Red-Light-Dependent Interaction of phyb with SPA1 Promotes COP1 SPA1 Dissociation and Photomorphogenic Development in Arabidopsis

Transcription

1 Research Article Red-Light-Dependent Interaction of phyb with SPA1 Promotes COP1 SPA1 Dissociation and Photomorphogenic Development in Arabidopsis Xue-Dan Lu 1, Chuan-Miao Zhou 2, Peng-Bo Xu 3, Qian Luo 1, Hong-Li Lian 1, * and Hong-Quan Yang 3, * 1 Key Laboratory of Urban Agriculture (South) Ministry of Agriculture and School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai , China 2 National Key Laboratory of Plant Molecular Genetics (NKLPMG), Institute of Plant Physiology and Ecology (SIPPE), Shanghai Institutes for Biological Sciences (SIBS), Shanghai , China 3 School of Life Sciences and Biotechnology, Shanghai Jiaotong University, Shanghai , China *Correspondence: Hong-Quan Yang (hqyang@sjtu.edu.cn), Hong-Li Lian (hllian@sjtu.edu.cn) ABSTRACT Arabidopsis phytochromes (phya-phye) are photoreceptors dedicated to sensing red/far-red light. Phytochromes promote photomorphogenic developments upon light irradiation via a signaling pathway that involves rapid degradation of PIFs (PHYTOCHROME INTERACTING FACTORS) and suppression of COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1) nuclear accumulation, through physical interactions with PIFs and COP1, respectively. Both phya and phyb, the two best characterized phytochromes, regulate plant photomorphogenesis predominantly under far-red light and red light, respectively. It has been demonstrated that SPA1 (SUPPRESSOR OF PHYTOCHROME A 1) associates with COP1 to promote COP1 activity and suppress photomorphogenesis. Here, we report that the mechanism underlying phybpromoted photomorphogenesis in red light involves direct physical and functional interactions between red-light-activated phyb and SPA1. We found that SPA1 acts genetically downstream of PHYB to repress photomorphogenesis in red light. Protein interaction studies in both yeast and Arabidopsis demonstrated that the photoactivated phyb represses the association of SPA1 with COP1, which is mediated, at least in part, through red-light-dependent interaction of phyb with SPA1. Moreover, we show that phya physically interacts with SPA1 in a Pfr-form-dependent manner, and that SPA1 acts downstream of PHYA to regulate photomorphogenesis in far-red light. This study provides a genetic and biochemical model of how photoactivated phyb represses the activity of COP1 SPA1 complex through direct interaction with SPA1 to promote photomorphogenesis in red light. Key words: phyb, photoactivation, SPA1, COP1, photomorphogenesis, phya Lu X.-D., Zhou C.-M., Xu P.-B., Luo Q., Lian H.-L., and Yang H.-Q. (2015). Red-Light-Dependent Interaction of phyb with SPA1 Promotes COP1 SPA1 Dissociation and Photomorphogenic Development in Arabidopsis. Mol. Plant. 8, INTRODUCTION Plants make use of light not only as the ultimate energy source but also as a crucial environmental signal that regulates multiple phases of plant growth and development (Deng and Quail, 1999; Neff et al., 2000; Jiao et al., 2007). Plants have evolved a set of sophisticated photoreceptors to perceive light signals, including the blue/ultraviolet-a ( nm) light-absorbing cryptochromes (CRY1 and CRY2) (Cashmore et al., 1999; Lin and Shalitin, 2003; Li and Yang, 2007) and phototropins (PHOT1 and PHOT2) (Briggs and Christie, 2002), UV-B ( nm) light-absorbing UVR8 (UV RESISTANCE LOCUS 8) (Rizzini et al., 2011), and the red ( nm) and farred ( nm) light-absorbing phytochromes (phya to phye) (Quail, 2002). It is known that cryptochromes and phytochromes act to regulate a variety of physiological processes, such as photomorphogenesis, entrainment of the circadian clock, stomatal development and movement, and floral initiation (Deng and Quail, 1999; Yang et al., 2000; Sang et al., 2005; Li and Yang, 2007; Liu et al., 2008; Kang et al., Published by the Molecular Plant Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS. Molecular Plant 8, , March 2015 ª The Author

2 2009; Wang et al., 2010; Yu et al., 2010; Li et al., 2011). Photomorphogenesis is one of the well-studied light responses, characterized by short hypocotyls and expanded cotyledons of plant seedlings. By contrast, dark-grown or etiolated seedlings exhibit a developmental response termed skotomorphogenesis, showing elongated hypocotyls with apical hook and closed cotyledons (McNellis and Deng, 1995). Phytochromes are the reversible photoswitches with a capacity to convert between two conformers. They are synthesized in their biologically inactive Pr form and mainly localized in cytoplasm (Quail, 2002). phyb is the major photoreceptor promoting photomorphogenesis under red light (McCormac et al., 1993; Reed et al., 1994; Quail, 2002), a process that requires the irradiation with red light to convert the Pr form of phyb to a biologically active Pfr form and the translocation of photoactivated phyb into the nucleus. But this process can be reversed by far-red light (Gil et al., 2000; Kircher et al., 2002). Likewise, phya is the predominant photoreceptor for sensing far-red light (McCormac et al., 1993; Quail et al., 1995). Under continuous far-red light, there is still a small fraction of phya in Pfr form, which can translocate into the nucleus and contribute to promote photomorphogenesis (Kendrick and Kronenberg, 1994; Kircher et al., 1999; Kim et al., 2000; Kircher et al., 2002). Notably, both photoactivation and the light-regulated nuclear accumulation of phytochromes are required for the most of their functions (Fankhauser and Chen, 2008; Li et al., 2011). Molecular genetic studies have clarified a considerable number of downstream events in phytochrome-mediated light signaling. Within the nucleus, activated Pfr forms of phytochromes interact physically with a group of transcription factors called phytochrome interacting factors (PIFs), triggering their phosphorylation, ubiquitination, and degradation, so as to regulate lightresponsive gene expression and modulate plant development (Bauer et al., 2004; Shen et al., 2005; Al-Sady et al., 2006; Shen et al., 2008; Bu et al., 2011; Leivar and Quail, 2011). Moreover, RING finger protein COP1 acts as the a E3 ligase responsible for the degradation of several photomorphogenesis-promoting transcription factors, such as HY5, HYH (HY5 HOMOLOG), LAF1 (LONG AFTER FAR-RED LIGHT 1), and HFR1 (LONG HYPOCOTYL IN FAR-RED 1), to represses de-etiolation under red/far-red light (Osterlund et al., 2000; Holm et al., 2002; Seo et al., 2003; Duek et al., 2004; Jang et al., 2005; Yang et al., 2005b; Lau and Deng, 2012). Phytochromes repress the activity of COP1 through mediating the COP1 nuclear exclusion (von Arnim et al., 1997; Osterlund and Deng, 1998; Subramanian et al., 2004). Interestingly, phytochromes are also the targets of COP1 for ubiquitination and degradation (Seo et al., 2004; Saijo et al., 2008; Jang et al., 2010). The ubiquitination of phyb by COP1 in vitro is facilitated by PIF proteins (Jang et al., 2010). Moreover, it has been recently demonstrated that PIL1 (PIF3- LIKE 1) is also involved in phyb signaling; the accumulation of PIL1 protein is regulated by direct phyb PIL1 and PIL1 COP1 interactions (Luo et al., 2014). SPA1 and three SPA1-related genes (SPA2 to SPA4) have been shown to act redundantly to repress seedling photomorphogenesis in Arabidopsis (Hoecker et al., 1998; Laubinger and Hoecker, 2003; Laubinger et al., 2004; Fittinghoff et al., 2006). The spa quadruple mutant seedlings exhibit a constitutively 468 Molecular Plant 8, , March 2015 ª The Author photomorphogenic phenotype, similar to the strong cop1 alleles (Deng et al., 1991; Hoecker and Quail, 2001; Laubinger and Hoecker, 2003; Laubinger et al., 2004; Zhu et al., 2008). All the SPA proteins are capable of direct interaction with COP1 and form a core complex with COP1, leading to the ubiquitination and degradation of the photomorphogenesispromoting factors, including HY5, LAF1 and HFR1, and repression of photomorphogenesis (Saijo et al., 2003; Seo et al., 2003; Yang et al., 2005a; Yang et al., 2005b). Moreover, it has been demonstrated that SPA and PIF proteins act synergistically to inhibit photomorphogenesis in the dark, and that COP1 SPA complex-mediated destabilization of HY5 is promoted by PIFs, with an interaction was detected between PI- F1and SPA1 (Xu et al., 2014). These findings suggest that SPAs are important regulators for integration with other components in light signaling. Recent studies have revealed an important role for SPA1 in cryptochrome signaling (Lian et al., 2011; Liu et al., 2011; Zuo et al., 2011). Specifically, blue-light-dependent CRY1 SPA1 interaction negatively regulates COP1 activity, at least in part, by promoting the dissociation of SPA1 from COP1, and thereby results in HY5 accumulation and photomorphogenesis (Lian et al., 2011; Liu et al., 2011). A more recent work revealed a new role for phyb to inhibit photomorphogenesis under far-red light by interacting with SPA1 (Zheng et al., 2013). The interaction between phyb and SPA1 promotes nuclear localization and accumulation of both proteins, resulting in enhanced rather than repressed activity of COP1 SPA1complex, and thus HY5 degradation in far-red light (Zheng et al., 2013). As it is well known that phyb is the major phytochrome responsible for photomorphogenesis under red light (Somers et al., 1991; Wagner et al., 1991; McCormac et al., 1993; Chen et al., 2004; Li et al., 2011), whether and how phyb SPA1 interaction is involved in phybmediated red light signaling remains unknown. Although SPA1 is shown to be in a complex that involves phya (Saijo et al., 2008), whether phya physically interacts with SPA1, and further regulates photomorphogenic development in far-red light, is still not clear. Here, we provide evidence showing that phyb directly interacts with SPA1 in a red-light-dependent manner in both yeast and plant cells. Furthermore, phyb SPA1 interaction facilitates the dissociation of SPA1 from COP1, resulting in stabilization of HY5 and thus promoting phyb-mediated photomorphogenesis in red light. Moreover, we verified the Pfr-form-dependent phya SPA1 interaction and demonstrated that SPAs are epistatic to PHYA in the regulation of photomorphogenesis in far-red light. These results, in junction with previous CRY SPA1 interaction studies (Lian et al., 2011; Liu et al., 2011; Zuo et al., 2011), suggest that SPA1 acts as a convergence point for the upstream blue, red, and far-red light signaling pathways activated by the cryptochrome and phytochrome photoreceptors. RESULTS Red-Light-Dependent phyb SPA1 Interaction SPA1 Specifically Interacts with the Photoactivated Pfr Form of phyb and phya in Yeast Cells It has been shown that SPA1 interacts directly with the C terminus of phyb and that such interaction antagonizes phya

3 Red-Light-Dependent phyb SPA1 Interaction Molecular Plant Figure 1. SPA1 Interacts Specifically with the Red-Light-Activated Pfr Form of phyb and phya in Yeast Cells. (A and C) Yeast two-hybrid prey constructs. All full-length and truncated forms of phyb (A) and phya (C) were fused with the GAL4-activation domain (AD). (B and D) Yeast two-hybrid assays showing redlight-dependent interactions between phyb (B) or phya (D) and SPA1. Full-length of SPA1 fused to a GAL4 binding domain was the bait construct. Yeast cells coexpressing the indicated combinations of constructs were grown on nonselective (SD-T-L) or selective media with 25 mm PCB (SD- T-L-H + PCB), in continuous red light (R, 3 mmol/ m 2 /s), far-red light (FR, 1 mmol/m 2 /s), or darkness (D). Yeast growth on the selective media indicated interactions. The pbridge and pgadt7 empty vectors were used as negative control. function to negatively regulate photomorphogenesis in far-red light (Zheng et al., 2013). However, whether phyb directly interacts with SPA1 in red light, and whether and how this interaction is involved in phyb-mediated red light signaling is largely unknown. To address these questions, we firstly utilized a yeast two-hybrid assay to test whether phyb interacts with SPA1 in red light, far-red light, and darkness, respectively, in the presence of the phytochrome chromophore, phycocyanobilin (PCB), which is required for phytochromes to convert between Pr and Pfr forms in yeast cells (Shimizu-Sato et al., 2002). We prepared a bait construct expressing the GAL4 binding domain fused to a full-length SPA1 protein, and prey constructs expressing the GAL4 activation domain fused to the full-length phyb, the N-terminal domain of phyb (phyb-n), and the C-terminal domain of phyb (phyb-c), respectively (Figure 1A). The results demonstrated that phyb interacted strongly with SPA1 in red light, but not in far-red light or darkness (Figure 1B). Moreover, phyb-n also interacted with SPA1 in a red-light-dependent manner, albeit not so strongly as the full-length phyb, while phyb-c constitutively interacted with SPA1 (Figure 1B). These results suggest that SPA1 specifically interacts with the biologically active Pfr form of phyb and that phyb may possess SPA1 binding sites within its N- and C-terminal domains. To investigate whether phya directly interacts with SPA1, we performed yeast two-hybrid assays by cotransforming the bait construct expressing GAL4 binding domain fused to full-length SPA1 protein together with prey constructs expressing the GAL4 activation domain fused to the full-length phya, the N-terminal domain of phya (phya-n), and the C-terminal domain of phya (phya-c) in yeast cells, respectively (Figure 1C). The results showed that phya interacted with SPA1 in red light but not in darkness or far-red light (Figure 1D). It has been shown that under the red light, the majority (88%) of phya photoconverts to the active Pfr state, while under continuous far-red light, 97% of phya is present in the inactive Pr state at a steady-state level (Mancinelli, 1994). The phya SPA1 interaction under far-red light was not detected in yeast two-hybrid assays (Figure 1D), likely due to the inactivation of most phya proteins in this condition. Deletion analysis further demonstrated that phya-c but not phya-n interacted with SPA1 (Figure 1D). SPA1 contains the N-terminal domain (SNT1) and the C- terminal domain (SCT1) (Lian et al., 2011). The results from b-galactosidase assays further showed that full-length SPA1, as well as the truncated form of SCT1, but not SNT1, interacted with phya-c (Supplemental Figure 1A and 1C). Moreover, phya-c can also interact with the C-terminal domains of the other three SPA proteins, SPA2, SPA3, and SPA4 (SCT2, SCT3, and SCT4) (Supplemental Figure 1B and 1C), suggesting that phytochromes may function to sequester all SPA quartet proteins. These data demonstrate that the interaction of phya with SPA1 is dependent on photoactivation of phya. Photoactivated phyb and phya Colocalize with SPA1 in Arabidopsis Protoplasts It has been shown that phyb and SPA1 constitutively colocalize in the nucleus in the dark, red, and far-red light, respectively (Zheng et al., 2013), which is puzzling, since only upon red light activation does phyb convert to Pfr conformer and is able to translocate into the nucleus to interact with its downstream factors such as PIFs, COP1, and PIL1 (Ni et al., 1999; Huq and Quail, 2002; Shimizu-Sato et al., 2002; Huq et al., 2004; Khanna et al., 2004; Oh et al., 2004; Leivar et al., 2008; Jang et al., 2010; Luo et al., 2014). We examined whether phyb and phya colocalize with SPA1 in Arabidopsis protoplasts in a light-dependent manner. N-terminal cyan fluorescent protein (CFP)-tagged SPA1 (CFP-SPA1) was transiently expressed in tobacco leaf epidermal cells together with the C-terminal yellow fluorescent protein (YFP)-tagged full-length and truncated forms of phyb and phya (phyb-yfp, phyb-n- YFP, phyb-c-yfp, phya-yfp, phya-n-yfp, and phya-c-yfp), respectively. The results showed that phyb colocalized with SPA1 both in darkness and red light, while phya colocalized Molecular Plant 8, , March 2015 ª The Author

4 Red-Light-Dependent phyb SPA1 Interaction Figure 2. phyb and phya Colocalize with SPA1 to the Nucleus in Arabidopsis Protoplasts upon Red and Red/Far-red Light, Respectively. (A) phyb translocalized into nucleus and colocalized with SPA1 under red light (20 mmol/m 2 /s) but not in darkness. Bars, 5 mm. The inner panels are enlarged portions of nucleus in their outer panels, respectively. (B) phya translocalized into nucleus and colocalized with SPA1 under far-red light (3 mmol/m 2 / s) and red light (20 mmol/m 2 /s) but not in darkness. Bars, 5 mm. The inner panels are enlarged portions of nucleus in their outer panels. To further investigate the possible red light dependency for the colocalization of phyb and SPA1, we transiently expressed the related CFP- and YFP-tagged proteins in Arabidopsis protoplasts. Confocal microscopy demonstrated that in darkness, the majority of phyb was distributed in cytoplasm while SPA1 was constitutively localized to nucleus. Upon red light irradiation, phyb translocated into nucleus and formed large and bright nuclear bodies (NBs). Red light-induced nucleus-distributed phyb colocalized with SPA1 in the same NBs, in contrast to the differential subcellular compartmentalization of phyb and SPA1 in darkness (Figure 2A). Likewise, colocalization assays in Arabidopsis protoplasts demonstrated that both far-red light and red light induced phya to translocate into nucleus and form NBs. As anticipated, the nuclear-localized phya also colocalized with SPA1 in the same NBs (Figure 2B). These data indicate that only photoactivated phyb or phya colocalizes with SPA1 in the nucleus in Arabidopsis. with SPA1 in darkness and far-red light (Supplemental Figure 2). Moreover, we found that phyb-n and phyb-c, as well as phya- N and phya-c colocalized with SPA1 in red light and far-red light, respectively (Supplemental Figure 2). Therefore, it appears that the colocalizations of SPA1 with phya and phyb are likely not light dependent in tobacco cells. Nevertheless, these data suggest that phyb and phya may interact with SPA1 through their both N-terminal and C-terminal domains. 470 Molecular Plant 8, , March 2015 ª The Author phyb Interacts with SPA1 in a Red- Light-Dependent Manner in Arabidopsis Given the red-light- and far-red-lightdependent phyb and phya SPA1 interactions in yeast cells and colocalization in Arabidopsis protoplasts, we further tested the possibility that photoactivated phyb and phya interact directly with SPA1 in plant cells in a bimolecular fluorescence complementation (BiFC) system. We fused phyb and phya to the N-terminal fragment of YFP (nyfp) and fused SPA1 to the C-terminal fragment of YFP (cyfp). Cotransformation of nyfp-phyb and SPA1-cYFP in tobacco leaf epidermal cells produced robust YFP fluorescence in nuclear speckles both in darkness and red light (Figure 3A), and cotransformation of nyfp-phya and SPA1-cYFP produced YFP fluorescence in nuclear speckles both in darkness and far-red light (Supplemental Figure 3). Although BiFC assays performed in

5 Red-Light-Dependent phyb SPA1 Interaction Molecular Plant Figure 3. phyb Interacts with SPA1 In Vivo and Red Light Enhances the phyb SPA1 Interaction in Arabidopsis. (A) BiFC assays of in vivo interactions of phyb SPA1 in red light (20 mmol/m 2 /s) and darkness. The indicated constructs were cotransformed into tobacco leaf epidermal cells. The images show overlays of fluorescence and light views. The right-hand panels show the nuclei labeled by red boxes in the adjacent panels at high magnification, respectively. Bars, 20 mm. (B) Coimmunoprecipitation assay showing that phyb interacts with SPA1 in Arabidopsis in a red-light-dependent manner. Seedlings overexpressing Myc-SPA1 in spa1 mutant background were grown in darkness for 5 d and then exposed to red light (20 mmol/m 2 /s) for 0, 10, 30, or 60 min. The endogenous phyb proteins were immunoprecipitated with anti-phyb antibody and the immunoblot was probed with anti-myc and anti-phyb antibody. tobacco leaves displayed no light dependency for phyb SPA1 and phya SPA1 interactions, the results suggest that phyb and phya interact with SPA1 directly in tobacco epidermal cells. To further verify the red-light-dependent association between phyb and SPA1 in Arabidopsis, we performed coimmunoprecipitation assay (CoIP) using transgenic spa1 mutant seedlings expressing Myc-SPA1 fusion protein (Myc-SPA1/spa1) (Yang and Wang, 2006). As shown in Figure 3B, Myc-SPA1 was hardly coimmunoprecipitated by the endogenous phyb in a darknessadapted control. In contrast, the coimmunoprecipitated Myc- SPA1 protein dramatically increased as the seedlings were exposed to red light for 10, 30, and 60 min. These results demonstrate that phyb interacts with SPA1 in a red-light-dependent manner in Arabidopsis. SPAs Are Genetically Epistatic to PHYB and PHYA in Regulating Photomorphogenesis under Red and Far-Red Light, Respectively To understand the roles of the red-light-dependent phyb SPA1 interaction in Arabidopsis, we investigated the genetic relationship of PHYB and SPAs. To do this, we generated the phyb spa1 spa2 triple, phyb spa1 spa2 spa4 quadruple, and phyb spa1 spa2 spa3 spa4 quintuple mutants, respectively, by genetic crossing. As shown in Figure 4A, phyb spa1 spa2 triple mutant displayed a slightly, while statistically significantly (Turkey s least significant difference [LSD] test, P % 0.01), shortened hypocotyl phenotype compared with phyb mutant under red light. Noticeably, phyb spa1 spa2 spa4 quadruple mutant showed stronger responsiveness to red light than phyb spa1 spa2 triple mutant, characterized by more shortened hypocotyls and expanded cotyledons. Interestingly, phyb spa1 spa2 spa3 spa4 quintuple mutant showed even stronger responsiveness to red light than phyb spa1 spa2 spa4 quadruple mutant, displaying significant shorter hypocotyls than phyb and fully opened cotyledons (Figure 4A and 4B). These results, in conjunction with the above demonstration that photoactivated phyb physically interacts with SPA1, suggest that SPAs act genetically downstream of PHYB to regulate photomorphogenesis under red light. The observation that the hypocotyls of phyb spa1 spa2 spa3 spa4 are statistically longer than those of spa1 spa2 spa3 spa4 can be due to many other photomorphogenesis-inhibiting factors downstream of PHYB, such as PIFs (Ni et al., 1999; Huq and Quail, 2002; Shimizu-Sato et al., 2002; Huq et al., 2004; Khanna et al., 2004; Oh et al., 2004; Leivar et al., 2008), COP1 (Jang et al., 2010) and PIL1 (Luo et al., 2014) (Figure 4A and 4B). It is likely that the enhancement of SPA-independent pathways mediated by these photomorphogenesis inhibitors leads to the promotion of hypocotyl elongation in phyb spa1 spa2 spa3 spa4. To examine whether PHYA and SPAs genetically interact, we constructed phya spa1 spa2 triple, phya spa1 spa2 spa4 quadruple, and phya spa1 spa2 spa3 spa4 quintuple mutants. Notably, the phya spa1 spa2 spa3 spa4 quintuple mutant exhibited similar phenotypes to spa1 spa2 spa3 spa4 mutant under far-red light, with a decrease by about 85% in hypocotyl length compared with phya. But the photomorphogenic phenotypes of phya spa1 spa2 triple mutant and phya spa1 spa2 spa4 quadruple mutant resembled that of phya mutant. Again, the etiolation phenotypes caused by PHYA mutation could not be rescued until all of the four SPA genes were mutated (Turkey s LSD test, P % 0.01) (Figure 4C and 4D). These results indicate that the SPAs are also epistatic to PHYA in the regulation of seedling photomorphogenesis under far-red light. These results, combined with the above demonstration that photoactivated phya physically interacts with SPA1, suggest that the four SPAs may act additively in mediating phya signaling. Molecular Plant 8, , March 2015 ª The Author

6 Red-Light-Dependent phyb SPA1 Interaction Figure 4. SPAs Are Epistatic to PHYB and PHYA to Regulate Photomorphogenesis under Red Light and Far-Red Light, Respectively. (A and C) Seedlings were grown on MS medium under red light (7 mmol/m 2 /s) (A) and far-red light (0.5 mmol/m 2 /s) (C) for 5 d before hypocotyl lengths were measured. Wild-type (WT), phyb, spa1 spa2 (spa12), phyb spa1 spa2 (phyb spa12), spa1 spa2 spa4 (spa124), phyb spa1 spa2 spa4 (phyb spa124), spa1 spa2 spa3 spa4 (spa1234), phyb spa1 spa2 spa3 spa4 (phyb spa1234), phya, phya spa1 spa2 (phya spa12), phya spa1 spa2 spa4 (phya spa124), phya spa1 spa2 spa3 spa4 (phya spa1234). Bars, 5 mm. (B and D) Quantification data of (A) and (C). Numbers indicate the hypocotyl lengths of the indicated plants. Data are means ± SD (n = 30). a to g indicate statistically significant differences between means for hypocotyl lengths of the indicated genotypes, as determined by Tukey s LSD test (P % 0.01). SPAs Act Downstream of phyb and phya to Regulate HY5 Accumulation in Red and Far-Red Light, Respectively It has been demonstrated that HY5 protein accumulates to high levels in spa1-3 mutant but decreases to low levels in SPA1 overexpressing plants (Saijo et al., 2003; Yang and Wang, 2006) and that SPA1 suppresses HY5 protein accumulation by facilitating the COP1-dependent ubiquitination and degradation of HY5 under far-red light (Saijo et al., 2003; Yang and Wang, 2006). On the basis of these studies, we reasoned that the enhanced photomorphogenic phenotype of spa1 spa2 spa3 spa4 and phyb spa1 spa2 spa3 spa4 might result from enriched abundance of HY5. To test this possibility, we examined protein levels of HY5 in seedlings grown under red light. The results demonstrated that the phyb spa1 spa2 spa3 spa4 quintuple mutant accumulated much higher levels of HY5 proteins than the phyb mutant under red light (Supplemental Figure 4A), indicating that the decreased HY5 accumulation caused by phyb mutation was largely rescued in the phyb spa1 spa2 spa3 spa4 quintuple mutant. The observation that HY5 protein accumulate less in phyb spa1 spa2 spa3 spa4 quintuple mutant than in spa1 spa2 spa3 spa4 mutant likely resulted from the presence of other factors such as COP1, which also acts downstream of PHYB and functions to promote HY5 degradation (Saijo et al., 2003; Jang et al., 2010). We also analyzed HY5 protein accumulation in phya single, spa1 spa2 spa3 spa4 quadruple, and phya spa1 spa2 spa3 spa4 quintuple mutants grown in far-red light, respectively, and found that HY5 accumulated to similar high levels in spa1 spa2 spa3 spa4 quadruple and phya spa1 spa2 spa3 spa4 quintuple mutants, but very low levels in phya mutant (Supplemental Figure 4B). Taken together, these results suggest that spa mutations enhance phyb- and phya-mediated stabilization of HY5 protein, confirming that SPAs act downstream of phyb and phya to repress photomorphogenesis in red and far-red light, respectively. 472 Molecular Plant 8, , March 2015 ª The Author The phyb SPA1 Interaction Suppresses COP1 SPA1 Interaction in Response to Red Light Based on the demonstrations that phyb physically interacts with SPA1 and that SPA1 is involved in phyb-mediated HY5 stabilization (Figures 1B, 2A, and 3; Supplemental Figure 4A), we then asked whether phyb interfere with the association of SPA1 with COP1 and restrain COP1 activity, so as to attenuate the degradation of HY5. To test this possibility, we performed CoIP assays using Myc-SPA1/spa1 (Yang and Wang, 2006) and Myc-SPA1/phyB seedlings grown in darkness for 5 d and treated with MG132 for 60 min before exposure to the red light for 0, 20, and 60 min, respectively. The results demonstrated that in the presence of phyb, the COP1 SPA1 interaction was reduced progressively along with the prolonged exposure to the red light at a fluence rates of 70 mmol/m 2 /s or 10 mmol/m 2 /s, with a dramatical reduce in the amount of COP1 coimmunoprecipitated with Myc-SPA1 in the red light compared with that in the dark (Figure 5A and Supplemental Figure 5A). In contrast, in the phyb mutant background, red light hardly inhibited the COP1 SPA1 interaction (Figure 5B and Supplemental Figure 5B). These data demonstrate that phyb promotes the dissociation of SPA1 from COP1 in response to red light in Arabidopsis. We further explored the effect of phyb on the association of SPA1 with COP1 in response to red light in yeast three-hybrid assays by using a bait construct expressing COP1, a prey construct expressing SPA1, and a full-length phyb bridge protein (Figure 6A). The COP1 SPA1 interaction in the presence of phyb protein decreased by about 20% and 80% after exposure to the red light for 1 h and 9 h, respectively, compared with dark treatment, when the yeast cells were grown in a synthetic dropout-trp-leu-met (SD-T-L-M) medium supplemented with PCB (Figure 6B). By contrast, in the absence of phyb, the COP1 SPA1 interaction in red light was as strong as that in the dark (Figure 6B). These data

7 Red-Light-Dependent phyb SPA1 Interaction Molecular Plant photoactivated phyb and PIFs (Ni et al., 1999; Huq and Quail, 2002; Shimizu-Sato et al., 2002; Huq et al., 2004; Khanna et al., 2004; Oh et al., 2004; Leivar et al., 2008), and the phybinduced nuclear exclusion of COP1 (von Arnim et al., 1997; Osterlund and Deng, 1998; Subramanian et al., 2004). Our study reveals that the interaction between phyb and SPA1 is also implicated in phyb-mediated red light signaling, supported by the following demonstrations: (1) phyb interacts directly with SPA1 in a red-light-dependent manner in yeast cells, and this interaction is likely mediated through both the N- and C-terminal domains of phyb (Figure 1A and 1B); (2) phyb colocalizes with SPA1 in the same NBs of Arabidopsis protoplasts upon red light irradiation (Figure 2A); and (3) phyb interacts directly with SPA1 in a red-light-dependent manner in plant cells (Figure 3). Our failure to detect phyb SPA1 or phya-spa1 interaction in our yeast two-hybrid assays under far-red light might be due to the insufficient amount of Pfr form of phyb or phya proteins (Figure 1). Since phyb SPA1 interaction is detected by CoIP in far-red light (Zheng et al., 2013), it is possible that the CoIP assay is more sensitive than the yeast two-hybrid assay. Figure 5. phyb Suppresses COP1 SPA1 Interaction in Response to Red Light In Vivo. (A) Transgenic seedlings overexpressing Myc-SPA1 in spa1 mutant background (phyb present) were grown in continuous darkness for 5 d and then exposed to red light (70 mmol/m 2 /s) for the indicated time. COP1, pulled down by immunoprecipitated Myc-SPA1, was progressively reduced with prolonged exposure to red light. The membrane was stripped and reprobed with anti-phyb antibody to detect the phyb protein levels. Relative band intensities were normalized for each panel and shown under each panel. (B) 5 d dark-grown transgenic seedlings overexpressing Myc-SPA1 in phyb mutant background (phyb absent) were exposed to red light (70 mmol/m 2 /s) for the indicated time. The abundance of COP1 coimmunoprecipitated by Myc-SPA1 remained similar. Relative band intensities were normalized and are shown under each panel. demonstrate that COP1 SPA1 interaction is inhibited by photoactivated phyb in yeast cells. We further explored whether the inhibition of COP1 SPA1 interaction resulted from red-light-induced interaction of phyb with SPA1. We prepared a bait construct expressing the coiled-coilcontaining domain of COP1 (CC1) (Figure 6A), which interacted with SPA1 but not with phyb in yeast two-hybrid assays (Figure 6C). In the presence of phyb, CC1-SPA1 interaction was suppressed by 58% and 62% when the yeast cells were exposed to the red light for 2 h and 7 h, respectively, compared with that in the dark (Figure 6A and 6D). However, when there was no phyb, CC1 SPA1 interaction was largely not affected by red light exposure (Figure 6A and 6D). These results suggest that the red-light-triggered interaction between phyb and SPA1 promotes the disassociation of COP1 from SPA1. DISCUSSION The Signaling Mechanism of phyb Involves Red-Light- Dependent Interaction with SPA1 It has been demonstrated that phyb signaling in red lightmediatedphotomorphogenesis involves the interactions between It is well established that phyb is the primary photoreceptor that mediates the red light inhibition of hypocotyl elongation (Somers et al., 1991; Wagner et al., 1991; Wagner et al., 1996). Interestingly, some studies have shown that phyb promotes hypocotyl elongation in far-red light (Wagner et al., 1991; Wagner et al., 1996; Hennig et al., 2001; Zheng et al., 2013). A recent work has demonstrated that phyb interacts with SPA1 to promote hypocotyl elongation under far-red light (Zheng et al., 2013). However, red-light-dependent interaction of phyb with SPA1, and the significance of such a interaction concerning the mode of action of phyb in promoting photomorphogenesis under red light have not been revealed in this work. These questions are important because phyb is the pivotal photoreceptor in mediating red light signaling (Somers et al., 1991; Wagner et al., 1991; McCormac et al., 1993). Our work provides answers to these questions by showing that phyb SPA1 interaction is red-lightdependent and SPAs act genetically downstream of PHYB to regulate photomorphogenesis under red light (Figure 4). Red-Light-Dependent Interaction of phyb and SPA1 Promotes the Dissociation of SPA1 from COP1 The present study demonstrates that phyb induces COP1 SPA1 dissociation in response to red light (Figures 5 and 6; Supplemental Figure 5). It is known that COP1 translocates from nucleus to cytoplasm upon light exposure and the nuclear exclusion of COP1 is rather slow (about 24 h) (von Arnim et al., 1997; Osterlund and Deng, 1998; Lau and Deng, 2012) and that phyb is responsible for mediating red-light-induced redistribution of COP1 (Osterlund and Deng, 1998). We showed that in the presence of phyb, the interaction of COP1 SPA1 is reduced by 40% upon 20 min red light irradiation (Figure 5A). Considering the rapid reduction of the COP1 SPA1 interaction by photoactivated phyb, we propose that COP1 nucleus depletion normally triggered by a relatively longer time of light exposure may contribute little, if anything, to the dissociation of COP1 from SPA1 in plant cells. Moreover, since COP1 was tagged to a nuclear localization signal sequence, it is very likely that COP1 constitutively localized to the nucleus in yeast cells. The results from our yeast three-hybrid assays further support Molecular Plant 8, , March 2015 ª The Author

8 Red-Light-Dependent phyb SPA1 Interaction Figure 6. Red-Light-Activated phyb Inhibits the COP1 SPA1 Interaction in Yeast Cells. (A) Yeast three-hybrid constructs expressing both the bait and the bridge proteins. COP1 or CC1 were fused with GAL4 binding domain (BD), respectively. P Met25, inducible promoter driving the expression of the bridge protein phyb. COP1 or CC1 fused to BD domain without bridge proteins were used as negative controls in yeast three-hybrid assays. CC1 fused to BD was also the bait construct of yeast two-hybrid assays. (B) Quantitative yeast three-hybrid analyses show that red-light-activated phyb repressed the interaction of COP1 SPA1. Yeast cells were cultured in SD-T-L-M medium with 25 mm PCB in darkness for 18 h (Dark), in darkness for 17 h and then exposed to red light (40 mmol/m 2 /s) for 1 h (Red 1 h) or in darkness for 9 h and then exposed to red light for 9 h (Red 9 h). The interaction values (Miller units) in darkness were set to 100%, and the relative interactions under red light were expressed as the percentage of the dark value. Data are mean ± SD (n = 3). (C) Yeast two-hybrid assays show that CC1 did not interact with phyb in either red light (R, 3 mmol/ m 2 /s) or darkness (D) in yeast cells. (D) Quantitative yeast three-hybrid analyses showing the red-light-dependent phyb inhibition of the CC1 SPA1 interaction. In the presence of phyb, exposure to red light (40 mmol/m 2 /s) for 2 h (Red 2 h) or 7 h (Red 7 h) resulted in a dramatic reduction of CC1 SPA1 interaction compared with that in darkness (Dark). In the absence of phyb, the CC1 SPA1 interaction was not affected by indicated red light treatment. that phyb represses COP1 SPA1 interaction in red light by a mechanism other than the mediation of COP1 redistribution (Figure 6A and 6B). Previous studies have shown that photoexcited CRY1 promotes the dissociation of COP1 from SPA1, which is mediated through both CRY1 SPA1 and CRY1 COP1 interactions (Lian et al., 2011; Liu et al., 2011). Given that phyb interacts with COP1 (Jang et al., 2010) and SPA1 (this study), we propose that both phyb SPA1 and phyb COP1 interactions might be involved in the inhibition of COP1 SPA1 association in red light. Our data in yeast cells demonstrated that photoactivated phyb also inhibited the interaction of SPA1 with CC1, which could not interact with phyb. These results verified that red-light-induced interaction of phyb with SPA1 is involved in the inhibition of COP1 SPA1 association (Figure 6A, 6C, and 6D). It is worth stressing that the phyb SPA1 interaction in far-red light and red light results in opposing physiological consequences through distinct mechanisms. Specifically, phyb interacts with SPA1 to facilitate the nuclear accumulation of SPA1 and COP1 in far-red light, thus repressing HY5 stabilization and photomorphogenesis (Zheng et al., 2013). In contrast, red-light-induced interaction of phyb with SPA1 results in the dissociation of SPA1 from COP1, which in turn represses COP1 activity, and increases the accumulation of HY5 proteins, leading to photomorphogenesis in red light (Figures 4, 5, and 6; Supplemental Figures 4 and 5). Therefore, our findings provide new insights into the biological significance of phyb SPA1 interaction and further expand the phyb signaling network in red light enhancement of photomorphogenic development. 474 Molecular Plant 8, , March 2015 ª The Author The Far-Red Light Signaling Mechanism of phya May Involve Light-Dependent Interaction with SPA1 It is worth noting that the spa1 mutant was obtained through screening for extragenic mutations that can suppress the elongated hypocotyl phenotype of phya-105, a weak phya mutant allele (Hoecker et al., 1998). It has been demonstrated that phya promotes SPA1 expression and SPA1 protein accumulation (Hoecker et al., 1999; Laubinger et al., 2006). Importantly, SPA1 is shown to regulate CRY signaling in blue light and phyb signaling in red light through blue-light- and redlight-dependent interactions with CRY and phyb, respectively (Lian et al., 2011; Liu et al., 2011; Zuo et al., 2011; this work). These above mentioned results strongly indicate that SPA1 is likely involved in phya signaling through direct interaction with phya. Indeed, our data demonstrate that phya interacts with SPA1 in a Pfr form-dependent manner in both yeast cells (Figure 1C and 1D) and Arabidopsis protoplasts (Figure 2B) and that SPAs act downstream of phya to repress HY5 protein accumulation and photomorphogenesis in far-red light (Figure 4C and 4D; Supplemental Figure 4B). The yeast two-hybrid assays indicate that both the N- and C-terminal domains of phyb interact with SPA1, whereas the C-terminal domain of phya, but not the N-terminal domain, interacts with SPA1 (Figure 1). Considering that both phyb N- and C-terminal domains interact with SPA1 in both yeast cells (Figure 1A and 1B) and plant cells (Supplemental Figure 2A), and both phya N- and C-terminal domains colocalize with SPA1 to the same nuclear speckles in tobacco epidermal cells (Supplemental Figure 2B), we speculate that like phyb, phya may also interact

9 Red-Light-Dependent phyb SPA1 Interaction Molecular Plant COP1 has been demonstrated to strongly interact with SPA1 in darkness, and that blue, red, and far-red light all dramatically inhibit the association of COP1 with SPA1 (Saijo et al., 2003). To date, CRY1 and phyb have been shown to be responsible for blue- and red-light-induced inhibition of COP1 SPA1 interaction, at least partly through blue- and red-light-triggered SPA1 CRY1 and SPA1 phyb interactions, respectively (Lian et al., 2011; Liu et al., 2011; this study). Considering that phya interacts with SPA1 (Figures 1C, 1D, and 2B; Supplemental Figures 1, 2B, and 3) and COP1 (Seo et al., 2004; Viczián et al., 2012), respectively, it is possible that phya may mediate farred-light-induced disassociation of COP1 from SPA1 through phya SPA1 interaction. Examination of this possibility will make it possible to establish SPA1 as a direct downstream factor of phya in mediating far-red light signaling. METHODS Figure 7. Red-Light-Activated phyb Interacts with SPA1 to Suppress the Formation and Activity of COP1 SPA1 Complex. (A) In darkness, the Pr form of phyb localizes to the cytoplasm while SPA1 interacts with COP1 in nucleus and facilitates the COP1-mediated ubiquitination and degradation of photomorphogenesis-promoting transcription factor HY5, inhibiting target gene expression and promoting skotomorphogenesis. (B) Upon red light irradiation, phyb converts to its biologically active Pfr form and translocates into nucleus while COP1 is exported to cytoplasm slowly. Pfr-phyB interacts with SPA1 in nucleus and impairs the interaction of SPA1 with COP1, alleviating the degradation of HY5 and initiating the photomorphogenic gene expression and photomorphogenesis. with SPA1 through both N- and C-terminal domains. The failure to detect the interaction of phya N-terminus with SPA1 in yeast twohybrid assays might result from the improper expression and/or conformation change of phya-n in yeast cells, which will need further investigations. A Model Describing the Actions of Red-Light-Activated phyb and SPA1 Interaction in Regulating Photomorphogenesis In this work, we demonstrated that other than PIFs (Ni et al., 1999; Huq and Quail, 2002; Shimizu-Sato et al., 2002; Huq et al., 2004; Khanna et al., 2004; Oh et al., 2004; Leivar et al., 2008), COP1 (Jang et al., 2010) and PIL1 (Luo et al., 2014), SPA1 acts as a new direct downstream component of phyb in mediating red light signaling. Based on previous studies and our findings, we propose a model whereby in darkness, the biologically inactive phyb (Pr form) resides in the cytoplasm, while nuclear-localized SPA1 forms an inhibitory complex with COP1 in the nucleus. Consequently, the positive regulator of photomorphogenesis, HY5, is ubiquitinated by the COP1 SPA1 complex and degraded by 26S proteasome, resulting in seedling skotomorphogenesis. Upon red light irradiation, photoactivated phyb (Pfr form) translocates into the nucleus and interacts with SPA1 directly in a red-light-dependent manner, consecutively resulting in the disassociation of COP1-SPA1 complex, repressing the ubiquitination and degradation of HY5, and promoting photomorphogenic development. In a long run, phyb may act to promote the depletion of COP1 from nucleus (Figure 7). Plant Growth and Light Sources The Arabidopsis materials and growth conditions were as described previously (Yang et al., 2001; Liu et al., 2008; Wang et al., 2010). Arabidopsis thaliana Columbia accession (Col) was used as the wild-type. Seeds were grown on Murashige and Skoog (MS) basal medium (Sigma-Aldrich) containing 2% sucrose after being sterilized with 20% bleaching water and then incubated at 4 C for 3 d. To facilitate germination of the seeds, plates were exposed to white light (150 mmol/m 2 /s) for 12 h before being transferred to continuous darkness, red light, or far-red light at indicated fluence rates at 21 C, respectively. An Li250 quantum photometer (Li-Cor, Lincoln, NE) was used to measure the fluence rates of light. Tobacco plants (Nicotiana benthamiana) were grown in a greenhouse under continuous white light. Construction of Mutants The construction of mutants of SPA genes such as spa1 spa2, spa1 spa2 spa4, spa1 spa2 spa3 spa4, and Myc-SPA1/spa1 has been described previously (Yang and Wang, 2006; Kang et al., 2009; Lian et al., 2011). phya- 211, phyb-9, cop1-4, and hy5 have been described in previous works (Yang et al., 2001; Wang et al., 2010; Jia et al., 2013). The following triple, quadruple, and quintuple mutants for genetic analyses were generated by sexual crosses: phyb spa1 spa2, phyb spa1 spa2 spa4, phyb spa1 spa2 spa3 spa4, phya spa1 spa2, phya spa1 spa2 spa4, and phya spa1 spa2 spa3 spa4. Likewise, Myc-SPA1/phyB plants were obtained by genetic crossing Myc-SPA1/spa1 with phyb mutant. Yeast Two-Hybrid Assays For the GAL4 yeast two-hybrid assays, the bait and prey vectors were cotransformed into yeast strain AH109 via the lithium acetate transformation procedure, as described in the yeast protocols handbook (Clontech Laboratories) and interaction assays with PCB were performed as described by Shimizu-Sato et al., The full-length of SPA1 was cloned into pbridge. Full-length phyb, the N-terminal domain of phyb (1 640 amino acids) and the C-terminal domain of phyb ( amino acids) (Jang et al., 2010), as well as full length phya, the N-terminal domain of phya (1 600 amino acids) and the C-terminal domains of phya ( amino acids) (Seo et al., 2004) were amplified by PCR and cloned into pgadt7 Vector. Three days after transformation, six yeast clones grown on synthetic dropout-trp-leu medium (SD-T-L) with even growth were chosen and incubated in liquid SD-T-L medium adding 25 mm PCB (Scientific Frontier) for 3 h and the culture was inoculated into SD-T-L-His medium with 25 mm PCB (SD-T-L-H + PCB) in continuous red light (3 mmol/m 2 /s), far-red light (1 mmol/m 2 /s), or darkness. The LexA yeast two-hybrid assays and the calculation of relative b-galactosidase activities were performed as described previously (Yang et al., 2001; Sang et al., 2005; Lian et al., 2011). The plexa bait constructs expressing SPA1, SNT1, SCT1, SCT2, SCT3, SCT4 were made in previous work (Lian Molecular Plant 8, , March 2015 ª The Author

10 et al., 2011), while the C-terminal domain of phya and the N-terminal domain of phya (Seo et al., 2004) were amplified by PCR and cloned into pjg4-5. Each assay was performed in triplicate and one of the representative assays is shown. Yeast Three-Hybrid Assays A yeast three-hybrid assay was performed as described previously (Lian et al. 2011) and interaction assays with PCB were performed as described by Shimizu-Sato et al., 2002 with minor modifications. Transformed colonies were selected on SD-T-L medium. Three independent clones with three respective replicates were used in each performance. Cell cultures in liquid SD-T-L-M medium with 25 mm PCB were placed in darkness for at least 9 h before being treated with red light (40 mmol/m 2 /s) for the indicated time or further incubated in darkness. All the cell cultures were incubated until the OD 600 was 0.8 with the conditional expression of the bridge proteins. Calculations of relative b-galactosidase activities were performed as described previously (Yang et al., 2001). At least three independent experiments were performed and the result of one representative is shown. Transient Transformation in Plant Cells Arabidopsis mesophyll protoplast preparation and transfection follow the protocols described previously (Sheen, 2001). The vectors for analyses of colocalization are the same as in the previous study (Liu et al., 2008; Lian et al., 2011). The vectors of BiFC (pxy104 and pxy106) were described previously (Yu et al., 2008). Full-length coding regions of phyb and phya were cloned into the pxy104-cyfp vector while SPA1 was coded into the pxy106-nyfp vector. Agrobacterium cells containing either constructs for colocalization or BiFC expression vectors were washed and resuspended in liquid MS medium and infiltrated into tobacco leaves from the lower epidermis. After incubation in darkness for 36 h and exposure to the light as indicated, the tobacco leaf cells were used to analyze the expression of various fluorescent proteins by confocal microscopy (Leica TCS SP5Ⅱ). CoIPs and Western Blot Analysis CoIPs were performed as reported before (Lian et al., 2011), with minor modifications. Plant materials were ground in liquid nitrogen and then the total proteins were extracted with lysis buffer (50 mm Tris-Cl, ph 7.5, 150 mm NaCl, 1 mm EDTA, 10% glycerol, 1% Triton X-100, 1 mm PefaBloc [Sigma-Aldrich], 13 protease inhibitor cocktail [Roche], and 50 mm MG132 [Merck]). For the phyb/myc-spa1 CoIP study, about 1.5 mg of total protein was incubated with 5 ml of anti-phyb antiserum bound to protein A-Sepharose beads (GE Healthcare) for 1 h. For the Myc-SPA1/COP1 Co-IP study, 40 ml of anti-myc agarose beads (Sigma-Aldrich) was added into the lysates from corresponding seedlings for 30 min. The beads were washed four times with lysis buffer and eluted by boiling with 23 SDS sample buffer for 5 min and then separated in 10% SDS-PAGE gel. Anti-Myc (Santa Cruz, 1:200 dilution), anti-phyb (homemade, 1:200 dilution) (Luo et al., 2014) and anti-cop1 (homemade, 1:500 dilution) (Lian et al., 2011) antibodies were used to detect Myc-SPA1, endogenous phyb, and COP1, respectively. For the detection of HY5 protein accumulation, approximately 30 mg of total proteins was extracted from 3 d red light- or far-red-light-grown seedlings. Samples were analyzed by fractionating in 12% SDS-PAGE gel and immunoblotted with anti-hy5 (homemade, 1:400 dilution) (Jia et al., 2013), anti-actin (Abmart, China, 1:2000 dilution) antibodies. SUPPLEMENTAL INFORMATION Supplemental Information is available at Molecular Plant Online. FUNDING This work was supported by grants from the National Natural Science Foundation of China ( to H.L.L, and , and to H.Q.Y), China Innovative Research Team, Ministry of Education, Shanghai Graduate Education and Innovation Program (Horticulture) and 111 Project (B14016). Red-Light-Dependent phyb SPA1 Interaction ACKNOWLEDGMENTS We thank the ABRC for Arabidopsis mutant seeds and Fangyuan Zhang for drawing the model in Figure 7. No conflict of interest declared. Received: October 13, 2014 Revised: November 13, 2014 Accepted: November 14, 2014 Published: December 30, 2014 REFERENCES Al-Sady, B., Ni, W., Kircher, S., Schäfer, E., and Quail, P.H. (2006). Photoactivated phytochrome induces rapid PIF3 phosphorylation prior to proteasome-mediated degradation. Mol. Cell 23: Bauer, D., Viczián, A., Kircher, S., Nobis, T., Nitschke, R., Kunkel, T., Panigrahi, K.C., Ádám, É., Fejes, E., and Schäfer, E. (2004). Constitutive photomorphogenesis 1 and multiple photoreceptors control degradation of phytochrome interacting factor 3, a transcription factor required for light signaling in Arabidopsis. Plant Cell 16: Briggs, W.R., and Christie, J.M. (2002). Phototropins 1 and 2: versatile plant blue-light receptors. Trends Plant Sci. 7: Bu, Q., Zhu, L., Dennis, M.D., Yu, L., Lu, S.X., Person, M.D., Tobin, E.M., Browning, K.S., and Huq, E. (2011). Phosphorylation by CK2 enhances the rapid light-induced degradation of phytochrome interacting factor 1 in Arabidopsis. J. Biol. Chem. 286: Cashmore, A.R., Jarillo, J.A., Wu, Y.J., and Liu, D. (1999). Cryptochromes: blue light receptors for plants and animals. Science 284: Chen, M., Chory, J., and Fankhauser, C. (2004). Light signal transduction in higher plants. Annu. Rev. Genet. 38: Deng, X.W., and Quail, P.H. (1999). Signalling in light-controlled development. Semin. Cell Dev. Biol. 10: Deng, X.W., Caspar, T., and Quail, P.H. (1991). cop1: a regulatory locus involved in light-controlled development and gene expression in Arabidopsis. Genes Dev. 5: Duek, P.D., Elmer, M.V., van Oosten, V.R., and Fankhauser, C. (2004). The degradation of HFR1, a putative bhlh class transcription factor involved in light signaling, is regulated by phosphorylation and requires COP1. Curr. Biol. 14: Fankhauser, C., and Chen, M. (2008). Transposing phytochrome into the nucleus. Trends Plant Sci. 13: Fittinghoff, K., Laubinger, S., Nixdorf, M., Fackendahl, P., Baumgardt, R.L., Batschauer, A., and Hoecker, U. (2006). Functional and expression analysis of Arabidopsis SPA genes during seedling photomorphogenesis and adult growth. Plant J. 47: Gil, P., Kircher, S., Adam, E., Bury, E., Kozma-Bognar, L., Schäfer, E., and Nagy, F. (2000). Photocontrol of subcellular partitioning of phytochrome-b: GFP fusion protein in tobacco seedlings. Plant J. 22: Hennig, L., Poppe, C., Sweere, U., Martin, A., and Schafer, E. (2001). Negative interference of endogenous phytochrome B with phytochrome A function in Arabidopsis. Plant Physiol. 125: Hoecker, U., and Quail, P.H. (2001). The phytochrome A-specific signaling intermediate SPA1 interacts directly with COP1, a constitutive repressor of light signaling in Arabidopsis. J. Biol. Chem. 276: Hoecker, U., Xu, Y., and Quail, P.H. (1998). SPA1: a new genetic locus involved in phytochrome A specific signal transduction. Plant Cell 10: Hoecker, U., Tepperman, J.M., and Quail, P.H. (1999). SPA1, a WDrepeat protein specific to phytochrome A signal transduction. Science 284: Molecular Plant 8, , March 2015 ª The Author 2015.