The Plant Journal (2013) 74, 638–651
doi: 10.1111/tpj.12153
Light and the E3 ubiquitin ligase COP1/SPA control the protein stability of the MYB transcription factors PAP1 and PAP2 involved in anthocyanin accumulation in Arabidopsis Alexander Maier1,†, Andrea Schrader1, Leonie Kokkelink1, Christian Falke1, Bastian Welter1, Elisa Iniesto2, Vicente Rubio2, € lskamp1 and Ute Hoecker1,* Joachim F. Uhrig2, Martin Hu 1 Botanical Institute and Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne, BioCenter, Zu€ lpicher Str. 47b, 50674 Cologne, Germany, and 2 Centro Nacional de Biotecnologıa-CSIC, Darwin, 3. Campus de la UAM. Cantoblanco, 28049 Madrid, Spain Received 28 December 2012; revised 7 February 2013; accepted 12 February 2013; published online 20 February 2013. *For correspondence (e-mail
[email protected]). † Present address: Institut für Entwicklungs- und Molekularbiologie der Pflanzen, Heinrich-Heine-Universität Düsseldorf, Universita€ tsstraβe. 1, 40225 Du€ sseldorf, Germany.
SUMMARY Anthocyanins are natural pigments that accumulate only in light-grown and not in dark-grown Arabidopsis plants. Repression of anthocyanin accumulation in darkness requires the CONSTITUTIVELY PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA-105 (COP1/SPA) ubiquitin ligase, as cop1 and spa mutants produce anthocyanins also in the dark. Here, we show that COP1 and SPA proteins interact with the myeloblastosis (MYB) transcription factors PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP)1 and PAP2, two members of a small protein family that is required for anthocyanin accumulation and for the expression of structural genes in the anthocyanin biosynthesis pathway. The increased anthocyanin levels in cop1 mutants requires the PAP1 gene family, indicating that COP1 functions upstream of the PAP1 gene family. PAP1 and PAP2 proteins are degraded in the dark and this degradation is dependent on the proteasome and on COP1. Hence, the light requirement for anthocyanin biosynthesis results, at least in part, from the light-mediated stabilization of PAP1 and PAP2. Consistent with this conclusion, moderate overexpression of PAP1 leads to an increase in anthocyanin levels only in the light and not in darkness. Here we show that SPA genes are also required for reducing PAP1 and PAP2 transcript levels in dark-grown seedlings. Taken together, these results indicate that the COP1/SPA complex affects PAP1 and PAP2 both transcriptionally and post-translationally. Thus, our findings have identified mechanisms via which the COP1/SPA complex controls anthocyanin levels in Arabidopsis that may be useful for applications in biotechnology directed towards increasing anthocyanin content in plants. Keywords: COP1, SPA1, PAP1, PAP2, anthocyanin, photomorphogenesis, protein degradation, ubiquitin ligase, Arabidopsis thaliana.
INTRODUCTION Light is one of the most important environmental factors to regulate growth, development and metabolism of plants. Light is sensed by plants through several classes of photoreceptors that include the red- and far-red light-sensing phytochromes, the blue/ultraviolet (UV)-A-perceiving cryptochromes and phototropins, and the UV-B-sensing photoreceptor UVR8 (Demarsy and Fankhauser, 2009; Nagatani, 2010; Chaves et al., 2011; Heijde and Ulm, 2012). Upon light activation, photoreceptors induce developmental and growth responses such as seedling de-etiolation, phototropism and the induction of flowering, as well as 638
changes in metabolism such as the biosynthesis of chlorophyll and anthocyanins (Kami et al., 2010). In the dark, light responses are suppressed by the activities of the CONSTITUTIVELY PHOTOMORPHOGENIC1/ SUPPRESSOR OF PHYA-105 (COP1/SPA) complex, a tetrameric complex consisting of two COP1 and two SPA proteins. In dark-grown plants, this complex acts as a CUL4/ DDB1-based ubiquitin ligase to ubiquitinate transcription factors that are required for the light response, thereby targeting them for degradation in the 26S proteasome (Hoecker, 2005; Yi and Deng, 2005; Zhu et al., 2008; Chen et al., © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd
COP1 controls PAP1 and PAP2 protein stability 639 2010). COP1 and the four members of the SPA protein family are WD-repeat proteins that interact with each other via their respective coiled-coil domains. The N-termini of COP1 and SPA proteins are distinct, with COP1 having a RING finger domain and SPA proteins containing a kinaselike domain. It is thought that both COP1 and SPA proteins are required for activity of the COP1/SPA complex. This conclusion is based on the finding that cop1 and spa1 spa2 spa3 spa4 quadruple mutants undergo constitutive photomorphogenesis and exhibit features of light-grown seedlings in complete darkness (Deng et al., 1991; Hoecker et al., 1999; Hoecker and Quail, 2001; Saijo et al., 2003; Laubinger et al., 2004). In the light, photoreceptors inhibit COP1/SPA function so that the targets of this ubiquitin ligase accumulate to initiate light responses. Recently, it has been shown that the inhibition of COP1/SPA by light involves a direct light-induced interaction between cryptochromes and SPA1 (Lian et al., 2011; Liu et al., 2011; Zuo et al., 2011). Moreover, light exposure causes rapid destabilization of SPA1 and SPA2 (Balcerowicz et al., 2011). Hence, evolution of the plant-specific SPA proteins may have allowed the placement of COP1 activity under the control of light. Several targets of the COP1/SPA ubiquitin ligase have been identified. These targets include the transcription factors LONG HYPOCOTYL 5 (HY5) and its homolog HYH, plus HFR1, LAF1, and STH and its homologs, which are involved in seedling de-etiolation and the shade avoidance response (Hoecker, 2005; Yi and Deng, 2005). Moreover, COP1/SPA controls the degradation of the floral inducer CONSTANS and thereby regulates photoperiodic flowering (Laubinger et al., 2006; Jang et al., 2008; Liu et al., 2008). In addition to these transcription factors, the photoreceptors phytochrome A and B and cryptochrome 2 are subject to COP1-/SPA-mediated degradation (Shalitin et al., 2002; Seo et al., 2004; Jang et al., 2010; Weidler et al., 2012). One feature of the constitutive photomorphogenesis phenotype of cop1 and spa mutants is the increased accumulation of anthocyanins (Deng et al., 1991; Hoecker et al., 1998; Laubinger et al., 2004). In Arabidopsis, anthocyanin accumulation occurs only in the light and this accumulation is further enhanced by a number of environmental stress factors, such as cold, drought, pathogen attack and nutrient depletion (Chalker-Scott, 1999). The biosynthesis of anthocyanins is regulated by transcription factors that induce the expression of structural genes that code for enzymes in the biosynthesis pathway (Broun, 2005; Petroni and Tonelli, 2011). These transcription factors include HY5 and a complex consisting of a WD-repeat protein, a bHLH protein and a myeloblastosis (MYB) protein (Ang et al., 1998; Koes et al., 2005; Lee et al., 2007; Shin et al., 2007; Feller et al., 2011). In Arabidopsis, the WD-repeat protein TTG1 acts together with the bHLH proteins GL3, EGL3 or TT8 and the PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP) protein family of R2R3
MYB transcription factors (PAP1, PAP2, MYB113 and MYB114) (Zhang et al., 2003; Zimmermann et al., 2004; Gonzalez et al., 2008). TTG1 and the bHLH proteins have additional functions in mucilage production and patterning of trichomes and root hairs (Pesch and Hülskamp, 2009). The PAP protein family, in contrast, is required specifically for anthocyanin accumulation and, therefore, provides specificity to the WD-repeat/bHLH/MYB complex (Borevitz et al., 2000; Tohge et al., 2005; Cominelli et al., 2008; Gonzalez et al., 2008; Lillo et al., 2008; Shi and Xie, 2010). The expression of PAP1, PAP2, TT8, GL3 and EGL3 is induced by light, while the expression of TTG1 is light-independent (Cominelli et al., 2008). PAP1 gene expression in seedlings predominates compared with the expression of the other three MYBs. Consistent with this finding, pap1 mutants and PAP1 RNAi lines show a strong reduction in anthocyanin levels (Teng et al., 2005; Gonzalez et al., 2008). Knock-down of all four MYB transcripts by RNAi abolished anthocyanin production, a finding that indicated functional redundancy among the MYB genes (Gonzalez et al., 2008). Indeed, overexpression of each of the four PAP-related MYBs led to an increase in anthocyanin levels in Arabidopsis or tobacco (Borevitz et al., 2000; Gonzalez et al., 2008; Velten et al., 2012). This situation confirms the functional conservation among these MYBs and, furthermore, shows that these MYB proteins are limiting for the production of anthocyanin. As PAP1 overexpression in the activation-tagging line pap1-D is sufficient to increase anthocyanin levels in lightgrown but not in dark-grown seedlings, it was suggested that light initiates additional processes necessary for anthocyanin accumulation (Cominelli et al., 2008). Here, we show that PAP1 and PAP2 proteins are degraded in the dark in a COP1/SPA-dependent fashion. This result leads us to suggest that light increases anthocyanin levels, at least in part, by stabilizing the PAP1 and PAP2 proteins. RESULTS The MYB transcription factors PAP1 and PAP2 interact with COP1 and SPA proteins To identify new potential substrates of the COP1/SPA E3 ubiquitin ligase we carried out a yeast two-hybrid screen with COP1, SPA1 and SPA4 as bait. As prey, we used libraries that were comprised of ca. 1200 Arabidopsis transcription factors (Paz-Ares, 2002; Castrillo et al., 2011). In these screens, the MYB transcription factors PAP1 and PAP2 were found as interactors of COP1 and/or SPA proteins. To confirm the interactions found in the screen, new GAD–PAP1 and GAD–PAP2 constructs were generated and tested for an interaction with the bait proteins in the yeast two-hybrid assay. PAP1 interacted with SPA1, SPA3 and SPA4, while PAP2 interacted with SPA1, SPA4 and COP1 (Figure 1a). No interaction between SPA2 and PAP1 or
© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), 74, 638–651
640 Alexander Maier et al.
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Figure 1. PAP1 and PAP2 interact with members of the COP1/SPA complex. (a) Yeast two-hybrid analysis of PAP1 and PAP2 interactions with COP1 and SPA proteins. Proteins were expressed as fusions with the GAL4 DNA-binding domain (BD) or activation domain (AD), as indicated. The empty vectors pGBKT7 and pGADT7, expressing BD and AD, respectively, served as negative controls. To suppress autoactivation 3-AT was added to the selective medium. (b) YFP–PAP1 and YFP–PAP2 are nuclear-localized proteins. Onion epidermal cells were transfected with the indicated constructs by particle bombardment. Co-bombarded CFP–talin was used as a marker to identify successfully transfected cells. The magnification is the same in all images. Bar = 100 lm. (c) Co-localization of CFP–SPA1 with YFP–PAP1 and YFP–PAP2 in nuclear speckles in transiently transfected onion epidermal cells. The magnification is the same in all images. Bar = 10 lm. (d) Co-localization of RFP–HA–COP1 with YFP–PAP2 in nuclear speckles in transiently transfected Arabidopsis cell suspension cultures. Bar = 10 lm. (e) HA–PAP1 and HA–PAP2 proteins interact with endogenous COP1 and SPA1 in Arabidopsis cell suspension cultures. Total protein was extracted from Arabidopsis cell cultures transfected with 35S:HA–PAP1 or 35S:HA–PAP2 constructs, respectively. HA–PAP proteins were immunoprecipitated using a-HA-conjugated agarose beads. Total protein (input) and pellet fractions (IP:HA) were analyzed by immunoblot using a-HA, a-COP1 and a-SPA1 antibodies. Non-transformed cells (Col-0) or cell cultures transformed with the empty vector (control) served as negative controls. (f) YFP–PAP2 interacts with RFP–HA–COP1. Leaves of N. benthamiana were infiltrated with agrobacteria carrying the indicated constructs. Total protein was extracted and immunoprecipitated using a-GFP-conjugated beads. Total protein (input) and pellet fractions (IP:YFP) were analyzed by immunoblot using a-HA and a-GFP antibodies.
© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), 74, 638–651
COP1 controls PAP1 and PAP2 protein stability 641 PAP2 was observed. As SPA1 and COP1 are the primary regulators of anthocyanin biosynthesis among the members of the COP1/SPA complex (Deng et al., 1991; Hoecker et al., 1998), we focused our further studies on the interaction of SPA1 and COP1 with the PAP proteins. To test whether PAP proteins interact with COP1 and/or SPA1 in planta, we assessed co-localization of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fusion proteins expressed in transfected onion cells. YFP– PAP1 and YFP–PAP2 fusion proteins were found to be localized in the nucleus (Figure 1b), as expected as the proteins are transcription factors. The YFP–PAP proteins were mostly distributed diffusely in the nucleus without forming distinct speckles, as evident when co-expressed with CFP (Figure 1c). In contrast, when YFP–PAP1 or YFP–PAP2 was co-expressed with CFP–SPA1, the YFP-tagged PAP proteins formed speckles that co-localized with CFP–SPA1. This finding shows that SPA1 co-expression recruited PAP1 and PAP2 into nuclear speckles and therefore it provides strong support for the idea of complex formation. CFP–COP1 coexpression did not recruit YFP–PAP1 or YFP–PAP2 into speckles when expressed in onion epidermal cells. However, when expressed in Arabidopsis cell suspension cells, COP1 co-localized with PAP2 (Figures 1d and S1). We then performed co-immunoprecipitation experiments to ask directly whether PAP proteins form a complex with COP1 and SPA proteins in planta. To this end, we expressed the proteins in Arabidopsis cell suspension cultures. Expression of HA-tagged PAP proteins led to a red colour formation, i.e. anthocyanin over-accumulation, in the transfected cells (Figure S2), and confirmed that the HA-tagged PAP proteins are functional. HA–PAP1 and HA–PAP2 co-immunoprecipitated the endogenous COP1 and SPA1 proteins (Figure 1e). This result shows that both proteins, PAP1 and PAP2, interact with COP1 and SPA1 in vivo. To confirm this result for PAP2 and to test a second expression system, we transfected tobacco leaves with YFP–PAP2 and RFP–HA–COP1 constructs. Also using this system, YFP–PAP2 co-immunoprecipitated RFP–HA–COP1 (Figure 1f). The WD40 domain of COP1 is essential for the interaction with the PAP2 protein All substrates of the COP1/SPA complex known so far were shown to interact with the WD-repeat domain of COP1. We therefore asked whether the COP1 WD-repeat domain is also essential for interaction with the PAP proteins and selected PAP2 to address this question. We tested the effect of a missense mutation in the WD-repeat domain (COP1K550E) that had been shown previously to disrupt the interaction of COP1 with HY5, HYH, STO and STH (Holm et al., 2001, 2002). The COP1K550E mutation also abolished the interaction with PAP2 (Figure 2a). This finding shows that an intact WD-repeat domain of COP1 is necessary for COP1–PAP2 interaction.
Next, we used the yeast three-hybrid system to test whether a known COP1-interacting domain can compete with PAP2 for binding to the WD-repeat domain of COP1. A conserved COP1-interacting domain (CID), VPE/D/G together with an upstream stretch of negatively charged residues, had been previously identified in STO, STH and HY5 to be responsible for the interaction with the WDrepeat domain of COP1 (Holm et al., 2001). We therefore expressed this CID fused to green fluorescent protein (GFP) (GFP–CID) from a methionine-repressible promoter (ProMet25). GFP served as a scaffold to allow proper folding of this small domain. The expectation was that co-expression of GFP–CID competes with other substrates for binding to COP1 and therefore reduces the binding of substrates to COP1 (Figure 2b). Pre-experiments showed that GFP–CID interacted with COP1 (Figure S3). We first tested this competition assay for interaction between COP1 and HY5 (Figure 2b,c). When yeast transformants that carried both plasmids, AD–COP1 and BD–HY5– ProMet25:GFP–CID, were grown in the presence of high methionine concentrations (100 lM), which suppress the expression of GFP–CID, COP1 showed strong interaction with HY5, as indicated by growth on His-deficient medium (Figure 2c, first row). When the methionine concentration was lowered from 100 lM to 20 lM, thereby allowing GFP–CID expression, yeast growth strongly decreased in these transformants. This result indicates that the COP1–HY5 interaction is weakened by co-expression of the GFP–CID competitor. Co-expression of ProMet25:GFP, in contrast, did not affect the COP1–HY5 interaction (Figure 2c, second row). All other negative controls (Figure 2c, rows 3–5) did not support growth on –His selection medium, confirming that yeast growth in AD– COP1 + BD–HY5 reflects the COP1–HY5 interaction. We next co-expressed GFP–CID with PAP2 to test whether GFP–CID could also compete with PAP2 for binding to COP1. Because BD–PAP2 showed strong autoactivation of the reporter genes, PAP2 was fused to the activation domain of Gal4 (AD–PAP2). A reduction in the methionine concentration that allows co-expression of GFP –CID led to a reduction in yeast growth in transformants that carried AD–PAP2 and BD–COP1–ProMet25:GFP–CID plasmids (Figure 2d, first row). Again, this growth reduction was specifically observed when GFP–CID was coexpressed and not when GFP was co-expressed from the methionine-repressible promoter (Figure 2d, second row). These results indicate that PAP2 and CID compete for binding to COP1. Taken together, these results suggest that PAP2 interacts with the WD-repeat domain of COP1 in a similar fashion as HY5, STO and STH. The increased anthocyanin accumulation in the cop1-4 mutant is dependent on PAP function To test whether the increased anthocyanin accumulation in the cop1 mutant is dependent on PAP function the cop1-4
© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), 74, 638–651
642 Alexander Maier et al.
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Figure 2. The WD40 domain of COP1 mediates the interaction with the PAP2 protein. (a) The COP1K550E missense mutation in the WD-repeat domain of COP1 disrupts PAP2 binding to COP1 in the yeast two-hybrid assay. Bait constructs were fused with the GAL4 DNA-binding domain (BD). Prey constructs were fused with the GAL4 activation domain (AD). GFP fused to BD or AD was used as negative controls. Interaction of bait and prey proteins was determined by growth on His-deficient medium (left) and by quantification of a-galactosidase activity (right). Error bars indicate the standard error of the mean (SEM) from 10 replicates. (b) Schematic representation of a yeast three-hybrid competition assay for substrate binding to the WD-repeat domain of COP1. The GFP-tagged COP1-interacting domain of STO (GFP–CID) is expressed from the Met-repressible promoter MET25. It is expressed at low methionine concentrations (left) and is expected to compete with substrates of COP1, e.g. BD–HY5, for binding to the WD-repeat domain of AD–COP1, thereby reducing expression of the HIS3 reporter gene. In the presence of high methionine concentrations (right), GFP–CID expression is repressed, thereby allowing strong interaction of AD–COP1 with the substrate protein, e.g. BD–HY5. (c,d) The COP1-interacting domain of STO (GFP–CID) competes with HY5 (c) and PAP2 (d) for binding to COP1. The indicated AD and BD fusion proteins were co-expressed with GFP–CID or GFP, respectively, which are under the control of the Met-repressible MET25 promoter. Yeast cells were plated on growth media supplemented with the indicated methionine (Met) concentrations.
mutation was crossed into PAP1 RNAi and Myb RNAi backgrounds. These RNAi lines harbour constructs that specifically target the mRNA of PAP1 or the mRNAs of all four
PAP1-related genes (PAP1, PAP2, Myb113 and Myb114), respectively (Gonzalez et al., 2008). Transgenic PAP1-RNAi seedlings showed a reduction in anthocyanin levels in the
© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), 74, 638–651
COP1 controls PAP1 and PAP2 protein stability 643
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Light exposure leads to an increase in HA–PAP1 and HA– PAP2 protein levels The physical interaction between PAP proteins and COP1/ SPA proteins and the epistatic relationship between Myb RNAi and the cop1 mutation both suggest that PAP proteins may be substrates of the COP1/SPA ubiquitin ligase. We therefore investigated whether PAP protein levels are regulated by light. To this end, we generated transgenic plants that harbour a 35S:HA–PAP1 or 35S:HA–PAP2 construct. We used the 35S promoter rather than the endogenous PAP promoters, because PAP1 and PAP2 transcript levels are induced by light (Cominelli et al., 2008) and would thus not allow us to distinguish clearly between transcriptional and post-transcriptional effects of light. All transgenic lines exhibited black seeds due to high accumulation of anthocyanin, a finding that indicated that the HA– PAP fusion proteins were functional (Figure S4). HA–PAP1 and HA–PAP2 protein levels were much higher in lightgrown seedlings when compared with dark-grown seedlings (Figures 4a, S8a). This light-induced increase in HA– PAP protein abundance was not due to a change in HA– PAP transcript levels (Figure 4b). This result suggests that light regulates HA–PAP1 and HA–PAP2 post-translationally, probably by causing stabilization of the HA–PAP proteins. We further investigated HA–PAP protein levels in transgenic seedlings that were grown in the dark for 4 days and then transferred to light. A 6-h exposure to light led to a strong increase in HA–PAP1 and HA–PAP2 protein levels. This increase was partly maintained over the 4 days of light exposure (Figures 5a,b and S8b), again without a dramatic change in the HA–PAP1 and HA–PAP2 transcript levels (Figure 5c). Figure 6(b) shows that HA–PAP1 and HA– PAP2 protein levels were increased already after a 2-h or 4-h irradiation of dark-grown seedlings.
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nin biosynthesis. These factors may include HYH, STH2, STH3/LZF1 and the PAP proteins.
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light while Myb RNAi seedlings failed to accumulate any anthocyanin, as had been reported previously (Figure 3a; Gonzalez et al., 2008). PAP1 RNAi cop1-4 seedlings displayed slightly lower anthocyanin levels in white light when compared with the cop1-4 mutant, whereas in the dark there was no significant effect of the PAP1 RNAi construct (Figure 3a). In contrast, Myb RNAi cop1-4 seedlings failed to accumulate much anthocyanin in the light or in the dark and thus exhibited a similar phenotype as Myb RNAi seedlings. This finding demonstrates that the cop14 mutation was incapable of enhancing anthocyanin levels in the absence of PAP gene function. The finding that knock-down of only one of the four PAP1-related transcripts (PAP1 RNAi) was insufficient to suppress anthocyanin accumulation in cop1-4 possibly reflects functional redundancy among PAP genes. In conclusion, anthocyanin accumulation in the cop1 mutant is fully dependent on PAP gene function, and suggests that COP1 functions upstream of PAP gene products in the light signalling pathway, leading to the biosynthesis of anthocyanins. The transcription factor HY5, which is necessary for a broad spectrum of light responses, is also known to promote anthocyanin biosynthesis in light-grown seedlings (Holm et al., 2002; Shin et al., 2007). Because HY5 is a substrate for the COP1/SPA complex (Osterlund et al., 2000), we asked whether the high anthocyanin accumulation in cop1 mutants may be caused exclusively by a hyperaccumulation of HY5 protein. We therefore examined anthocyanin levels in a cop1-4 hy5-215 double mutant (Figure 3b). In light-grown seedlings, anthocyanin levels in cop1 hy5 were lower than those in the cop1 single mutant, but were considerably higher than those in the hy5 single mutant. This result shows that the cop1 mutation causes an increase in anthocyanin abundance even in a hy5 mutant background. Hence, other factors in addition to HY5 must be involved in the COP1-dependent regulation of anthocya-
Figure 3. The increased anthocyanin accumulation caused by the cop1-4 mutation is fully dependent on the PAP1 gene family and partially dependent on HY5. (a, b) Anthocyanin levels in 60 seedlings (a) and 90 seedlings (b) of the indicated genotypes grown in the dark or white light for 4 days. PAP1 RNAi transgenic seedlings carry a construct specifically targeting the PAP1 mRNA, while Myb RNAi seedlings carry a construct targeting all four PAP-related mRNAs.
© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), 74, 638–651
644 Alexander Maier et al. Figure 4. Light increases HA–PAP1 and HA–PAP2 protein levels in transgenic 35S:HA–PAP1 and 35S:HA–PAP2 seedlings. (a) Immunoblot analysis of HA–PAP1 and HA– PAP2 protein levels in transgenic 35S:HA–PAP1 and 35S:HA–PAP2 lines. Seedlings were grown in the dark or in white light for 4 days. HA–PAP1 and HA–PAP2 proteins levels were determined using an a-HA antibody. Col wild-type seedlings were used as a negative control to show the specificity of the a-HA antibody. HSC-70 protein levels served as a loading control. (b) Transcript levels of HA–PAP1 and HA–PAP2 in the lines shown in A. Levels were determined using primers specifically amplifying the transgene-encoded transcripts and are shown relative to those of UBQ10. Error bars indicate the standard error of the mean (SEM).
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Figure 5. Exposure of dark-grown seedlings to light increases HA–PAP1 and HA–PAP2 protein levels. (a, b) HA–PAP1 (a) and HA–PAP2 (b) protein levels in seedlings of 35S:HA–PAP1 and 35S:HA–PAP2 transgenic lines that were grown for 4 days in darkness (0 h) and then transferred to continuous white light for 6 h, 1 day or 4 days. As a control seedlings were kept in the dark for an additional 4 days. HA–PAP1 and HA– PAP2 proteins were detected using an HA-specific antibody. HSC-70 protein levels served as a loading control. (c) Transcript levels of HA–PAP1 and HA–PAP2 in the lines shown in (a) and (b). Levels were determined using primers specifically amplifying the transgeneencoded transcripts and are shown relative to those of UBQ10. Error bars indicate the standard error of the mean (SEM).
© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), 74, 638–651
COP1 controls PAP1 and PAP2 protein stability 645 Figure 6. HA–PAP1 and HA–PAP2 proteins are degraded in the proteasome. (a) Four-day-old light-grown 35S:HA–PAP1 and 35S:HA–PAP2 seedlings were treated with 50 lM MG132 (+) or mock-treated (–). HA–PAP1 and HA –PAP2 protein levels were determined using an HA-specific antibody. Coomassie staining of the membrane served as a loading control. (b, c) Four-day-old dark-grown 35S:HA–PAP1 (a) or 35S:HA–PAP2 (b) seedlings were transferred to white light (Wc) for 2 or 4 h while treated with MG132 as described in (a). The asterisks marks an unspecific band detected by the a-HA antibody. HSC-70 protein levels served as a loading control.
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Figure 7. The cop1-4 mutation stabilizes the HA–PAP2 protein and leads to a strong increase in anthocyanin levels. (a) HA–PAP2 protein levels in 35S:HA–PAP2 (line 8/8) and 35S:HA–PAP2 cop1-4 (line 8/8) seedlings grown in the dark or white light for 4 days. HA–PAP1 and HA–PAP2 protein levels were determined using an HA-specific antibody. HSC-70 protein levels served as a loading control. (b) HA–PAP2 transcript levels of the lines shown in (a). Levels were determined using primers specifically amplifying the transgene-encoded transcripts and are shown relative to those of UBQ10. Error bars indicate the standard error of the mean (SEM). (c) Anthocyanin levels in seedlings of the indicated genotypes grown in the dark or white light for 4 days. The error bars indicate the SEM.
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PAP proteins are degraded by the 26S proteasome We tested whether ubiquitination and subsequent degradation in the proteasome are involved in the regulation of PAP protein levels. Indeed, application of the proteasome inhibitor MG132 caused a strong increase in the HA–PAP1 and HA–PAP2 protein levels (Figure 6a). Also, when seedlings were transferred from dark to light, MG132 treatment caused a stronger increase in HA–PAP1 and HA–PAP2 protein levels when compared with the mock control (Figures 6b, c and S8c). Degradation of PAP2 in the dark depends on COP1 To analyze whether COP1 is involved in the regulation of PAP1 and PAP2 protein levels the 35S:HA–PAP1- and 35S: HA–PAP2-overexpressing lines were crossed with the cop1-4 mutant. 35S:HA–PAP1 cop1-4 seedlings could not be analyzed because the PAP1 transgene was silenced in all obtained F3 generations. HA–PAP2 protein levels in dark-grown seedlings were elevated strongly in the cop1 mutant when compared with the COP1 wild-type back-
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ground, without a change in HA–PAP2 transcript levels (Figures 7a,b and S8d). HA–PAP2 protein levels in darkgrown cop1 mutant seedlings were as high as those in light-grown COP1 wild-type seedlings. These results demonstrate that COP1 is required for the destabilization of the HA–PAP2 protein in the dark. Consistent with this finding, the effect of HA–PAP2 overexpression on anthocyanin accumulation was considerable higher in plants with the cop1-4 mutant background than in the COP1 wild-type background, in particular in dark-grown seedlings (Figure 7c). Taken together, these results show that light via COP1 regulates PAP2 protein stability. As COP1/SPA and PAP2 interact physically with each other, it is most likely that PAP2 is a substrate of the COP1/SPA ubiquitin ligase. We therefore performed in vitro ubiquitination assays using recombinant maltose-binding protein-tagged COP1 and GST–PAP2 proteins. GST–PAP2 was non-specifically monoubiquitinated by E2, but was not ubiquitinated by recombinant COP1 (Figure S5). This result suggests to us that COP1-mediated polyubiquitination of PAP2 requires additional co-factors that were not present in the in vitro assay.
© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), 74, 638–651
646 Alexander Maier et al. Indeed, COP1 requires SPA proteins, CUL4, DDB1 and RBX1 for polyubiquitination of substrates in planta (Lau and Deng, 2012). Hence, addition of these co-factors may be necessary to observe polyubiquitination of PAP proteins in vitro. Overexpression of PAP1 and PAP2 increases anthocyanin levels The 35S:HA–PAP seedlings showed higher anthocyanin levels than the wild type not only in white light but also in the dark (Figure 8a). In contrast, the activation-tagged
Figure 8. Overexpression of HA–PAP1 and HA– PAP2 proteins results in increased anthocyanin levels in the dark or white light. (a) Anthocyanin levels in seedlings of the indicated genotypes grown in the dark or white light for 4 days. The error bars indicate the SEM. (b) Total PAP1 and PAP2 transcript levels in seedlings of the indicated genotypes that were grown as in A. PAP1 and PAP2 transcript levels were determined using primers amplifying parts of the PAP1 or PAP2 ORF, respectively. These primers, therefore, amplify the endogenous PAP1 or PAP2 transcripts as well as the HA–PAP1 or HA–PAP2 transcripts, respectively. (c,d) Total PAP1 transcript levels (c) and anthocyanin levels (d) in seedlings of Col-0 WT, pap1-D, the strong overexpressor 35S:HA–PAP1 line 8/3 and the weaker overexpressor 35S:PAP1 line 17/12. Seedlings were grown as in (a). Transcripts were detected as described in (b).
(a)
(b)
(c)
pap1-D overexpression line exhibited an increase in anthocyanin accumulation only in the light and not in the dark, as reported previously (Figure 8a; Cominelli et al., 2008). These results suggest that some HA–PAP1 and HA–PAP2 proteins escape COP1-mediated degradation in the dark. Indeed, HA–PAP immunodetection clearly recognizes the presence of HA–PAP1 or HA–PAP2 protein, respectively, in extracts of dark-grown seedlings (Figures 4a, 5a,b and 7a). We first tested whether the addition of the HA-tag to the PAP proteins may be responsible for residual accumulation of HA–PAP proteins in dark-grown seedlings because the
(d)
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COP1 controls PAP1 and PAP2 protein stability 647 HA-tag might lead to stabilization of the protein. To this end, we generated transgenic 35S:PAP1 lines that expressed only the open-reading frame of PAP1. These lines showed de-repression of anthocyanin accumulation in dark-grown seedlings similar to the 35S:HA–PAP1 lines (Figure S6), a finding that indicated that the HA-tag does not alter PAP protein function. Next, we asked whether overexpression of HA–PAP1 and HA–PAP2 may be the reason for escaping COP1-mediated degradation. Indeed, the HA–PAP1 and HA–PAP2 overexpression lines exhibited more than 1000-fold higher total PAP1 or PAP2 transcript levels, respectively, when compared with the wild type in the dark (Figure 8b). This result suggested to us that the strong overexpression of HA–PAP1 and HA–PAP2 might lead to a very high biosynthesis rate of HA–PAP1 and HA–PAP2 proteins that cannot be fully counteracted by COP1-mediated degradation. To test this possibility, we determined the total PAP1 transcript levels in the activation-tagging line pap1-D and in a PAP1 overexpression line that showed lower anthocyanin levels (L17/12). Both lines showed a hyperaccumulation of anthocyanin only in lightgrown seedlings and not in those seedlings grown in the dark, when compared with the wild type (Figure 8a,d; Cominelli et al., 2008). Both lines showed considerably lower PAP1 transcript levels than the previously examined 35S:HA–PAP1 lines, accumulating only 10- to 50-fold higher PAP1 transcript levels than the wild type in darkgrown seedlings (Figure 8c). This result indicates that these moderately increased PAP1 transcript levels were not capable of causing a hyperaccumulation of anthocyanin in dark-grown seedlings, probably because the produced PAP1 protein is degraded through the activity of the COP1/SPA complex. Consistent with this interpretation, light-grown seedlings in which COP1/SPA activity is inhibited by light exhibited a significant accumulation of anthocyanin at these moderate PAP1 transcript levels, as shown for the light-grown wild-type seedlings (Figure 8a,c,d). Taken together, these results therefore suggest that the observed accumulation of anthocyanin in dark-grown 35S: HA–PAP1 and 35S:HA–PAP2 seedlings is due to the strong overexpression of the transgenes, thereby saturating the COP1-mediated degradation of the HA–PAP1 and HA–PAP2 proteins. Regulation of PAP1 and PAP2 transcript levels by COP1 and SPA genes PAP1 and PAP2 transcript levels are up-regulated by light (Cominelli et al., 2008). We therefore investigated whether the COP1/SPA complex also affects PAP transcript levels. Dark-grown cop1-6 (Figure 9) and cop1-4 mutant (Figure 8b) seedlings showed only slightly elevated PAP1 and PAP2 transcript levels when compared with wild-type seedlings. In contrast, transcript levels of the chlorophyll a/b binding protein CAB that we analyzed as a control were
Figure 9. PAP1 and PAP2 transcript abundance in cop1 and spa mutants. Seedlings were grown for 4 days in the dark or continuous white light. Error bars indicate the standard error of the mean (SEM).
very strongly up-regulated in dark-grown cop1-4 mutants (Figure S7). In contrast, dark-grown spa mutants showed much higher PAP1 and PAP2 transcript levels than did the wild type. Hence, SPA genes are clearly involved in suppression of PAP1 and PAP2 expression in dark-grown seedlings. DISCUSSION In Arabidopsis, the production of anthocyanins is a feature of light-grown plants and does not occur in the dark. This repression of anthocyanin accumulation in the dark requires the COP1/SPA ubiquitin ligase, as it has been shown that cop1 and spa mutants produce anthocyanins also in the dark (Deng et al., 1991; Hoecker et al., 1998; Laubinger et al., 2004). Here, we have addressed the light requirement for anthocyanin production and have shown that light exposure causes a stabilization of the MYB transcription factors PAP1 and PAP2, which are positive regulators of anthocyanin biosynthesis genes. We show that PAP1 and PAP2 proteins are degraded in the dark and that this degradation is dependent on COP1. Because COP1 and
© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), 74, 638–651
648 Alexander Maier et al. SPA proteins interact directly with the PAP proteins, our results strongly suggest that PAP proteins are substrates of the COP1/SPA ubiquitin ligase. Anthocyanin biosynthesis is controlled by a number of transcription factors that act as master regulators to coordinate the expression of structural genes in the anthocyanin biosynthesis pathway (Petroni and Tonelli, 2011). One of these key regulators comprises the four-member PAP protein family. PAP proteins are a major factor in the control of anthocyanin production, as RNAi-mediated knock-down of the four PAP transcripts leads to a loss of anthocyanin production (Gonzalez et al., 2008). It is therefore evident that a regulation of PAP protein stability would have a major effect on the production of anthocyanins. A PAP-transcription-independent regulation of anthocyanin production by light has been proposed by Rowan et al. (2009), who observed that in 35S::PAP1 overexpressors low light/high temperature treatment reduced anthocyanin levels despite ongoing high expression of PAP1. Our results show that PAP protein levels are controlled post-translationally by light via the COP1/SPA complex. When expressed from a constitutive promoter, PAP1 and PAP2 protein levels were much lower in dark-grown than in light-grown seedlings, suggesting that darkness causes an enhanced degradation of these PAP proteins. Indeed, PAP proteins were subject to proteasome-mediated degradation and, moreover, PAP protein degradation in the dark was not observed in the cop1 mutant. Taken together, our results demonstrate that PAP1 and PAP2 are degraded in the dark in a COP1-dependent fashion. Thus, as light inactivates the COP1/SPA ubiquitin ligase, the light requirement for anthocyanin biosynthesis results, at least in part, from the light-mediated stabilization of PAP1 and PAP2. A similar mechanism for COP1-mediated control of a MYB transcription factor was recently reported to be present in apple, suggesting that this mechanism is conserved in dicots. MdCOP1 was shown to interact with the MdMYB1 transcription factor that results subsequently in degradation of MdMYB1 in the dark and in reduced anthocyanin accumulation in the peel of apple fruits (Li et al., 2012). Hence, future modifications in COP1-mediated degradation of MYB transcription factors involved in anthocyanin production might be a very efficient and specific approach to increase anthocyanin levels, while maintaining otherwise normal light signalling; this factor would be important for applications in biotechnology. In addition to the PAP proteins, other transcription factors act as positive regulators to induce the expression of structural genes in the anthocyanin biosynthesis pathway. These transcription factors include HY5 and its homolog HYH, and members of the STO protein family of B-box transcription factors. They have also been shown to be degraded in the dark and to be targets of the COP1/SPA ubiquitin ligase (Osterlund et al., 2000; Holm et al., 2002;
Datta et al., 2008). Hence, the COP1/SPA ubiquitin ligase allows a co-ordinated control of anthocyanin biosynthesis by targeting not only one, but several, key regulators of anthocyanin biosynthesis. This co-ordinated regulation probably allows stricter control and better fine tuning of anthocyanin accumulation in response to a change in light conditions. COP1 and SPA1 interacted with PAP1 and PAP2 in vitro and in vivo and, moreover, SPA1 could recruit PAP1 and PAP2 into nuclear speckles. We mapped the PAP2interacting domain to the WD-repeat domain of COP1: the K550 residue in the COP1 WD-repeat domain that was previously shown to be important for binding HY5, STO and STH (Holm et al., 2001) was also essential for the interaction with PAP2. This finding suggests that these transcription factors interact with the same domain of COP1 and, therefore, it supports our model that PAP1 and PAP2 are substrates of the COP1/SPA ubiquitin ligase. Indeed, our yeast three-hybrid studies showed that the COP1-interacting domain of STH was capable of competing with PAP2 for binding to COP1, thereby reducing the COP1–PAP2 interaction. Mutagenesis studies on the COP1–STO and COP1–HY5 interactions suggested that K550 forms a direct salt bridge with a negatively charged residue in STO and HY5 (Holm et al., 2001). It is therefore likely that a similar salt bridge is formed between COP1 and PAP2. Dark-grown HA–PAP2-expressing lines exhibited very high anthocyanin levels only in a cop1 mutant background and not in a COP1 wild-type background (Figure 7c). This observation is consistent with our finding that COP1 destabilizes the HA–PAP2 protein. However, there was some anthocyanin produced in the dark in the presence of wildtype COP1, which suggests that some HA–PAP2 protein escaped COP1-mediated degradation. This finding is in contrast with the PAP1-overexpressing line pap1-D, which fails to accumulate any detectable anthocyanin in the dark (Cominelli et al., 2008; our study). Our transcript analysis shows that the different behaviour of pap1-D and 35S::HA– PAP1 lines is probably due to the much stronger overexpression of PAP1 in our HA–PAP1 lines when compared with the pap1-D line. Hence, saturation of COP1-mediated degradation in our HA–PAP1 lines may allow anthocyanin production in dark-grown seedlings. However, we cannot exclude the possibility that strong overexpression of PAP1 also displaces HY5, STO or STH proteins from COP1 and subsequently reduces the degradation of these transcription factors that then could contribute to the synthesis of anthocyanins in dark-grown seedlings. In any case, our results show that it is possible to circumvent the light requirement for anthocyanin production by strong overexpression of PAP1 or PAP2. This evidence suggests that the transcriptional regulation of PAP1 and PAP2 by light also contributes to the promoting effect of light on the
© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), 74, 638–651
COP1 controls PAP1 and PAP2 protein stability 649 biosynthesis of anthocyanins, as was suggested by Cominelli et al. (2008). Our results show that SPA genes are also necessary to down-regulate PAP1 and PAP2 transcript levels in darkgrown seedlings, a finding that suggests that SPA genes are also necessary for the transcriptional control of PAP1 and PAP2 by light. Although cop1 mutations did not cause any drastic misregulation of PAP transcript levels, this result may be due to the hypomorphic nature of the cop1 alleles used. Hence, light via the COP1/SPA complex regulates PAP1 and PAP2 on two mechanistic levels: (i) light increases the transcript levels of these PAP transcription factors, probably through an increase in PAP1 and PAP2 gene expression; and (ii) it increases the protein stability of the PAP proteins. Such a dual mode of regulation has also been observed for HY5, which is transcriptionally and posttranslationally regulated by light (Osterlund et al., 2000). Our finding that mutants that carry a hypomorphic cop1 allele also show a PAP-dependent increase in anthocyanin production in the dark, despite showing no change in PAP1 and PAP2 transcript levels suggests that stabilization of PAP1 and PAP2 proteins is sufficient to allow an increase in anthocyanin levels. However, the assessment of the relative contributions of COP1-/SPA-mediated transcriptional and post-translational control of PAP1 and PAP2 for the production of anthocyanins will require further analysis. EXPERIMENTAL PROCEDURES Plant materials, growth conditions and plasmid constructions The following mutants and transgenic lines have been described previously: spa1-100 (Fittinghoff et al., 2006), spa triple and quadruple mutants (Laubinger et al., 2004; Rolauffs et al., 2012), cop1-4, cop1-6, (McNellis et al., 1994), PAP1 RNAi and Myb RNAi (Gonzalez et al., 2008), pap1-D (Borevitz et al., 2000) and hy5-215 (Oyama et al., 1997). To generate PAP1 RNAi, Myb RNAi, 35S:HA– PAP1 and 35S:HA–PAP2 plants in the cop1-4 mutant background, the respective progenitors were crossed and the obtained F2 and F3 plants were selected for resistance to BASTA (35S:HA–PAP constructs) or kanamycin (RNAi constructs) and the cop1-4 mutant adult and seedling phenotypes. All plasmid constructions for the generation of transgenic plants are provided in the Methods S1. Arabidopsis seeds were surface sterilized and cold treated as described before (Fittinghoff et al., 2006). Seeds were plated on Murashige and Skoog (MS, Serva, http://www.serva.del) medium supplemented with 1% sucrose. Cold-treated seeds were exposed to white light for 3 h and then transferred to continuous light conditions (40 lmol m 2 s 1 white light from cool-white fluorescent tubes) or continuous darkness at 21°C for 4 days or as indicated. For treatment with MG132, 4-day-old dark-grown seedlings were transferred from solid to liquid MS medium containing 50 lM MG132 or 0.5% dimethyl sulphoxide (DMSO) (mock), respectively, and vacuum infiltrated for 10 min. They were then kept in the dark for 15 min before shifting to continuous white light as indicated.
Arabidopsis thaliana cell suspension cultures were described in Shahriari et al. (2010) and were grown in cell culture medium (MS, 4 ml/l Gamborg’s B5 vitamins [Sigma Aldrich, www.sigma aldrich.com], 3% sucrose, 1 mg/l 2,4-dichlorophenoxyacetic acid) at 22°C in the dark while shaking at 120 rpm.
Analysis of anthocyanin content Seedlings were transferred to reaction tubes containing 300 ll of extraction buffer [18% (v/v) 1-propanol; 1% (v/v) HCl]. The samples were boiled for 3 min and subsequently incubated in the dark at room temperature for 24 h. After 15 min of centrifugation 100 ll of the supernatants was transferred to a 96-well microtitre plate. The anthocyanin content was determined spectrophotometrically by calculating the difference in absorbance at 535 nm and 650 nm (A535 nm–A650 nm), as determined by a Tecan Infiniteâ 200 plate reader (Tecan, www.tecan.com).
Protein–protein interaction assays To identify SPA1, SPA4 and COP1 interactors, the REGIA and RR yeast two-hybrid transcription factor libraries (Paz-Ares, 2002; Castrillo et al., 2011) were screened using SPA1–pDEST32, SPA4– pDEST32 and COP1–pDEST32 or COP1–GBKT7 as baits. In yeast three-hybrid experiments, pBridge vectors coding for the binding domain (BD), BD–COP1 or BD–HY5 were used in co-transformation with AD–COP1, AD–PAP2 or AD–GFP. Details on the interaction protocols are provided in Methods S1. For in vivo protein–protein interaction assays, Arabidopsis cell suspension cultures or leaves of N. benthamiana were transfected with A. tumefaciens carrying expression plasmids. Details on vectors constructions and the pull down assays are provided in Methods S1.
Localization, co-localization experiments and fluorescence microscopy Co-localization experiments after bombardment of onion epidermal cells were carried out as described by Zhu et al. (2008). The co-transformation of Arabidopsis cell cultures was described before (Berger et al., 2007) and representative pictures were generated by confocal laser-scanning microscopy (CLSM) 9 days (YFP –PAP2) or 5 days (YFP–PAP1) after the transfection. CLSM was performed using a Leica TCS-SP2 confocal microscope (DMRE7) equipped with the Leica software Lite 2.05 (LCS, Leica Microsystems, www.leica-microsystems.com). For two different fluorescence channels sequential scanning between frames was applied starting with the laser with the higher wavelength. The step size was 1–2 lm for each stack. Each picture for the stack is the average of three pictures. Z-stacks were merged for representation using the named software.
Protein extraction from Arabidopsis seedlings and immunoblot analysis Approximately 200 mg of seedling tissue was snap frozen in liquid nitrogen and ground to a fine powder. Laemmli buffer (2 9 ) was added in a ratio of 1:2 (v/w, 2 9 Laemmli buffer/tissue). Samples were boiled for 5 min and centrifuged for 1 min at 10 600 g. Protein concentrations were determined using the Amido Black assay as described previously (Balcerowicz et al., 2011). Protein samples were separated by SDS–PAGE and blotted onto polyvinylidene difluoride (PVDF) membranes. After transfer, membranes were blocked with Rotiblock (Roth, Karlsruhe, Germany) and incubated with the respective primary antibody followed by a
© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), 74, 638–651
650 Alexander Maier et al. horseradish peroxidase (HRP)-conjugated secondary antibody, with subsequent visualization on a LAS-4000 Mini bioimager (GE Healthcare Life Sciences, www.gelifesciences.com) (Fuji). Signal intensities were quantified using Multi-Gauge software (GE Healthcare Life Sciences, www.gelifesciences.com) (Fuji) and shown as mean standard error (SE) of at least two biological replicates. Commercial antibodies used were a-HA (Roche), aHSC70 (Stressgen, www.bioxys.com/i_Stressgen/antibodies.htm), a-mouse IgG–HRP (Sigma, www.sigmaaldrich.com), and a-rat IgG –HRP (Santa Cruz Biotechnology, www.scbt.com). a-COP1 and aSPA1 antibodies were described previously in Balcerowicz et al. (2011).
RNA isolation and transcript analysis RNA isolation and quantitative polymerase chain reaction (qPCR) experiments were performed as described previously (Balcerowicz et al., 2011). Two biological replicates were used per sample, and each was analyzed in duplicate. The results were analyzed by the DDCt method using UBQ10 as a normalizer. Gene-specific primer sequences are provided in Table S1.
Accession numbers PAP1 (At1g56650), PAP2 (At1g66390), COP1 (At2g32950), SPA1 (At2g46340), SPA2 (At4g11110), SPA3 (At3g15354), SPA4 (At1g53090), HY5 (At5 g11260).
ACKNOWLEDGEMENTS We thank Gabriele Fiene as well as the students Anna Bartsch, € ber and Laura Rupprecht for excellent Vicky Tilmes, Linda Gru technical assistance, Klaus Menrath and the green house staff for expert care of our plants, Alan Lloyd for providing PAP1 RNAi and Myb RNAi seed, Roman Ulm for providing hy5-215 and cop1-4 hy5-215 seed and the Nottingham Arabidopsis Stock Centre for pap1-D seed. We thank Franziska Turck for providing the spotted RR yeast two-hybrid library and for help with screening. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 635-TPC2 to U.H. and SFB 635-TPA2 to M.H.), a grant € r Bildung und Forschung (BIOfrom the Bundesministerium fu DISC-WIZPLANT to J.U.) and a grant from the Spanish Ministry of Economy and Competitiveness (MINECO; BIO2010-18820 to V.R.).
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article. Figure S1. Localization of RFP-HA, RFP-HA-COP1, YFP, YFP-PAP1 and YFP-PAP2 in co-transfected Arabidopsis cell suspension cultures. Figure S2. Overexpression of PAP proteins leads to increased anthocyanin accumulation in Arabidopsis cell suspension cultures. Figure S3. The COP1-interacting domain (CID) of STO interacts with COP1 in the yeast two-hybrid system and this interaction requires an intact WD40 domain. Figure S4. Transgenic plants expressing 35S:HA-PAP1 exhibit black seeds. Figure S5. In vitro ubiquitination assay using recombinant MBPCOP1 and GST-PAP2. Figure S6. Anthocyanin levels in 35S::PAP1 transgenic lines. Figure S7. PAP1, PAP2 and CAB transcript abundance in wild-type (Col-0) and cop1-4 mutant seedlings. Figure S8. Quantification of immunoblot data shown in Figures 4–7.
Table S1. Sequences of primers used for Real time PCR and cloning (5′ to 3′). Methods S1 Supplemental methods.
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© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), 74, 638–651