Arabidopsis COP1 shapes the temporal pattern of CO accumulation conferring a photoperiodic flowering response
2008; Springer Nature; Volume: 27; Issue: 8 Linguagem: Inglês
10.1038/emboj.2008.68
ISSN1460-2075
AutoresSeonghoe Jang, Virginie Marchal, Kishore C. S. Panigrahi, Stephan Wenkel, Wim J. J. Soppe, Xing‐Wang Deng, Federico Valverde, George Coupland,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoArticle3 April 2008Open Access Arabidopsis COP1 shapes the temporal pattern of CO accumulation conferring a photoperiodic flowering response Seonghoe Jang Seonghoe Jang Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Virginie Marchal Virginie Marchal Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Kishore C S Panigrahi Kishore C S Panigrahi Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Stephan Wenkel Stephan Wenkel Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, GermanyPresent address: Department of Plant Biology, Carnegie Institution of Washington, 260 Panama Street, Stanford, CA 94305, USA Search for more papers by this author Wim Soppe Wim Soppe Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Xing-Wang Deng Xing-Wang Deng Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT, USA Search for more papers by this author Federico Valverde Federico Valverde Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC, Universidad de Sevilla, Sevilla, Spain Search for more papers by this author George Coupland Corresponding Author George Coupland Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Seonghoe Jang Seonghoe Jang Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Virginie Marchal Virginie Marchal Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Kishore C S Panigrahi Kishore C S Panigrahi Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Stephan Wenkel Stephan Wenkel Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, GermanyPresent address: Department of Plant Biology, Carnegie Institution of Washington, 260 Panama Street, Stanford, CA 94305, USA Search for more papers by this author Wim Soppe Wim Soppe Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Xing-Wang Deng Xing-Wang Deng Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT, USA Search for more papers by this author Federico Valverde Federico Valverde Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC, Universidad de Sevilla, Sevilla, Spain Search for more papers by this author George Coupland Corresponding Author George Coupland Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Author Information Seonghoe Jang1, Virginie Marchal1, Kishore C S Panigrahi1, Stephan Wenkel1, Wim Soppe1, Xing-Wang Deng2, Federico Valverde3 and George Coupland 1 1Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany 2Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT, USA 3Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC, Universidad de Sevilla, Sevilla, Spain *Corresponding author. Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Carl von Linne Weg 10, Cologne 50829, Germany. Tel.: +49 221 5062 205; Fax: +49 221 5062 207; E-mail: [email protected] The EMBO Journal (2008)27:1277-1288https://doi.org/10.1038/emboj.2008.68 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info The transcriptional regulator CONSTANS (CO) promotes flowering of Arabidopsis under long summer days (LDs) but not under short winter days (SDs). Post-translational regulation of CO is crucial for this response by stabilizing the protein at the end of a LD, whereas promoting its degradation throughout the night under LD and SD. We show that mutations in CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a component of a ubiquitin ligase, cause extreme early flowering under SDs, and that this is largely dependent on CO activity. Furthermore, transcription of the CO target gene FT is increased in cop1 mutants and decreased in plants overexpressing COP1 in phloem companion cells. COP1 and CO interact in vivo and in vitro through the C-terminal region of CO. COP1 promotes CO degradation mainly in the dark, so that in cop1 mutants CO protein but not CO mRNA abundance is dramatically increased during the night. However, in the morning CO degradation occurs independently of COP1 by a phytochrome B-dependent mechanism. Thus, COP1 contributes to day length perception by reducing the abundance of CO during the night and thereby delaying flowering under SDs. Introduction Exposure to light influences many aspects of the plant life cycle, a process referred to as photomorphogenesis. Light promotes seed germination and seedling growth, thereby ensuring that young plants are exposed to an optimal environment for photosynthesis. Photomorphogenesis also has important functions in the development of adult plants (Neff et al, 2000). The mechanisms controlling adult photomorphogenic traits such as control of flowering in response to day length are less well understood than those that occur in the seedling. However, a genetic pathway that promotes flowering of Arabidopsis in response to long days (LDs) has been defined (Searle and Coupland, 2004; Imaizumi and Kay, 2006). Within this pathway, the transcriptional regulator CONSTANS (CO) has an important function by promoting flowering specifically under LDs. Here, we demonstrate that the ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a major regulator of seedling photomorphogenesis (Deng et al, 1992), negatively regulates CO protein abundance in the vascular tissue of adult plants as part of the mechanism by which Arabidopsis discriminates between LD and SD during flowering-time control. CO is a major regulator of photoperiodic flowering. Mutations in CO delay flowering specifically under LD, whereas its overexpression from a viral promoter causes extreme early flowering under LD and SD. CO contains two B-box-type zinc-finger motifs near its N terminus and a CCT (CONSTANS, CONSTANS-LIKE, TOC1) domain at its C terminus (Putterill et al, 1995). The latter domain is plant specific, but shows similarity to the DNA-binding domain of the HAP2 subunit of the CCAAT box-binding complex, suggesting that CO might bind to DNA directly (Wenkel et al, 2006). The closely related genes FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF) are highly and rapidly increased in expression in response to CO expression (Samach et al, 2000; Wigge et al, 2005; Yamaguchi et al, 2005). These genes encode RAF kinase inhibitor-like proteins that exert an effect as potent inducers of flowering (Kardailsky et al, 1999; Kobayashi et al, 1999). CO activates FT in the companion cells of the phloem within the vascular tissue, and FT protein is then proposed to move through the phloem sieve elements to the shoot apical meristem (An et al, 2004; Corbesier et al, 2007; Jaeger and Wigge, 2007; Mathieu et al, 2007), where it changes gene expression patterns and induces flowering (Abe et al, 2005; Wigge et al, 2005; Searle et al, 2006). The mechanism by which CO activity is controlled by day length involves both transcriptional and post-translational regulation. CO transcription is regulated by the circadian clock so that its expression rises around 12 h after dawn and stays high until the following dawn (Suarez-Lopez et al, 2001). Exposure to light between 10 and 14 h after dawn further promotes CO transcription through the activity of the photoreceptor FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) and its interacting partner GIGANTEA (GI) (Suarez-Lopez et al, 2001; Imaizumi et al, 2003; Sawa et al, 2007). At the post-translational level, CO protein is stabilized when plants are exposed to light, whereas in darkness CO protein is rapidly degraded through ubiquitination and the activity of the proteasome. These mechanisms combine to ensure that a peak in CO protein abundance occurs under LDs when plants are exposed to light between 10 and 16 h after dawn, whereas under SD, when plants are exposed to darkness during this interval, CO protein does not accumulate (Valverde et al, 2004). The importance of ubiquitination and degradation of CO protein by the proteasome in these processes was demonstrated by use of proteasome inhibitors. These regulatory steps ensure that transcription of FT and TSF occurs under LDs but not under SDs. The photoreceptors required for post-translational regulation of CO have been characterized. Mutations in the genes encoding the photoreceptors phytochrome A (phyA) and cryptochrome 2 (cry2) delay flowering, and these mutations also reduce the accumulation of CO protein (Valverde et al, 2004). Similarly, far-red light or blue light promotes flowering and stabilizes CO protein, and these regions of the spectrum activate phyA and cry2, respectively. In contrast, red light delays flowering and reduces the accumulation of CO protein. This response appears to be mainly controlled by phytochrome B (phyB), because phyB mutations cause early flowering and allow increased accumulation of CO protein. COP1 is a major negative regulator of photomorphogenic responses, so that cop1 mutants undergo photomorphogenesis in darkness in the absence of photoreceptor activation (Deng et al, 1991). COP1 encodes a RING finger protein with a coiled-coil motif and WD40 repeats (Deng et al, 1992), and exerts an effect as a ubiquitin ligase that promotes the degradation of transcription factors implicated in seedling photomorphogenesis (Osterlund et al, 2000). In mammalian and plant cells, COP1 seems to exert an effect as part of a complex that also includes DEETIOLATED 1 (DET1), DAMAGED DNA-BINDING PROTEIN 1 (DDB1), cullin 4A and RING BOX 1 (RBX1) (Chory et al, 1989; Schroeder et al, 2002; Wertz et al, 2004; Hoecker, 2005; Chen et al, 2006). However, the SUPPRESSOR OF PHYTOCHROME A-105 1 (SPA) family of proteins is plant specific, related in sequence to COP1 and modulates the ubiquitin ligase activity of COP1. SPA proteins contain a coiled-coil domain and WD40 repeats related to those of COP1 as well as a kinase-like domain not present in COP1 (Hoecker et al, 1999). Quadruple mutants in which the four SPA genes are mutated exhibit a phenotype similar to that of cop1 mutants (Laubinger et al, 2004). Furthermore, SPA1 and COP1 physically interact and SPA1 modulates COP1 activity in vitro (Hoecker and Quail, 2001; Saijo et al, 2003; Seo et al, 2003). Several protein targets for COP1 are transcription factors that regulate seedling photomorphogenesis (Osterlund et al, 2000; Seo et al, 2003; Duek et al, 2004; Jang et al, 2005; Yang et al, 2005). Each of these proteins was shown based on mutagenesis studies to have a function in the regulation of seedling growth in response to light. COP1 targets each of these proteins for degradation in the dark, but in the light COP1 activity is suppressed allowing these transcription factors to accumulate and promote seedling photomorphogenesis. In addition to these roles in seedling development, COP1 also influences photomorphogenesis of adult plants. Although null mutant alleles of COP1 cause seedling lethality, plants homozygous for weaker cop1 alleles are viable. These plants are early flowering, particularly under SDs, indicating that COP1 is required for the suppression of flowering (McNellis et al, 1994). Furthermore cop1 mutants, but not wild-type (WT) plants, flower in darkness if provided with sugar (Nakagawa and Komeda, 2004). In addition, spa1 mutants flower early and SPA proteins modulate CO abundance so that in spa1 spa3 spa4 triple mutants 16 h after dawn under LDs increased levels of CO protein were detected (Ishikawa et al, 2006; Laubinger et al, 2006). Here, we analysed the role of COP1 in the light regulation of flowering time by genetic and molecular studies. We show that COP1 represses CO activity in the vascular tissue, and reduces CO protein levels particularly under SDs and in the dark, thereby facilitating a flowering response to day length. Results Genetic and spatial interactions between COP1 and CO in the regulation of flowering time Previously cop1 mutants were shown to flower earlier than WT plants under short days (SDs) and at a similar time to WT plants under LDs (Mcnellis et al, 1994). Under our conditions, cop1–4 mutants flowered dramatically earlier than WT plants under SDs, as shown previously, but in addition flowered earlier than WT plants under LDs. The cop1–4 mutant produced around 53 leaves fewer than WT plants before flowering under SDs, whereas under LDs the difference between mutant and WT was around 5 leaves (Figure 1A–C; Supplementary Table 1). Therefore, the photoperiod response of cop1–4 mutants was severely reduced so that they flowered after forming only 3 leaves more under SDs than LDs, whereas WT plants formed around 45 leaves more under SDs. Figure 1.Genetic characterization of the interaction between CO and COP1. (A, B) cop1–4 mutants flowered earlier than wild-type Columbia plants irrespective of photoperiod, and the co-10 mutation suppresses the extreme effect of the cop1–4 mutation on flowering time under 16 h LD (A) and 8 h SD (B). (C) Flowering times in LD and SD of genotypes shown in (A, B). Flowering time is expressed as total leaf number (TLN) at flowering. (D) COP1 expression under the phloem-specific promoter SUC2 largely rescued the early-flowering cop1–4 mutant phenotype. The plants were grown under SD. (E) Simultaneous expression of CO and COP1 in the phloem tissue. SUC2:CO SUC2:HA:COP1 transgenic plants flowered later than SUC2:CO transgenic plants. (F) SUC2:COP1 caused late flowering of wild-type Columbia plants under LD. (G) Flowering times expressed as TLN at flowering under LD and SD of genotypes shown in (D–F). Download figure Download PowerPoint In WT plants, CO promotes early flowering under LDs but not SDs. To test whether the early flowering of cop1–4 mutants under SDs was caused by activation of CO under these conditions, the cop1–4 co-10 double mutant was constructed and its flowering time was measured. The double mutant flowered after forming around 30 leaves more than cop1–4 mutants under SDs, demonstrating that CO has an important role in the early flowering of cop1–4 mutants under SDs (Figure 1B and C). Nevertheless, cop1–4 co-10 plants formed 20 leaves fewer than co-10 plants under these conditions, indicating that part of the early flowering of the cop1–4 mutant occurs independently of CO. Under LDs, cop1–4 co-10 plants also flowered at a time intermediate between co-10 and cop1–4 (Figure 1A and C). These genetic results suggest that COP1 exerts an effect as a negative regulator of CO under SDs, so that CO promotes flowering of cop1–4 mutants but not WT plants under SDs. CO is expressed only in the vascular tissue and exerts an effect in the phloem companion cells to activate the transcription of the flowering-time gene FT (Takada and Goto, 2003; An et al, 2004). To test whether COP1 also regulates flowering from the phloem, COP1 or HA:COP1 was expressed from the SUC2 promoter, which is active specifically in the phloem companion cells (Imlau et al, 1999). The SUC2:COP1 transgene was introduced into WT Columbia plants and into cop1–4 mutants, whereas SUC2:HA:COP1 was introduced into SUC2:CO plants. SUC2:COP1 delayed flowering of cop1–4 mutants under LDs and SDs, and of WT plants under LDs (Figure 1D–G). Therefore, COP1 exerts an effect in the companion cells, where CO is expressed, to delay flowering. However, SUC2:COP1 cop1–4 plants still flower earlier than WT plants under SDs, suggesting that COP1 expression in companion cells is not sufficient to completely rescue the early-flowering phenotype of cop1–4 mutants, and therefore that COP1 probably also exerts an effect in additional cell types to delay flowering. The observation that SUC2:HA:COP1 delays flowering of SUC2:CO plants under LDs and SDs supports the idea that the delay of flowering associated with SUC2:COP1 is at least in part caused by reduction of CO activity (Figure 1G). Taken together, the flowering-time phenotypes of plants misexpressing COP1 in the phloem are consistent with the idea that COP1 exerts an effect in the phloem companion cells to repress the promotion of flowering by CO. COP1 reduces FT mRNA levels FT transcription is activated by CO and likely represents a direct target of CO protein (Samach et al, 2000; Wigge et al, 2005). Therefore, if COP1 exerts an effect to repress CO activity this should be reflected in reduced FT mRNA levels. In WT plants grown under SDs, CO mRNA is present during the night but FT mRNA is not expressed, because CO protein is rapidly degraded in the dark (Suarez-Lopez et al, 2001; Valverde et al, 2004). The effects of COP1 on CO transcription and CO activity were tested by analysing CO and FT mRNA levels at 4-h intervals for 24 h in SD-grown plants of different genotypes (Figure 2A and B). In WT Columbia, CO mRNA was detected during the night under SDs, but was absent in co-10 and cop1–4 co-10 plants as expected due to the T-DNA insertion present in the CO gene in the co-10 allele (Materials and methods). In cop1–4 mutants, the CO mRNA pattern is similar to that observed in WT plants but rises earlier, appearing weakly 8 h after dawn, whereas in WT plants CO mRNA was first detected 12 h after dawn. In contrast, FT mRNA was detected in cop1–4 mutants but not WT plants, consistent with the early flowering of these mutants under SDs. Similarly, under LDs, FT mRNA levels were much higher in cop1–4 plants than in WT plants consistent with the earlier flowering of the mutants under these conditions (Supplementary Figure 1). However, under LDs, CO mRNA was consistently detected at lower levels in cop1–4 mutants than in WT plants (Supplementary Figure 1). The expression of FT mRNA in cop1–4 mutants requires CO activity, as demonstrated by the absence of FT mRNA in cop1–4 co-10 plants (Figure 2A). These results are consistent with the idea that COP1 delays flowering of WT plants under SDs, and to a lesser extent under LDs, by repressing CO activity and thereby preventing FT expression. Figure 2.Effect of COP1 on CO and FT mRNA levels. (A) CO and FT mRNA analysis in wild-type (WT) Columbia, cop1–4 mutants, co-10 mutants and cop1–4 co-10 double-mutant plants under 8 h SDs. (B) Quantification of the mRNA levels shown in (A). Expressed as a ratio between UBQ10 mRNA level and FT or CO mRNA level. (C) COP1 and FT mRNAs in cop1–4 mutants and two SUC2:COP1 cop1–4 transformants. All plants were grown under SD and harvested 16 h after dawn. (D) FT, COP1 and CO mRNAs in SUC2:CO and three SUC2:CO SUC2:HA:COP1 transformants. All plants were grown under SD and harvested 8 h after dawn. (E) CO and FT mRNAs in WT Columbia plants and in a SUC2:COP1 Columbia transformant. All plants were grown under LD and harvested at 4-h intervals. All genotypes are in the accession Columbia, and in (C, D) the numbers represent independent transgenic plants. In (A, E) 2-week-old seedlings were sampled, whereas in (C, D) rosette leaves of 3-week-old plants were harvested. Download figure Download PowerPoint The abundance of FT mRNA was also tested in transgenic plants expressing COP1 or HA:COP1 mRNAs at high levels in the phloem companion cells from the SUC2 promoter (Figure 2C–E). CO and FT mRNA levels were compared through a LD time course in SUC2:COP1 and WT plants. CO mRNA levels were very similar in both genotypes at all times, whereas FT mRNA levels were severely reduced in SUC2:COP1 plants (Figure 2E), consistent with the overexpression of COP1 in phloem companion cells leading to a reduction in CO activity at the post-transcriptional level. Similarly, 16 h after dawn under SDs, when FT mRNA reaches peak levels in cop1–4 mutants (Figure 2A), SUC2:COP1 cop1–4 plants displayed severely reduced levels of FT mRNA (Figure 2C). Finally, in SUC2:HA:COP1 SUC2:CO plants the level of FT mRNA was lower than in the SUC2:CO progenitor plants, but the level of CO mRNA was unaffected (Figure 2D). Therefore, analysis of FT mRNA in these transgenic plants supports the conclusion that COP1 delays flowering by repressing at the post-transcriptional level the capacity of CO to promote FT transcription in the phloem companion cells. COP1 and CO physically interact in vitro and in vivo The observation that the ubiquitin ligase COP1 represses CO-mediated activation of FT suggested that CO might be a substrate for COP1. To test this hypothesis, we first investigated whether COP1 was able to physically interact with CO. In the yeast two-hybrid system, we detected no interaction between CO and COP1, although an interaction between COP1 and the CO-related protein CO-LIKE3 (COL3) was previously detected by this method (Datta et al, 2006), and we were able to confirm this interaction. Therefore, whether COP1 and CO interact in vitro was tested using a co-immunoprecipitation assay (Figure 3). COP1 attached to the GAL4 activation domain (GAD:COP1) and CO were made in an in vitro transcription/translation system and combined. GAD:COP1 was precipitated with anti-GAD antibody and CO was co-precipitated with GAD:COP1 (Figure 3). In contrast, CO was not co-immunoprecipitated with GAD alone. These experiments suggest that CO interacts with COP1 in the GAD:COP1 fusion protein. Fragments of CO were also combined with GAD:COP1 to determine which regions of CO are required for the interaction with COP1. Two segments of CO were tested: one containing the region between amino acids 107 and 373, which was called COΔB-box because it did not contain the zinc-finger B-boxes found at the N terminus of CO, and a second containing the region between amino acids 1 and 331, which was named COΔCCT, because the CCT domain near the C terminus of CO was removed. In vitro precipitation experiments demonstrated that COΔB-box was co-immunoprecipitated with GAD:COP1, whereas COΔCCT was not. Therefore, the N-terminal region containing the B-boxes is not required for interaction with COP1, suggesting that the interaction with COP1 is mediated by the C-terminal region of CO that contains the CCT domain. Figure 3.In vitro interaction between CO and COP1 detected by co-immunoprecipitation. 35S-methionine-labeled CO, COΔB-box or COΔCCT was incubated with 35S-methionine-labeled GAD:COP1 or GAD and co-immunoprecipitated with anti-GAD antibodies. Supernatant fractions and pellet fractions were resolved by SDS–PAGE and visualized by autoradiography using a phosphorimager. Quantification of the fractions of prey proteins that were co-immunoprecipitated by the indicated bait proteins GAD:COP1 or GAD. Error bars denote the standard error of the mean of two replicate experiments. Download figure Download PowerPoint Whether the interaction between CO and COP1 also occurred in vivo in plant cells was tested using fluorescent resonance energy transfer (FRET). Microprojectile bombardment was used to co-express cyan fluorescent protein (CFP):COP1 and yellow fluorescent protein (YFP):CO in leaf epidermal cells of Arabidopsis. CFP:COP1 and YFP:CO colocalized to the nucleus and also colocalized in speckles within the nucleus (Figure 4A and B). Physical interaction of CFP:COP1 and YFP:CO was tested by measuring FRET using photoacceptor bleaching, as previously described (Wenkel et al, 2006) (Figure 4C and D). Quantification of FRET signals demonstrated that FRET occurred between YFP:CO and CFP:COP1 both in the nucleus and specifically in nuclear speckles (Figure 4C and D). In control experiments using YFP and CFP, YFP:CO and CFP or YFP and CFP:COP1 FRET was detected at significantly lower levels (Figure 4C). These experiments demonstrate that YFP:CO and CFP:COP1 colocalize and physically interact in the nuclei of plant cells. Figure 4.CO protein physically interacts with COP1 in plant cells. (A) Transient co-expression of 35S:YFP:CO and 35S:CFP:COP1 constructs. A 35S:dsRED construct was cotransformed to highlight the transformed cell. The arrows represent the nucleus in which CO and COP1 are colocalized. (B) Enlargement of the nucleus shown in each of the panels represented in (A). (C) Quantification of FRET in vivo between CFP:CO and YFP:COP1. YFP:CO detected as an increase in CFP fluorescence after photobleaching of YFP. Quantification of FRET efficiencies after acceptor photobleaching measured in nuclei and nuclear speckles. Data are mean±s.d. of 10–20 cells from three separate experiments. (D) Visualization of increase in CFP fluorescence after YFP photobleaching. Left-hand panel, cells expressing CFP:COP1 and YFP, which exerts an effect as a negative control. Right-hand panel, cells expressing CFP:COP1 and YFP:CO. Scale bar: 6 μm in (A) and 8 μm in (D). Download figure Download PowerPoint COP1 and phyB have complementary roles in repressing CO protein levels under LDs and SDs The genetic and molecular experiments described earlier supported the hypothesis that COP1 negatively regulates CO activity at the post-transcriptional level. Therefore, we tested the effect of COP1 on CO protein levels. First, CO protein abundance was examined in nuclei of WT Columbia, co-10, transgenic 35S:CO and cop1–4 plants harvested 16 h after dawn under LDs, when CO protein is expected to be at highest abundance (Valverde et al, 2004) (Figure 5A). As shown previously, CO was clearly detectable in 35S:CO transgenic plants that overexpress the protein, but was below the level of detection in nuclei of WT plants. However, in cop1–4 mutants, CO was clearly detected, suggesting that in WT plants COP1 has a major function in reducing CO protein levels at this time. Strong support that the protein detected in cop1–4 mutants was indeed CO protein came from the analysis of cop1–4 co-10 double mutants, in which the protein detected in cop1–4 mutants was no longer present (Figure 5A). Figure 5.Detection of CO protein in cop1–4, cop1–6 phyB-9 and spa1–7 plants. (A) CO protein was detected in 35S:CO transgenic plants and cop1–4 mutants, but not in WT Columbia, co-10 or cop1–4 co-10 mutants. Plants were grown under 16 h LDs and harvested 16 h after dawn. (B, C) CO protein in cop1–4 mutants under 16 h LD or 8 h SD. Numbers above each lane represent hours after dawn that the sample was harvested. Light bar represents day; dark bar represents night. (D) CO protein detection in cop1–6 and cop1–6 phyB-9 plants grown under SDs. Samples were harvested 6 and 16 h after dawn. The reduction in CO protein at 6 h in cop1–6 plants (see also (C)) does not occur in cop1–6 phyB-9 plants. (E) CO protein detection in spa1–7 mutants under 8 h SD. Numbers and bars as described for (B, C). In WT plants, CO protein could not be detected and therefore is not included as control ((A); Valverde et al, 2004). For all panels, histone 3a was used as a loading control. Download figure Download PowerPoint CO mRNA shows a diurnal rhythm in abundance in WT plants and in cop1–4 mutants, therefore the diurnal pattern of CO protein abundance was tested under LDs and SDs in cop1–4 mutants (Figure 5B and C). Under SDs of 8 h light, cop1–4 mutants flower dramatically earlier than WT plants (Figure 1) and CO protein was present for most but not all of the diurnal cycle (Figure 5C). CO was strongly detected soon after dawn, was absent or present at much lower abundance 4 and 8 h after dawn, and then was present strongly for the remainder of the night from 10 to 24 h after dawn. The appearance of CO protein from 10 h after dawn is likely due to an increase in CO mRNA levels, as the abundance of CO mRNA increased steeply between 4 and 14 h after dawn in the same plants used for the protein analysis (Supplementary Figure 2). In contrast, CO mRNA abundance fell between 14 and 24 h after dawn, whereas CO protein levels were high throughout this time. This comparison suggests that impairing COP1 function causes CO protein to be relatively stable in the dark. However, the steep decline in CO protein abundance between 0.5 and 4 h after dawn suggests that a second post-translational mechanism, independent of COP1, might negatively regulate CO protein levels in the morning. Under LDs of 16-h photoperiods, CO protein was detected from dawn until 4 h into the photoperiod, was undetectable 6 h after dawn and then was present for the remainder of the photoperiod and throughout the night (Figure 5B). This pattern was similar to that detected under SDs, but the protein was detectable for longer and was only absent at one time point, 6 h after dawn. The broader peak in CO protein under LDs is likely due to CO mRNA being expressed for longer under LDs, as previously described (Suarez-Lopez et al, 2001; Imaizumi et al, 2003). The photoreceptor phyB was previously shown to promote the degradation of CO protein early in the day in 35S:CO plants, and this was proposed to contribute to the inhibitory effect of phyB on flowering time (Valverde et al, 2004). To test whether phyB is respon
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