PubMed Google Scholar). Seedlings were grown in the dark for 3 days and subsequently transferred to the following light conditions: 0.01 μmol m−2 s−1 FRc for 0–10 h (PORA transcript analysis), 2.3 μmol m−2 s−1 FRc for 0–28 h (for CHStranscript analysis), and 10 min of 10 μmol m−2s−1 FR followed by transfer to darkness for 0–18 h (forCAB transcript analysis). Tissue was harvested in green safe light, and total RNA was isolated using the RNeasy Plant Miniprep kit (Qiagen). Five μg of total RNA was separated on a formaldehyde-containing agarose gel and was subsequently transferred to a nylon membrane (Micron Separations). The membrane was hybridized with the following 32P-labeled probes recognizing light-regulated genes: a 170-base pair fragment from the 5′-untranslated region of the Arabidopsis PORA gene (25Armstrong G.A. Runge S. Frick G. Sperling U. Apel K. Plant Physiol. 1995; 108: 1505-1517Crossref PubMed Scopus (218) Google Scholar), a 0.5-kilobase pair BamHI-SacI fragment corresponding to the Arabidopsis CAB3 coding region, and a 0.95-kilobase pair EcoRI cDNA fragment coding for the Arabidopsis CHS gene. To normalize for unequal loading, membranes were subsequently hybridized with a 18 S rRNA probe from pea. Signals were quantified by PhosphorImager (Molecular Dynamics) analysis. We employed the yeast two-hybrid system to test the possibility that SPA1 physically interacts with COP1. Fig. 1 shows that COP1 selectively bound full-length SPA1, as well as a truncated form of SPA1 lacking the C-terminal 333 amino acids (SPA1-NT696). These results indicate that SPA1 and COP1 interact in yeast and that the domain responsible for binding of COP1 lies in the N-terminal domain of SPA1. To verify the interaction detected in yeast, we conducted an in vitrobinding assay based on co-immunoprecipitation of COP1 with the GAD-SPA1 fusion protein using antibodies raised against the Gal4 activation domain (GAD) segment of the GAD-SPA1 fusion protein. Prey proteins and GAD-tagged bait proteins were synthesized by coupled in vitro transcription and translation. In these reactions prey proteins were fully radioactively labeled with [35S]methionine, while bait proteins were only partially labeled by the addition of a mixture of unlabeled and35S-labeled methionine to facilitate resolving of the co-immunoprecipitated proteins. COP1 bound strongly to GAD-SPA1, but not to GAD, indicating that COP1 selectively interacted with SPA1 (Fig.2A). We quantitatively determined the proportion of added COP1 that was co-immunoprecipitated with the bait by PhosphorImager analysis and found that GAD-SPA1 bound 18.6% of the added COP1 protein, while GAD bound 0.3% of the added COP1 (Fig. 2B). The molar ratio of bound COP1 to immobilized GAD-SPA1 or GAD was calculated as well (Fig.2C). These data indicate that COP1 bound to GAD-SPA1 with 190-fold higher apparent affinity than to GAD. We also performed the reciprocal co-immunoprecipitation experiment, using GAD-COP1 as the bait and SPA1 as the prey. As shown in Fig. 2,D–F, SPA1 strongly and selectively bound to COP1 also in this configuration. To define the COP1-interacting region in SPA1, we tested truncated versions of SPA1 for in vitro binding to COP1 (Fig.3, A–C). In these experiments, we used 35S-labeled SPA1 and SPA1 deletion derivatives as prey and GAD-COP1 and GAD as bait. SPA1-NT696 lacking the C-terminal region, including all four WD-repeats, bound to COP1 as efficiently as the full-length SPA1 protein, indicating that the WD-repeat domain of SPA1 is not necessary for the interaction with COP1. These results are consistent with the COP1 binding activity of SPA1-NT696 that we observed in the yeast two-hybrid assay (Fig. 1). Removal of 151 more amino acids, including the predicted coiled-coil domain of SPA1, (SPA1-NT545), abolished COP1 binding activity. The data suggest that the coiled-coil domain of SPA1 may be the COP1-interacting domain. We therefore tested whether the coiled-coil domain of SPA1 is sufficient for binding of COP1. Indeed, the putative coiled-coil domain of SPA1 alone (SPA1-cc) displayed similar binding activities compared with the full-length SPA1 protein, allowing COP1 to co-immunoprecipitate 25% of the added SPA1-cc protein. None of the SPA1 proteins showed strong interaction with the control bait GAD, indicating that the observed binding activities of SPA1-cc and SPA1-NT696 were specific for COP1. We also conducted the reciprocal experiment using GAD-tagged SPA1 deletion derivatives as bait and 35S-labeled COP1 as prey. GAD-SPA1, GAD-SPA1-NT696, and GAD-SPA1-cc strongly co-immunoprecipitated COP1 (22, 20, and 29% of the added COP1, respectively), while GAD-SPA1-NT 545 and GAD did not show strong interaction with COP1 (binding of 0.6 and 0.5% of the added COP1, respectively) (data not shown). Thus, the data from the reciprocal experiment support the conclusion that the coiled-coil domain of SPA1 is necessary and sufficient for binding of COP1 in vitro. To determine the domain in COP1 necessary for interaction with SPA1, we then employed deletion derivatives of COP1 in the in vitrobinding assay with full-length SPA1 (Fig.4, A–C). Here, we used GAD-SPA1 and GAD as bait and 35S-labeled truncated COP1 proteins as prey. Because the COP1 protein contains a putative coiled-coil domain, we first tested whether this domain is important for binding of SPA1. A deletion derivative of COP1 lacking the coiled-coil domain (COP1-Δcc) did not exhibit any detectable interaction with SPA1, indicating that the coiled-coil domain of COP1 is necessary for the SPA1 binding activity of COP1. We were unable to investigate whether the coiled-coil domain of COP1 is sufficient for interaction with SPA1, because the coiled-coil domain alone could not be expressed in vitro. However, the N-terminal 268 amino acids of COP1 comprising the predicted RING finger and coiled-coil domains (COP1-NT268) displayed strong and selective interaction with SPA1, binding 17% of the added SPA1 protein. These results indicate that the N terminus of COP1 is sufficient for binding to SPA1. Because we previously only described the morphological phenotype of the spa1 mutant, we wished to investigate whether spa1 mutations also affect the expression of known phyA-regulated genes. Moreover, a branching of the phyA signaling pathway was suggested by the isolation of mutants (fhy1, hfr1/rep1) that were altered in the expression of only a subset of genes regulated by light (1 2, 13, 26). We therefore wished to place SPA1 within this branched network of phyA signaling. We compared transcript levels of light-regulated genes in dark-grownspa1 mutant and wild-type seedlings exposed to FR for only several hours. We selected three genes that were shown to be regulated by separate branches of the phyA signaling pathway (13Soh M.-S. Kim Y.-M. Han S.-J. Song P.-S. Plant Cell. 2000; 12: 2061-2074Crossref PubMed Scopus (106) Google Scholar, 26Barnes S.A. Quaggio R.B. Whitelam G.C. Chua N.-H. Plant J. 1996; 10: 1155-1161Crossref PubMed Scopus (55) Google Scholar): the light-inducible genes coding for chalcone synthase (CHS), the chlorophyll ab-binding protein (CAB), and the light-repressible gene encoding protochlorophyllide oxidoreductase A (PORA). Fig. 5, Aand B, show that CHS and CAB mRNA abundance was considerably higher in light-exposed spa1mutants than in wild-type. Here a small but reproducible difference between spa1 mutant and wild-type seedlings was also observed in the dark. This effect, however, was fully dependant on phyA, as it was no longer observed in the phyA-deficient spa1–2 phyA-101 double mutant (Fig. 5D), suggesting that germinating Arabidopsis seeds may contain active phyA and/or active SPA1. Thus, CHS and CAB genes appeared hypersensitive to FR in the absence of functional SPA1, and this effect was evident as early as 2 h after the initiation of light treatment. Transcript levels of the light-repressible PORAgene exhibited an enhanced down-regulation by FRc in spa1mutants when compared with wild-type (Fig. 5C). This differential response was detectable after an FRc irradiation of at least 4 h. Changes in the rate of transcript synthesis, however, may have been initiated earlier but not immediately observed due to the time required for the degradation of mRNA produced in the preceding dark treatment. Taken together, these results indicate thatspa1 mutations enhance light responses of all three examined phyA-regulated genes. We therefore conclude that SPA1 is not involved in a known branched part of the phyA signaling pathway. In this study, we have investigated the possibility that the phytochrome signaling factors SPA1 and COP1 are physically interacting proteins. Using two experimental approaches, the yeast two-hybrid system and an in vitro interaction assay, we have provided evidence that SPA1 directly binds to COP1. The observed strength and selectivity of the SPA1-COP1 interaction together with in vivo findings reported here and previously that both proteins act as repressors of light-regulated responses in the plant (17Hoecker U. Xu Y. Quail P.H. Plant Cell. 1998; 10: 19-33PubMed Google Scholar, 27Deng X.W. Casper T. Quail P.H. Genes Dev. 1991; 5: 1172-1182Crossref PubMed Scopus (435) Google Scholar, 28Deng X.W. Quail P.H. Plant J. 1992; 2: 83-95Crossref Scopus (123) Google Scholar) suggest that this interaction may be functionally significant to SPA1-mediated phyA signal transduction. The isolation of various types of light signaling mutants has led to the hypothesis that separate photoreceptor-specific segments of the light signaling pathway converge further downstream in a common pathway shared by several photoreceptors (4Deng X.-W. Quail P.H. Semin. Cell Dev. Biol. 1999; 10: 121-129Crossref PubMed Scopus (172) Google Scholar, 5Neff M.M. Fankhauser C. Chory J. Genes Dev. 2000; 14: 257-271Crossref PubMed Google Scholar). It has been proposed that COP1 may be a protein where signals from several photoreceptors come together, because COP1 appears to respond to Rc, FRc, and blue light (20McNellis T.W. Deng X.-W. Plant Cell. 1995; 7: 1749-1761Crossref PubMed Scopus (192) Google Scholar). We have now provided the first molecular evidence that COP1 may indeed be such a convergence point by showing that a protein apparently dedicated to signaling from an individual photoreceptor physically interacts with COP1. Hence, our data suggest that SPA1 may be a component responsible for linking a phyA-specific input pathway to COP1 (Fig. 6). Similar convergence points have been identified in proteins PIF3, PKS1, and NDPK2, which bind to both phyA and phyB and appear to represent signaling intermediates shared by phyA and phyB (29Ni M. Tepperman J.M. Quail P.H. Cell. 1998; 95: 657-667Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar, 30Ni M. Tepperman J.M. Quail P.H. Nature. 1999; 400: 781-784Crossref PubMed Scopus (372) Google Scholar, 31Zhu Y. Tepperman J.M. Fairchild C.D. Quail P.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13419-13424Crossref PubMed Scopus (111) Google Scholar, 32Fankhauser C. Yeh K.C. Lagarias J.C. Zhang H. Elich T.D. Chory J. Science. 1999; 284: 1539-1541Crossref PubMed Scopus (330) Google Scholar, 33Choi G. Yi H. Lee J. Kwon Y.-K. Soh M.S. Shin B. Luka Z. Hahn T.R. Song P.S. Nature. 1999; 401: 610-613Crossref PubMed Scopus (253) Google Scholar). Thus, light signal transduction appears to occur through a complex network that involves interactions of photoreceptor-specific components with shared partners at at least two different points in the pathway. While SPA1 and COP1 both function as nuclear repressors of photomorphogenesis in Arabidopsis, they appear to be oppositely regulated by light: COP1 activity is high in the dark and reduced by light, while SPA1 function requires light. At least two nonmutually exclusive models may be evoked to reconcile the light regulation of SPA1 and COP1 with the observed physical interaction of the two proteins. First, SPA1, once activated by light in a phyA-dependent fashion, may increase COP1 repressor activity by producing SPA1·COP1-containing protein complexes in the cell that cause stronger repression than COP1 does alone. It has been established that the WD-repeat domain of COP1 functions as an autonomous repressor of photomorphogenesis (24Torii K.U. McNellis T.W. Deng X.-W. EMBO J. 1998; 17: 5577-5587Crossref PubMed Scopus (113) Google Scholar). The findings that SPA1 contains a WD-repeat domain that is highly similar in sequence to that of COP1 and, moreover, is essential for SPA1 function in vivo (16Hoecker U. Tepperman J.M. Quail P.H. Science. 1999; 284: 496-499Crossref PubMed Scopus (226) Google Scholar) support the idea that SPA1 and COP1 may act in concert in repressing photomorphogenesis. An alternative possibility for SPA1 function may be that active SPA1, when bound to COP1, may interfere with the light-induced inactivation of COP1, thus prolonging its repressive activity. One mechanism by which light inactivates COP1 is the light-induced depletion of COP1 from the nucleus (21von Arnim A.G. Deng X.-W. Cell. 1994; 79: 1035-1045Abstract Full Text PDF PubMed Scopus (364) Google Scholar). This process requires at least 24 h of light exposure and is considered too slow to account for rapid COP1-mediated changes observed in response to light (34von Arnim A.G. Osterlund M.T. Kwok S.F. Deng X.-W. Plant Physiol. 1997; 114: 779-788Crossref PubMed Scopus (107) Google Scholar). Therefore, the existence of an additional, early inactivation mechanism has been proposed. Our findings that spa1 mutations cause early changes in the expression of the light-regulated genes CHS,CAB, and PORA suggest that, in this model, SPA1 more likely modulates an early COP1 inactivation mechanism. Our deletion analyses aimed at defining the domains in SPA1 and COP1 responsible for the interaction of the two proteins indicate that the putative coiled-coil domain of SPA1 most likely binds to the presumed coiled-coil domain of COP1. To investigate whether otherArabidopsis proteins exist with a coiled-coil domain similar to that of SPA1, we searched the Arabidopsis data base containing the complete sequence of the Arabidopsis genome. We detected only two hypothetical proteins with significant sequence similarity (GenBankTM accession numbers T08190 andAAF87859), both exhibiting 32% identity with the amino acid sequence of the SPA1 coiled-coil domain. These hypothetical proteins are indeed predicted to form coiled-coil structures and, moreover, show additional high sequence similarity to the WD-repeat region of SPA1. Hence, if these proteins are indeed synthesized in the cell, they might represent COP1-interacting proteins as well. Interestingly, COP1 function requires its coiled-coil domain for homodimerization, which in turn is necessary for the COP1-HY5 interaction (24Torii K.U. McNellis T.W. Deng X.-W. EMBO J. 1998; 17: 5577-5587Crossref PubMed Scopus (113) Google Scholar). Thus, the coiled-coil domain of COP1 may be a possible site for regulation of COP1 activity by interacting proteins such as SPA1. Several other COP1-interacting proteins have been identified of which three proteins (CIP1, CIP4, CIP7) also appear to bind to the coiled-coil domain of COP1 (35Matsui M. Stoop C.D. von Arnim A.G. Wei N. Deng X.-W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4239-4243Crossref PubMed Scopus (65) Google Scholar, 36Yamamoto Y.Y. Matsui M. Ang L.H. Deng X.-W. Plant Cell. 1998; 10: 1083-1094Crossref PubMed Scopus (112) Google Scholar, 37Yamamoto Y.Y. Deng X.-W. Matsui M. Plant Cell. 2001; 13: 399-411Crossref PubMed Scopus (36) Google Scholar). There is evidence that CIP4 and CIP7 may function as positive regulators of light-regulated genes (36Yamamoto Y.Y. Matsui M. Ang L.H. Deng X.-W. Plant Cell. 1998; 10: 1083-1094Crossref PubMed Scopus (112) Google Scholar, 37Yamamoto Y.Y. Deng X.-W. Matsui M. Plant Cell. 2001; 13: 399-411Crossref PubMed Scopus (36) Google Scholar). It will be interesting to investigate the interplay of the various COP1-binding proteins. Taken together, we have provided evidence that SPA1 may physically link an upstream phyA-specific branch of the light signaling pathway to the repressor COP1. A future biochemical analysis of the SPA1·COP1 complex in planta under different light conditions and in photoreceptor-deficient backgrounds will shed new light on the mechanisms involved in phyA signaling that are as yet not understood. We thank N. Wei for the gift ofCHS and CAB probes, G. Armstrong for the gift of the PORA probe, X.-W. Deng for providing us with the COP1 cDNA, and P. Westhoff and members of the laboratory for helpful discussions." @default.
• W1516409823 created "2016-06-24" @default.
• W1516409823 creator A5057290282 @default.
• W1516409823 creator A5065470504 @default.
• W1516409823 date "2001-10-01" @default.
• W1516409823 modified "2023-10-11" @default.
• W1516409823 title "The Phytochrome A-specific Signaling Intermediate SPA1 Interacts Directly with COP1, a Constitutive Repressor of Light Signaling inArabidopsis" @default.
• W1516409823 cites W1551393329 @default.
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