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. 2020 Jul 30;184(2):1097–1111. doi: 10.1104/pp.20.00439

The Histone-Modifying Complex PWR/HOS15/HD2C Epigenetically Regulates Cold Tolerance1,[OPEN]

Chae Jin Lim a,b,2, Junghoon Park a,b,2, Mingzhe Shen c, Hee Jin Park a,b, Mi Sun Cheong c, Ki Suk Park b, Dongwon Baek c, Min Jae Bae b, Ahktar Ali a,b, Masood Jan a,b, Sang Yeol Lee c, Byeong-ha Lee d, Woe-Yeon Kim c, Jose M Pardo e, Dea-Jin Yun b,3,4
PMCID: PMC7536694  PMID: 32732349

A histone-modifying complex regulates the expression of cold-responsive genes and the freezing tolerance of plants.

Abstract

Cold stress is a major environmental stress that severely affects plant growth and crop productivity. Arabidopsis (Arabidopsis thaliana) HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE15 (HOS15) is a substrate receptor of the CULLIN4-based CLR4 ubiquitin E3 ligase complex, which epigenetically regulates cold tolerance by degrading HISTONE DEACETYLASE2C (HD2C) to switch from repressive to permissive chromatin structure in response to cold stress. In this study, we characterized a HOS15-binding protein, POWERDRESS (PWR), and analyzed its function in the cold stress response. PWR loss-of-function plants (pwr) showed lower expression of cold-regulated (COR) genes and sensitivity to freezing. PWR interacts with HD2C through HOS15, and cold-induced HD2C degradation by HOS15 is diminished in the pwr mutant. The association of HOS15 and HD2C to promoters of cold-responsive COR genes was dependent on PWR. Consistent with these observations, the high acetylation levels of histone H3 by cold-induced and HOS15-mediated HD2C degradation were significantly reduced in pwr under cold stress. PWR also interacts with C-repeat element-binding factor transcription factors to modulate their cold-induced binding to the promoter of COR genes. Collectively, our data signify that the PWR-HOS15-HD2C histone-modifying complex regulates the expression of COR genes and the freezing tolerance of plants.

Cold stress is one of the major environmental factors that seriously limits the growth and productivity of plants. To overcome this constraint, plants have developed effective ways to increase resistance to cold stress and freezing. Cold acclimation is a process that increases freezing tolerance upon exposure to low but nonfreezing temperatures. This process involves the activation or expression of cold-regulated (COR) genes and consequent physiological and biochemical changes in response to cold stress (Guo et al., 2018; Ding et al., 2019). Over the past two decades, various effectors and regulators of stress signaling pathways have been identified (Guo et al., 2018; Liu et al., 2018b; Zhang et al., 2019; Tang et al., 2020). One of the best-characterized mechanisms is the C-REPEAT ELEMENT-BINDING FACTORS (CBFs; also known as DEHYDRATION RESPONSIVE ELEMENT-BINDING proteins [DREB]: CBF1/DREB1B, CBF2/DREB1C, and CBF3/DREB1A) transcription factor-dependent cold signaling pathway (Chinnusamy et al., 2007; Guo et al., 2018; Liu et al., 2018a). These CBF transcription factors are APETALA2-like DNA-binding domain proteins that bind to the conserved C-REPEAT ELEMENT/DEHYDRATION RESPONSIVE ELEMENT (CRT/DRE) on the promoter regions of COR genes, such as COR15A, COR47, and COR78, and induce the expression of these genes, leading to freezing tolerance (Chinnusamy et al., 2007; Guo et al., 2018; Liu et al., 2018a).

Control of gene expression in the cold signaling pathway is also epigenetically regulated through the structural changes of chromatin (Park et al., 2018b; Ding et al., 2019; Chang et al., 2020). Heterochromatin and euchromatin are reversibly interchanged to repress or activate gene expression, respectively (Chen and Tian, 2007). These structural changes in chromatin are regulated by posttranslational modifications of histone, such as histone acetylation, methylation, ubiquitination, sumoylation, and phosphorylation. These modifications are accomplished by covalent modification of the N-terminal tails of core histones (Nathan et al., 2006; Sridhar et al., 2007; Luo and He, 2020). The structural change of chromatin mediated by histone acetylation is reversible and greatly influences the regulation of gene expression (Chen and Tian, 2007; Clapier and Cairns, 2009). Histone acetylation catalyzed by histone acetyltransferases reduces the charge interaction of histone and DNA, leading to exposure of the DNA and facilitated binding of transcription factors (Verbsky and Richards, 2001; Clapier and Cairns, 2009). Histone acetylation enhances gene transcription, whereas histone deacetylation catalyzed by histone deacetylases (HDACs) leads to chromatin compaction and subsequent gene silencing (Chen and Tian, 2007).

Arabidopsis (Arabidopsis thaliana) HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE15 (HOS15) is a WD40-repeat protein that shares high sequence similarity with human (Homo sapiens) TRANSDUCIN-BETA-LIKE1 (TBL1), an intrinsic component of the repressor silencing mediator for retinoic acid receptor and thyroid hormone receptor/nuclear receptor corepressor (SMRT/NCoR) protein complex that is involved in histone acetylation (Zhu et al., 2008). In Arabidopsis, POWERDRESS (PWR) is predicted to be a plant NCoR1 homolog (Wang and Brendel, 2004). Using immunoprecipitation and tandem mass spectrometry analyses, we found that PWR interacts with HOS15 (Park et al., 2018a). Several reports have described that HOS15 functions in a complex with PWR and histone deacetylases (Park et al., 2018a, 2018b; Suzuki et al., 2018; Mayer et al., 2019). In addition, HOS15 has been characterized to regulate the transcription of several genes, including GIGANTEA, a floral promoter in photoperiod-dependent flowering (Park et al., 2019), DA1-RELATED PROTEIN3, a negative regulator of cell proliferation (Suzuki et al., 2018), and WRKY53, a transcription factor involved in leaf senescence (Chen et al., 2016). Moreover, the transcription levels of some target genes of HOS15 were up-regulated in pwr mutants (Chen et al., 2016; Suzuki et al., 2018; Mayer et al., 2019; Park et al., 2019). Besides this role as a corepressor with PWR and histone deacetylases, HOS15 also functions as a substrate receptor for the CULLIN4 (CUL4)-based ubiquitin E3 ligase complex, CRL4. In response to cold stress, HOS15 mediates the degradation of HISTONE DEACETYLASE2C (HD2C) and switches the chromatin structure from repressive to permissive form, thereby acting as a positive regulator of cold stress (Park et al., 2018b). This facilitates the recruitment of CBFs for the expression of COR genes and the development of cold tolerance (Park et al., 2018b). However, the presumed role of PWR as a component of CRL4 ubiquitin E3 ligase in cold stress signaling has not yet been tested.

In this study, we report that PWR forms a complex with HOS15 and HD2C and that the PWR-HOS15-HD2C complex epigenetically controls freezing tolerance in plants. Indeed, PWR regulates COR gene expression similarly to HOS15 and the loss-of-function pwr mutant phenocopies the hos15 mutant, with no additive effects in the double mutant. Moreover, PWR is required for the binding of HOS15 and HD2C to the COR gene promoters. The cold-induced acetylation of histone H3, facilitated by HOS15-mediated HD2C degradation, is greatly reduced in the pwr mutants. Furthermore, PWR directly interacts with CBFs, and cold-induced binding of CBFs to COR chromatin is dependent on PWR. Collectively, our data provide mechanistic insight into how a histone-modifying PWR-HOS15-HD2C complex and CBFs coregulate cold-responsive COR gene expression to promote freezing tolerance in plants.

RESULTS

PWR and HOS15 Function in the Same Pathway in Response to Cold

PWR is a homolog of the repressor complex protein NCoR1 of mammals that in Arabidopsis interacts with HOS15 (Park et al., 2018a; Suzuki et al., 2018; Mayer et al., 2019). Loss-of-function mutants hos15-1 (C24) and hos15-2 (Columbia-0 [Col-0]) have a freezing-sensitive phenotype (Zhu et al., 2008; Park et al., 2018b). Based on these facts, we hypothesized that PWR, being a HOS15-binding protein, would also be involved in cold stress response. To investigate this hypothesis, pwr-2 (SALK_071811C) and pwr-3 (SALK_006823) homozygote mutants were isolated (Supplemental Fig. S1). Mutant pwr-2 bears a T-DNA inserted at the second exon of PWR, and pwr-3 has a T-DNA inserted at the first intron of PWR (Supplemental Fig. S1A). Both pwr mutants exhibited morphological phenotypes similar to those of the hos15-2 mutant. Similarities included short and blunt-ended siliques, small plant size, shorter hypocotyl lengths, and early-flowering phenotypes (Supplemental Fig. S1, D–G; Yumul et al., 2013; Kim et al., 2016). These common morphological phenotypes support the possibility that PWR and HOS15 proteins function together in the same developmental processes. To observe the phenotypes under freezing stress, 2-week-old seedlings grown on Murashige and Skoog (MS) agar were exposed to freezing temperature before and after cold acclimation (4°C, 7 d). Under both conditions, pwr mutants were sensitive to freezing compared with the wild type (Fig. 1A; Supplemental Fig. S2A). The survival rates were calculated by counting numbers of seedlings from Figure 1A and Supplemental Figure S2A. Survival rates of pwr mutants were significantly lower than those of the wild type (Fig. 1B; Supplemental Fig. S2B). Additionally, the freezing tolerance of plants grown in soil was reduced in the pwr mutants compared with the wild type (Fig. 1C; Supplemental Fig. S2C). Consistent with these freezing-sensitive phenotypes, pwr-2, pwr-3, and hos15-2 mutants displayed substantially higher electrolyte leakage than the wild type at freezing temperatures regardless of cold acclimation conditions (Fig. 1D; Supplemental Fig. S2D). To test the cold stress responses in pwr mutants, we investigated the transcript level of COR genes in pwr-2 and pwr-3 lines. Cold-induced expression of COR genes was significantly decreased in the pwr-2 and pwr-3 mutants compared with the wild type (Fig. 2A). We also examined whether the expression levels of the CBF transcription factors that regulate the expression of cold-responsive genes through direct binding to the promoter regions of COR genes (Chinnusamy et al., 2007) were altered in the pwr mutants. The transcript levels and protein abundance of CBF transcription factors were not changed significantly in the pwr mutants compared with the wild type (Fig. 2). These results suggested that PWR, similarly to HOS15, contributes to cold stress signaling by regulating the expression of the COR genes but not of CBFs. Furthermore, we found that the pwr-3 hos15-2 double mutant exhibited the same phenotype as each single mutant (Supplemental Fig. S3), suggesting that HOS15 and PWR regulate cold stress signaling in the same pathway.

Figure 1.

Figure 1.

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PWR is involved in freezing tolerance. A, Freezing phenotypes of 2-week-old wild-type (WT; Col-0), two pwr mutants (pwr-2 and pwr-3), and hos15-2 (positive control) seedlings grown on MS medium under long-day conditions. Two-week-old plants grown on MS medium plates pretreated with cold (4°C for cold acclimation) for 7 d were exposed to −9°C freezing stress, followed by recovery at 23°C for 4 d. Photographs were taken before (control) and after exposure to the −9°C freezing stress. B, Survival rates of plants were calculated from A. The data are means ± sd (n = 25). Asterisks indicate statistically significant differences from Col-0 by Student’s t test (*P < 0.05 and **P < 0.01). Similar results were obtained in three independent experiments. C, Three-week-old plants grown on soil were cold acclimated at 4°C for 7 d and then exposed to freezing at the indicated temperatures. Photographs were taken after the recovery at 23°C for 5 d. D, Electrolyte leakage assay of acclimated plants was performed at the indicated temperatures. The data are means ± sd of biological replicates (n = 3).

Figure 2.

Figure 2.

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PWR positively regulates the expression of COR genes. A, COR but not CBF gene expression is reduced in pwr-2 and pwr-3 mutants. The transcript levels of cold-induced genes were quantified by reverse transcription quantitative PCR (RT-qPCR) in Col-0 and pwr mutants. cDNA was obtained from 2-week-old plants treated at 4°C for the indicated times. ACTIN2 was used to normalize the expression of COR and CBF genes. Data represent means ± sd from three biological replicates with three technical repeats each. Asterisks indicate statistically significant differences from Col-0 by Student’s t test (**P < 0.01). B, Cold-induced CBF protein accumulation was not significantly changed in the hos15-2, pwr-2, and pwr-2 hos15-2 mutants compared with the wild type (WT). Total proteins were extracted from 2-week-old wild-type (Col-0), hos15-2, pwr-2, and pwr-2 hos15-2 plants treated with cold stress (4°C) for 6 h and applied to immunoblots with anti-CBF antibody cross-reacting with CBF1 to CBF3 (left). The relative band intensity of CBF proteins was calculated from blots of three biological repeats using Image Lab software (right). Values are means ± sd.

PWR Is Involved in HOS15-Mediated Degradation of HD2C

To understand the function of the PWR protein, we compared the sequences of Arabidopsis PWR with those of PWR homologs within plant species and of NCoR1 and NCoR2 of mammals. The Swi3, Ada2, N-Cor, and TFIIIB (SANT) domains and the N-terminal nuclear localization signals (NLS) were highly conserved, but the intervening sequences between these functional domains were relatively variable between proteins (Supplemental Figs. S4A and S5). The SANT domains of the Arabidopsis PWR protein are highly homologous to those of human NCoR1 (Kim et al., 2016). However, NCoR has the SANT domains closer to the N terminus, whereas PWR has them in the C-terminal half of the protein. Outside these conserved domains, alignment analysis of the full amino acid sequence of PWR showed little similarity (13.1% similarity and 7.05% identity) to the NCoR1 protein (Supplemental Fig. S5).

The HOS15 protein acts as a substrate receptor in the CUL4-based E3 ligase complex targeting HD2C in the nucleus for proteasome-dependent degradation (Park et al., 2018b). The NCoR protein, a PWR homolog in animals, is degraded by TBL1, a HOS15 homolog, upon a specific signal by the thyroid hormone (Perissi et al., 2008). Therefore, the amount of PWR protein, a HOS15-interacting protein in plants, was determined in the hos15-2 mutant under cold stress. PWR protein and transcript levels were unchanged by cold stress in the wild type (Supplemental Fig. S6A). Unexpectedly, the amount of PWR protein in the hos15-2 mutant decreased while the transcript level increased (Supplemental Fig. S6B). Thus, we reasoned that PWR binding to HOS15 did not result in PWR degradation but could instead affect the stability of HD2C, which also interacts with HOS15. Hence, we tested whether cold-induced degradation of HD2C protein (Park et al., 2018b) was facilitated by PWR. Even though the transcript level of HD2C was not affected in hos15-2 and pwr mutants, cold-induced degradation of the HD2C protein was impaired in pwr mutants, similarly to the hos15-2 mutant (Fig. 3A). The physical interaction of PWR with HD2C was tested using a split-luciferase (LUC) complementation assay, which is based on the reconstituted LUC activity when two proteins fused with N- and C-terminal LUC fragments (NLuc and CLuc) physically interact in vivo (Chen et al., 2008). Nicotiana benthamiana leaves that were transiently coexpressing CLuc-PWR and HD2C-NLuc displayed luminescence signals above background but not as intense as the positive control (Fig. 3B; Supplemental Fig. S7A). The interaction of PWR with HD2C was further tested in a yeast two-hybrid assay. In contrast to the split-LUC assay (Fig. 3B), the yeast two-hybrid assay did not show an interaction of PWR with HD2C (Fig. 3C). Together, these results suggest that the PWR interaction with HD2C is indirect, probably limited by the amount of adaptor protein(s) of plants with no yeast homologs. In line with this hypothesis, a coimmunoprecipitation (Co-IP) assay in Arabidopsis showed that the interaction of PWR and HD2C in the wild type was diminished in the hos15-2 mutant (Fig. 3D), indicating that HOS15 is required for the interaction of PWR and HD2C. In other words, HOS15 acts as a bridge for HD2C-PWR complex formation. To test whether the interaction between HOS15, HD2C, and PWR was altered by cold stress, the Co-IP assay was repeated after treatment with the proteasome inhibitor MG132 to prevent HD2C protein degradation under cold stress and with cycloheximide (CHX) to prevent de novo protein synthesis. The interaction between HOS15, HD2C, and PWR did not differ significantly with and without cold stress (Fig. 3E). Again, the amount of HD2C coimmunoprecipitated by PWR was substantially reduced in the hos15-2 mutant background compared with the wild type. In these experiments, the amount of preexisting PWR (input) was always lower in the hos15-2 mutant compared with the wild type and the hd2c-1 mutant, regardless of the temperature, suggesting that HOS15 may enhance PWR stability. Consistent with the nuclear localization of HOS15 and HD2C (Park et al., 2018b), GFP-fused PWR was predominantly localized in the nucleus (Supplemental Fig. S8). From these results, PWR appears to regulate HD2C stability through HOS15 rather than by direct interaction with the HD2C protein. This is consistent with the demonstrated role of HOS15 as a substrate receptor for the CUL4-based ubiquitin E3 ligase complex under cold stress (Park et al., 2018b).

Figure 3.

Figure 3.

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Cold-induced degradation of HD2C protein is impaired in hos15-2 and pwr mutants. A, Reduction of HD2C protein levels upon cold stress. Total protein was extracted from 2-week-old Arabidopsis wild-type (WT; Col-0), hos15-2, pwr-2, pwr-3, and hd2c-1 plants exposed to cold (0°C) for 24 h and then separated by SDS-PAGE. The hd2c-1 mutant was used as a control for anti-HD2C antibody specificity. Coomassie Brilliant Blue (CBB) staining was used as a loading control (top). The relative band intensity of HD2C proteins was calculated from blots of three biological repeats using Image Lab software (middle). Asterisks indicate a statistically significant difference from Col-0 by Student’s t test (**P < 0.01). Relative HD2C mRNA levels in pwr-2 and pwr-3 mutants were no different from wild-type plants upon cold stress. HD2C mRNA expression in all tested plants was checked by RT-qPCR analysis (bottom). Data represent means ± sd from three biological replicates with three technical repeats each. B, LUC complementation imaging assay using N. benthamiana leaves. N. benthamiana leaves were coinfiltrated with Agrobacterium tumefaciens strains carrying HD2C-NLuc or NLuc (vector) and CLuc-PWR or CLuc (vector). Bioluminescence was captured using a CCD camera. Leaves expressing CLuc-SGT1b and RAR1-NLuc were used as positive controls. C, Yeast strain PJ69-4A cotransformed with pDEST22 (AD) PWR-pDEST22 (AD), or HOS15-pDEST22 (AD) and pDEST32 (BD) or HD2C-pDEST32 (BD) were spotted on Trp-, Leu-, and His-deficient medium (−TLH). Plates were photographed after incubation at 30°C for 5 d. D, Co-IP assay using 2-week-old Arabidopsis wild-type (Col-0), hos15-2, and hd2c-1 plants. Protein extracts (Input) were immunoprecipitated (IP) with anti-HD2C antibody, and immunoblots were developed with anti-PWR and anti-HD2C antibodies. E, Co-IP assay using 10-d-old Arabidopsis wild-type (Col-0), hos15-2, hd2c-1, and pwr-2 plants. Plants were treated with or without cold (0°C) for 12 h in the presence of CHX (100 µm) and the proteasome inhibitor MG132 (50 µm). Protein extracts (Input) were immunoprecipitated with anti-PWR antibody, and blots were probed with anti-PWR, anti-HD2C, and anti-HOS15 antibodies. The relative intensities of bands in the Co-IP experiments (D and E) were calculated using Image Lab software. Numbers below each band indicate the normalized intensities relative to the protein abundance in the control sample (Col-0) at 22°C, which was assigned the arbitrary value of 1.

PWR Is Required for the Association of HOS15 and HD2C to the Promoter Regions of COR Genes

HD2C and HOS15 indirectly bind to the CRT/DRE region of COR gene promoters to which CBF proteins associate (Park et al., 2018b). When HD2C is degraded by HOS15 in response to cold stress, histones are acetylated to promote COR gene expression for cold tolerance (Park et al., 2018b). To determine the effect of PWR on the association of HD2C and HOS15 with COR gene promoters, we performed chromatin immunoprecipitation (ChIP) assays with two amplicon regions of COR15A. Amplicon COR15A-II (region II) contains the CBF-binding cis-element, and COR15A-I (region I) was used as a mock sequence (Fig. 4A). HD2C appeared to bind the amplicon region II in the wild type (Col-0) at 22°C, and cold treatment reduced the amount of HD2C associated with this region (Fig. 4B; Park et al., 2018b). The association of HD2C to the CBF-binding region in the COR15A promoter was significantly lower in the hos15 and pwr single and double mutants under warm temperature compared with the wild type and was reduced further upon cold stress to reach a low level similar to that of the wild type (Fig. 4B). HOS15 also bound to the same region of the COR15 promoter as HD2C (i.e. COR15A-II), while its association with COR15A-II increased in the cold condition (Fig. 4C). However, the cold-induced association of HOS15 protein to the COR15A-II region was decreased in pwr mutants (Fig. 4C), suggesting that PWR enhances the recruitment of HOS15 to the COR15A promoter under cold, which then results in HD2C degradation.

Figure 4.

Figure 4.

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PWR is required for HOS15 function. A, Promoter scheme of the COR15A gene. Region II was predicted to contain CBF-binding cis-elements (white boxes), whereas region I was not. B, ChIP assays were performed with anti-HD2C antibody in wild-type (WT), hos15-2, pwr-3, and pwr-3 hos15-2 plants treated with cold (0°C) for 24 h. C to F, Chromatin from 2-week-old plants treated with cold (4°C, 24 h) was immunoprecipitated with anti-HOS15 (C), anti-acetylated histone H3 (AcH3; D), anti-H3K9Ac (E), and anti-H3K14Ac (F) antibodies. The amount of DNA in the immunoprecipitate complex was determined by RT-qPCR and is presented as the fold enrichment after normalization with a mock control using the ACTIN7 gene promoter. Data represent means ± sd from three biological replicates with three technical repeats each (n = 3). Asterisks indicate statistically significant differences from Col-0 by Student’s t test (*P < 0.05 and **P < 0.01). Similar results were obtained from three independent experiments.

HD2C deacetylates histone H3 (Luo et al., 2012a; Buszewicz et al., 2016). As PWR indirectly interacts with HD2C and regulates COR gene expression (Figs. 2A and 3, B–E), we investigated how PWR is functionally linked with HD2C in terms of chromatin-dependent regulation of COR gene expression. In the wild type, the level of acetylated H3 on COR15A and COR78 promoters was increased by cold treatment, as expected from relaxed chromatin (Fig. 4D; Supplemental Fig. S9C). However, cold treatment failed to induce the acetylation of H3 on COR genes in hos15-2 and pwr plants (Fig. 4D; Supplemental Fig. S9C). Similarly, the levels of H3K9Ac and H3K14Ac, which are regulated by PWR and HD2C (Luo et al., 2012a; Tasset et al., 2018), were significantly increased upon cold in the wild type but not in hos15-2 and pwr mutants (Fig. 4, E and F). Taken together, these results indicate that the PWR-HOS15 complex regulates the expression of COR genes through HD2C protein stability and histone acetylation in response to cold.

PWR Promotes the Binding of CBF Proteins to COR Promoter Regions

We have shown that HOS15 likely bridges a complex of PWR and HD2C that associates with CRT/DRE regions of COR genes, where the binding of CBF proteins enhances COR gene expression (Figs. 3 and 4; Supplemental Fig. S9B; Park et al., 2018b, 2019; Mayer et al., 2019). To test the interaction of PWR and CBF proteins, yeast two-hybrid, Co-IP, and split-LUC complementation assays were performed (Fig. 5, A–C; Supplemental Fig. S7B). The results demonstrated that all three CBF isoforms directly interacted with PWR but not with HD2C. Next, we examined whether HOS15 and PWR affected the binding of CBF proteins to COR gene promoters in response to cold stress (Fig. 5D; Supplemental Fig. S10). Cold treatment (4°C, 24 h) greatly enhanced the binding of CBF proteins to the CRT/DRE regions of COR15A and COR78 in the wild type (Col-0). However, cold-induced CBF binding was drastically reduced in hos15-2 and pwr mutants (Fig. 5D; Supplemental Fig. S10), indicating that HOS15 and PWR facilitate the binding of CBF to the chromatin of COR genes in response to cold stress. In addition, CBF binding to the CRT/DRE region of the COR15 gene promoter was not significantly different between the pwr-3 hos15-2 double mutant and each single mutant plant (Supplemental Fig. S11). These results suggest that the PWR-HOS15 complex is required for the binding of CBF transcription factors to CRT/DRE regions of COR gene promoters during cold stress.

Figure 5.

Figure 5.

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PWR facilitates the binding of CBF proteins to the COR promoter. A, Yeast split-ubiquitin assay showing direct binding of PWR to CBF1, CBF2, and CBF3 proteins. Serially diluted yeast cells expressing Nub-PWR or Nub (vector) and CBF1-Cub-RUra3P, CBF2-Cub-RUra3P, CBF3-Cub-RUra3P, or Cub-RUra3P (vector) were spotted on selective media (both His, Trp, and uracil [−HTU] and −HTU + 5-fluoroorotic acid [FOA]) and then incubated at 30°C for 4 d. Samples spotted on nonselective medium (−HT) were used as a loading control. B, Co-IP assays using 2-week-old Arabidopsis wild-type, cbf1,2,3-1 triple mutant, and pwr-2 plants treated at 4°C for 6 h. Protein extracts (Input) were immunoprecipitated (IP) with anti-PWR antibody, and immunoblots were developed with anti-PWR and anti-CBF antibodies. C, Split-LUC assay using N. benthamiana leaves. PWR-NLuc, HD2C-NLuc, or NLuc (vector) and CLuc-CBF1, CLuc-CBF2, CLuc-CBF3, or CLuc (vector) were transiently coexpressed in N. benthamiana leaves using A. tumefaciens, and LUC images were captured using a CCD camera. D, ChIP assays were performed with anti-CBF antibody cross-reacting with CBF1 to CBF3 in wild-type (WT), hos15-2, pwr-2, and pwr-3 plants treated at 4°C for 24 h to determine the amounts of CBF binding to the COR15A gene promoter or nonbinding region by RT-qPCR. IgG was used as a technical control (gray bars). Data represent means ± sd from three biological replicates with three technical repeats each (n = 3). Asterisks indicate statistically significant differences from Col-0 by Student’s t test(**P < 0.01). Similar results were obtained from three independent experiments.

DISCUSSION

The HOS15-PWR Complex Modulates Various Signaling Pathways by Regulating Histone Acetylation on Target Genes

Chromatin structure is determined by a number of histone modifications that strongly affect gene expression under developmental programs and in response to environmental stresses (Hollender and Liu, 2008; Clapier and Cairns, 2009). The mechanisms and constituent proteins for chromatin modification are extensively conserved in eukaryotic organisms, including plants, animals, and yeast, which indicate ancient mechanisms that evolved from a common ancestor (Chen and Tian, 2007). Among epigenetic regulations, histone acetylation appears to directly control gene expression, as HDACs and histone acetyltransferases interact with corepressors or coactivators to form complexes that regulate the transcription of genes through changes in chromatin properties (Pandey et al., 2002; Hollender and Liu, 2008).

HOS15 is a plant homolog of human TRANSDUCING BETA-LIKE1 X-LINKED (TBL1X) and TBL1X RECEPTOR1 (TBL1XR1), which are core components of the SMRT/NCoR1 corepressor complex (Perissi et al., 2008; Park et al., 2018b). In mammals, this corepressor complex interacts with HDAC3 and negatively regulates gene expression by decreasing the level of histone acetylation of the target gene promoter (Rosenfeld et al., 2006). In Arabidopsis, based on phylogenetic analysis, RPD3-like type class I of HDACs, comprising HDA6, HDA7, HDA9, and HDA19, are most closely related to HDAC3 of mammals (Pandey et al., 2002; Hollender and Liu, 2008; Alinsug et al., 2009). HOS15 was demonstrated to interact with HDA6, HDA9, and HDA19 (Park et al., 2018b, 2019; Mayer et al., 2019). Moreover, HOS15-deficient mutants share a common phenotype with the hda9 mutant, including the small plant size, blunt siliques, early flowering, and short hypocotyls (Park et al., 2018b, 2019; Suzuki et al., 2018). The hda9 mutant is also less sensitive to dehydration and salt stress, suggesting that HDA9 negatively affects the salt and drought stress responses (Zheng et al., 2016). PWR is a SANT domain-containing protein that interacts with HDA9 and functions as a repressor in flowering, leaf senescence, and thermomorphogenesis (Yumul et al., 2013; Chen et al., 2016; Kim et al., 2016; Suzuki et al., 2018; Tasset et al., 2018; Mayer et al., 2019). The pwr mutants showed morphological phenotypes similar to those of hos15-2 and hda9 mutants (Supplemental Fig. S1; Yumul et al., 2013; Kim et al., 2016; Park et al., 2019). PWR is required for H3K9 acetylation on thermomorphogenic genes and the hypocotyl elongation induced by high ambient temperature (Tasset et al., 2018). In addition, both PWR and HDA9 interact with HOS15 (Park et al., 2018a, 2019; Mayer et al., 2019). Moreover, the target spectra of the three proteins seem to overlap, and they are involved in ion homeostasis and the response to stresses that mainly include cold, dehydration, abscisic acid (ABA)-mediated response, and oxidative stress (Mayer et al., 2019; Park et al., 2019). Hence, there is ample evidence supporting that PWR interacts with HOS15 and HDA9 to regulate various stress responses. Here, we have shown that a similar complex with a different geometry of partners, namely PWR, HOS15, and HD2C, plays a role in defining chromatin structure at the COR gene promoters, thereby contributing to cold-induced chromatin remodeling.

The epistatic effects of hos15-2 and hda9 mutations, together with microarray analyses, suggested that HOS15 had additional functions independent of HDA9 (Mayer et al., 2019). HOS15 also interacts with HDA6 and HDA19, which are RPD3-like type class I HDACs (Park et al., 2018b). Both HDA6 and HDA19 are involved in ABA- and salt-mediated transcriptional responses and negatively regulate the expression of stress-responsive genes (Chen and Wu, 2010; Zhu et al., 2019). Our findings demonstrate the involvement of the PWR-HOS15 complex in the cold stress signaling pathway by regulating HD2C protein abundance, which appears to be mediated by the substrate receptor activity of HOS15 in the CUL4 E3 ligase complex (Park et al., 2018b). The HD2-type histone deacetylase HD2C functions as a transcriptional repressor by promoting chromatin compaction (Wu et al., 2003). The hd2c loss-of-function mutant exhibits globally increased levels of H3K9K14 acetylation and H3K4 dimethylation and a decreased level of H3K9 dimethylation (Luo et al., 2012a). HD2C is involved in ABA and abiotic stress responses (Sridha and Wu, 2006; Buszewicz et al., 2016), and the transcript levels of HD2C are reduced upon ABA and salt treatments (Luo et al., 2012a). HD2C-deficient mutants show low germination and survival rates, while HD2C overexpressors are insensitive to ABA, salt, and drought treatments (Sridha and Wu, 2006; Colville et al., 2011; Luo et al., 2012a). HD2C interacts with DNA METHYLTRANSFERASE2 (Song et al., 2010) as well as with HDA6 and HDA19 to regulate stress-inducible genes (Luo et al., 2012a, 2012b). Genes encoding ABA INSENSITIVE3 and ABA receptors PYL4, PYL5, and PYL6, which are positive regulators of ABA signaling, seem to be direct targets of HDA19 (Ryu et al., 2014; Mehdi et al., 2016). Moreover, the expression of DELTA1-PYRROLINE-5-CARBOXYLATE SYNTHASE, encoding a rate-limiting enzyme in Pro biosynthesis, is enhanced in hda19 (Ueda et al., 2017). In summary, HDACs interact with several cofactors and play a role in various environmental stress responses. Taken together, these data signify that the PWR-HOS15 module is involved in a variety of stress responses through the interaction with several HDACs.

Plant PWR Functions Differently from Animal NCoR1

SMRT/NCoR1 corepressor complexes have been well studied in association with nuclear hormone receptors in animals (Oberoi et al., 2011). SMRT/NCoR1 repressor complexes are recruited to ligand-unbound retinoic acid receptor and thyroid hormone receptor, which bind to response elements of target genes. Upon ligand binding, SMRT/NCoR1 and the E1A C-terminal binding protein are degraded by the TBL1XR1- and TBL1X-dependent proteasome, respectively (Perissi et al., 2008). In these processes, TBL1X and TBL1XR1 function as E3 ubiquitin ligases for the recruitment of the ubiquitin-proteasome system to degrade corepressors, leading to the activation or transcription of specific nuclear receptor-controlled genes. By contrast, the plant homolog of TBL1, HOS15, was found to act as a substrate receptor for the CUL4-based ubiquitin E3 ligase to degrade the plant-specific histone deacetylase HD2C rather than degrading PWR, the plant homolog of NCoR (Fig. 3A; Supplemental Fig. S6B; Park et al., 2018b). Our finding that PWR interacts indirectly with HD2C through HOS15 is consistent with the cold-induced degradation of HD2C being reduced in pwr mutants, similar to hos15-2 (Fig. 3). Surprisingly, HOS15 seems to enhance the stability of PWR. As exemplified by the Co-IP experiments shown in Figure 3, D and E, and Supplemental Figure S6B, the amount of preexisting PWR before treatments with the inhibitors of protein synthesis and degradation, CHX and MG132, were always lower in the hos15-2 mutant compared with the wild type and the hd2c-1 mutant, regardless of the temperature. Presumably, PWR gains stability through the interaction with HOS15. The low overall sequence similarity between PWR and NCoR proteins may explain their analogous functions through distinct molecular mechanisms. Only two SANT domain regions (SANT1 and SANT2) of PWR share conserved primary sequence with NCoR (52% and 63% similarity, respectively; Kim et al., 2016), whereas the full amino acid sequence of PWR shares only 13.1% similarity and 7.05% identity with the full peptide sequence of NCoR protein. The SANT1 and SANT2 domains of NCoR are necessary for HDAC activation and the binding to unmodified histone tails, respectively (Yu et al., 2003). However, the SANT2 domain of PWR does not bind to unmodified histone but only to the modified histone (Kim et al., 2016). Mammalian NCoR1 has three repressor domains next to the SANT domain for the repression activity, but those domains are not found in PWR. Thus, the plant proteins PWR and HOS15 seem to have similar functions to the mammalian homologs but different operating mechanisms.

The HOS15-PWR Complex Is a Coactivator with the CBF Transcription Factors

CBF transcription factors play a predominant role in promoting COR gene expression and the adaptation to severe cold stress (Chinnusamy et al., 2007; Liu et al., 2018a). Neither PWR nor HOS15 affected the transcriptional induction of CBF genes upon cold treatment, but these proteins physically interacted with all three CBF proteins (Figs. 2 and 5, A–C; Park et al., 2018b). Our results indicate that the PWR-HOS15 complex is strongly associated with the CTR/DRE elements on the COR gene promoters, which are the binding elements for the CBF transcription factors (Fig. 4C; Supplemental Fig. S9B; Park et al., 2018b). In addition, CBF association with the COR genes was greatly reduced in the hos15-2, pwr-2, and pwr-3 mutants (Fig. 5D; Supplemental Fig. S10). All these findings suggest that the PWR-HOS15 complex interacts with the CBF transcription factors and that the complex is required for the association of CBF with the promoter region of the COR genes to induce expression, at least in part by promoting HD2C degradation and chromatin aperture upon cold stress. Thus, the PWR-HOS15 complex can be considered a CBF-dependent coactivator.

In ada2b and general control nonderepressible5 (gcn5) mutants, which are deficient in histone acetyltransferases, COR gene induction by cold stress is delayed and their expression levels are reduced (Vlachonasios et al., 2003). However, the ada2b mutant (Wassilewskija-2 ecotype) showed increased freezing tolerance, indicating that ADA2b may function in the repression of freezing tolerance through histone acetylation (Vlachonasios et al., 2003). Interestingly, despite the observation that the hos15-1 mutant (C24 ecotype) had a freezing-sensitive phenotype, the expression of the COR genes was increased. Conversely, in the hos15-2 mutant (Col-0 ecotype), the freezing-sensitive phenotype correlated with lower COR gene expression (Zhu et al., 2008; Park et al., 2018b). Thus, the cold-signaling mechanism in ada2b mutants in the Col-0 background would be interesting to study to clarify the differences among these ecotypes. In addition, GCN5, which displays histone acetyltransferase activity, is recruited by CBF1 through the transcriptional coactivator ADA2 to increase the expression of COR genes (Mao et al., 2006). Here, we show that COR gene expression was reduced in hos15-2, pwr-2, and pwr-3 mutants during cold acclimation. In these mutants, H3 acetylation levels at the COR gene promoter regions were reduced (Figs. 2A and 4D; Supplemental Fig. S9C). In addition, HD2C protein stability was negatively regulated by the PWR-HOS15 complex. We suggest that the PWR-HOS15 complex is important for the formation of an epigenetic complex to regulate histone acetylation and deacetylation. Subsequently, this altered chromatin structure affects the expression of COR genes during cold acclimation.

Epigenetic regulation plays an important role in many aspects of biological processes. In this study, we have discovered a complex composed of PWR, HOS15, HD2C, and CBF transcription factors that regulate COR gene expression and cold tolerance. Our earlier report (Park et al., 2018b) and our findings here have been incorporated into the model shown in Figure 6. In normal conditions, PWR-HOS15 forms a complex with HD2C by which the COR chromatin is hypoacetylated and the COR gene expression is repressed. In response to cold stress, PWR-HOS15 recruits CUL4 to form a CRL4HOS15 complex for HD2C degradation, resulting in the enhanced acetylation of H3 on the COR chromatin. Consequently, the COR chromatin is changed from a repressive (closed) form to an active (open) form. Next, CBF transcription factors can easily access the open region of the COR promoters to induce the expression of COR genes for cold tolerance. The interaction of PWR-HOS15 with CBFs further facilitates the recruitment of these transcription factors needed for enhanced COR gene expression. Basal levels of CBFs expressed under temperate conditions might help in recruiting PWR-HOS15 to COR genes (Park et al., 2018b).

Figure 6.

Figure 6.

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Working model for the PWR-HOS15-HD2C complex in response to cold stress. In normal growth conditions, the PWR-HOS15-HD2C complex negatively regulates the expression of COR genes through histone deacetylation and chromatin compaction. Under cold stress, proteasome-dependent degradation of HD2C is mediated by the PWR-HOS15 complex, resulting in increased H3 acetylation on the COR gene chromatin. Thus, the structure of chromatin changes from heterochromatin (closed chromatin) to euchromatin (open chromatin). In addition, the PWR-HOS15 complex recruits CBF transcription factors to the COR gene promoter to start transcription and mount freezing tolerance. Basal levels of CBFs expressed under temperate conditions might help in recruiting HOS15 to COR genes.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana) Col-0 background plants were used for this study. The T-DNA insertion mutants pwr-2 (SALK_071811), pwr-3 (SALK_006823), hd2c-1 (SALK_129799), and hos15-2 (GK_785B10) were obtained from the Arabidopsis Biological Resource Center (https://abrc.osu.edu/) and the Nottingham Arabidopsis Stock Centre (http://arabidopsis.info/). Double mutants pwr-2 hos15-2 and pwr-3 hos15-2 were generated by crossing. Seeds of the wild type (Col-0) and mutants were surface sterilized with 70% (v/v) ethanol and 3% (v/v) bleach (sodium hypochlorite solution) and stratified at 4°C for 3 d in the dark. All plants were grown on one-half-strength MS medium plates or soil at 23°C ± 1°C using a 16-h-light/8-h-dark photoperiod with 100 µmol m−2 s−1 light intensity.

Freezing Tolerance Assay and Electrolyte Leakage Measurements

The freezing tolerance assay was performed as previously described (Park et al., 2018b) with some modifications. Briefly, 2-week-old wild-type plants and mutants grown on one-half-strength MS 0.9% (w/v) agar medium containing 1.5% (w/v) Suc (pH 5.7) under long-day conditions at 23°C ± 1°C were untreated or cold acclimated by incubation at 4°C ± 1°C for 7 d. Plants were then placed in a RUMED 4001 freezing chamber adjusted to −1°C and programmed to reduce the temperature to 1°C for 1 h for the nonacclimated condition and to 1°C for 2 h for cold-acclimated plants. When the desired temperatures were reached, cold-treated plants were removed from the chamber. Then, the plants were covered with ice and kept in a 4°C ± 1°C dark room for 12 h. The plates were then incubated in the presence of light at 23°C. Survival was assessed after 4 d. For the freezing tolerance assay in soil, 7-d-old wild-type and mutant seedlings grown on one-half-strength MS medium were transferred to soil and cultivated for 2 weeks. These 3-week-old plants were either not treated or were acclimated to cold by incubation at 4°C ± 1°C for 7 d in the RUMED 4001 freezing chamber that was programmed to decrease in temperature to 1°C for 0.5 h for the nonacclimated condition and maintained for 1 h, and it dropped to the indicated temperatures. After cold treatment in the chamber, plants were covered with ice in the dark for 24 h at 4°C ± 1°C. After the ice melted, plants were placed in the presence of light at 23°C. The survival rates were determined 4 d later at 23°C.

To measure electrolyte leakage, 3-week-old plant rosette leaflets were incubated in 200 μL of deionized water in a model AP28R programmed freezing bath (Poly Science) for the indicated time periods. The amount of electrolyte leakage was measured as previously described (Park et al., 2018b).

RNA Extraction and Quantitative RT-qPCR Analysis

Total RNA from 2-week-old plants was extracted with the RNeasy Plant Mini Kit (Qiagen) and treated with RNase-free DNase (Sigma-Aldrich). cDNA was synthesized by SuperScript II reverse transcriptase (Invitrogen). RT-qPCR was performed using the SYBR Green PCR Master Mix Kit (Bio-Rad). The gene-specific primer sequences are provided in Supplemental Table S1. The relative expression levels were analyzed by the comparative cycle threshold (ΔΔCt) method.

Split-LUC Complementation Imaging Assay

The assay was performed with modified protocols as previously described (Chen et al., 2008). The open reading frame regions of the PWR, CBF1, CBF2, CBF3, and HD2C genes were cloned into vectors for split-LUC complementation, which were modified and used for the Gateway system from pCambia1300-NLuc and pCambia1300-CLuc. The indicated constructs were mobilized into Agrobacterium tumefaciens GV3101. The transformed GV3101 cells were grown in Luria-Bertani medium at 30°C overnight. Cells were then washed once with wash buffer (10 mm MgCl2 and 10 mm MES) and resuspended in infiltration buffer (100 µm acetosyringone, 10 mm MgCl2, and 10 mm MES). After 4 h of incubation, bacterial suspensions (OD600 = 0.5) were infiltrated into Nicotiana benthamiana leaves using a needleless syringe. After 3 d, luciferin (1 mm) was sprayed onto the leaves, and light according to LUC activity was detected by an iXon CCD imaging apparatus (Andor Technology).

Yeast Two-Hybrid Assay

For yeast two-hybrid assays, HOS15, HD2C, and PWR were cloned into pDEST22 (AD) and pDEST32 (BD). These plasmids were cotransformed into Saccharomyces cerevisiae strain PJ69-4A by polyethylene glycol-mediated heat shock (42°C) transformation using the protocol of the manufacturer (Clontech). The growth of cotransformed yeast cells was observed on Trp, Leu, and His nutrient-deficient medium. Transformation and growth of the yeast cell control was checked on Trp and Leu nutrient-deficient medium. Plates were photographed after a 5-d incubation at 30°C.

The split-ubiquitin assay was performed with modified protocols as previously described (Reichel and Johnsson, 2005). In brief, PWR and CBFs were cloned into pMet-GWY-Cub-URA3p-CYC1 and pCup-NuI-GWY-CYC1 vectors modified from the original pMet and pCub vectors, respectively. Constructs were transformed into S. cerevisiae strain JD53 by polyethylene glycol-mediated heat shock (42°C). Interactions between pairs of proteins were analyzed by yeast cell growth on Trp, His, and uracil nutrient-deficient medium containing 5-fluoroorotic acid (1 mg mL−1; Zymo Research). Plates were photographed 3 to 5 d after incubation at 30°C.

Protein Extraction and Western-Blot Analysis

Total proteins were isolated from 2-week-old Arabidopsis seedlings by using extraction buffer containing 150 mm NaCl, 1 mm EDTA, 0.5% (v/v) Nonidet P-40, 3 mm dithiothreitol, 100 mm Tris-Cl (pH 7.5), and protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 1 μg mL−1 aprotinin, 1 μg mL−1 pepstatin, 5 μg mL−1 leupeptin, 5 μg mL−1 antipain, 5 μg mL−1 chymostatin, 2 mm NaF, 2 mm Na2VO3, and 50 mm MG132). For immunoblotting, the membranes were incubated with the appropriate primary antibody, either anti-PWR, anti-HD2C (Agrisera), anti-HOS15, anti-tubulin (Sigma), and anti-CBFs cross-reacting with CBF1 to CBF3, for 2 h at room temperature or overnight at 4°C. The membranes then were developed using peroxidase-conjugated secondary anti-rabbit or anti-mouse antibody (Santa Cruz Biotechnology), and the antigenic proteins were detected with enhanced chemiluminescence using the appropriate reagent (Bio-Rad).

Co-IP Assay

For the Co-IP assay, total proteins were extracted from 2-week-old Arabidopsis seedlings or 10-d-old Arabidopsis plants treated with or without cold (0°C) for 12 h in the presence of CHX (100 µm) and the proteasome inhibitor MG132 (50 µm). For immunoprecipitation, the protein extracts were centrifuged twice at 12,000 rpm for 15 min at 4°C and incubated with anti-PWR or anti-HD2C (Agrisera) antibody fused to protein A agarose beads for 2 h at 4°C with gentle rotation. After washing with 1× phosphate-buffered saline, proteins were mixed with 4× SDS buffer and heated at 90°C at 4 min. Protein was separated by SDS-PAGE, followed by immunoblotting. The immunoblots were developed using appropriate primary antibodies, including anti-PWR, anti-HD2C, anti-CBFs, and anti-HOS15, for 2 h at 23°C ± 1°C or overnight at 4°C. The membranes were developed by peroxidase-conjugated secondary anti-rabbit or anti-mouse antibody (Santa Cruz Biotechnology), and the antigenic proteins were detected with enhanced chemiluminescence using the appropriate reagent (Bio-Rad).

Subcellular Localization

PWR was fused at the N terminus of GFP in pMDC83. Both PWR-GFP and NLS-dsRed fluorescent protein (RFP) were transiently coexpressed in N. benthamiana leaves using A. tumefaciens GV3101. The fluorescence signals of GFP and RFP were captured using a confocal laser scanning microscope (Olympus FV1000).

ChIP Assay

The ChIP assay was performed as previously described (Saleh et al., 2008; Park et al., 2018b) with slight modifications. Two-week-old Arabidopsis plants were treated with 1% (v/v) formaldehyde for 15 min under vacuum to fix the chromatin structure and subsequently with 0.1 m Gly for 5 min of incubation to stop the cross-linking reaction. Nuclei were isolated with nuclei lysis buffer (150 mm NaCl, 1 mm EDTA, 50 mm HEPES [pH 8], 1% [v/v] Triton X-100, 0.1% [w/v] SDS, 0.1% [w/v] deoxycholate, 1 mm phenylmethylsulfonyl fluoride, 10 mm sodium butyrate, 1 μg mL−1 pepstatin A, and 1 μg mL−1 aprotinin), and then nuclear proteins were extracted and sonicated using a BioruptorII device (BMS) to fragment the chromosomal DNA. Immunoprecipitation was performed using salmon sperm DNA/protein A agarose beads (Upstate Biotechnology) fused to anti-HD2C antibody (Agrisera), anti-HOS15 antibody, anti-CBFs antibody, anti-acetylated H3 antibody (Millipore), anti-acetylated H3K9 antibody (Millipore), or anti-acetylated H3K14 antibody (Millipore). Beads treated with anti-rabbit IgG, anti-rat IgG, or anti-mouse IgG were used as a negative control in each ChIP assay. The beads were then washed twice with low-salt buffer (150 mm NaCl, 0.1% [w/v] SDS, 1% [v/v] Triton X-100, 2 mm EDTA, and 20 mm Tris-Cl [pH 8]), once with high-salt buffer (500 mm NaCl, 0.1% [w/v] SDS, 1% [v/v] Triton X-100, 2 mm EDTA, and 20 mm Tris-Cl [pH 8]), once with LiCl buffer (250 mm LiCl, 1% [v/v] Nonidet P-40, 1% [w/v] deoxycholate, 1 mm EDTA, and 10 mm Tris-Cl [pH 8]), and twice with TE buffer (10 mm Tris-Cl [pH 8] and 1 mm EDTA). The immunocomplexes were eluted with elution buffer (0.1 m NaHCO3 and 1% [w/v] SDS), and the elution products were reverse cross-linked by incubation at 65°C overnight. RNase A and proteinase K were then added to each tube, and the DNA was purified by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation. The precipitated DNA were dissolved in TE buffer and determined by RT-qPCR with the primers listed in Supplemental Table S1. An ACTIN7 DNA fragment was used for normalization.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis GenBank databases under the following accession numbers: AT3G52250 (PWR), AT5G67320 (HOS15), AT5G03740 (HD2C), AT4G25490 (CBF1), AT4G25470 (CBF2), AT4G25480 (CBF3), AT2G42540 (COR15A), and AT5G52310 (COR78).

Supplemental Data

The following supplemental materials are available.

Acknowledgments

We thank Dr. Ray A. Bressan and Hans J. Bohnert for their valuable discussion on this research.

Footnotes

1

This work was supported by the Next Generation Bio-Green21 Program, Rural Development Administration, Republic of Korea (grant no. PJ01318201 to D.-J.Y. and grant no. PJ01318205 to J.M.P.), the National Research Foundation of Korea funded by the Korean Government (grant no. 2019R1A2C2084096 to D.-J.Y.), and the Global Research Lab (grant no. 2017K1A1A2013146 to D.-J.Y.).

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