COLD-REGULATED GENE 27 Integrates Signals from Light and the Circadian Clock to Promote Hypocotyl Growth in Arabidopsis

One-sentence summary: COR27 inhibits the biochemical activity of HY5, and up-regulates PIF4 expression especially in the afternoon to promote hypocotyl elongation. ABSTRACT Light and the circadian clock are two essential external and internal cues affecting seedling development. COLD-REGULATED GENE 27 (COR27), which is regulated by cold temperatures and light signals, functions as a key regulator of the circadian clock. Here, we report that COR27 acts as a negative regulator of light signaling. COR27 physically interacts with the CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1)-SUPPRESSOR OF PHYTOCHROME A 1 (SPA1) E3 ubiquitin ligase complex, and undergoes COP1-mediated degradation via the 26S proteasome system in the dark. cor27 mutant seedlings exhibit shorter hypocotyls, while transgenic lines overexpressing COR27 show elongated hypocotyls in the light. In addition, light induces the accumulation of COR27. On one hand, accumulated COR27 interacts with ELONGATED HYPOCOTYL 5 (HY5) to repress HY5 DNA binding activity. On the other hand, COR27 associates with the chromatin at the PHYTOCHROME INTERACTING FACTOR 4 (PIF4) promoter region and up-regulates PIF4 expression in a circadian clock-dependent manner. Together, our findings reveal a mechanistic framework thereby COR27 represses photomorphogenesis in the light and provide insights toward how light and the circadian clock synergistically control hypocotyl growth.


INTRODUCTION
In nature, a seed germinating in the soil will first undergo skotomorphogenesis (seedling development in the dark), which is characterized by an elongated hypocotyl, a closed apical hook and small, unfolded cotyledons to provide little resistance against soil particles, until the seedling reaches the soil surface. Upon light exposure, seedlings then switch to the light-mediated developmental program known as photomorphogenesis. These two distinct developmental processes enable the proper and healthy development of a young seedling (Jiao et al., 2007;Song et al., 2020).
During the transition from the dark-to the light-driven developmental process, the expression of approximately one-third of genes throughout the Arabidopsis (Arabidopsis thaliana) genome is significantly altered, indicating that light orchestrates massive transcriptomic reprogramming in plants (Ma et al., 2001).
COP1 not only represses photomorphogenesis but also mediates diverse cellular and physiological development responses, including the control of light input to the circadian clock (Huang et al., 2014). COP1 controls the period length of circadian clock-mediated gene expression at the transcriptional level (Millar et al., 1995) and promotes the degradation of clock or clock-regulated components such as EARLY FLOWERING 3 (ELF3), GIGANTEA (GI) and CONSTANS (CO) during the night at the post-translational level (Yu et al., 2008;Jang et al., 2008). Similar to light, the circadian clock also mediates hypocotyl elongation. Multiple circadian clock components, such as CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1 (TOC1), ELF3, ELF4 and LUX ARRHYTHMO (LUX) participate in the regulation of hypocotyl growth (Schaffer et al., 1998;Wang and Tobin, 1998;Más et al., 2003;Nusinow et al., 2011), suggesting that the circadian clock and light signaling synergistically mediate seedling development after seed germination. All these morning-or evening-phased central clock components control seedling hypocotyl growth by regulating the bHLH-type transcription factor PHYTOCHROME INTERACTING FACTOR 4 (PIF4) at the transcriptional and/or protein levels under diurnal conditions (Niwa et al., 2009;Nusinow et al., 2011;Zhu et al., 2016). PIF4 promotes hypocotyl elongation mainly by regulating downstream auxin signaling, which promotes cell elongation (Sun et al., 2013). In addition to the circadian clock, multiple signaling cascades (phytochromes, cryptochromes, UV-B, low and high ambient temperature) converge on PIF4 and the PIF4-mediated auxin signal transduction pathway to control hypocotyl growth (Huq and Quail, 2002;Ma et al., 2016;Pedmale et al., 2016;Jung et al., 2016;Legris et al., 2016;Hayes et al., 2017;Dong et al., 2020;Jiang et al., 2020;Yan et al., 2020). Thus, PIF4 functions as a central hub, acting downstream of multiple signaling pathways, to gate seedling growth.
COLD-REGULATED GENE 27 (COR27), which is regulated by both low temperatures and light signals, acts as a night-time repressor of the circadian clock (Li et al., 2016;Wang et al., 2017). The CCA1-LHY morning complex associates with the COR27 promoter and regulates its rhythmic expression. COR27 binds to TOC1 and PSEUDO-RESPONSE REGULATOR 5 (PRR5) chromatin to repress their transcription in the afternoon and at night (Li et al., 2016). In this study, we show that the COP1-SPA1 complex interacts with COR27 and promotes its proteolysis via the 26S proteasome system in the dark. COR27 negatively regulates photomorphogenic development in the light. Our biochemical studies reveal that light-induced COR27 not only negatively modulates HY5 activity but also binds to the promoter regions of PIF4 and up-regulates its transcription to maintain its proper expression in the afternoon. Our study thus provides a mechanistic framework for COR27 in controlling the biochemical activity of HY5 and circadian clock-regulated PIF4 transcription in repressing seedling development.

COR27 Physically Interacts with the COP1-SPA1 Complex
COP1 is a central repressor of light signaling (Deng et al., 1991;1992;Huang et al., 2014;Han et al., 2020). To identify novel components of light signaling, we carried out a yeast two-hybrid screen using COP1 as a bait. This screen identified COR27 as a COP1-interacting protein in yeast cells (Figures 1A and 1B). Further domain mapping analysis revealed that the C-terminal region (COR27-C, 121-246), but not the Nterminal half (COR27-N, 1-120), of COR27 interacted with COP1 ( Figures 1A and 1B).
We continued to perform yeast two-hybrid assays to identify the domain within COP1 that was responsible for interaction with COR27. None of the COP1 truncation fragments interacted with COR27, COR27-N or COR27-C ( Figures 1A and 1B). These data suggest that the overall structure of COP1 and the C-terminal portion of COR27 are required for their protein-protein interaction.
COP1 and SPA form stable core complexes in plants (Zhu et al., 2008). We thus tested whether COR27 interacts with SPA proteins (SPA1-SPA4) by yeast two-hybrid assays. COR27 specifically interacted with SPA1, but not with SPA2, SPA3 or SPA4 ( Figure 1C). COR27-N, but not COR27-C, interacted with SPA1 ( Figure 1C), suggesting that the N-terminal of COR27 is required for its association with SPA1.
To verify the interaction between COR27 and the COP1-SPA1 complex, we employed a bimolecular fluorescence complementation (BiFC) assay and fused COR27 with a split N-terminal of Yellow Fluorescent Protein (YFP) (YFP N ), COP1 or SPA1 with a split C-terminal of YFP (YFP C ). We observed strong YFP signals when we transiently co-expressed COR27-YFP N and COP1-YFP C , or COR27-YFP N and SPA1-YFP C in Nicotiana benthamiana leaves. The negative controls did not produce any detectable YFP signal ( Figure 1E). Next, we performed in vitro pull-down assays using recombinant His-COR27 and Maltose Binding Protein (MBP)-COP1. His-COR27 successfully pulled down MBP-COP1, but not negative control MBP ( Figure 1F), suggesting that COR27 interacts with COP1 in vitro. To verify the COR27 and COP1-SPA1 interaction in vivo, we further performed co-immunoprecipitation (Co-IP) assays using YFP-COR27 transgenic seedlings or N. benthamiana leaves transiently co-  Figure 1G). Similarly, YFP-COR27 transiently co-expressed in N. benthamiana leaf cells, together with Flag-SPA1 or YFP-GST, immunoprecipitated Flag-SPA1, but not YFP-GST (negative control) ( Figure 1H). Together, these data firmly demonstrate that COR27 associates with the COP1-SPA1 complex in planta.

COR27 Undergoes COP1-Mediated Degradation in the Dark
COR27 is induced by blue light and accumulates during the day but decreases in the night under long-day (LD) conditions (Li et al., 2016). In agreement, YFP-COR27 transgenic seedlings grown in white light accumulated markedly more YFP-COR27 compared to those grown in the dark (Figure 2A). YFP-COR27 abundance gradually decreased in white light-grown YFP-COR27 transgenic seedlings upon transfer to darkness at the indicated time points (0, 1, 3, 6, 12 and 24 h) ( Figure 2B), suggesting that COR27 is subjected to degradation in the dark. As an E3 ubiquitin ligase complex, COP1-SPA1 ubiquitinates a number of downstream substrates and promotes their degradation through the 26S proteasome system in the dark (Huang et al., 2014;Hoecker, 2017). Since COP1-SPA1 interacts with COR27, which becomes degraded in the dark (Figures 1 and 2A and 2B), we thus examined whether COR27 degradation depended on the 26S proteasome system and COP1. Treating YFP-COR27 seedlings with the proteasome inhibitor MG132 clearly stabilized YFP-COR27 in dark-grown seedlings ( Figure 2C), demonstrating that COR27 is indeed subjected to 26S proteasome system-mediated degradation. Next, we introduced the cop1-4 and cop1-6 mutations into YFP-COR27 transgenic lines by genetic crossing. Immunoblot assays revealed that loss of COP1 function led to the accumulation of YFP-COR27 in darkgrown seedlings ( Figure 2D). Consistent with these results, we detected significantly stronger YFP signal in YFP-COR27 cop1-4 and YFP-COR27 cop1-6 seedlings when compard to YFP-COR27 transgenic lines grown in the dark ( Figures 2E and 2F). Taken together, these results suggest that COP1 promotes the degradation of COR27 via the 26S proteasome system in etiolated seedlings.

COR27 Acts as a Negative Regulator of Light Signaling
To explore the role of COR27 in light signaling, we identified a T-DNA insertion mutant (namely, cor27-3) with much lower COR27 expression. We also generated two additional independent cor27 mutant lines (namely, cor27-4 and cor27-5) by Clustered To verify these results, we carried out a BiFC assay using N. benthamiana leaves. We clearly detected strong YFP signal when transiently co-expressing both COR27-YFP N and HY5-YFP C ( Figure 5C). However, the negative controls (COR27-YFP N and YFP C and YFP N and HY5-YFP C ) did not produce any YFP signal ( Figure   5C). We further employed a Co-IP assay using transgenic plants co-expressing YFP-COR27 and HA-HY5 or YFP-GST and HA-HY5. YFP-COR27, but not YFP-GST, immunoprecipitated the HA-HY5 protein in N. benthamiana leaves ( Figure 5D).
Together, these data suggest that COR27 physically interacts with HY5.

COR27 Represses HY5 Biochemical Activity
To assess the biological significance of the COR27-HY5 interaction, we tested whether COR27 affects the biochemical activity of HY5. GST-HY5, but not GST (negative control), was able to bind to the biotin-labeled FHY1 and CHS promoter sub-fragments in EMSA assays, which is consistent with previous studies ( Lin et al., 2018).
Although COR27 alone did not affect FHY1pro:LUC and CHSprpo:LUC expression, the activation of HY5 on these two reporters markedly decreased in the same experimental system when HY5 and COR27 were transiently co-expressed ( Figures 6E to 6G). The transcript and protein levels of HY5 were comparable in Col-0, cor27-3 and myc-COR27 (Supplemental Figure 5), suggesting that COR27 may not affect HY5 at either the transcriptional or protein levels in Arabidopsis. Together, these data suggest that COR27 can repress the binding of HY5 to its target sites, thereby interfering with its transcriptional activity toward target genes.
Next, we examined the genetic relationship between COR27 and HY5. cor27-3 displayed shorter hypocotyls, whereas hy5-215 exhibited dramatically elongated hypocotyls. The hypocotyl length of cor27-3 hy5-215 double mutant seedlings was longer than that of Col-0 and cor27-3, but was slightly shorter than that of hy5-215

COR27 Associates with the PIF4 Promoter and Up-Regulates its Transcription
Considering 19, IAA29 and YUCCA8 (YUC8) (PIF4-regulated genes) showed reduced transcript levels in cor27-3 but higher levels in transgenic seedlings over-expressing myc-COR27 ( Figures 8D to 8G). When tested in pif4-2 and cor27-3 pif4-2 mutants, the expression of these genes reached comparable that were much lower than those seen in Col-0. In addition, their transcript levels in myc-COR27 pif4-2 seedlings were clearly reduced compared with those measured in myc-COR27 seedlings ( Figures 8D to 8G). These findings suggest that COR27 positively controls PIF4 and PIF4-controlled genes.
COR27 is a transcriptional regulator and associates with the chromatin of PRR5 and TOC1 to repress their expression (Li et al., 2016). We thus performed chromatin immunoprecipitation (ChIP)-qPCR analysis to test whether COR27 associates with the genomic region of PIF4. As shown in Figures 8B and 8C, COR27 did bind to the PIF4 promoter regions in vivo. Together, these data indicate that COR27 associates with the PIF4 promoter and up-regulates its transcription.

COR27 Genetically Acts Upstream of PIF4
Our biochemical assays indicate that COR27 acts upstream of PIF4; thus, a mutation in PIF4 should be epistatic to a loss of COR27 function. To this end, we examined the genetic link between COR27 and PIF4. pif4-2 had shortened hypocotyls under white and red light conditions, which is consistent with a previous study (Huq and Quail, 2002). cor27-3 showed shorter hypocotyls than Col-0 but significantly longer hypocotyls than pif4-2 ( Figures 9A to 9D). The hypocotyl length of the cor27-3 pif4-2 double mutant was indistinguishable from that of pif4-2 ( Figures 9A to 9D), indicating that COR27 acts upstream of PIF4 with respect to hypocotyl growth. Moreover, the hypocotyl length of GFP-PIF4 cor27-3 seedlings was similar to that of GFP-PIF4 seedlings, suggesting that the function of COR27 is dependent on PIF4 in the regulation of hypocotyl growth. Myc-COR27 pif4-2 seedlings had significantly shorter hypocotyls than myc-COR27 seedlings, both however markedly longer than the cor27-3, pif4-2 and cor27-3 pif4-2 genotypes ( Figures 9A to 9D). The long hypocotyl length of myc-COR27 and myc-COR27 pif4-2 might be caused by ectopic over-expression of myc-COR27, thereby promoting hypocotyl elongation independently of PIF4.

DISCUSSION
Extensive studies have documented that numerous components of light signaling and the circadian clock synergistically contribute to control hypocotyl growth, demonstrating that external light signals and internal circadian rhythms functionally work in concert to regulate seedling development (Schaffer et al., 1998;Wang and Tobin, 1998;Más et al., 2003;Nusinow et al., 2011). COR27 has been reported to play pleotropic roles in the circadian clock, photoperiodic flowering and freezing tolerance (Mikkelsen and Thomashow, 2009;Li et al., 2016;Wang et al., 2017). In this study, we show that COR27 acts as a negative regulator of light signaling. COR27 is degraded in a COP1-dependent manner via the 26S  Collectively, COR27 is regulated by light, the circadian clock and low temperatures. The COP1-SPA1 E3 ubiquitin ligase complex promotes the degradation of COR27 via the 26S proteasome system at night. Light-accumulated COR27, on the one hand, interacts with HY5 and inhibits its action; on the other hand, it binds to the chromatin of the PIF4 locus, which subsequently leads to an increase in PIF4 transcription in the afternoon. Consequently, these molecular regulatory events serve to promote hypocotyl elongation in plants ( Figure 10).

Transgenic plants
We introduced the binary constructs pEarleyGateway-myc-COR27 and pEarleyGateway-YFP-COR27 into Agrobacterium strain GV3101 by the freeze-thaw method. We transformed Arabidopsis wild type (Col-0) plants by the floral dip method (Clough and Bent, 1998). Transgenic plants were selected on MS medium containing 20 mg/L Basta.

Hypocotyl Length Measurements
For hypocotyl length measurements, we sowed surface-sterilized seeds sown on MS medium before stratification at 4°C in the dark for 3 d. To induce uniform germination, we exposed seeds to white light for 8 h at 22°C. We then transferred all seeds into constant darkness, white, blue, red, or far-red light conditions in LED growth chambers (Percival Scientific) at 22°C. 4-d old seedlings were photographed using a camera (Canon, EOS80D), and hypocotyl lengths were measured by using ImageJ software.

Yeast One-Hybrid (Y1H) and Yeast Two-Hybrid (Y2H) Assays
We used the constructs FHY1pro: LacZ (Li et al., 2010) and BBX31pro:LacZ (Heng et al., 2019) in yeast one hybrid assays. We co-transformed the respective combinations of AD-fusion vectors and LacZ reporters into yeast strain EGY48. We selected and grew transformants on SD/-Trp-Ura dropout medium according to the Yeast Protocols Handbook (Clontech). We used a liquid assay protocol to measure yeast colony β-galactosidase activity (Thermo Fisher Scientific).
To confirm protein-protein interactions, we co-transformed the respective combinations of bait and prey constructs into yeast strain Y2H Gold (Clontech). We also co-transformed the empty pGADT7 and pGBKT7 vectors in parallel as negative controls. After growth on SD/-Trp-Leu and SD/-Trp-Leu-His-Ade dropout medium, we tested protein-protein interactions using selective SD/-Trp-Leu-His-Ade dropout medium supplied with X-α-Gal and Aureobasidin A (AbA, Clontech). We checked for interaction after 3 d of incubation at 30°C. We performed all yeast transformations as described in the Yeast Protocols Handbook (Clontech). To detect COR27 interactions with SPA or HY5 proteins, we performed yeast two hybrid assays using the Matchmaker LexA Two-Hybrid System (Clontech). We co-transformed the respective combinations of pLexA and pB42AD fusion plasmids into yeast strain EGY48 containing p8op-LacZ plasmid. We co-transformed the empty pLexA and pB42AD vectors in parallel as negative controls. We selected and grew transformants on SD/-His-Trp-Ura dropout medium at 30℃. We grew transformants on SD/-His-Trp-Ura dropout medium containing 80 mg/L X-gal for blue color development.

BiFC assays
We generated the constructs pSPYNE-35S-COR27, pSPYCE-35S-COP1, pSPYCE-35S-SPA1 and pSPYCE-35S-HY5 as described above and introduced them into Agrobacterium strain GV3101. We grew the resulting colonies overnight in LB medium at 28ºC, pelleted the cells by centrifugation and resuspended the pellet in infiltration buffer (10 mM MgCl2, 150 mM Acetosyringone, 10 mM MES PH 5.6) to a final cell density equivalent to OD600=0.6. We infiltrated the cell suspensions containing the indicated transformant pairs into N. benthamiana leaves. We detected YFP fluorescence using a Carl Zeiss confocal laser scanning microscope (LSM510 Meta) 24 h after infiltration.

Co-immunoprecipitation assays (Co-IP)
For co-IP assays, we collected 4-d-old dark-grown Col-0 and 35S:YFP-COR27 seedlings and then ground them to a fine powder in liquid nitrogen. We added 400 µL protein extraction buffer (containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM and 100 mM NaCl, immediately neutralized by 2 M Tris-HCl, pH 9.0, and 100 mM NaCl, and concentrated using Strata Clean Resin (Stratagene) prior to immunoblot analysis.

In vitro Pull-Down Assays
For in vitro pull-down assays, we cloned the full-length CDS sequence of COR27 in the pET28a vector and introduced the construct into E. coli strain BL21 to produce recombinant COR27. We mixed 1 μg purified His-COR27 fusion protein with 20 μL His beads in His binding buffer (20 mM Tris-HCl pH 7.5, 250 mM NaCl, 10% glycerol, and 1 mM PMSF). We incubated the mixtures at 4°C for 3 h. We then added 1 μg purified Maltose Binding Protein (MBP), or MBP-COP1 fusion protein to the mixtures before an additional incubation of 1 h at 4°C. After four washes with binding buffer, we boiled the pellet fraction with 5× SDS protein loading buffer. We detected input and pull-down prey proteins by immunoblot analysis using anti-His (1:5,000 (v/v), Sigma-Aldrich, #H1029-2ML) and anti-MBP (1:5,000 (v/v), New England Biolabs, #E8031S) monoclonal antibodies.

Immunoblot Analysis
For immunoblot analysis, we collected wild-type or mutant Arabidopsis seedlings and extracted total proteins using protein extraction buffer (100 mM NaH2PO4, 10 mM Tween-20 (PBST) for 10 min, we incubated the membrane with secondary antibody (1:10,000 (v/v), Sigma-Aldrich, #A0545) for 1 h at room temperature. After three washes with PBST for 10 min again, we exposed the film using a Bio-Rad illumination detection device through ECL prime Western Blotting detection reagent (GE Healthcare, #RPN 2232).

Electrophoretic Mobility Shift Assay (EMSA)
We cloned the full-length HY5 CDS sequence into the pGEX-4T vector and introduced the resulting construct into E. coli strain BL21 (Invitrogen) to produce recombinant HY5 protein. Next, we used biotin-labeled probes and a Light Shift Chemiluminescent EMSA kit for EMSA (Thermo Fisher Scientific) as described previously (Xu et al., 2016;Lin et al., 2018). The promoter sub-fragments of FHY1 (143 bp, -280 to -138 bp) and CHS (150 bp, -547 to -398 bp) upstream of ATG were amplified by PCR. For biotin labeling, we mixed these purified PCR products with biotin and incubated them under UV light for 30 min. We then incubated purified His-COR27, GST-HY5, or GST proteins as indicated, together with 40 fmol biotin-labeled probes in a 20 μL reaction mixture at 25°C for 20 min, followed by separation on 6% native polyacrylamide gels in 0.5× Tris Borate EDTA (TBE) buffer. We electroblotted the resolved proteins onto Hybond N + (Millipore) nylon membranes in 0.5× TBE for 40 min, and detected the labeled probes. The probes used in this study are listed in Supplemental Data Set 1.

Dual-Luciferase Reporter System
We PCR-amplified promoter sub-fragments for FHY1 and CHS and cloned them into the pGreenII 0800-LUC vector (Hellens et al., 2005)

Quantitative RT-PCR and Semi-Quantitative PCR Analysis
We extracted total RNA from Arabidopsis samples using the RNeasy Plant Minikit (Qiagen), and synthesized first-strand cDNAs from 2 μg total RNA using the 5× All-In-One RT Master Mix cDNA synthesis system (Applied Biological Materials) according to manufacturer's instructions. For qPCR, we performed all reactions with SYBR Green PCR Master Mix (Takara) in a 20 μL reaction mixture and run on the StepOnePlus Real-time PCR detection system (Applied Biosystems) following the manufacturer's instructions. We used the Arabidopsis housekeeping gene PROTEIN PHOSPHATASE 2A (PP2A) as a reference gene. All assays for target genes were conducted with three biological repeats, each with three technical repeats. The quantification of threshold cycle (CT) value analysis was achieved using the 2(-ΔCT) method. For semi-quantitative qPCR, cDNA was combined with PCR Mix (Takara, #R040A). The PCR products were loaded onto a 1% agarose gel for electrophoresis.
The primers used in this study are listed in Supplemental Data Set 1.

Statistical Analysis
Statistical

AUTHOR CONTRIBUTIONS
W.Z., H.Z., F.L., X.Z., and D.X performed the research. D.X., and X.W.D designed the project, analyzed the data and wrote the article.