DREB1A/CBF3 Is Repressed by Transgene-Induced DNA ...DREB1A/CBF3 Is Repressed by Transgene-Induced...

DREB1A/CBF3 Is Repressed by Transgene-Induced DNAMethylation in the Arabidopsis ice1-1 Mutant[OPEN]

Satoshi Kidokoro,a,1 June-Sik Kim,b,1 Tomona Ishikawa,a Takamasa Suzuki,c Kazuo Shinozaki,b andKazuko Yamaguchi-Shinozakia,2

a Laboratory of Plant Molecular Physiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo113-8657, JapanbGene Discovery Research Group, RIKEN Center for Sustainable Resource Science, Tsukuba, Ibaraki 305-0074, JapancCollege of Bioscience and Biotechnology, Chubu University, Matsumoto-cho, Kasugai, Aichi, 478-8501, Japan

ORCID IDs: 0000-0003-3311-4736 (S.K.); 0000-0002-4703-609X (J.-S.K.); 0000-0003-2898-8869 (T.I.); 0000-0002-1977-0510 (T.S.);0000-0002-6317-9867 (K.S.); 0000-0002-0249-8258 (K.Y.-S.)

DREB1/CBFs are key transcription factors involved in plant cold stress adaptation. The expression of DREB1/CBFs triggersa cold-responsive transcriptional cascade, after which many stress tolerance genes are expressed. Thus, elucidating themechanisms of cold stress–inducible DREB1/CBF expression is important to understand the molecular mechanisms of plantcold stress responses and tolerance. We analyzed the roles of a transcription factor, INDUCER OF CBF EXPRESSION1 (ICE1),that is well known as an important transcriptional activator in the cold-inducible expression of DREB1A/CBF3 in Arabidopsis(Arabidopsis thaliana). ice1-1 is a widely accepted mutant allele known to abolish cold-inducible DREB1A expression, and thisevidence has strongly supported ICE1-DREB1A regulation for many years. However, in ice1-1 outcross descendants, weunexpectedly discovered that ice1-1 DREB1A repression was genetically independent of the ice1-1 allele ICE1(R236H).Moreover, neither ICE1 overexpression nor double loss-of-function mutation of ICE1 and its homolog SCRM2 alteredDREB1A expression. Instead, a transgene locus harboring a reporter gene in the ice1-1 genome was responsible for alteringDREB1A expression. The DREB1A promoter was hypermethylated due to the transgene. We showed that DREB1A repressionin ice1-1 results from transgene-induced silencing and not genetic regulation by ICE1. The ICE1(R236H) mutation has alsobeen reported as scrm-D, which confers constitutive stomatal differentiation. The scrm-D phenotype and the expression ofa stomatal differentiation marker gene were confirmed to be linked to the ICE1(R236H) mutation. We propose that the currentICE1-DREB1 regulatory model should be revalidated without the previous assumptions.

INTRODUCTION

Cold stress is an environmental condition that affects plantgrowth, development, and productivity. Under cold stress con-ditions, the expression of numerous genes that function in thestress response and in tolerance is induced in various plantspecies.Theproductsof thesegenes function toenhance freezingstress toleranceand to regulategeneexpressionundercoldstressconditions (Thomashow, 1999; Yamaguchi-Shinozaki and Shi-nozaki, 2006). The dehydration-responsive element (DRE)/C-re-peat with the common core motif A/GCCGAC has been identifiedas a cis-acting promoter element that regulates gene expressionin response to both cold and dehydration stresses in plants(Baker et al., 1994; Yamaguchi-Shinozaki and Shinozaki, 1994).Three transcription factors, DREB1A/CBF3, DREB1B/CBF1,and DREB1C/CBF2, bind to the DRE, activating the expressionof many downstream cold-inducible genes. Overexpression ofDREB1/CBFs improves stress tolerance to freezing, drought,

and high salinity in transgenic Arabidopsis (Arabidopsis thaliana;Jaglo-Ottosen et al., 1998; Liu et al., 1998). More than 100 targetgenes of DREB1s have been identified by transcriptome anal-yses (Maruyama et al., 2009; Park et al., 2015). Many of theproducts of these target genes have been reported to function inthe acquisition of stress tolerance and in the further regulation ofstress responses. Moreover, double and triple genome-editedDREB1 mutants presented a severe reduction in freezing tol-erance (Jia et al., 2016; Zhao et al., 2016). Thus, these threeDREB1 transcription factors reportedly act as master switchesin cold-inducible gene expression (Yamaguchi-Shinozaki andShinozaki, 1994).Since all three DREB1 genes are rapidly and significantly in-

duced by cold stress, their induction is considered to be the firstswitch in the cold-responsive expression of numerous genes(Yamaguchi-Shinozaki and Shinozaki, 1994). Therefore, eluci-dating the mechanisms of DREB1 induction in response to coldstress is important. Some transcription factors have been iden-tified to regulate the cold-inducible expression of DREB1s.CALMODULIN BINDING TRANSCRIPTION ACTIVATOR3/Arabi-dopsis thalianaSIGNAL-RESPONSIVEGENE1 (CAMTA3/AtSR1),along with CAMTA1 and CAMTA2, has been indicated to activatethe expression of CBF1/DREB1B and CBF2/DREB1C (Dohertyet al., 2009). The expression of many cold-inducible genes, in-cluding DREB1s and their downstream genes, has also beenrevealed tobe regulatedby thecircadianclock.CCA1and itsclose

1 These authors contributed equally to this work.2 Address correspondence to [email protected] author responsible for the distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is Kazuko Yamaguchi-Shinozaki ([email protected]).[OPEN]Articles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.19.00532

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https://orcid.org/0000-0003-3311-4736https://orcid.org/0000-0002-4703-609Xhttps://orcid.org/0000-0003-2898-8869https://orcid.org/0000-0002-1977-0510https://orcid.org/0000-0002-6317-9867https://orcid.org/0000-0002-0249-8258http://orcid.org/0000-0003-3311-4736http://orcid.org/0000-0002-4703-609Xhttp://orcid.org/0000-0003-2898-8869http://orcid.org/0000-0002-1977-0510http://orcid.org/0000-0002-6317-9867http://orcid.org/0000-0002-0249-8258http://crossmark.crossref.org/dialog/?doi=10.1105/tpc.19.00532&domain=pdf&date_stamp=2020-03-21mailto:[email protected]://www.plantcell.orgmailto:[email protected]://www.plantcell.org/cgi/doi/10.1105/tpc.19.00532http://www.plantcell.org

homolog LHY, which are key components of the circadian os-cillators and morning-expressed MYB transcription factors, havebeen shown to bind to the promoter regions ofDREB1s, and cold-inducible expression of DREB1s has been reported to be signif-icantly reduced incca1 lhydoublemutantplants (Dongetal., 2011;Kidokoro et al. 2017), suggesting that the key circadian compo-nents, such as CCA1 and LHY, also function as importanttranscriptional activators in the cold-responsive expression ofDREB1s.We recently revealed thatplants recognizecoldstressastwo different signals, rapid and gradual temperature decreases,and that each of the three DREB1 genes is differently induced inresponse to these two stress signals. CAMTA3 and CAMTA5respond to a rapid temperature decrease and induce the ex-pression of DREB1B and DREB1C. By contrast, CCA1 and LHYstrongly induce the expression of DREB1A and DREB1C in re-sponse to rapid and gradual temperature decreases (Kidokoroet al. 2017). The presence of the two different signaling pathwaysleading to theexpressionofDREB1s in response tocoldstresshasmade it difficult to elucidate the regulatory mechanisms of theirexpression.

TheMYC-like basic helix-loop-helix transcription factor INDUCEROF CBF EXPRESSION1/SCREAM (ICE1/SCRM) is also a well-knownregulatorofDREB1/CBFexpression.An ice1-1mutantwasfirst isolated in a screen formutations that impair the cold-inducedtranscription of a firefly luciferase (LUC) reporter gene driven bytheCBF3/DREB1A promoter (Chinnusamy et al., 2003). The cold-inducible expression of endogenous CBF3/DREB1A clearly de-creased in the ice1-1mutant, but thatofDREB1BandDREB1Cdidnot. The ice1-1 mutant showed a significant decrease in plantchilling and freezing tolerance. Moreover, overexpression of theICE1 gene in the wild-type Arabidopsis plants enhanced the ex-pressionof theCBF/DREB1 regulon in response tocoldstressandimproved the freezing stress tolerance of the transgenic plants. Itwasconcluded that themutationofoneaminoacid residue,Arg, atamino acid 236 to His (R236H) in the ICE1 protein caused thisdecreased expression of CBF3/DREB1A (Chinnusamy et al.,2003).

Kanaoka et al. (2008) isolated a scrm-Dmutant whose stomataldevelopment was abnormal. In this mutant, nearly all cells in theepidermis developed into guard cells. The authors revealed thatthis phenotype was caused by the same missense mutation(R236H) in the same ICE1 protein by using map-based cloning. Inthe scrm-D mutant, increased expression of stomatal differenti-ation marker genes such as FAMA and EPF1was tightly linked tothe ICE1(R236H) mutation (Pillitteri et al. 2011). The ICE1(R236H)mutation dominantly and semidominantly affects DREB1A ex-pression and stomatal development, respectively, but how thismutation results in two different phenotypes is unclear. Addi-tionally, a T-DNA insertion double mutant of ICE1 and its ho-mologous gene,SCRM2/ICE2, did not exhibit stomatal differentiationin the epidermis, which was opposite to the effect observed in thescrm-D mutant (Kanaoka et al., 2008). This double mutationcausedaslightdecrease in theexpressionof all threeCBF/DREB1genes (Kim et al., 2015), while the ice1-1mutant showed a strongdecrease in only CBF3/DREB1A expression (Chinnusamy et al.,2003).

Because the DREB1A promoter contains several typical MYC-type transcription factor binding sequences (CANNTG), it is

possible that ICE1 targets these sequences and regulates cold-inducibleDREB1Aexpression (Chinnusamyetal., 2003;Kimet al.,2015). In addition, various reports have shown that the activity ofthe ICE1 protein is modulated by various posttranslationalmodifications, such as phosphorylation, SUMOylation, and ubiq-uitination, to regulate cold stress tolerance (Dong et al., 2006;Miuraet al., 2007, 2011;Dingetal., 2015). Thus,many factorshavebeen reported to regulate the cold-responsive expression ofDREB1 genes. Among these factors, the cold-inducible expres-sion of DREB1A is activated by both ICE1 and circadian com-ponents, while that of DREB1B and DREB1C is activated byCAMTAs and circadian components. Therefore, the mechanismunderlying the regulation of the cold-inducible expression ofDREB1A may differ from that of the other two DREB1s, but thissupposition has not yet been clarified.In this study, we focused on the regulatory mechanism of the

cold-inducible expression of DREB1A/CBF3 and tried to analyzethe role of ICE1 in the regulation of this expression. However, weunexpectedly discovered that DREB1A repression in ice1-1 isgenetically independent of the known ICE1(R236H) mutation.Using genomic analysis, we deduced that a T-DNA allele from theice1-1 genome is associated with DREB1A repression. Oursubsequent analyses demonstrated that DREB1A repression inice1-1 is achieved by DNA methylation-mediated gene silencingtriggered by T-DNA, not genetic regulation.

RESULTS

An R236H Mutation within ICE1 Is Independent ofDREB1A Repression

To identify the cis-acting elements involved in the cold-inducibleexpression of DREB1A, we generated transgenic Arabidopsisplants that express an emerald luciferase (ELUC) reporter genedriven by four tandem repeats of theDREB1A promoter fragment(2143 to 255 bp from the transcription start site), including twoconserved sequences (boxes V and VI) among the promoters ofthree DREB1s, and its minimal promoter (257 to 1118 bp) andnamed it 1AR:ELUC (Figure 1A). We detected obvious cold-inducible expression of the ELUC gene in the generated1AR:ELUC plants in the Col-0 background, indicating thatthe 1AR fragment governs the cold-inducible expression ofDREB1A (Figure 1B). ICE1 has been assumed to be a candidatetranscription factor that targets E-box sequences (CANNTG)within the DREB1A promoter and regulates the cold-inducibleexpression of DREB1A (Chinnusamy et al., 2003). 1AR:ELUCcontains one E-box sequence (CACCTG); therefore, to in-vestigate whether ICE1 can regulate cold-responsive tran-scription via the fragment, we crossed an 1AR:ELUC plant withan ice1-1mutant plant. The F2 population segregated into threesubgroups that exhibited different rosette phenotypes, the wildtype, intermediate (heterozygous), and ice1-1 (homozygous), atan ;1:2:1 ratio (Figure 1C). We observed the epidermal de-velopment of these three subgroups and found that they showednormal development, increased stomata, and a stomata-onlyphenotype similar to that of ice1-1 or scrm-D, respectively(Figure 1D). These results were consistent with the reported

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effect of the ICE1(R236H) mutation, which is known to stronglyupregulate ICE1 target genes, including EPF1 (Kanaoka et al.,2008; Pillitteri et al. 2011).

We then analyzed the expression of 1AR:ELUC under coldstress conditions at 4°C for 3 h in the three F2 subgroups(Figure 1E). Because the coding sequence of ELUC that we in-troduced is not homologous to that of firefly LUC, which wasoriginally included in the ice1-1mutant as a reporter gene, ELUCexpression could be specifically analyzedbyquantitativeRT-PCR(RT-qPCR). We expected that ELUC expression was represseddepending on the presence of the ICE1(R236H) mutation. How-ever, the expression of ELUC did not seem to be associated withthe observed rosette phenotypes. Many F2 plants homozygousfor ICE1(wild type) presented unexpectedly low levels of ELUCexpression—levels that were similar to those in the ice1-1mutant.Moreover, someF2plantswith ICE1(R236H)presentedhigh levelsof ELUC expression—levels that were as high as those in the wildtype (1AR:ELUC) plants (Figure 1E). To confirm the expression of

endogenous ICE1/SCRM target genes in the three classes of F2plants,wemeasured theexpressionofDREB1AandEPF1 in thoseplants. The expression ofDREB1Awas unexpectedly low inmanyF2 plants with homozygous ICE1(wild type) in response to coldstressbutwashighly induced in someF2plantswith ICE1(R236H;Figure 1E). These expression patterns ofDREB1A in the F2 plantswere similar to thoseof theELUC reporter genesdrivenby1AR.Bycontrast, the expression of EPF1, one of the target genes of ICE1in stomatal development, was increased in the heterozygousplants and greatly increased in the homozygous R236H plants(Figure 1E). These results implied that DREB1A repression in theice1-1 mutant is genetically independent of the ICE1(R236H)mutation, while the elevated expression of EPF1 is tightly asso-ciated with this mutation as well as with stomatal development.We further analyzed the cold-inducible expression of DREB1A

in the scrm-D mutant plants and two lines of transgenic plants(Col-0) harboring the ICE1 genomic fragment prepared from theice1-1mutantgenomicDNA (Figure2A).Comparedwith that in the

Figure 1. Dissociation of DREB1A Repression from the ice1-1 Allele.

(A)Schematic diagramof the reporter constructs. TheELUC reporter gene is drivenby four tandem repeats of an 89-bp fragment aroundboxesVandVI anda minimal promoter sequence of the DREB1A promoter. Nos-T indicates the nopaline synthase terminator.(B)Expressionof theELUC reporter gene in response tocold stress. The transcript level of eachgene in the transgenicArabidopsis seedlingswasmeasuredbyRT-qPCR.Two representative lines are shown. Thebars refer tomeans6 SDs; experimentswereperformed in triplicate. Line 2wasused for crossingwiththe ice1-1 mutant plants.(C)Process flow of the F2 population generated by an 1AR:ELUC3 ice1-1 cross. Two-week-old seedlings of the F2 population grown on agarmedium areshown. Bar 5 10 mm.(D) Abaxial rosette leaf epidermis of 1AR:ELUC, ice1-1, scrm-D, and the F2 population generated by an 1AR:ELUC 3 ice1-1 cross. Bars 5 20 mm.(E)Related gene expression in F2 individuals evaluated byRT-qPCR. ICE1 alleles were determined by the apparent rosette and stomatal phenotypes. n.d.,not detected; WT, wild type.

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wild type (Col-0) plants, EPF1 expression in the scrm-D mutantand transgenic plants was significantly induced, whereas theexpression levels ofDREB1A did not significantly change in theseplants. Therefore, we concluded that the DREB1A repression inthe ice1-1mutant is not due to the ICE1(R236H) mutation; rather,the repression is attributed to unknown independent geneticvariation.

To analyze the effect of ICE1 loss of function on DREB1 ex-pression, we obtained T-DNA insertion alleles of ICE1 (ice1-2) andits homolog SCRM2 (scrm2-1) and generated a double loss-of-function mutant of ICE1 and SCRM2 (ice1-2 scrm2-1; Kanaokaet al., 2008). These single and double mutant plants were grownon germination medium (GM) agar plates with ice1-1 and the

wild-type plants. Among these plants, ice1-1 showed growthinhibition, and ice1-2 scrm2-1 exhibited more severe growth in-hibition, aspreviously reportedbyKanaokaetal. (2008;Figure2B).Using the plants grown on the agar plates, we examined theexpression of EPF1 and found that its expression was reduced inice1-2 and more reduced in ice1-2 scrm2-1 (Figure 2C). Theexpression levels of the threeDREB1genes and their downstreamgenes (COR15A, RD29A, and GolS3) in these plants were sub-sequently analyzed at 4°C for 24 h (Figure 2D). The expression ofDREB1A in ice1-2, scrm2-1, and ice1-2 scrm2-1 was not signif-icantly changed, except for a decrease in expression in ice1-2scrm2-1 when treated at 4°C for 1 h. By contrast, DREB1A ex-pression remained at extremely low levels in the ice1-1 mutant

Figure 2. Cold-Induced Expression of DREB1s and Their Downstream Genes in Mutant Plants of ICE1 and Its Homolog.

(A) Expression of EPF1 and DREB1A in ice1-1, scrm-D, and two lines of transgenic plants (Col-0) harboring the ICE1(R236H) genomic DNA fragmentprepared from the genomic DNA extracted from the ice1-1 mutant.(B) Plant growth of the mutant plants of ICE1 and its homolog SCRM2/ICE2. Two-week-old seedlings grown on agar medium are shown. Bar5 10 mm.(C) and (D)Gene expression in single anddoublemutant plants of ICE1 andSCRM2/ICE2. Gene expression levels ofEPF1 under unstressed conditions (C)and those of three DREB1s and their downstream genes under cold stress conditions (D) were detected.The transcript level of eachgenewasmeasuredbyRT-qPCR.Thebars refer tomeans6 SDs; experimentswereperformed in triplicate. Theasterisks indicatesignificant differences (**P < 0.01 or *P < 0.05 according to Student’s t test; Supplemental Data Set) in the expression of each gene in the transgenic plantscompared with the wild-type (WT; Col-0) plants.

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http://www.plantcell.org/cgi/content/full/tpc.19.00532/DC1

during the24h, evenunder cold stress conditions. TheexpressionpatternsofDREB1BandDREB1C in all thesemutantswere similarto those in the wild type during the 24 h under cold stress con-ditions. These results indicated that among the three DREB1genes, only DREB1A showed decreased expression in ice1-1compared with that in the wild-type plants. By contrast, the ex-pression levels of the DREB1-downstream genes COR15A,RD29A, andGolS3were slightly but significantly decreased in theice1-1 and double mutant plants.

In addition, we tested the effects of overexpression of ICE1 intransgenic Arabidopsis plants. Using the cauliflower mosaic virus(CaMV) 35S promoter, we generated two ICE1 overexpressionlines that presented high levels of ICE1 expression (Figure 3A).Compared with the vector control plants, neither overexpressionline presented significant increases in the expression of the threeDREB1 genes or their downstream genes, while the expression ofEPF1 was slightly but significantly increased under the controlconditions (Figures 3A and 3B). We examined the freezing tol-eranceof theoverexpression linesand found that theseplantsalsodidnot showsignificantdifferences in stress tolerance (Figures3Cand 3D). These results suggest that the overexpression of ICE1using the 35S promoter does not confer significant effects on the

expression ofDREB1 or its downstream genes, nor does it conferfreezing stress tolerance in Arabidopsis plants.

A T-DNA Insertion on Chromosome 1 of ice1-1 Is Associatedwith DREB1A Repression

To understand the genetic characteristics of the novel locus forDREB1A repression, we propagated a new F2 population from anice1-13 Col-0 cross. Eight of the 11 F2 plants that exhibited theICE1(wild type) rosette phenotype showed repressed DREB1Aexpressionas the ice1-1plantdid,andwesubsequently foundtwoheterozygous ICE1(R236/ wild type) plants having Col-0–likeDREB1A expression (Figure 4A). These results are consistentwith our observations in the 1AR:ELUC 3 ice1-1 progeny (Fig-ure 1), again providing strong evidence thatDREB1A repression isnot due to the ICE1(R236H) allele. DREB1A expression was an-alyzed further in the self-pollinated F3 generation by pooling sixto eight plants into a sample. The progeny of the F2 plants withCol-0–like DREB1A expression uniformly presented similar fullyactivated DREB1A expression, indicating that they were nullsegregants (Figure 4B). By contrast, the progeny of the F2 plantswith repressed DREB1A presented variable DREB1A expression.

Figure 3. Cold-Induced Expression of DREB1s and Their Downstream Genes in ICE1-Overexpressing Plants.

(A)and (B)Geneexpression in ICE1-GFP–overexpressingplants.Geneexpression levelsof ICE1andEPF1underunstressedconditions (A)and thoseof thethree DREB1s and their downstream genes under cold stress conditions (B)were detected. The transcript level of each gene wasmeasured by RT-qPCR.The bars refer to means 6 SDs; experiments were performed in triplicate.(C) and (D)Freezing tolerance of ICE1-GFPoverexpressionplants. Nonacclimated seedlingswere treated at29°C for 0.5 h.Representative images (C) andsurvival rates (D) of plants after recovery are shown. The bars refer to means 6 SDs; experiments were performed in triplicate.The asterisks indicate significant differences (**P < 0.01 according to Student’s t test; Supplemental Data Set) in the expression of each gene in thetransgenic plants compared with that of the vector control (VC) plants, in which only GFP was expressed under the CaMV 35S promoter.


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Figure 4. Identification of the NICE1 Locus.

(A)Cold-induced expression ofDREB1A in the F2progeny of an ice1-13Col-0 cross. The lines indicatedwith arrowswere used for genome resequencing.(B)Cold-inducedexpressionofDREB1A in the bulkedF3progeny (six to eight seedlings) of the F2 individuals in (A). Thewhite, light gray, and dark gray barsindicatenull segregantsandheterogeneousandhomogenousmutantsofNICE1, respectively. The transcript levelof eachgenewasmeasuredbyRT-qPCR.The bars refer to means6 SDs; experiments were performed in triplicate. The different letters above the bars designate significant differences (two-tailedt test with Bonferroni–Holm correction, P < 0.05; Supplemental Data Set).(C)Chromosomal distribution of SNPs and genetic loci related to DREB1A repression in ice1-1. The gray fills and green bars refer to the numbers of SNPsdetected in total and of SNPswith perfect association withDREB1A repression in a bin (Mbp), respectively. The approximate loci of the ICE1 andDREB1Agenes and of the two discovered T-DNAs (Ch1-T, Ch5-T) are given. Chr, chromosome.(D) and (E)Schematic view of the Ch1-T (D) and Ch5-T (E) loci. The gray bar and open arrows indicate the TE and genes, respectively. The primer sets andpositions for confirming the T-DNA insertion are shown with small black arrows (a to f). BD, T-DNA border; LB, T-DNA left border; RB, T-DNA right border.(F)Genotypes of the two transgenes in the F2 individuals from the ice1-13Col-0 cross in (A). LUC CDS refers to amplification of the transgenic luciferasecoding DNA sequence.

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Two of the progeny showed similar repression to that of ice1-1,while the other six showed only modest repression of DREB1A,indicating that the pooled plants were heterogeneous (Figure 4B).Thus far, the observed segregation ratio was 2:6:3 in the F2ICE1(wild type) plants, suggesting that theDREB1A repression ofice1-1 is regulated by a single genetic locus in a dominant-negative manner (x2, P > 0.95). This is the same characteristicthat the original ice1-1 report mentioned (Chinusamy et al., 2003).We named this novel locus New ICE1 (NICE1).

To characterize the NICE1 locus within the Arabidopsis ge-nome,we resequenced the individual genomes of ice1-1, scrm-D,six of the F2 plants described above, two homozygous NICE1mutants (NICE11/1), oneheterozygousNICE1mutant (NICE11/2),and three null segregants (NICE12/2). More than 4000 biallelicsingle-nucleotide polymorphisms (SNPs) were detected fromeach resequenced genome, and they were filtered based on theknown NICE1 genotypes. The filtration yielded eight SNPs ofcytosine-to-thymine conversion. These SNPs were gatheredwithin a range of 8.0 to 10.5 Mbp on chromosome 1 (Figure 4C),which was a different chromosome than those containing the lociof DREB1A (chromosome 4) and ICE1 (chromosome 3). Amongthe eight SNPs, four were found in each coding region of fourgenes, and the other four were located within intergenic regions(Supplemental Figure 1A). TheSNPs from thecoding regionswerefurther evaluated for their association withDREB1A repression bythe use of the other nonsequenced F2 segregants. However, onlyincomplete association was observed in all of these cases, in-dicating that the detected SNPs are nearNICE1but are not causalalleles for the DREB1A repression of ice1-1 (SupplementalFigure 1B).

We therefore attempted to investigate the other possibility:a T-DNA insertion. The original report of ice1-1 indicated that theice1-1 genome harbors a single T-DNA locus for reporter geneexpression, but the locus has not been elucidated (Chinnusamyet al., 2003). To identify the T-DNA locus, we prepared another setof genome sequencing data from ice1-1 in the paired-end form.We searched for the T-DNA locus by screening abnormal sin-gleton mapping features in the Arabidopsis reference genome,and the candidates were verified by Sanger sequencing analysis.Wediscovered twoT-DNAloci fromthe ice1-1sequencingdataonchromosome 1 and chromosome 5 and named them Ch1-T andCh5-T, respectively (Figures 4C to 4E). Ch1-T was positioned inthe middle of a transposable element (TE) on chromosome 1(AT1TE25865) and overlapped with the range where the eightcandidate SNPs accumulated (Figure 4D). Ch5-T was positionedin the 39 untranslated region of a protein-coding gene (AT5G45760)on chromosome 5 and was accompanied by a 204-bp genomicdeletion (Figure4E).OurSanger sequencinganalysis revealed thatCh1-T contains at least two copies of the reporter gene (DREB1Apromoter-driven LUC), each at the left and right borders of theT-DNA in an inverted repeat form.Ch5-Twas found to contain onecopy of the reporter gene, and theDREB1A promoter was dividedinto two fragments and flanked the T-DNA (Figures 4D and 4E).Both T-DNA sequences remain incomplete, since the extendedsequences from the T-DNA borders could not be extended anyfurther. To investigate the association between these two T-DNAloci and DREB1A repression, the genotypes of Ch1-T and Ch5-Twere analyzed in the 11 ICE1(wild type) F2plants described above

(Figure 3A). In contrast to the candidate SNPs, the Ch1-T geno-type showed a complete association with DREB1A repression(Figure 4F). Notably, the germline distributed as the progenitor ofice1-1 (CS67845) did not contain either of the two ice1-1 T-DNAs,although its genome still harbored the ectopicLUCgene aspart ofthe reporter gene (Figure 4F).For further validation,weanalyzed the expressionofDREB1A in

BC4F2 segregating plants derived from the backcross of ice1-1 toCol-0. The association between Ch1-T and DREB1A repressionwas also maintained in the BC4F2 population, although the ex-pression of two neighboring homologs, DREB1B and DREB1C,was not affected by Ch1-T (Figures 5A and 5B). The associationoccurred in a dominant-negative manner (Figure 5C), as our F2population suggested (Figures 4A and 4B) and the original ice1-1report indicated (Chinnusamy et al., 2003). The Ch5-T genotypeswere not associated with DREB1A repression (Figure 5B).Moreover, the BC4F2 plants harboring only Ch5-T presented solidLUC induction in response tocold treatment, indicating thatCh5-Tcontains theactive reporter gene (Figure5B).Overall, we identifieda T-DNA on chromosome 1 (Ch1-T) as the NICE1 locus re-sponsible for thedominant-negativeDREB1A regulationof ice1-1.

Induced DNA Methylation of the DREB1A PromoterRepresses Its Activity

The next question was how the single T-DNA allele regulates bothnative and transgenic DREB1A promoters on remote chromo-somes. We first suspected the regional influence of the T-DNAinsertion on the behavior of nearby genes, which probably influ-ences subsequent DREB1A promoter activity. However, twoneighboring genes on either side (AT1G22710 and AT1G22720)are relatively distant (>4 kb) from the NICE1 locus (Figure 4D),and the NICE1 genotype seemed to have little effect on the ex-pression of these geneswhether the plants were treatedwith coldstress or not (Supplemental Figure 2). In addition, the expressionof AT1G22710 was downregulated in ice1-1, while the samedownregulation was also observed in the scrm-D mutant,showing that AT1G22710 regulation is independent of DREB1Arepression (Supplemental Figure 2). We attempted to recon-struct the NICE1 transgene in transgenic plants to analyze themechanism of DREB1A repression. Cold-induced DREB1A ex-pressionwasnot altered in transgenicArabidopsis plants towhicha TE (AT1TE25865) or a TEwithDREB1Apro:LUCwas introduced(Supplemental Figure3).On theotherhand,wecouldnotobtainanytransgenic plants when we introduced a TE (AT1TE25865) with aninverted repeat of DREB1Apro:LUC similar to the NICE1 locus(Supplemental Figure 3).DNAmethylation was another hypothesis, inspired by previous

reports describing the silencing of both a transgene and the as-sociated endogenous gene by ectopically induced DNA meth-ylation (Sidorenko and Peterson, 2001; Gong et al., 2002; Wanget al., 2011). 5-Methylcytosine (5mC) is a prominent form ofmethylated DNA in eukaryotes and is a stable but reversibleepigenetic mark responsible for various biological processes andsilencing of repetitive genomic features, including TEs (Zhanget al., 2018). Most DNA methylation in mammals occurs at CGsites, whereasDNAmethylation in plants occurs at every cytosinebase via multiple specified pathways for each sequence context,


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CG, CHG, and CHH (where H is A, C, or T). Plants also haveevolved an RNA-directed DNA methylation (RdDM) pathway,which is capable of triggering de novo 5mC accumulation inparticular genomic regions from a remote methylated origin withDNA sequence similarity across the genome (Matzke et al., 2015;Zhang et al., 2018). Accordingly, we hypothesized that theNICE1locus, including the transgenic DREB1A promoter, underwentrapid 5mC-mediated silencing and triggered RdDM to silence thenative DREB1A promoter.

We first evaluated the 5mC levels of the DREB1A promoterattributed to NICE1 allelic variants. The DREB1A promoter in theNICE1 transgene contains the 1-kb DREB1A promoter region(from 21007 to 119 nucleotides), harboring both a TE fragment(AT4TE60970; from 2861 to 2759) and the 1AR fragment (from2143 to255),whichcan regulate thecold-inducibleexpressionofDREB1A (Figure 6A). Local bisulfite sequencing was conductedto analyze the 39 region of the promoter including 1AR (from2350to 211), which also benefited the assessment of the 5mC levelsof the native and transgenic DREB1A promoters separately(Figure 6A). The analyzed DREB1A promoter region of the Col-0 plant was barely methylated, indicating that the DREB1A pro-moter is not a major target of Arabidopsis DNA methylation(Figures 6B and 6C). By contrast, NICE11/1 andNICE11/2 plantsdisplayed obvious 5mC accumulation in the same promoter re-gion of both the transgenic and native DREB1A loci. This 5mCaccumulation was observed in all three cytosine contexts, andthe induced levels were comparable between NICE11/1 andNICE11/2 (Figures 6B and 6C). This hypermethylation level re-covered toCol-0–like levels in theNICE2/2 plants (Figures 6B and6C). We subsequently evaluated 5mC levels in the promoter

regions of DREB1B and DREB1C. Although these two homologsneighbor DREB1A and share multiple homologous parts in theirpromoter region (Shinwari et al., 1998), the two promoter regionswere barely methylated in all NICE1 genotypes (SupplementalFigure 4), as their activity was not altered by NICE1 genotypes(Figure 5B). Our results indicated that the DREB1A promoterbecomes hypermethylated coincidently with the T-DNA allele ofthe NICE1 locus.Next, we investigated the influence of hypermethylation on

DREB1A promoter activity. 5-aza-29-Deoxycytidine (5azaC) isa 5mC inhibitor that reduces global 5mC levels and releases 5mC-sensitive transcription in plant cells (Wang et al., 2011; Ikeda et al.,2017). Col-0 and NICE11/1 seedlings were grown in variousconcentrations of 5azaC, after which cold-induced DREB1Aexpression in the seedlings was measured (Figure 6D). The re-pressed DREB1A expression in NICE11/1 was significantly re-covered by the addition of 5azaC in a dose-dependent manner,and the highest concentration of 5azaC (4 mg L21) specificallyrecovered DREB1A expression to a level comparable to that ofCol-0. Similar DREB1A recovery by 5azaC was observed undernoncold conditions, indicating that the promoter hypermethylationin NICE11/1 plants also repressed the basal expression ofDREB1A (Figure 6D). The parental ice1-1 plants had a similarhypermethylated DREB1A promoter, and the repressed DREB1Aexpression was significantly recovered by 5azaC treatment(Supplemental Figure 5). By contrast, the DREB1A expression inCol-0 was not affected by 5azaC applications, regardless of coldtreatment (Figure 6D). Accordingly, our results indicate thatDREB1A promoter activity is 5mC sensitive and that promoterhypermethylation in ice1-1 actually represses the expression of

Figure 5. DREB1A Repression Is Dominantly Associated with a T-DNA Insertion in Chromosome 1.

(A) T-DNA insertions in BC4F2 segregants of ice1-13Col-0. Three segregants in four genotypes are shown. The primer sets and positions are described inFigures 4D and 4E. CDS, coding sequence.(B)Cold-inducible gene expression ofDREB1s and luciferase in the BC4F2 segregants. The bars refer to the means6 SDs; experiments were performed intriplicate.(C) Cold-inducible expression of DREB1A in the Ch1-T segregants. The transcript level was measured by RT-qPCR. Bars refer to the means6 SDs fromtriplicate experiments. The different letters above the bars indicate significant differences (two-tailed t test with Bonferroni–Holm correction, P < 0.01;Supplemental Data Set).

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DREB1A under both cold and noncold conditions. The observedhypermethylation levels that were similar between NICE11/1 andNICE11/2 supported the dominant-negative effect of the NICE1T-DNA allele on DREB1A expression.

RdDM Participates in DREB1A Promoter Hypermethylationby NICE1

Wenext investigatedwhether theRdDMmachineryparticipates inDREB1A repression. Previous studies have reported that thedysfunction of core RdDM components such as DOMAINS RE-ARRANGED METHYLTRANSFERASE2 (DRM2) or NUCLEARRNA POLYMERASE D1 (NRPD1), which is the largest subunitof RNA polymerase IV, can be used to relieve RdDM-mediatedhypermethylation and associated promoter repression (Yamamuroet al., 2014; Tang et al., 2016). We introduced the drm2 or nrpd1mutation into NICE11/1 by crossing and then measured theDREB1A activity. The repressed DREB1A expression was signifi-cantly recovered in both the drm2 NICE11/1 and nrpd1 NICE11/1

doublemutantsunderbothcoldandnoncoldconditions (Figure7A).The drm2 and nrpd1 single mutants had little effect on cold-inducible DREB1A expression (Supplemental Figure 6). In addi-tion, the effect of an RdDM-independent DNA methyltransferaseCHROMOMETHYLASE3 (CMT3; Cao et al., 2003) was analyzed inthe sameway, although the doublemutant cmt3 NICE11/1 did notdisplay any significantDREB1A recovery (Figure 7A; SupplementalFigure6).Wesubsequentlyassessedthe5mClevelsof theDREB1Apromoter in the double mutant plants. Consistent with the re-covered activity of the promoter, compared with the NICE11/1

single mutant, the drm2 NICE11/1 and nrpd1 NICE11/1 plantspresented largely reduced 5mC levels in all cytosine contexts,whereas the cmt3 NICE11/1 plants presented only subtle changesin 5mC levels (Figures 6B, 6C, 7B, and C7C).BecauseRdDM targets are guided by small RNAs (sRNAs) from

the methylated origin (Matzke et al., 2015; Zhang et al., 2018), weanticipated that theNICE11/1 plants would accumulate unnaturalsRNAs originating from the DREB1A promoter. sRNA gel blotswere conducted with Col-0, NICE11/1, and nrpd1 NICE11/1

plants (Figure 7D). Using a probe of the 1AR promoter region, wedetected a clear sRNA accumulation signal from NICE11/1, but

Figure 6. Hypermethylation of the Repressed DREB1A Promoter.

(A) Schematic view of native and transgenicDREB1A promoters in ice1-1.The open and closed bars indicate the intergenic and coding regions,respectively. The gray shaded and striped boxes indicate the TE fragment(AT4TE60970) and the 1AR region within the DREB1A promoter, re-spectively. The horizontal bar of the transgene represents the anonymousbackbone sequences of the T-DNA. The bisulfite sequencing target region

is indicatedbya redbar.Distances (bp) from theDREB1A transcription startsite are given.(B) and (C)Cytosinemethylation levels in theDREB1A promoter of thewildtype (Col-0) and the F2 segregants generated by an ice1-13Col-0 cross.(B) Bars indicate the relative positions (x axis) and the methylation levels (yaxis) of each cytosine in the promoter. (C) Cumulative 5mC levels bycytosine context evaluated from local bisulfite sequencing. The differentletters above the bars indicate significant differences (two-tailed Fisher’sexact test; P < 0.001; Supplemental Data Set). TSS, transcription start site.(D) Cold-responsive DREB1A transcript levels of plants grown undervarious concentrationsof 5azaC.The transcript levelwasmeasuredbyRT-qPCR. The bars refer to the means 6 SDs of triplicate experiments. Theasterisks indicate significant differences (P < 0.01 according to Student’st test; Supplemental Data Set) from the Col-0 data under the same con-ditions. SomeP-values are shown in red; these values were determined byFisher’s exact test (C) and Welch’s pairwise t test (D).


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the signal was not detected from nrpd1 NICE11/1 or Col-0. Thisfinding indicated that NICE11/1 causes the plants to generatesRNAs of the DREB1A promoter in a polymerase IV–dependentmanner, which is in concordance with current knowledge of theRdDM pathway (Matzke et al., 2015; Zhang et al., 2018). Overall,our results support the participation of the RdDM machinery inDREB1A repression in ice1-1 (Supplemental Figure 7).

DISCUSSION

The expression of DREB1A/CBF3 is the key step of the cold-responsive transcriptional cascade, after which a large number ofcold-inducible genes are expressed. In this study, to elucidate therole of ICE1 in the cold-inducible expression of DREB1A, weanalyzed the relationship between DREB1A repression and theice1-1 mutation (R236H). Unexpectedly, we found that DREB1Arepression was not due to the ice1-1 mutation (R236H). ice1-1(Chinnusamy et al., 2003) and scrm-D (Kanaoka et al., 2008) havethe same missense mutation (R236H) of the basic helix-loop-helix–type transcription factor ICE1/SCRM, and both mutantplants typically present obvious defects in leaf and stomataldevelopment. However, their genetic behaviors seemingly con-trast: the ICE1(R236H) mutation in ice1-1 showed a dominant-negative effect on DREB1A expression, while the same mutationin scrm-D showed a semidominant positive effect on the ex-pression of the downstream genes involved in stomatal de-velopment. This inconsistency has been explained by a model inwhich ICE1could functionasaconvergencepoint integrating coldand other signal response pathways (Ding et al., 2015; Barrero-Giland Salinas, 2017), although no convincing evidence to supportthe model has been provided. Recently, it was reported that themissensemutation R236H of SCRM/ICE1 in scrm-D increases itsstability because its ability to interact withMITOGEN-ACTIVATEDPROTEIN KINASE3 (MPK3) and MPK6 that negatively regulateICE1/SCRMprotein stability is abolished (Putarjunan et al., 2019).These results are consistentwith the semidominant positive effectexhibited by scrm-D, but are not consistent with the dominant-negative effect on DREB1A expression exhibited by ice1-1. Ourstudy revealed that DREB1A transcription was differentially af-fected in these two scrm-D and ice1-1 mutant plants; cold-induced DREB1A expression was repressed in ice1-1, whereasthis expression was not repressed in scrm-D (Figure 2A). More-over, we have provided a clear answer to this conflict by indicatingthat two mutant phenotypes of ice1-1 are able to be separated inthe backcrossing progeny (Figures 1C to 1E). We demonstratedthat a transgene position in chromosome 1 (NICE1) drives the

Figure 7. Participation of RdDM Machinery in DREB1A Repression.

(A) Cold-inducible DREB1A transcription recovery via dysfunction ofRdDM components. The transcript level was measured by RT-qPCR. Thebars refer to themeans6 SDs of triplicate experiments. The different lettersabove the bars indicate significant differences under the same conditions(two-tailed t test with Bonferroni–Holm correction, P < 0.01; SupplementalData Set).(B) and (C) Cytosine methylation levels in the DREB1A promoters ofNICE11/1plantswithdifferentDNAmethylationcomponentdysfunctions. (B)

Bars indicate the relative positions (x axis) andmethylation levels (y axis) ofeach cytosine within the promoter. (C)Cumulative 5mC levels by cytosinecontext evaluated from local bisulfite sequencing. The different lettersabove the bars refer to significant differences (two-tailed Fisher’s exacttest, P < 0.001; Supplemental Data Set). Some P-values are shown in red.TSS, transcription start site.(D) sRNA gel blot analysis of NICE11/1 and nrpd1 NICE11/1 plants. Non-sRNA bands stained with ethidium bromide (EtBr) and mature miR167detection were used as controls.

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repression of DREB1A expression (Figures 4 and 5) via RdDMmachinery (Figures 6 and 7). Thus, our results indicated thatthe ice1-1 mutation is irrelevant to DREB1A transcriptionalregulation.

The roles of ICE1 in the cold stress regulatory pathway are notnegligible. After the discovery of the ice1-1 mutant (Chinnusamyet al., 2003), many reports by multiple research groups haveagreed with the regulatory pathway (Lee et al., 2005; Miura et al.,2007;Kimet al., 2015) or have supported thepositive roles of ICE1in plant cold stress tolerance by the use of transgenic plantsoverexpressing ICE1 or ICE1 homologs (Miura et al., 2011; Xuet al., 2014; Huang et al., 2015). However, the dysfunction of ICE1and its homolog ICE2/SCRM2 in the ice1-2 scrm2-2/1 doublemutant resulted in only marginal effects on DREB1 expression(Kim et al., 2015), and most of the other reports did not analyzeDREB1 expression. Furthermore, we observed little effect onDREB1 expression in the single and double T-DNAmutant plants(ice1-2,scrm2-1, and ice1-2scrm2-1; Figure2D), incontrast to theobvious defects in EPF1 regulation and subsequent stomataldevelopment in the double mutant plants (Figure 2C; Kanaokaet al., 2008). The expression levels of three DREB1 downstreamgenes were slightly but significantly decreased in the doublemutant and ice1-1 mutant plants. However, these expressionpatterns were inconsistent with the expression patterns of theDREB1 genes, indicating that the decreased expression of thedownstream genes could be independent of the regulation ofDREB1 expression. Their decreased expression might be due tothe severe growth inhibition caused by the abnormal stomataldevelopment of ice1-1 and ice1-2 scrm2-1 or due to other sig-naling pathways independent of DREB1/CBF. In fact, ICE1 wasrecently reported to function in various signaling pathways in-cluding abscisic acid signaling (Wei et al., 2018; Hu et al., 2019;MacGregor etal., 2019). Theeffectsof theseotherpathwaysmightaffect theexpressionof theDREB1downstreamgenes regardlessof DREB1 expression. In addition, we could not detect any in-creased expression of the DREB1 genes or DREB1 downstreamgenes, and we did not observe any improvements in the freezingstress tolerance of transgenic Arabidopsis plants overexpressingICE1whenwe used the CaMV 35S promoter to overexpress ICE1(Figure 3). Considering that the ICE1(R236H) mutation is not re-lated to the repression of DREB1A expression and that neitherICE1 overexpression nor ice1-2 scrm2-1 altered DREB1A ex-pression, we propose that the present ICE1-DREB1 regulatorymodel should be carefully revalidated without the previous as-sumption. By contrast, the scrm-D stomatal phenotype and ex-pression of the stomatal differentiation marker gene EPF1 wereconfirmed to be linked to the ICE1(R236H) mutation.

The T-DNA and RdDM-mediated regulation indicated that theDREB1A repression of ice1-1 is another case of transgene-induced silencing. This phenomenon is widely observed duringplant genetic engineering and occasionally suppresses the trans-gene itself and other cotransfected transgenes (Matzke et al.,1994; Daxinger et al., 2008; Mlotshwa et al., 2010). Some trans-geneswere reported to cause homology-dependent endogenousgene silencing in transgenic plants (Sidorenko and Peterson,2001; Wang et al., 2011), similar to how NICE1 caused silencingof DREB1A (Figures 6 and 7). However, compared with mosttransgene-induced silencing that gradually occurs in generations,

NICE1-induced silencing has strong characteristics in terms of itsdistinctively instant effects onDREB1A expression (Figures 4 and5). The structure of the NICE1 transgene locus is intriguing andcontains an inverted repeat of reporter genes (Figure 4D), whichoffers hints to interpret the instantDREB1A repression. Themaize(Zeamays)MuDR transposon suppressorMuk locus generates aninverted repeat transcript homologous to MuDR, which is pro-cessed into sRNAs and initiates the heritable suppression ofMuDR (Slotkin et al., 2005). In Arabidopsis, previous reports ontargeted de novo DNA methylation demonstrated that ex-pressing inverted repeats of the target promoter sequence issufficient to provoke hypermethylation of the promoter and si-lencing of the following gene in a single generation (Kanno et al.,2004; Kinoshita et al., 2007). Therefore, it is suggested that theinverted repeat structure of NICE1 potentially acceleratestransgene-induced silencing via rapid sRNA generation throughthe allocated pathway.The instant recovery of DREB1A after losing the NICE1 trans-

gene implied that theDREB1Apromoter becamea target of active5mC removal (Figures 4 and 6). The first report of Arabidopsisactive DNA demethylase REPRESSOR OF SILENCING1 (ROS1)described that ros1dysfunctionbrought instant repressionofbothtransgenic and native RD29A promoters (Gong et al., 2002). Thisanalogous feature suggested that ROS1 functions against transgene-induced silencing. Considering its enzyme activity, ROS1 wouldbe a factor that supports the rapid demethylation and recovery oftheDREB1Apromoter activity in theoutcrossingprogeny that lackNICE1. With these unique features, future studies on NICE1 interms of gene silencing and RdDM would provide a deeper un-derstanding of transgene-induced silencing and its regulation.Our results indicated that ICE1 isnot involved in the regulationof

cold-inducible expression ofDREB1A/CBF3. The question arisesas to what kind of transcription factors regulate DREB1A/CBF3expression. The central oscillators of the circadian clock, CCA1and LHY, play an important role in inducing the expression ofDREB1A under cold conditions (Dong et al., 2011; Kidokoro et al.,2017). Considering that the expression of DREB1A exhibiteda circadian rhythm under cold conditions, the major regulatoryfactors involved in cold-inducible DREB1A expression may beclock-related factors such as CCA1 and LHY. However, evenin cca1 lhy double mutants, the cold-inducible expression ofDREB1A persisted considerably and continued to exhibit a cir-cadian rhythm, which indicates that other clock-related factorsmay be involved in DREB1A expression. Furthermore, CCA1 andLHY are known to function as repressors of the expression ofseveral core clock genes such as TOC1, PRR5, PRR7, and PRR9at 22°C (Nagel et al., 2015; Kamioka et al., 2016; Shalit-Kanehet al., 2018). The mechanisms by which circadian clock–relatedfactors including CCA1 and LHY activate the cold-specific ex-pressionofDREB1Ahavenot yetbeenclarified.Wespeculate thatunknown factors can alter the function of circadian clock–relatedfactors in response to cold stress and that these unknown factorsprobably form complexeswith the circadian clock–related factorsunder cold stress conditions. We expect that these factors will beclarified in the near future.


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METHODS

Plant Materials

The Arabidopsis (Arabidopsis thaliana) seeds of ice1-1 (CS67843; Chin-nusamy et al., 2003), the known parental line of ice1-1 (CS67845; Chin-nusamyet al., 2003), ice1-2 (SALK_003155;Kanaoka et al., 2008), scrm2-1(CS836083; Kanaoka et al., 2008), drm2 (SALK_150863; Yamamuro et al.,2014), cmt3 (SALK_148381; Cao et al., 2003), and nrpd1 (SALK_128428;Yamamuro et al., 2014) were obtained from the Arabidopsis BiologicalResources Center. The scrm-D (Kanaoka et al., 2008) seeds were kindlyprovided by Keiko U. Torii (University of Washington). For the transgenicArabidopsis plants, the 5030-bp genomic region (including the 2284-bppromoter region) of ice1-1was amplified and cloned into theKpnI andNotIsites of a pGreen0029 vector (Hellens et al., 2000). The subsequent pro-cesses of transfection and antibiotic selection were in accordance withprevious processes (Kidokoro et al., 2017). The plants were grown on GMagar plates at 226 1°Cunder a 16-h-light/8-h-dark cycle and aphoton fluxdensity of 506 10 mmol m22 s21 of white light. The oligomers applied arepresented in the Supplemental Table.

Cold Treatment and Plant RNA Extraction

For thecoldstress treatment, 12-d-oldwholeseedlingsonagarplatesweregradually chilled in the 4°C cold chamber for 3 h (Kidokoro et al., 2017). Thetreatments were started at 2 h after dawn. Four to eight plants, dependingon the plant size, were pooled to obtain a single sample for RNA prepa-ration. The plant total RNA extraction was conducted with RNAiso plus(Takara Bio) and supplemental DNase treatment. The extracted RNA wassubjected to RT-qPCR and RNA gel blot assays.

RT-qPCR

cDNAwas synthesized from the plant total RNA by a High-Capacity cDNAReverse Transcription Kit (Applied Biosystems). RT-qPCRwas performedusing the QuantStudio 3 Real-Time PCR system and software version 1.2(Applied Biosystems). Power SYBR Green Master Mix (Applied Bio-systems) was used for amplification. Arabidopsis IPP2 was used as thequantitative control for the template. For each biological replicate in all RT-qPCR experiments, three independent RNA samples were analyzed. Theoligomers applied are presented in Supplemental Table.

Whole-Genome Resequencing

A total of 1mgofArabidopsis genomicDNA for each samplewas subjectedto Illumina sequencing library construction according to the TruSeq DNAsample preparation guide (Illumina). Single-read sequencing was per-formed on a NextSeq 500 system, resulting in an average of 34.5 millionreads (75 nucleotides) from each library. Themanually cleaned reads weremapped to The Arabidopsis Information Resource (TAIR10) using bwaversion 0.7.5a (Li and Durbin, 2009). Genetic variants of the samples werecalled using BCFtools version 1.3.1 (Li, 2011). Only biallelic SNPs sup-ported bymore than six readswere retained for the analysis. Supplementalpaired-end sequencing was performed on a HiSeq 2500 system, whichoutput 23.4 million reads (23 101 nucleotides) of the ice1-1 genome. Thecleaned readsweremapped to TAIR10 using bwa, and abnormal singletonmapping featureswere retrieved to estimatewhere T-DNAwaspositioned.Todetermine thebasepair-resolutionbordersof theT-DNA locus, targetedassemblybyTASRversion1.6.2 (WarrenandHolt, 2011)wasperformedonthe flanking genomic region where singleton mapping features wereobserved.

Local Bisulfite Sequencing

A total of 0.5 mg of Arabidopsis genomic DNA was subjected to bisulfitetreatment and subsequent Sanger sequencing analysis. The bisulfitetreatment and DNA purification steps were achieved with an EpiTect Bi-sulfite Kit (Qiagen). Each region of interest was amplified from the bisulfite-treated DNA, and the amplicon was sequenced individually upon sub-cloning into a pGEM-T vector system (Promega).More than 12 independentclones were sequenced for each sample data. The oligomers applied arepresented in Supplemental Table.

5azaC Treatment

Sterilized Arabidopsis seeds were sown on GM agar plates that weresupplemented with 5azaC (A2232, Tokyo Chemical Industry). The samevolume of dimethyl sulfoxide solvent was supplemented for the mockcondition. The seedlings were grown for 12 d under the same growthconditions described above and then subjected to the cold treatmentdescribed above to evaluate the treatment effects.

sRNA Gel Blots

The details of the procedure followed that in a previous report by Schwabet al. (2006). In brief, 30 mg of total RNA per lane was run on a 17% (w/v)acrylamide gel that was supplemented with 7 M urea, after which the RNAwas transferred to a Nytran SPC membrane (GE Healthcare). The blotswere hybridized using 32P-end–labeled oligonucleotide probes of theDREB1Apromoter sequence,which included boxes V and VI (SupplementalTable). Hybridizationwas performed in PerfectHyb Plus HybridizationBuffer(Sigma-Aldrich) at 38°C overnight.

Freezing Tolerance Test

The freezing treatmentwasperformedaspreviouslydescribedbyKidokoroet al. (2015), withminormodifications. Ten-day-oldwhole seedlings grownon agar plates were chilled in a 22°C cold chamber for 2 h. After thegeneration of ice nuclei, the temperature was lowered by 21°C h21 until29°C was reached. The plates were then transferred to 4°C and thawedovernight. After recovery at 22°C for a week, the seedlings that generatednew leaves were counted as having survived.

Accession Numbers

The raw sequence data from the whole genome resequencing analysiswere deposited in National Center for Biotechnology Information ShortRead Archive under a specific accession number (PRJNA489736). Se-quence data from this article can be found in the Arabidopsis GenomeInitiative database under the following accession numbers: ICE1(AT3G26744), SCRM2 (AT1G12860), DREB1A (AT4G25480), DREB1B(AT4G25490), DREB1C (AT4G25470), EPF1 (AT2G20875), COR15A(AT2G42540), RD29A (AT5G52310), GolS3 (AT1G09350), NRPD1(AT1G63020), DRM2 (AT5G14620), CMT3 (AT1G69770), and IPP2(AT3G02780).

Supplemental Data

Supplemental Figure 1. The candidate SNPs were rejected byadditional derived cleaved amplified polymorphic sequences (dCAPS)(Supports Figure 4.).

Supplemental Figure 2. NICE1 alleles have little effect on theexpression of flanking genes (Supports Figure 4.).

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Supplemental Figure 3. Cold-induced DREB1A expression in trans-genic plants transformed with TE or TE and DREB1Apro:LUC (Sup-ports Figure 4.).

Supplemental Figure 4. Cytosine methylation levels of the DREB1Band DREB1C promoters by NICE1 genotype (Supports Figure 6.).

Supplemental Figure 5. Effects of 5azaC treatment on DREB1Aexpression in ice1-1 (Supports Figure 6.).

Supplemental Figure 6. Dysfunctional effects of DNA methylationcomponents on DREB1A expression (Supports Figure 7.).

Supplemental Figure 7. A working model of 5mC-mediated DREB1Arepression and recovery via the Arabidopsis ice1-1mutation (SupportsFigures 4-7.).

Supplemental Table. Oligomers used in this study.

Supplemental Data Set. P values of statistical analyses in this study.

ACKNOWLEDGMENTS

We thank Yuriko Tanaka (University of Tokyo), Tomomi Shinagawa, AyamiFuruta (Chubu University), and Saho Mizukado and Fuyuko Shimoda(RIKEN) for providing excellent technical assistance and Etsuko Toma(University of Tokyo) for providing skillful editorial assistance. We alsothank Tetsuji Kakutani (University of Tokyo) for the fruitful discussions andvaluable suggestions concerning DNA methylation to prepare the articleand Keiko U. Torii (University of Washington) for kindly providing scrm-Dmutant seeds. This work was supported by the Japan Society for thePromotion of Science (Grants-in-Aid for Scientific Research for YoungScientists [B]17K15413 to S.K., for Scientific Research [A]; 18H03996 toK.Y.-S., and for ScientificResearchon InnovativeAreas15H05960 toK.Y.-S.), and by RIKEN (Special Postdoctoral Researcher program and theIncentive Research Project to J.-S.K.).

AUTHOR CONTRIBUTIONS

S.K., J.-S.K., and K.Y.-S. designed the study. S.K., J.-S.K., and T.I.performed the experiments and analyzed the data. T.S. contributed tothegenome resequencing.S.K., J.-S.K., K.S., andK.Y.-S.wrote thearticle.All of the authors discussed the results and commented on the article.

Received July 12, 2019; revisedDecember 17, 2019; accepted February 3,2020; published February 7, 2020.

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