COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1), a ubiquitin E3 ligase, is a central negative regulator of photomorphogenesis. However, how COP1 activity is regulated by post-translational modifications remains largely unknown. Here we show that SUMO (small ubiquitin-like modifier) modification enhances COP1 activity. Loss-of-function siz1 mutant seedlings exhibit a weak constitutive photomorphogenic phenotype. SIZ1 physically interacts with COP1 and mediates the sumoylation of COP1. A K193R substitution in COP1 blocks its SUMO modification and reduces COP1 activity in vitro and in planta. Consistently, COP1 activity is reduced in siz1 and the level of HY5, a COP1 target protein, is increased in siz1. Sumoylated COP1 may exhibits higher transubiquitination activity than does non-sumoylated COP1, but SIZ1-mediated SUMO modification does not affect COP1 dimerization, COP1-HY5 interaction, and nuclear accumulation of COP1. Interestingly, prolonged light exposure reduces the sumoylation level of COP1, and COP1 mediates the ubiquitination and degradation of SIZ1. These regulatory mechanisms may maintain the homeostasis of COP1 activity, ensuing proper photomorphogenic development in changing light environment. Our genetic and biochemical studies identify a function for SIZ1 in photomorphogenesis and reveal a novel SUMO-regulated ubiquitin ligase, COP1, in plants.
Sumoylation is a post-translational modification in which SUMO (small ubiquitin-like modifier) peptides are covalently attached to a SUMO consensus motif (ψKxE/D; ψ a large hydrophobic amino acid residue; K, the acceptor lysine; x, any amino acid; E/D, glutamate or aspartate) in target proteins through a series of biochemical steps involving activation (E1), conjugation (E2), and ligation (E3) enzymes [1,2]. SUMO conjugation can be reversed by SUMO-specific proteases [3]. In yeast and metazoans, sumoylation has been implicated in several aspects of cellular functions, including chromatin remodeling, DNA repair, nuclear/cytoplasmic transport, transcription, and the cell cycle [4]. The PIAS [Protein inhibitors of activated STATs (signal transducer and activator of transcription) ]-type SUMO E3 ligase, SIZ1 [SAP (scaffold attachment factor, acinus, PIAS), and Miz1 (Msx2-interacting zinc finger) ], regulates abiotic stress responses (i. e. , responses to heat, cold, drought, and salt stresses), hormone signaling (i. e. , abscisic acid, salicylic acid, and auxin pathways), nutrient (i. e. , phosphate, nitrogen, and copper) homeostasis, and development (i. e. , flowering time and female gametophyte development) in Arabidopsis [5,6]. Increasing evidence indicates that the SUMO and ubiquitin systems are tightly associated. Sumoylation antagonizes ubiquitination by competing for acceptor K residues or promotes ubiquitination by recruiting SUMO-targeted ubiquitin ligases (STUbLs) to sumoylated substrates in yeast, mammals, and plants [7,8]. Moreover, the PIAS family of SUMO E3 ligases positively or negatively regulates ubiquitin ligase activity by SUMO modification of the SUMO-regulated ubiquitin ligases (SRUbL) in humans [9,10]. In Arabidopsis, SUMO modifications regulate the protein stability of DELLA, ICE1 (inducer of CBF/DREB1 expression), ABI5 (ABA insensitive 5), MYB30, NIA1/2 (nitrate reductase), SLY1 (SLEEPY1) and SnRK1 (Snf1-related protein kinase 1) likely by antagonizing or promoting ubiquitination [11–17]. SRUbLs may also exist in plants; however, they remain to be identified. Dark-grown seedlings have an elongated hypocotyl, closed cotyledons, and an apical hook (skotomorphogenesis). In the light, Arabidopsis seedlings undergo photomorphogenesis and exhibit short hypocotyls and open cotyledons with no apical hooks [18]. The ubiquitin E3 ligase COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1), a central repressor of photomorphogenesis, mediates the ubiquitination and degradation of positive regulators of photomorphogenesis, such as HY5 (ELONGATED HYPOCOTYL 5), HYH (HY5 HOMOLOGUE), LAF1 (LONG AFTER FAR-RED LIGHT 1), HFR1 (LONG HYPOCOTYL IN FAR RED 1), STH3 (SALT TOLERANCE HOMOLOG 3) /BBX2 and PIL1 (PHYTOCHROME INTERACTING FACTOR 3-LIKE1) [19–24]. SPA (SUPPRESSOR OF PHYA-105) and PIFs (PHYTOCHROME INTERACTING FACTORs) interact with COP1, and enhances its E3 ligase activity [21,25,26]. CSU1 (COP1 SUPPRESSOR1), a RING-finger E3 ubiquitin ligase, regulates COP1 homeostasis by ubiquitinating and degrading COP1 in darkness [27]. CSU2 and FIN219 interact with COP1, and negatively regulate its E3 ligase activity and protein level, respectively [28,29]. In response to lights, phyA (phytochrome A), phyB, CRY1 (crytochrome 1) and CRY2, repress COP1 activity through modulating COP1-SPA1 complex [30–34], Reduced COP1 activity in the lights causes the accumulation of HY5 and the transcriptomic reprogramming of HY5 target genes [35,36]. However, post-translational modifications that regulate COP1 activity are largely unknown. Recent study has revealed that SUMO modification of phyB represses red light signaling, at least partly, through inhibiting interaction between phyB and PIFs [37]. In this study, we demonstrate that SIZ1 negatively regulates photomorphogenesis, at least partly, through promoting COP1 ubiquitin E3 ligase activity by SUMO modification, and that COP1 in turn mediates the ubiquitination and degradation of SIZ1. Our results reveal a novel regulatory mechanism of COP1 and SIZ1 in photomorphogenesis.
The observation that loss-of-function siz1 mutant seedlings showed a short-hypocotyl phenotype under white light prompted us to evaluate the light responsiveness of siz1-2. The siz1-2 seedlings exhibited a short-hypocotyl phenotype under darkness and red, blue, and far-red light conditions (Fig 1A and 1B). Expression of ProSIZ1: SIZ1-GFP in siz1-2 plants (complemented lines referred to as SSG) [38] suppressed the short-hypocotyl phenotype of siz1-2 under these conditions, indicating that mutation of SIZ1 is responsible for the short-hypocotyl phenotype (Fig 1A and 1B). The short-hypocotyl phenotype of siz1-2 was due to a reduction in cell elongation but not in cell number (S1 Fig). The expression of light-inducible genes, CAB (CHLOROPHYLL A/B BINDING PROTEIN) and RBCS (RUBISCO SMALL SUBUNIT) [39,40], and a light-repressed gene, PORA (PROTOCHLOROPHYLLIDE OXIDOREDUCTASE A) [41], was up- and down-regulated, respectively, in siz1-2 during the transition from darkness to light, indicating that the regulation is stronger in siz1-2 than in wild type (Fig 1C). In addition to the short-hypocotyl phenotype, siz1-2 seedlings had unfolded apical hooks in darkness (Fig 1D) and exhibited more opened cotyledons than did wild-type seedlings in dark and light conditions (Fig 1E). These results suggest that SIZ1 negatively regulates photomorphogenesis. To determine if SIZ1-mediated SUMO1/2 modification is involved in the regulation of hypocotyl elongation, we determined the hypocotyl length of sum1 and sum2 double mutant under darkness and red, blue, and far-red light conditions (Fig 2). SUMO1 and 2 have redundant functions, and the sum1 sum2 double knockout mutant is embryo lethal [42]. Therefore, we used a viable weak allele, sum1-1 amiR-SUM2, in which SUMO2 expression is down-regulated by RNAi in the sum1-1 knockout mutant background [43]. Similar to siz1-2, sum1-1 amiR-SUM2 seedlings exhibited a short-hypocotyl phenotype under dark and light conditions, suggesting that SUMO1/2 modification regulates hypocotyl elongation (Fig 2). Elevated SA levels in siz1-2 cause dwarfism, and the dwarf phenotype is substantially suppressed by expression of NahG, a bacterial salicylate hydroxylase [44]. To determine if the short-hypocotyl phenotype of siz1-2 is due to increased SA levels, the hypocotyl lengths of five-day-old wild-type, siz1-2, NahG, and NahG siz1-2 seedlings were compared under darkness and red, blue, and far-red light conditions (Fig 2). The expression of NahG did not affect hypocotyl elongation, but the siz1-2 and NahG siz1-2 seedlings exhibited an identical short-hypocotyl phenotype in all tested conditions, indicating that the elevated SA level in siz1-2 did not contribute to the short-hypocotyl phenotype. Since siz1-2 seedlings exhibit a weak cop1-like phenotype, we examined whether SIZ1 physically interacted with COP1 to regulate its activity. To do so, we performed a bimolecular fluorescence complementation (BiFC) assay. YFP fluorescence signals were detected in the nucleus of N. benthamiana cells that coexpressed COP1-YFPN (fused with the N-terminal half of YFP) and SIZ1-YFPC (fused with the C-terminal half of YFP) under light (1 h light exposure) and dark conditions (Fig 3A and S2 Fig). Consistently, SIZ1-GFP was coimmunoprecipitated with Myc-COP1 (Fig 3B). These results indicate that SIZ1 physically interacts with COP1 in the nucleus. SUMOplot (http: //www. abgent. com/sumoplot) analysis predicted the presence of three potential sumoylation motifs [45] (VK14PD, IK193ED, and WK653SD) in COP1 (S3 Fig), suggesting that COP1 may be a SUMO substrate. To test this possibility, we performed an in vitro sumoylation assay to determine SUMO modification of COP1 as described previously [15]. Anti-FLAG and anti-SUMO1 antibody detected slow migrating multiple bands above original COP1 protein in the reaction containing SUMO E1 (His6-SAE1b and His6-SAE2), SUMO E2 (His6-SCE1), and His6-SUMO1-GG, but not in the reaction lacking His6-SUMO1-GG, suggesting that COP1 is a possible SUMO substrate (Fig 3C). To further confirm COP1 is sumoylated in vivo, we performed an in vivo sumoylation analysis as described previously [38]. We co-expressed Myc-COP1 with FLAG-SUMO1 or FLAG-SUMO1AA (a conjugation-deficient mutant) in Arabidopsis protoplasts or N. benthamiana leaves. Myc-COP1 was immunoprecipitated with anti-Myc antibody and the immunoprecipitated proteins were detected with anti-FLAG antibody. Higher molecular weight sumoylated COP1 bands were detected when Myc-COP1 and FLAG-SUMO1 were co-expressed, but not in Myc-COP1 and FLAG-SUMO1AA co-expressing cells (Fig 3D and S4A Fig). Moreover, to confirm that sumoylation of COP1 occurs in planta, we generated an Myc-COP1 overexpression transgenic line (referred to as 35S-Myc-COP1) and performed an in vivo sumoylation analysis. Anti-SUMO1 antibody detected higher molecular weight sumoylated COP1 bands (Fig 3E). Furthermore, we evaluated if sumoylation of COP1 changes in response to light. Since light exposure promotes nucleus to cytosol re-localization of COP1 [46], we monitored the sumoylation status of COP1 in the nucleus under dark (0 h) and light (12 h) conditions (Fig 3F). Under darkness, sumoylated COP1 bands were detected in the Myc-COP1 and FLAG-SUMO1 co-expressing nuclear fraction, but the sumoylation level of COP1 was substantially reduced in response to 12 h of light exposure. Taken together, these data demonstrate that COP1 is a SUMO substrate, and sumoylation level of COP1 is regulated by light. To test if SIZ1 mediates SUMO conjugation of COP1, we cotransformed Myc-COP1 and FLAG-SUMO1 into Arabidopsis protoplasts isolated from wild-type or siz1-2 plants, and analyzed the sumoylation status of COP1. Whereas COP1-SUMO1 conjugate was detected in the wild type, substantially lower levels were present in siz1-2 (Fig 3G), indicating that SIZ1 facilitates the sumoylation of COP1. The lower levels of sumoylated COP1 in siz1-2 may be due to another SUMO E3 ligase (s) that facilitates the residual SUMO modification of COP1. Alternatively, it is also possible that E1 and E2 contribute to basal levels of COP1-SUMO conjugation, since E1 and E2 alone mediate the sumoylation of COP1 in vitro (Fig 3C). K-to-R substitutions in sumoylation motifs block SUMO conjugation [47]. To elucidate the sumoylation motifs of COP1, SUMO conjugation of Myc-COP1K14R, Myc-COP1K193R, and Myc-COP1K653R were evaluated in Arabidopsis protoplasts. The K193R substitution blocked COP1-SUMO1 conjugation, but K14R or K653R substitutions did not (Fig 3H). Unfortunately, we could not confirm the effect of K193R substitution in vitro, due to anti-FLAG antibody detected long smear bands above the original purified MBP-COP1K193R-FLAG, which would strongly affect subsequent in vitro sumoylation analysis. COP1-SUMO1 conjugation was also blocked by the K193R substitution in N. benthamiana leaves (S4B Fig). These results indicate that SIZ1 mediates sumoylation of COP1 and that K193 is critical for SUMO conjugation. Since SIZ1 mediates SUMO modification of COP1 and siz1-2 seedlings exhibit a weak cop1-like phenotype (Figs 1 and 3), we hypothesis that SIZ1-mediated SUMO modification may enhances COP1 activity. To test this possibility, we first determined the effect of sumoylation of COP1 in planta. Myc-COP1 and Myc-COP1K193R (a non-sumoylated form) overexpressing Arabidopsis transgenic plants were generated, and two independent lines with similar levels of transgene expression for each construct were selected for further phenotypic analysis (S6 Fig). 35S-Myc-COP1 seedlings exhibited longer hypocotyls than did the wild type under white light conditions (Fig 4A), confirming a previous report [48]. Under 30 μmol m-2 s-1 fluence rate of white light conditions, the hypocotyls of 35S-Myc-COP1K193R seedlings were longer than that of the wild type, but shorter than that of 35S-Myc-COP1 (Fig 4B). Under relatively higher fluence rate of white light conditions (50 and 80 μmol m-2 s-1), while 35S-Myc-COP1 seedlings still exhibit longer hypocotyls than wild type, the hypocotyls of 35S-Myc-COP1K193R seedlings were of similar length as those of the wild type, suggesting that non-sumoylated Myc-COP1K193R exhibits lower activity than Myc-COP1 (Fig 4A and 4B). Next, total protein extracts isolated from 35S-Myc-COP1,35S-Myc-COP1K193R, Col-0 and cop1-4 seedlings were used as E3s to perform an in vitro HY5 ubiquitination assay as described previously [49,50] (Fig 4C). Ubiquitination of HY5 was stronger in the reaction containing protein extracts isolated from 35S-Myc-COP1 than in that containing extracts from 35S-Myc-COP1K193R. We did not detected ubiquitination of HY5 in the reaction containing Col-0 protein extracts due to short expose time for the blot (Fig 4C). Next, we overexpressed COP1 in siz1-2 plants by crossing the COP1 overexpression line, COP1 OE, with siz1-2 (referred to here as COP1 OE siz1-2), and tested if the siz1 mutation reduces COP1 OE effect on hypocotyl elongation. Overexpression of COP1 causes a long-hypocotyl phenotype under light conditions (Fig 4D and 4E) [48]. Consistent with our hypothesis, the hypocotyl length of COP1 OE siz1-2 was substantially shorter than that of COP1 OE, suggesting that COP1 activity is reduced in siz1-2 (Fig 4D and 4E). Finally, we compared the COP1 ubiquitin E3 ligase activity of Col-0 and siz1-2. Total protein extracts from the wild type mediated the ubiquitination of HY5 more efficiently than did those from siz1-2 (Fig 4F). Collectively, these results demonstrate that SIZ1-mediated SUMO conjugation promotes COP1 E3 ubiquitin ligase activity. Furthermore, genetic interaction between COP1 and SIZ1 was analyzed. The hypocotyl length of cop1-4 [51] was shorter than that of the wild type and siz1-2 under dark and light conditions (Fig 4G and 4H and S7 Fig). The cop1-4 siz1-2 double mutant exhibited a short-hypocotyl phenotype similar to cop1-4 in both dark and light conditions, suggesting that SIZ1 regulates hypocotyl elongation partly through COP1 (Fig 4G and 4H and S7 Fig). SIZ1 enhances COP1 activity (Fig 4) and COP1 mediates ubiquitination and degradation of HY5 [19]. Therefore, we tested if the protein level of HY5 is up-regulated by mutation of SIZ1. Anti-HY5 antibody [52] revealed that endogenous HY5 was more abundant in siz1-2 than in the wild type under light conditions (Fig 5A). Consistent with previous report [19], higher level of HY5 was accumulated in cop1-4, and the HY5 level in cop1-4 was similar to that of cop1-4 siz1-2, suggesting that higher level of HY5 in siz1-2 is possibly due to reduced COP1 activity rather than increased transcription of HY5 in siz1-2 (Fig 5A). Consistent with this hypothesis, the level of HY5 expression was similar in the wild type and siz1-2 (Fig 5B). These results indicate that SIZ1 negatively regulates HY5 protein abundance. Consistent with the increased HY5 level in siz1-2, the expression level of the cell elongation-related direct target genes of HY5, i. e. , EXP2 (EXPANSIN2), EXT3 (EXTENSIN3), XTR6 (XYLOGLUCAN ENDOTRANSGLYCOSYLASE6), and XTH17 (ENDOTRANSGLUCOSYLASE/HYDROLASE17) [36,53], was down-regulated in siz1-2 compared to the wild type (Fig 5C). The loss-of-function hy5-215 seedlings exhibited a long-hypocotyl phenotype under light conditions [54]. To identify genetic interactions between HY5 and SIZ1, we generated the hy5-215 siz1-2 double mutant through genetic crossing. The short-hypocotyl phenotype of siz1-2 was partially suppressed by hy5-215 under various light conditions (Fig 5D and 5E), suggesting that the accumulation of HY5 in siz1-2 at least partly accounts for the short-hypocotyl phenotype under light conditions. In addition to HY5, COP1 also mediates the ubiquitination and degradation of HYH, LAF1, HFR1, STH3/BBX2 and PIL1 [19–24]. Thus, it is possible that the protein levels of these positive regulators of photomorphogenesis are higher in siz1-2, which may causes a weak cop1-like phenotype of siz1-2. SIZ1-mediated SUMO modification may regulate COP1 expression, COP1 stability, nuclear accumulation level, interaction with other proteins and/or affects its enzymatic activity. The level of COP1 expression was not significantly affected by the siz1-2 mutation, indicating that SIZ1 does not regulate the transcription of COP1 (Fig 6A). Anti-COP1 antibody [30] revealed that endogenous COP1 protein abundance was similar in the wild type and siz1-2, indicating that the reduced COP1 activity in siz1-2 was not due to reduced levels of COP1 (Fig 6B). Since COP1 functions as a homodimer [55], we examined whether SUMO modification affected COP1 dimerization. Immunoprecipitation analysis showed that Myc-COP1 and Myc-COP1K193R (a non-sumoylated form) immunoprecipitated the same amount of FLAG-COP1, indicating that sumoylation of COP1 did not significantly affect COP1 dimerization (Fig 6C). Next, we examined if SUMO modification enhanced the COP1-HY5 interaction using in vitro co-immunoprecipitation assays. Sumoylation of COP1 did not enhance the substrate accessibility of COP1 under dark and light conditions (Fig 6D). Moreover, the siz1 mutation did not affect the level of nuclear COP1 under dark and light conditions (Fig 6E). Finally, we evaluated if SUMO conjugation regulates transubiquitination acitivity of COP1. In vitro sumoylated and non-sumoylated MBP-COP1-FLAG were used as E3s to perform an in vitro HY5 ubiquitination assay. Anti-GST and anti-ubiquitin antibodies detected higher level of ubiquitinated proteins in the reaction containing sumoylated COP1 than did non-sumoylated COP1 (Fig 6F), suggesting that SIZ1-mediated SUMO modification may enhances transubiquitination activity of COP1. Although the biological functions of SIZ1 have been extensively characterized, the mechanism that regulates SIZ1 activity is largely unknown. Our finding that COP1 interacted with SIZ1 (Fig 3A and 3B) prompted us to determine if COP1 mediates the ubiquitination and degradation of SIZ1. To determine if COP1 promotes degradation of SIZ1, we analyzed the decay kinetics of SIZ1-GFP in the presence or absence of Myc-COP1. The degradation of SIZ1 was more rapid in the presence of Myc-COP1 (Fig 7A). Moreover, the COP1-promoted SIZ1 degradation was inhibited by MG132, a 26S proteasome inhibitor (Fig 7B, upper panel). In agreement with a previous report [21], MG132 also repressed the degradation of COP1 (Fig 7B, middle panel). SIZ1 expression was not affected in cop1-4, indicating that COP1 does not regulate SIZ1 transcription (Fig 7C). Next, we evaluated if COP1 mediates the ubiquitination of SIZ1. MBP-COP1-FLAG was used as the E3 ligase in an in vitro SIZ1 ubiquitination assay. Anti-Myc and anti-ubiquitin antibody revealed that COP1 facilitates ubiquitination of SIZ1 in vitro (Fig 7D). These results suggest that COP1 negatively regulates SIZ1 protein stability through ubiquitination and subsequent 26S proteasome-dependent degradation.
Loss-of-function siz1-2 and sum1-1 amiR-SUM2 seedlings exhibited a short-hypocotyl phenotype under dark and light conditions (Figs 1 and 2), suggesting that SIZ1-mediated SUMO1/2 modification negatively regulates photomorphogenesis. SIZ1 physically interacts with COP1 and facilitates its SUMO modification (Fig 3). COP1 exhibits higher activity compared to that of non-sumoylated COP1, COP1K193R, and the COP1 activity was attenuated in siz1-2 (Fig 4). The reduced COP1 activity in siz1 resulted in higher levels of HY5, stronger down-regulation of cell elongation-related HY5 direct target genes (Fig 5A and 5C). These results strongly suggest that SIZ1-mediated SUMO modification enhances COP1 activity; thus, COP1 is a plant SUMO-regulated ubiquitin ligases (SRUbL). The hypocotyl length of cop1-4 siz1-2 was slightly shorter than that of cop1-4 (Fig 4G and 4H and S7 Fig), suggesting that SIZ1 regulates hypocotyl elongation through at least two pathways, a COP1-dependent and -independent pathway. Recently, it has been shown that SUMO modification of phyB negatively regulate photomorphogenesis under red light [37]. OTS1 (OVERLY TOLERANT TO SALT1) mediate desumoylation of phyB, but whether the SUMO modification is facilitated by SIZ1 remains to be determined. However, it should be noted that cop1-4 is a weak allele, which expresses a partially functional truncated COP1 protein (1–282 aa, which contains the K193 sumoylation motif) [51]. Therefore, it is also possible that the partial COP1 activity in cop1-4 is further attenuated in cop1-4 siz1-2. It has been demonstrated that COP1 is regulated by different mechanisms: (1) light, FIN219 and cold induces nuclear-to-cytoplasmic export of COP1, but ethylene enhances nuclear retention of COP1 in the light [29,56–58]; (2) CSU1, FIN219 and heat shocks reduce protein abundance of COP1 [27,29,59]; (3) SPA positively regulates COP1 activity may through enhancing substrate recruitment [21,25]; (4) phyA, phyB, CRY1 and CRY2 repress COP1 activity by affecting COP1-SPA complex in the lights [30–34]; (5) PIF1 not only enhances substrate recruitment, but also increases autoubiquitination and transubiquitination activities of COP1 [26]. The present study reveals that SIZ1-mediated sumoylation does not regulate COP1 protein abundance, dimerization of COP1, or nuclear-to-cytoplasmic translocation (Fig 6). SUMO modification possibly increased transubiquitination activity of COP1 in vitro (Fig 6F), but did not affect substrate recruitment (Fig 6D). At the molecular level, sumoylation affects protein-protein interactions and target protein conformation [60,61]. We speculate that the enhanced transubiquitination activity of COP1 by sumoylation possibly due to conformational change of COP1 or increased COP1-E2 interaction. Recent study has demonstrated that CSU2 negatively regulates COP1 activity through their coiled-coil domains [28]. SUMO modification site, K193, is located in the coiled-coil domain of COP1. Thus, it is also possible that the sumoylation inhibits interaction between CSU2 and COP1 in vivo, but remain to be elucidated in the near future. It has been shown that a certain amount of COP1 is present in the nucleus even under prolonged light exposure, and plays a critical role in regulating development [27,51,58,62–64]. Interestingly, prolonged light exposure reduced sumoylation levels of nuclear-localized COP1 (Fig 3F), which results in decreased COP1 activity. Thus, we hypothesize that light-induced reduction of COP1-SUMO levels is required for the maintenance of moderate COP1 activity under light conditions to ensure the tight regulation of photomorphogenesis. The reduced sumoylation level of COP1 under light conditions may be due to decreased SIZ1-mediated sumoylation and/or increased SUMO protease (s) -mediated desumoylation, but the details of the mechanisms by which light regulates the balance between sumoylation and desumoylation remain to be elucidated. Collectively, our study demonstrates that SIZ1-mediated sumoylation negatively regulates photomorphogenesis, at least partly, through enhancing COP1 activity. Interestingly, COP1 in turn mediates the ubiquitination and 26S proteasome-dependent degradation of SIZ1 (Fig 8). This feedback repression of SIZ1 activity by COP1 may reflect the requirement of tightly regulated COP1 activity for proper photomorphogenic development.
Seeds of wild type, siz1-2 (Salk_065397), cop1-4, COP1 OE, hy5-215, sum1-1 amiR-SUM2 [43], SSG [38], NahG, and NahG siz1-2 [44] were in the Columbia ecotype. cop1-4 siz1-2, hy5-215 siz1-2, and COP1 OE siz1-2 were generated by genetic crossing and the double mutants were identified as described in previous reports [51,54]. To generate 35S-Myc-COP1 and 35S-Myc-COP1K193R, pBI121-Myc-COP1 and pBI121-Myc-COP1K193R were transformed into Col-0 by the Agrobacterium-mediated floral dip method [65], respectively, and homozygous T3 plants were used. After surface sterilization, seeds were sown on Murashige and Skoog growth medium (1/4× Murashige and Skoog basal salts, 1% sucrose, and 0. 75% agar). After 3 days of incubation at 4°C in darkness, the seeds were exposed to 6 h of white light to induce germination, and then transferred to light chambers containing red, blue, or far-red light emitting diodes at the indicated fluence rates. White light (100 μmol m-2 s-1 unless indicated otherwise) was provided using white fluorescent lamps. To analyze hypocotyl length, cotyledon angle, and hypocotyl cell length and cell numbers, seedlings were photographed with a camera (Canon) or a cool CCD camera coupled to an Olympus BX53 microscope and the images were analyzed with NIH ImageJ software (http: //rsbweb. nih. gov/ij/). Total RNA was extracted from seedlings with TRIZOL reagent (RNAiso Plus, TaKaRa) and reverse transcribed using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). qRT-PCR was then performed with SYBR Premix ExTaq (TaKaRa) according to the manufacturer’s instructions. Three biological replicates were performed. The relative expression level of each gene was normalized to that of Ubiquitin-Conjugation Enzyme (UBC). Primer sequences are listed in S1 Table. COP1-YFPN and SIZ1-YFPC plasmids together with the proper control plasmids (empty YFPN or YFPC vector plasmids) were transformed into Agrobacterium tumefaciens strain GV3101 and infiltrated into Nicotiana benthamiana leaves as previously described [66]. After incubation at 22°C for 3 days under 16 h white light (100 μmol m-2 s-1) /8 h darkness, YFP fluorescence was detected with a fluorescence microscope (Olympus BX53) under darkness or after 1 h white light (100 μmol m-2 s-1) exposure. For co-immunoprecipitation of COP1 and SIZ1, Myc-COP1 and SIZ1-GFP were separately expressed in Col-0 protoplasts to avoid degradation of SIZ1 by COP1. Immunoprecipitation was carried out using a mixture of COP1 and SIZ1 protein extracts. The mixture of protein extracts was immunoprecipitated with anti-Myc-conjugated agarose beads (Sigma, F-2426) and co-immunoprecipitated SIZ1-GFP proteins were detected with anti-GFP antibody (Clontech, 632375). To analyze the sumoylation effect in the interaction between HY5 and COP1,1 μg of GST-HY5 protein purified from E. coli [52], as bait, was incubated with the protein extracts isolated from N. benthamiana co-expressing Myc-COP1 with FLAG-SUMO1 or FLAG-SUMO1AA. The mixture of protein extracts was immunoprecipitated with anti-Myc-conjugated agarose beads (Sigma, F-2426) and co-immunoprecipitated GST-HY5 proteins were detected with anti-GST antibody (Abcam, ab19256). To determine the sumoylation status of COP1, proteins were extracted in a buffer composed of 50 mM Tris-Cl (pH 7. 4), 150 mM NaCl, 1 mM EDTA (pH 8. 0), 1 mM DTT, 20 mM NEM, 1% TritonX-100, and 1× complete protease inhibitor mixture (Roche, 04693159001). Then, the protein extracts were immunoprecipitated with anti-Myc-conjugated agarose beads (Sigma, F-2426) for 3 h. Next, the beads were washed with protein extraction buffer four times, and the immunoprecipitated proteins were eluted with 2× SDS loading buffer for immunoblot analysis. The sumoylated form of COP1 was identified with anti-FLAG antibody (Sigma, F3165) or anti-SUMO1 antibody (Abcam, ab5316). An in vitro sumoylation assay was performed as described previously [15] with minor modifications. Briefly, 50 ng of His6-AtSAE1b, 50 ng of His6-AtSAE2,50 ng of His6-AtSCE1,8 μg of His6-AtSUMO1-GG, and 100 ng of MBP-COP1-FLAG were incubated in 30 μl of reaction buffer (20 mM HEPES pH7. 5,5 mM MgCl2,2 mM ATP) for 3 h at 30°C. Sumoylated MBP-COP1-FLAG was detected with anti-FLAG (Sigma, F3165) and anti-SUMO1 antibody (Abcam, Ab5316). An in vitro ubiquitination assay was performed as described previously [50] with minor modifications. E3s, recombinant wheat E1,500 ng purified E2,5 μg ubiquitin, and 100 ng substrates were incubated in 30 μl of reaction buffer (50 mM Tris-Cl (pH 7. 4), 10 mM MgCl2,5 mM ATP, and 2 mM DTT) for 3 h at 30°C. The substrates were detected with anti-His (Sigma, H9658), anti-GST (Abcam, ab19256), or anti-Myc (Sigma, M4439) antibody, and the ubiquitination was determined with anti-ubiquitin (Sigma, U5379) antibody. Nuclear protein extraction was carried out with the CelLytic PN Extraction Kit (Sigma, CELLYTPN1) as described previous [62]. To generate pSPYNE-COP1-YFPN, full-length COP1 cDNA without the termination codon was amplified with gene-specific primers COP1-F-SpeI/COP1nt-R-XhoI. The COP1 cDNA was inserted in-frame at the SpeI/XhoI sites of the pSPYNE-35S vector [66]. To generate pSPYCE-SIZ1-YFPc, full-length SIZ1 cDNA without the termination codon was amplified with gene-specific primers SIZ1-F-XmaI/SIZ1-R-SpeI and ligated into the pBluescript vector. pBluescript-SIZ1 was digested with SmaI and SpeI, and the SIZ1 cDNA was inserted in-frame at the HpaI/SpeI sites of the pSPYCE-35S vector [66]. To generate pCambia1302-SIZ1-GFP, pBluescript-SIZ1 was digested with SmaI and SpeI. The pCambia1302 vector was digested with NcoI, to generate blunt ends, and then with SpeI. The insert was then ligated into the pCambia1302 vector. To generate pMAL-C2-MBP-COP1-FLAG, full-length cDNA of COP1 without the termination codon was amplified with gene-specific primers COP1-5’-SmaI/COP1nt-3’-XhoI, and ligated into the pBluescript vector. pBluescript-COP1 was digested with SmaI and XhoI, and inserted in-frame at the SmaI and XhoI sites of the pMAL-C2-MBP-FLAG vector. To generate pMAL-C2-MBP-SIZ1-Myc, full-length cDNA of SIZ1 without the termination codon was amplified with gene-specific primers SIZ1-F-XbaI/SIZ1-R-ClaI, and ligated into the pBluescript vector. pBluescript-SIZ1 was digested with XbaI and ClaI, and inserted in-frame at the XbaI and ClaI sites of the pMAL-C2-MBP-MYC vector. To generate p326-Myc-COP1, the full-length cDNA of COP1 was amplified with gene-specific primers COP1-F-HindIII/COP1-R-XhoI, and ligated into the pBluescript vector. pBluescript-COP1 was digested with HindIII and XhoI, and inserted in-frame at the HindIII and XhoI sites of the p326-35S-nMyc vector. pBluescript-COP1 was used as template with primer pairs COP1 K14R-F/COP1 K14R-R, COP1 K193R-F/COP1 K193R-R, and COP1 K653R-F/COP1 K653R-R, to generate pBluescript-COP1K14R, pBluscript-COP1K193R, and pBluescript-COP1K653R, respectively. pBluescript-COP1K14R, pBluescript-COP1K193R, and pBluescript-COP1K653R were digested with HindIII and XhoI, and inserted in-frame at the HindIII and XhoI sites of the p326-35S-nMyc vector to generate p326-35S-Myc-COP1K14R, p326-Myc-COP1K193R, and p326-Myc-COP1K653R, respectively. p326-Myc-COP1 and p326-Myc-COP1 K193R were digested with SpeI and XhoI, and the digested products were inserted into the SpeI and XhoI sites of the pBI121-35S-nMyc vector to generate pBI121-Myc-COP1 and pBI121-Myc-COP1K193R, respectively. To generate p326-FLAG-SUMO1 and pBI121-FLAG-SUMO1, SUMO1 cDNA was amplified with gene-specific primers SUMO-F-XbaI and SUMO-R-XhoI, and inserted into the XbaI and XhoI sites of the p326-35S-nFLAG and pBI121-35S-nFLAG vector. To generate p326-FLAG-SUMO1AA and pBI121-FLAG-SUMO1AA, SUMO1 cDNA was amplified with gene-specific primers SUMO-F-XbaI and SUMOAA-R-XhoI, and inserted into the XbaI and XhoI sites of the p326-35S-nFLAG and pBI121-35S-nFLAG vector. All primer sequences are listed in S1 Table.
In darkness, the ubiquitin E3 ligase COP1 accumulates in the nucleus and mediates ubiquitination and degradation of positive regulators of photomorphogenesis, such as HY5. In response to light, COP1 activity is reduced to ensure proper photomorphogenic development. However, post-translational modifications that regulate COP1 activity are largely unknown. We have found that the Arabidopsis SUMO E3 ligase SIZ1 negatively regulates photomorphogenesis. Genetic and biochemical lines of evidence demonstrate that SIZ1-mediated SUMO modification of COP1 enhances its E3 ubiquitin ligase activity, which causes increased ubiquitination and degradation of HY5. In response to the light, sumoylation level of COP1 is decreased, which may also contributes to the reduction of COP1 activity in the light. Moreover, COP1 mediates ubiquitination and 26S proteasome-dependent degradation of SIZ1 and this feedback repression may ensure the moderate levels of COP1 activity. Our study established a post-translational regulatory modular consisting of SIZ1-mediated sumoylation and COP1-mediated ubiquitination that tightly regulate photomorphogenesis.
plant anatomy
plant growth and development
enzymes
plant embryo anatomy
enzymology
plant physiology
developmental biology
plant science
sumoylation
immunoprecipitation
seedlings
plants
extraction techniques
ligases
research and analysis methods
protein extraction
plant embryogenesis
plant development
proteins
ubiquitination
precipitation techniques
biochemistry
photomorphogenesis
post-translational modification
embryogenesis
biology and life sciences
hypocotyl
organisms
fruit and seed anatomy
An Arabidopsis SUMO E3 Ligase, SIZ1, Negatively Regulates Photomorphogenesis by Promoting COP1 Activity