The E3 Ubiquitin Ligase COP1 Regulates Thermosensory Flowering by Triggering GI Degradation in Arabidopsis

Floral transition is influenced by environmental factors such as light and temperature. Plants are capable of integrating photoperiod and ambient temperature signaling into their developmental program. Despite extensive investigations on individual genetic pathways, little is known about the molecular components that integrate both pathways. Here, we demonstrate that the RING finger–containing E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) acts as an integrator of photoperiod and ambient temperature signaling. In addition to the role in photoperiodic destabilization of CONSTANS (CO), COP1 also regulates temperature sensitivity by controlling the degradation of GIGANTEA (GI). COP1-impaired mutants showed reduced sensitivity to low ambient temperature. Notably, COP1 is more stabilized at low temperature and accelerates GI turnover in a 26S proteasome-dependent manner. The direct association of GI with the promoter of FLOWERING LOCUS T (FT) was reduced because of its ambient temperature-dependent protein stability control, and thus COP1-triggered GI turnover delays flowering at low temperatures via a CO-independent pathway. Taken together, our findings indicate that environmental conditions regulate the stability of COP1, and conditional specificity of its target selection stimulates proper developmental responses and ensures reproductive success.

ACTIN-RELATED PROTEIN 6 (ARP6) substituting H2A with H2A.Z and PHYTOCHROME INTERACTING FACTOR 4 (PIF4) have also emerged as additional regulators of temperature-dependent flowering. Histone variant H2A.Z-containing nucleosomes wrap DNA more tightly, reducing accessibility of transcriptional regulatory proteins (activators and repressors) to associated target genes [9][10][11] . Notably, the histone exchange is dependent on ambient temperature. Low temperatures trigger H2A.Z incorporation, whereas H2A.Z occupancy is significantly decreased at high temperature in selective loci 12 . Consistently, ARP6-deficient mutants with reduced incorporation of H2A.Z show early flowering similar to plants grown at high temperatures 12,13 . Thus, H2A.Z depletion at high temperature appears to confer transcriptional competence and allows plants to regulate a proper temperature response by recruiting appropriate transcriptional regulators 12,13 . Indeed, upon high temperature-induced H2A.Z eviction, the FT promoter becomes more accessible for binding by the high temperature-responsive bHLH transcription factor PIF4 to stimulate floral transition 8 .
The SHORT VEGETATIVE PHASE (SVP) MADS-box transcription factor is another crucial regulator of temperature-dependent flowering 6,14 . Low ambient temperatures trigger SVP accumulation, which negatively regulates FT by binding to vCArG motifs in its promoter 6 . SVP actions are fine-tuned along with FLOWERING LOCUS M (FLM). The FLM gene undergoes alternative splicing, generating four splice isoforms including FLM-β and FLM-δ 15,16 . The relative abundance of each splice variant is determined by the ambient temperature: FLM-β accumulates at low temperature, whereas high temperature triggers production of FLM-δ 15,16 . The FLM-β variant forms a transcriptional repressor complex together with SVP to regulate FT, SOC1, and TWIN SISTER OF FT (TSF), suppressing floral transition at low temperature 15 . In contrast, high temperature-induced FLM-δ acts as a dominant-negative regulator and disrupts the formation of a functional complex between FLM-β and SVP 15,16 .
Arabidopsis is capable of integrating light and temperature signaling into its developmental program. Photoperiod-promotive floral transition is suppressed at low temperatures 4 . In contrast, high temperature accelerates flowering, even in short day-grown plants 17 . Furthermore, temperature-insensitive phenotypes have been observed in photoreceptor mutants 4,18 , suggesting that light and temperature signaling are intricately interconnected. Despite the importance of crosstalk between light and temperature signaling, the underlying molecular mechanisms remain to be elucidated.
The RING-finger E3 ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) is a central regulator of light-dependent physiological processes such as photomorphogenesis, circadian oscillation, and floral transition [19][20][21] . In this work, we report that COP1 is also involved in ambient temperature-dependent flowering. COP1 is stabilized at low ambient temperatures and promotes degradation of GI that directly regulates FT expression. Consistently, COP1-deficient mutants exhibited reduced sensitivity to changes in ambient temperatures in an FT-dependent manner. Our findings provide biological insights into how temperature and photoperiod signals are integrated.

Results
cop1 mutants are insensitive to changes in ambient temperature in flowering. Ambient temperature signaling is closely intertwined with photoperiod signaling 7,17,22,23 . In a preliminary attempt to investigate light and temperature signaling crosstalk, we assessed light signaling mutants for their sensitivity to low ambient temperature. Of the light-signaling mutants examined, the COP1-impaired cop1-6 mutant showed reduced sensitivity to changes in ambient temperature under long day (LD) conditions. Flowering of wild-type (Col-0) plants was substantially delayed at 16 °C with a flowering time ratio (16 °C /23 °C ) of approximately 2.7 ( Fig. 1a,b), which was consistent with previous observations 4 . However, cop1-6 mutant exhibited early flowering independent of ambient temperatures, with a flowering time ratio of approximately 1.3 (Fig. 1a,b).
To determine the role of COP1 in thermosensory flowering, we obtained additional cop1-4 mutant and measured flowering time under different temperature conditions 21 . cop1-4 mutant exhibited a significant decrease in the sensitivity to low ambient temperature similar to cop1-6 mutant (Fig. 1b), suggesting that COP1 is involved in ambient temperature-dependent floral transition. The slight differences of cop1-4 and cop1-6 in temperature sensitivity might be due to allelic variations 20 . Because of its strong phenotypes, we employed cop1-6 mutant for subsequent analyses.
FT is required for COP1 action. Ambient temperature signals are integrated into the flowering integrator FT, and thus we next examined whether COP1 affects FT expression to achieve proper developmental responses to ambient temperatures.
FT expression was substantially suppressed at low temperatures in wild-type plants with a ratio (16 °C /23 °C ) of 0.2, whereas the temperature-dependent changes in FT expression were significantly reduced in cop1-6 mutant (Fig. 2a). SOC1 is another flowering integrator that acts partially downstream of FT 24 . To examine whether the altered expression of FT was relevant to a flowering pathway, we analyzed expression of SOC1. Similar to FT expression, transcript accumulation of SOC1 was also reduced in wild-type plants at low ambient temperature, but the change was significantly reduced in cop1-6 mutant (Fig. 2b).
To further evaluate the genetic hierarchy between FT and COP1, we crossed ft-10 with cop1-6. Although a stunted growth phenotypes were observed (Fig. S1), the flowering phenotype of the cop1-6 Scientific RepoRts | 5:12071 | DOi: 10.1038/srep12071 ft-10 double mutant was comparable to that of ft-10 regardless of ambient temperatures (Fig. 2c), indicating that FT acts downstream of COP1 in a thermosensory pathway. COP1 stability is controlled by ambient temperature. We next asked how COP1 perceives ambient temperature signals. To that end, we measured transcript accumulation of COP1 in seedlings grown at different ambient temperatures. Low temperature resulted in reduced transcript accumulation of COP1, whereas expression of COP1 was unchanged at high temperatures (Fig. S2). Since reduced expression of COP1 cannot explain the early flowering phenotype of cop1-6 mutant at low temperatures, it can be considered that COP1 expression might be associated with feedback regulation of protein accumulation. It is likely that at low ambient temperature, COP1 transcription is reduced to maintain homeostasis for increased COP1 levels.
To test this hypothesis, we employed the 35S:COP1-TAP transgenic plants 21 grown at different ambient temperatures. Notably, COP1 protein levels increased significantly at low temperature compared with its levels at 23 °C (Fig. 3a). The temperature effect on COP1 accumulation was observed in both light and dark periods of the day, while it was more prominent in the dark (Fig. 3b). COP1 stability seems to be controlled by multiple environmental factors including light and temperature, and the distinct degradation patterns of COP1 might be owing to the different destructive mechanisms depending on the surrounding environments.
To minimize the secondary effect caused by reduced growth rates at low temperature, we transferred 23 °C -grown plants to 16 °C and vice versa and incubated them for 2 days (Fig. 3a). Short-term exposure to low temperature also stimulated accumulation of COP1. In contrast, COP1 degradation was unchanged at high temperature (Fig. S3), indicating that the stability of COP1 is primarily regulated by low ambient temperature. Floral transition at low ambient temperature is regulated in part by COP1-GI module. CO is a representative proteolytic target of COP1, and thus we tested if CO activity is regulated by ambient temperatures. However, transcript and protein accumulation of CO were largely independent of changes in ambient temperature ( Fig. S4 and S5). In addition, it has been previously demonstrated that misexpression of CO do not influence temperature sensitivity in floral transition 4,25 , suggesting that CO is not a component of the thermosensory pathway.
COP1 is also involved in the regulation of GI stability along with ELF3 21 . We therefore investigated whether COP1 regulates protein turnover of GI at low temperatures. To this end, we employed the 35S:GI-HA transgenic plants 26 grown at different ambient temperatures to monitor GI accumulation. The stability of GI protein was markedly reduced at 16 °C compared with that at 23 °C (Fig. 4a), and the reduced protein accumulation was recovered by treatment with MG132, a potent inhibitor of the 26S proteasome (Fig. 4b), suggesting that an ubiquitination process underlies temperature-dependent GI accumulation. To further support a role of COP1 in GI turnover at low temperature, the 35S:GI-HA transgenic plants were crossed with cop1-6 mutant. In the 35S:GI-HA cop1-6 plants, GI accumulation was elevated compared with that of 35S:GI-HA plants and was not sensitive to changes in ambient temperature (Fig. 4c), indicating that COP1 is an unequivocal regulator of GI turnover at low temperature.
The ELF3 adaptor protein is also proteolytically degraded by COP1 in order to control GI turnover 21 . However, proteolysis of ELF3 was not increased at low temperatures ( Fig. S6), although it appeared that ELF3 is required for the proper response to changes in ambient temperature ( Fig. S7 and S8) 7 . Different molecular mechanisms likely underlie regulation of ELF3 activity at different ambient temperatures (see Discussion).
GI binds directly to the FT promoter 27 . Thus, we investigated whether the binding of GI to the FT promoter is regulated by ambient temperature. The 35S:GI-HA transgenic plants were grown at 23 °C and 16 °C . Total protein extracts from the control and 35S:GI-HA transgenic plants were immuno-precipitated with an anti-HA antibody. DNA bound to epitope-tagged GI proteins was analyzed by quantitative PCR (qPCR) assays. The ChIP analysis showed that the FT promoter was enriched by GI (Fig. 5a) as previously reported 27 . However, the GI association with the FT promoter was substantially reduced at low temperature, possibly due to reduced stability of GI protein (Fig. 5a). The reduced GI binding to the FT promoter at low temperature was impaired in cop1-6 mutant (Fig. 5b). To further convince this observation, we analyzed FT expression in gi-2 mutant at low ambient temperature. While FT expression was substantially reduced at 16 °C in wild-type background, constitutively reduced FT expression was observed in gi-2 mutant regardless of ambient temperatures (Fig. 5c), indicating the significant role of COP1-GI module in thermosensory flowering.
To confirm the genetic relationship between GI and COP1, we crossed between gi-2 and cop1-6 mutants. Under normal growth conditions, the flowering time of gi-2 cop1-6 mutant was comparable to that of gi-2 mutant (Fig. 6a). Furthermore, the temperature responsiveness of gi-2 cop1-6 and gi-2 mutants was also comparable, indicating that they act on the same genetic pathway.
Taken together, COP1 is a central regulator that integrates photoperiod and temperature signaling. In addition to its photoperiodic regulation of CO, COP1 also regulates temperature-dependent GI stability (Fig. 6b). At low temperature, COP1 is stabilized and triggers GI degradation even in inductive LD conditions. COP1 stability regulated by both photoperiod and ambient temperature facilitates to make integrative decisions for determining timing to flower, and the conditional specificity of COP1 to its targets ensures proper developmental transitions in plants.

Discussion
Controlled protein turnover and temperature signaling. Covalent attachment of ubiquitin proteins is a common means of modulating protein stability. The ubiquitination process is mediated by the sequential action of three enzymes: ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). The Arabidopsis genome encodes 2 E1, > 37 E2, and > 1,500 E3 enzymes 28,29 . Consistent with the large number of enzymes in the ubiquitination process, they are To minimize secondary effects caused by growth rate difference, 10-day-old seedlings grown at 16 °C or 23 °C under LDs were transferred to different ambient temperatures and incubated for 2 days. (b) Accumulation of COP1 at 16 °C in light and dark. Twelve-dayold seedlings grown under LDs were harvested at ZT20 or ZT4. COP1-TAP proteins (arrowheads) were detected immunologically using an anti-MYC antibody. Low molecular bands (arrows) might be degradation products. A part of Coomassie blue-stained gel (C) is shown as a loading control. Bands from three blots were quantified using Image J software and averaged (right panels).
involved in diverse aspects of plant growth and development including cell cycle progression, hormone responses, and plant responses to biotic and abiotic stresses 30-32 . Although no E3 ligases involved in thermosensory flowering have been identified to date, controlled protein turnover is likely crucial for temperature-dependent floral transition. For example, protein turnover of SVP is accelerated at high temperature, and SVP degradation is dependent on the 26S proteasome activity 15 .
In this study, we report that COP1 is implicated in ambient temperature-dependent floral transition. COP1 is more stabilized at low temperature and subsequently accelerates protein turnover of GI, which in turn down-regulates FT expression to determine proper timing to flower 27 . Consistently, cop1 and gi mutants are almost insensitive to changes in ambient temperatures.
It seems likely that GI and SVP antagonistically regulate FT expression by binding to the FT promoter depending on ambient temperatures. At low temperatures, SVP protein is stabilized, while GI is degraded by a COP1-mediated ubiquitination process. At high temperatures, a reverse pattern would be relevant for early flowering, whereby GI would be stabilized with reduced SVP activity. Collectively, balanced transcriptional regulation of FT is achieved by coordinated control of the transcriptional activator GI and repressor SVP, and temperature-dependent protein turnover is a key molecular mechanism underlying reestablishment of balanced accumulation of these proteins.
Integration of photoperiod and temperature information. Ambient temperature and photoperiod pathways are closely intertwined. Late flowering in short days can be overcome by growing at high temperature 17 . In contrast, long day-triggered early flowering is compromised at low temperature 4 . Furthermore, these two genetic pathways are largely dependent on the flowering pathway integrator FT 7,23 , suggesting the presence of intensive crosstalk.
Our results suggest that regulation of COP1 stability is at the center of this crosstalk. COP1 is a well-known E3 ligase that participates in photoperiod flowering, which is particularly stabilized during night and rapidly degrades accumulated CO 19 . This study provides evidence that COP1 is also involved in thermosensory flowering. At low temperature, COP1 is more stabilized and down-regulates GI stability. Since GI directly binds to the FT promoter 27 , the GI abundance is closely associated with transcript accumulation of FT in a CO-independent manner. Notably, increased stability of COP1 was also relevant with light period, thereby serving to integrate photoperiod and ambient temperature signaling. (c) Low temperature-triggered GI turnover in cop1-6. Bands from three blots were quantified using Image J software and averaged. Statistical significance of the measurements was determined by a Student's t-test (*P < 0.05) or one-way anova with Fisher's post hoc test (P < 0.05). The numbers above the bars indicate the ratio of expression at 16 °C and 23 °C (16 °C /23 °C ).
At low temperatures, protein stability of ELF3 is largely unaffected despite the enhanced activity of COP1 (Fig. S6). It seems contrary to the previous observation that COP1 induces proteolysis of ELF3 concomitant with GI degradation 21 . However, these results may suggest that ELF3 is either necessary but not sufficient for COP1-mediated ubiquitination of GI, or that ELF3 is not a rate-limiting factor for the activity of COP1.
It is known that CO is not associated with thermosensory flowering 4,25 . Consistently, we also showed that protein turnover of CO is not responsive to changes in ambient temperatures. However, it is currently Collectively, we show that COP1 perceives light and temperature signals at the level of protein accumulation. At normal growth temperature, COP1 primarily induces CO degradation during night-time. At low temperature, COP1 is substantially stabilized under both light and dark conditions, triggering GI turnover without affecting CO stability. These results demonstrate that COP1 acts as a central player in the integration of ambient temperature and photoperiod signaling and ensures proper floral transition to minimize reproductive failure.
Flowering time was measured by counting the total number of rosette leaves at flowering initiation. A total of 40 to 50 plants were measured and averaged for each plant group. Statistical significance was determined using Student's t-test (**P < 0.01; *P < 0.05).
Quantitative real-time RT-PCR analysis. Total RNA was extracted using TRI agent (Takara Bio, Singa, Japan) according to the manufacturer's recommendations. Reverse transcription (RT) was performed using Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Dr. Protein, Seoul, South Korea) with oligo(dT18) to synthesize first-strand cDNA from 2 μ g of total RNA. Total RNA samples were pretreated with an RNAse-free DNAse. cDNAs were diluted to 100 μ L with TE buffer, and 1 μ L of diluted cDNA was used for PCR amplification.
Quantitative RT-PCR reactions were performed in 96-well blocks using the Step-One Plus Real-Time PCR System (Applied Biosystems). The PCR primers used are listed in Table S1. The values for each set of primers were normalized relative to the EUKARYOTIC TRANSLATION INITIATION FACTOR 4A1 (eIF4A) gene (At3g13920). All RT-qPCR reactions were performed in three technical replicates using total RNA samples extracted from three independent biological replicate samples. The comparative Δ Δ C T method was employed to evaluate relative quantities of each amplified product in the samples. The threshold cycle (C T ) was automatically determined for each reaction by the system set with default parameters. The specificity of the RT-qPCR reactions was determined by melt curve analysis of the amplified products. A melting curve was constructed by measuring the fluorescence continuously when heating from 60 to 95 °C at the rate of 0.5 °C /sec. Immunoblot analysis. Harvested plant materials were ground in liquid nitrogen, and total cellular extracts were suspended in SDS-PAGE sample loading buffer. The protein samples were then analyzed by SDS/PAGE (10% gels) and blotted on to Hybond-P+ membranes (Amersham-Pharmacia). The epitope-tagged proteins were immunologically detected using anti-MYC or anti-HA antibodies (Millipore, Billerica, MA, USA).

Chromatin immunoprecipitation (ChIP).
ChIP assays were performed as previously described 35 .
Briefly, epitope-tagged transgenic plants, anti-MYC and anti-HA antibodies (Millipore) and salmon sperm DNA/protein A agarose beads (Millipore) were used for chromatin immunoprecipitation. DNA was purified using phenol/chloroform/isoamyl alcohol and sodium acetate (pH 5.2). The level of precipitated DNA fragments was quantified by quantitative real-time PCR using specific primer sets (Table S2). Values were normalized according to input DNA level. Values for control plants were set to 1 after normalization against eIF4a for quantitative PCR analysis.