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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
The Plant Cell, Vol. 32: 1035–1048, April 2020,
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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
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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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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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