Molecular Regulation of CBF Signaling in Cold Acclimation

TRPLSC 1675 No. of Pages 15

Review

Molecular Regulation of CBF Signaling inCold Acclimation

Yiting Shi,1,2 Yanglin Ding,1,2 and Shuhua Yang1,*

HighlightsPlants withstand freezing stress bytriggering cold acclimation processes;the CBF-dependent pathway has acentral role in cold acclimation inplants.

A cold acclimation mechanism is pro-posed by which the cold signal istransduced from the membrane tothe nucleus, leading to a series of bio-chemical and physiological changes inthe cell and the induction of cold-responsive genes.

A subset of transcriptional regulators isinvolved in CBF-dependent and -inde-pendent pathways that regulate cold-regulated (COR) gene expression.

Several protein kinases are importantregulators of the CBF signaling path-way, including SNF1-related proteinkinases (SnRK2s), receptor-like pro-tein kinases, and mitogen-activatedprotein kinases (MAPKs).

The interplay of cold, light, and phyto-hormone signaling is important for bal-ancing freezing tolerance and plantgrowth.

1State Key Laboratory of PlantPhysiology and Biochemistry, Collegeof Biological Sciences, ChinaAgricultural University, Beijing 100193,China2These authors contributed equally

*Correspondence:[email protected] (S. Yang).

Cold stress restricts plant growth, development, and distribution. Understand-ing how plants transduce and respond to cold signals has long been a topic ofinterest. Traditional genetic and molecular analyses have identified C-repeat/DREB binding factors (CBFs) as key transcription factors that function in coldacclimation. Recent studies revealed the involvement of pivotal protein kinasesand transcription factors in CBF-dependent signaling, expanding our knowl-edge of cold signal transduction from perception to downstream gene expres-sion events. In this review, we summarize recent advances in our understandingof the molecular regulation of these core components of the CBF cold signalingpathway. Knowledge of the mechanism underlying the ability of plants tosurvive freezing temperatures will facilitate the development of crop plantswith increased freezing tolerance.

General Effects of Cold Stress on PlantsCold stress is a major environmental stress that limits the geographical location of plants innature, and may significantly reduce crop production. When a plant encounters low temper-atures, a series of cellular responses and molecular strategies can be activated that allow theplant to adapt to cold stress (see Glossary). To understand the effects of cold stress on plants,one has to distinguish between chilling stress (0–15�C) and freezing stress (<0�C) [1,2]. Chillingstress causes the membrane to rigidify, destabilizes protein complexes, and impairs photo-synthesis [3], whereas freezing stress causes more serious injuries to the plant. Freezingtemperatures promote ice formation in the apoplast (intercellular spaces) of plant tissues[4]. The accumulation of intercellular ice physically disrupts the cell membrane [5], causinga decrease in the water potential outside the cell, and leading to severe cell dehydration [4,5]. Inresponse to freezing stress, plants in temperate climates, such as winter wheat (Triticumaestivum), rye (Secale cereale L.), barley (Hordeum vulgare), and oat (Avena sativa), haveevolved sophisticated cold acclimation mechanisms that improve plant freezing toleranceupon exposure to nonfreezing temperatures [2,6].

Understanding Cold Acclimation in PlantsCold acclimation involves an array of physiological and biochemical modifications. The fieldtook a step forward with the discovery of cold-regulated (COR) genes, which function in theplant response to low temperatures and were first described by Guy and colleagues in 1985 [7].In Arabidopsis thaliana, COR genes include the COR, low-temperature induced (LTI), respon-sive to desiccation (RD), and early dehydration-inducible (ERD) genes. Some of these genesencode key enzymes for osmolyte biosynthesis that increase freezing tolerance via theaccumulation of cryoprotective proteins and soluble sugars, thus repairing cold-rigidifiedmembranes and stabilizing cellular osmotic potential [6]. CBF transcription factors, alsoknown as dehydration responsive element (DRE) binding factor1 (DREB1) proteins, are criticalfor cold acclimation in higher plants [8,9]. CBF proteins recognize the CRT/DRE cis-element,

Trends in Plant Science, Month Year, Vol. xx, No. yy https://doi.org/10.1016/j.tplants.2018.04.002 1© 2018 Elsevier Ltd. All rights reserved.

mailto:[email protected]

https://doi.org/10.1016/j.tplants.2018.04.002


GlossaryC-repeat/DREB binding factor(CBF) transcription factors: alsoknown as DREB1s, these AP2 familytranscription factors mediate plantfreezing tolerance by binding to CRT/DRE cis-elements containing theconserved CCGAC sequence in CORgenes and upregulating theirexpression.Cold acclimation: a processwhereby plants acquire freezingtolerance after exposure tononfreezing temperatures. The keyroles of cold acclimation are tostabilize the membrane and tomaintain the osmotic potential ofplant cells to help them resistfreezing injury. The acclimationmechanism is conducted throughtranscriptional reprogramming ofstress-responsive genes, which altersmetabolic homeostasis according tothe severity of the cold stress andthe plant species.Cold-regulated (COR) genes:genes that are regulated by lowtemperature. In Arabidopsis thaliana,COR genes are classified as lowtemperature-induced (LTI), cold-inducible (KIN), responsive todesiccation (RD), and earlydehydration-inducible (ERD) genes,some of which encode osmolyte andcryoprotective proteins that protectplant cells against freezing injury.Cold stress: stress caused bytemperatures below normal growthconditions, which can be classifiedas chilling stress (0–15�C) or freezingstress (below 0�C). Chilling stressusually has deleterious effects onphotosynthesis, membrane fluidity,reactive oxygen species (ROS)homeostasis, and energy metabolismin tropical and subtropical plants.Freezing stress results in intercellularice formation, which leads topermanent membrane lesions andstructural damage to the cell.Post-translational modifications(PTMs): the covalent and generallyenzymatic modification of proteinsduring or after translation. PTMshave a fundamental role in proteomicchanges by regulating proteinstructure, activity, and localization.Such modifications includephosphorylation, glycosylation,ubiquitination, sumoylation, S-nitrosylation, methylation, and

which contains the conserved CCGAC sequence and is found in the promoters of a subset ofCOR genes [9–11]. When plants are exposed to nonfreezing low temperatures, CBF genes arerapidly induced within 15 min, followed by the activation of downstream target COR genes,known as the CBF regulon [9,11–13].

Three CBF family members, CBF1–CBF3, are tandemly arranged in an 8.7-kb region ofchromosome 4 of the arabidopsis genome [11,13]. The functions of CBF genes have beencharacterized by overexpressing a dominant negative form of CBF2 or using antisense, RNAi,or CRISPR/Cas9 technology, in addition to overexpressing these genes [14–18]. Cold-accli-mated cbf1,2,3 (cbfs) triple-mutant arabidopsis seedlings, which were generated via CRISPR/Cas9, were found to be more sensitive to freezing stress than were cold-acclimated cbf2, cbf3,and cbf1,3 mutants [15,16], indicating that CBF genes are critical for cold acclimation andexhibit functional redundancy. However, some contradictory conclusions were reached inthese studies: (i) one study reported that a T-DNA insertion mutation of CBF2 showedenhanced freezing tolerance [17], whereas another study found that cbf2 was slightly suscep-tible to freezing stress [15]; and (ii) both the cbf1,3 RNAi lines produced in [17] and the null allelecbf1,3 double mutant obtained in [16] were less tolerant to freezing stress than was the wild-type. However, the cbf1 cbf3 double mutant generated in [17] exhibited increased freezingtolerance. The reasons for these discrepancies remain unknown. It might be that truncatedCBF1/3 proteins are produced that interrupt the normal functioning of CBF2 [19]. Alternatively,the chromosome structure or crucial regulatory elements in some of these lines might bedestroyed due to the deletion of a large DNA fragment, leading tomisregulation of the remainingCBF. Indeed, the relationships among CBFs are complex and merit further exploration.

CBFs have been isolated in many plant species, such as rice, tomato (Solanum lycopersicum),B. napus, wheat, barley, andmaize (Zeamays), suggesting that CBF is conserved in plants thatboth have and lack the capacity to acclimate to cold temperatures [20,21]. Overexpression ofCBF genes results in the induction ofCOR gene expression and increased freezing tolerance inmany plant species [8,22–26]. Phylogenetic analysis of the arabidopsis CBF proteins and theirorthologs in various plants suggests that CBFs are phylogenetically conserved across thesespecies (Figure 1). Although CBF family proteins are primarily involved in regulating COR geneexpression, other functions have been noted for individual CBF members. For example, CBF2has been shown to negatively regulate the expression of CBF1 and CBF3 in arabidopsis [17].CBF4, a uniquemember of the CBF transcription factor family, has a role in plant drought stresstolerance [27]. Bioinformatic analysis comparing the transcriptional profiles of individual cbfsingle mutants identified both overlapping and distinct target genes for each CBF protein[15,16,28]. Thus, analyzing the functional divergence of different CBF genes in stressresponses would be a challenging topic for further research. Studies conducted in Arabidopsisthaliana populations provide direct evidence that natural variation at the CBF locus has animportant role in the adaptive evolution of plant freezing tolerance [29–32]. For example,promoter polymorphisms of CBFs affect the expression of CBF genes, while point mutationsor deletions inCBF coding regions alter the structure or transactivation of CBF proteins to affectCOR expression in accessions from warm habitats [33–35]. Further insight into the naturalselection of the CBF pathway would enhance our understanding of the importance of freezingtolerance in the evolution of arabidopsis ecotypes.

The Transcriptional Regulatory Network Underlying Cold AcclimationOver the past two decades, various transcription factors have been identified that regulateCBFexpression by recognizing different cis-elements in their promoters under cold conditions(Figure 2 and Table 1). DNA sequence analysis of CBF promoters identified two motifs (ICEr1

2 Trends in Plant Science, Month Year, Vol. xx, No. yy


acetylation. PTMs influence almost allaspects of cellular action.Transcriptional regulation: aprocess whereby cells control theexpression of genes in part throughtranscription factors, transcriptionalregulatory proteins that bind toparticular sites of the genome andthereby enhance or repress thetranscription of genes. This processallows the cell to appropriatelyrespond to environmental,developmental, or other signals.

AtCBF2 (Arabidopsis thaliana)




BoCBF1b (Brassica oleracea)

BoCBF3 (Brassica oleracea)

BoCBF1a (Brassica oleracea)



MtCBF2 (Medicago truncatula)


LeCBF3 (Solanum lycopersicum)




ZmDREB1.8 (Zea mays)










OsDERB1F (Oryza sa va)

OsDREB1E (Oryza sa va)

OsDREB1G (Oryza sa va)

OsDREB1C (Oryza sa va)

OsDREB1B (Oryza sa va)

OsDREB1D (Oryza sa va)

OsDREB1A (Oryza sa va)

OsDREB1H (Oryza sa va)

HvCBF11 (Hordeum vulgare)



HvCBF4A (Hordeum vulgare)









46

97

99

99

99

39

95

98

85

76

56

56

51

52

63

62

85

65

94

72

75

99

42

95

82

93

92

94

70

68

72

86

82

57

74

70

95

94

100

100

100

96

(See figure legend on the bottom of the next page.)

Trends in Plant Science, Month Year, Vol. xx, No. yy 3


Cold stress

Protein kinase Light Hormone

OST1

ICE1

MYC MYB CM2 G-box/E-box BRRE EBS CBS CarG-box CBF genes

MYB15 CAMTA3 CESTA EIN3 CCA1/LHY SOC1

PRRsTranscrip onal regula on

BZR1/BES1

1PIF3,4,7

MPK3/6 CaM PhyB BR ETH

Circadian Flowering

??

Ca2+

Figure 2. Transcriptional Regulation of C-Repeat/DREB Binding Factor (CBF) Genes during Cold Acclimation in Arabidopsis. CBFs act as mastertranscription factors that regulate a complex regulatory network. Under normal temperatures,CBF expression is maintained at low levels to ensure normal plant growth.Upon exposure to low temperatures, CBFs are induced rapidly by transcription factors, such as inducer of CBF expression 1 (ICE1), calmodulin-binding transcriptionactivator 3 (CAMTA3), brassinazole-resistant 1/brassinosteroid insensitive 1-EMS-supressor 1 (BZR1/BES1), CESTA (CES), and circadian clock associated 1/lateelongated hypocotyl (CCA1/LHY), or repressed by MYB15, phytochrome-interacting factors (PIFs), ethylene insensitive 3 (EIN3), and suppressor of constansoverexpression 1 (SOC1). In addition, pseudo-response regulators (PRRs) act either directly or indirectly as negative regulators of CBFs. The cold-activated proteinkinases open stomata 1 (OST1) and MPK3/6 phosphorylate and regulate the stability of ICE1, thus modulating CBF expression. MPK6 phosphorylates MYB15 torelease the repression of CBF expression. CAMTA proteins bind calmodulin (CAM), which is thought to integrate calcium signaling with CBF gene expression.PhytochromeB (PhyB) and PIFs are required for photoperiodic and/or light regulation of CBF expression. Stabilized PIFs bind to the CBF promoters and suppress theirexpression. Transcription factors involved in hormone signaling pathway, including brassinosteroids (BRs) and ethylene (ETH), alsomodulateCBF expression. TheCBFgenes are induced by BZR1/BES1 and CES of the BR signaling pathway, but are repressed by EIN3 of the ETH signaling pathway. CCA1/LHY, two important factorsregulating the circadian clock, directly activate CBF expression. The flowering time component SOC1 is required for the downregulation of CBFs. PRRs are suggestedto have negative effects on CBF transcription under low temperature stress. The detailed functions of these transcription factors are discussed in the main text.

and ICEr2) that are required for cold induction [36]. The best-characterized transcriptionalactivator to date is inducer ofCBF expression1 (ICE1), a MYC-like basic helix-loop-helix (bHLH)transcription factor [37]. ICE1 binds to the MYC-binding sites (CANNTG) [38] in the promotersof CBF1–3, thereby promoting their expression under cold stress [287_TD$DIFF][37,40]. The ice1 mutantwas originally isolated in a genetic screen for plants with abnormal regulation of CBF3–LUCexpression in the cold [37]. The ice1mutant exhibits impaired freezing tolerance and significantdefects in the induction of CBF genes, whereas overexpression of ICE1 enhances the cold-induced upregulation of CBF1–3 [37,39]. ICE2, a paralog of ICE1, has functionally redundant

Figure 1. Phylogenetic Tree of C-Repeat/DREB Binding Factors (CBFs) from Arabidopsis and OtherSpecies. The phylogenetic tree was constructed based on the amino acid sequence alignments from seven species.Homology searches were performed using BLAST with default parameters at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). This tree was constructed using MEGA7.0 with the Neighbor-Joining method. Numbers above/below branchesrepresented bootstrap values (from 1000 replicates). The arabidopsis CBFs are illustrated in red.


http://blast.ncbi.nlm.nih.gov/Blast.cgi

http://blast.ncbi.nlm.nih.gov/Blast.cgi


Table 1. Proteins Functioning in the CBF Pathway in Arabidopsis

Factor Protein characteristics Direct target(s) Effect on freezingtolerance(negative/positive)

Refs

CRLK1/2 Protein kinases MEKK1-MKK2-MPK4 Positive [95,99,100]

MPK3/6 Protein kinases ICE1, MYB15 Negative/Positive [50,94,95]

CRPK1 Protein kinase 14-3-3 proteins Negative [101]

OST1 Protein kinase ICE1, BTF3, BTF3L Positive [39,93]

HOS1 E3 ubiquitin ligase ICE1 Negative [90]

SIZ1 SUMO E3 ligase ICE1 Positive [89]

EBF1/2 E3 ubiquitin ligases PIF3, EIN3 Positive [53]

14-3-3l/k General regulator factors CBF1, CBF3 Negative [101]

BTF3/3L b-subunits of a nascentpolypeptide-associatedcomplex (NAC)

CBF1, CBF2, CBF3 Positive [93]

ICE1 Transcription factor CBF1, CBF2, CBF3 Positive [37,40,108]

ICE2 Transcription factor CBF1, CBF2, CBF3 Positive [40,41]

CAMTA3 Transcription activator CBF1, CBF2 Positive [45,47]

BZR1/BES1 Transcription factors CBF1, CBF2 Positive [60]

CCA1/LHY Transcription factors CBF1, CBF2, CBF3 Positive [62]

CESTA Transcription factors CBF1, CBF2, CBF3 Positive [59]

EIN3 Transcription factor CBF1, CBF2, CBF3 Negative [54]

PIF3/4/7 Transcription factors CBF1, CBF2, CBF3 Negative [53,109]

SOC1 Transcription factor CBF1, CBF2, CBF3 Negative [67]

PRRs Pseudo-response regulators CBF1, CBF2, CBF3 (director indirect)

Negative [65]

FVE Homolog of the mammalianretinoblastoma-associatedprotein

COR15A, COR47 (director indirect)

Negative [68]

PhyB photoreceptor PIF4, PIF7 Negative [81,109]

roles in regulating CBF1 expression [40,41]. ICE1 is constitutively expressed in leaves, stems,and other tissues [37]. Interestingly, ICE1/2 are specifically expressed in the stomatal cells ofleaf tissues [41,42], prompting speculation that ICE1/2 are specific regulators of the CBF-CORpathway in stomata in land plants. A previous study showed that low temperatures reducestomatal apertures by inducing apoplastic calcium uptake by guard cells in Commelinacommunis [43]. It would be interesting to test whether CBF signaling pathways participatein regulating stomatal development and movement during cold acclimation.

Another group of transcriptional activators that regulates CBFs is calmodulin-binding tran-scription activator (CAMTA) proteins, which contain conserved calmodulin-binding sites [44].CAMTA3 protein was initially found to bind to the CM2motif (CGCG-box) of theCBF2 promoterand activate its expression [45,46]. CAMTA1 and CAMTA2 are positive regulators of the cold-induced expression of CBF1–3 [45,46]. Moreover, CAMTA proteins (CAMTA1–5) function astranscriptional activators that regulate the expression of CBF1 and CBF2 [47]. Interestingly,CAMTA3 and CAMTA5 regulate the expression of CBF1 in response to rapid (but not gradual)



decreases in temperature [47]. Considering that low temperatures result in a rapid increase incytosolic calcium levels, this finding links calcium signaling and cold induction of the CBFpathway. It remains to be determined how the cold signal is transduced to activate CAMTAs,and which components are involved in the post-translational modification (PTM) and/orare cofactors of CAMTA at low temperature. Investigations into these questions would expandour current understanding of the CBF signaling pathway in plants.

Some transcriptional repressors also directly suppress the cold-induced activation of CBFs,including MYB15 [48] and Phytochrome-interacting factors (PIFs) [49]. MYB15, a member ofthe R2R3 family of MYB transcription factors, regulatesCBF expression under low temperatureconditions [48]. A recent study showed that MPK6 interacts with, and phosphorylates, MYB15to reduce its binding to CBF promoters [50], thus releasing the inhibitory effect of thistranscription factor on CBF gene expression. PIFs are a subset of bHLH transcription factorsthat have central roles in the phytochrome-mediated light-signaling network [49,51]. PIF4 andPIF7 bind to the G-box motifs in the CBF1 and CBF2 promoters and the E-box motif in theCBF3 promoter under long-day conditions [52]. Additionally, PIF3 associates with the G-boxand E-box motifs in the promoters of all three CBF genes, repressing their transcription underboth dark and low temperature conditions [53]. These observations suggest that negativetranscription factors have important roles in maintaining the homeostasis of CBF-regulatedtranscriptional networks during cold acclimation.

Plants have evolved sophisticated strategies in which hormone and cold signaling pathways arecoordinated to better adapt to freezing stress. Ethylene insensitive 3 (EIN3) is a key transcriptionfactor involved in ethylene signaling that negatively regulates freezing tolerance in arabidopsis,partially through affecting the expression ofCBFs by binding to the EBSmotifs in their promoters[54]. Two F-box proteins EBF1/2 of the ethylene-signaling pathway positively regulate CBFexpression by mediating the degradation of the transcriptional repressors EIN3 and PIF3[53,54]. Consistently, ethylene-insensitive plants grown on agar plates displayed enhancedfreezing tolerance [53,54]. These results support the notion that ethylene negatively regulatesfreezing tolerance. By contrast, another study showed that an arabidopsis ACS octuple mutantgrown in soil pots was deficient in ethylene biosynthesis, was hypersensitive to freezing, andexhibited reduced induction of CBF and COR genes under cold treatment [55]. Moreover,overexpression of tomato ethylene response factor 2 (TERF2/[288_TD$DIFF]LeERF2) enhances plant freezingtolerance through promoting ethylene biosynthesis and the ethylene signaling pathway [56],suggestingthatethylenehasapositive role in regulatingplant freezing tolerance. Itappears that thefunctionof ethylene inplant freezing tolerance isdifferentwhenplants aregrown in soil versus agarplates, implying that the humidity or the decreasing rates of the temperature may impact thefreezing tolerance. Thus, the exact roles of ethylene in freezing tolerance in different growthconditions and plant species might be diverse and require further investigation.

Jasmonic acid (JA) is a lipid-derived plant hormone that positively regulates CBF signaling bymediating the interaction between JAZ1/4 protein and ICE1/2, thus modulating the transcrip-tional activity of ICE proteins and CBF1–3 gene expression [57]. Brassinosteroids (BRs) are aclass of plant steroid hormones that also have a role in promoting freezing tolerance [58]. Thebin2-3 bil1 bil2mutant, a loss-of-function mutant of GSK3-like kinases involved in BR signaling,showed increased freezing tolerance compared with the wild-type [59,60]. BZR1/BES1, whichact as downstream transcription factors in the BR-signaling pathway, positively regulatefreezing tolerance by promoting CBF1 and CBF2 expression through binding to the BRREand E-box motifs in their promoters [60]. The BR-controlled bHLH transcription factor CESTAregulates CBF expression by recognizing G-box motifs in their promoters [59]. Further studies



of the interaction between hormone signaling and cold signaling are likely to provide novelinsights into how plants precisely coordinate growth and freezing tolerance.

CBF1–3 expression is not only responsive to low temperatures, but is also gated by thecircadian clock [61–63]. The cold-induced upregulation of CBF genes depends on the time ofday [61]. Both clock regulatory elements and cold-responsive elements have been identified inCBF promoters [62,64]. Consistent with this observation, the morning-expressed MYB tran-scription factors CCA1 and LHY bind to evening elements (EE) and CCA1-binding site (CBS)motifs in the CBF promoters, and the cold-induced expression of CBFs is significantly reducedin the cca1 lhy double mutant [62]. Moreover, mutations in PRR5, PRR7, and PRR9, encodingkey components of the circadian oscillator, result in the upregulation ofCBFs [65], implying thatthe circadian clock is an essential cue for the full induction of CBFs at low temperatures. Thefloral activators SOC1 and FVE mediate the direct link between cold signaling and floweringtime [66]. SOC1 is a MADS-box transcription factor that negatively regulates CBF expressionthrough directly binding to the CArG boxes in their promoters [67], while FVE, an arabidopsishomolog of the retinoblastoma-associated protein, is a negative regulator of the CBF pathway[68]. Thus, the CBF signaling pathway integrates many internal and environmental cues, suchas low temperature, the circadian clock, light, and flowering time (Figure 2).

Cold treatment induces massive reprogramming of the plant transcriptome [69,70]. Genome-wide expression profiling of wild-type versus cbfs plants revealed that CBF proteins extensivelyregulate the expression of genes involved in stress responses, carbohydrate and lipid metab-olism, cell wall modification, transcription factors, kinases, and calcium (Ca2+ [286_TD$DIFF]) and hormonalsignaling [15,16]. Analysis of CBF-overexpressing lines and a cbfs triple mutant revealed thatfew hundreds of the approximately 4000COR genes are regulated by CBFs [14–16], indicatingthat some CBF-independent transcription factors are involved in modulating COR expression.Various transcriptional activators that function in parallel with CBFs, including HSFC1, ZAT12,ZF, ZAT10, RAV1, CZF1, and HY5, help regulate COR genes by bypassing CBF signaling[12,14,71]. BZR1 regulates COR gene expression via both CBF-dependent and -independentpathways in addition [60]. Thus, CBF-independent pathways may have major roles in modu-lating COR gene expression. However, how different transcription factors fine-tune CORexpression to establish cold acclimation remains to be fully elucidated.

Perception of the Cold SignalThe current model of cold-sensing mechanisms in plants is that cold shock leads to changes inmembrane fluidity and rearrangement of the cytoskeleton that trigger Ca2+ influx, which in turnactivates the expression of downstreamCOR genes [72]. The transient receptor potential (TRP)superfamily of cation channels is involved in thermosensation in mammals [73]. However,because no TRP orthologs have been identified in land plants, a fundamental question iswhether calcium channels are involved in cold signal perception. The identification of COLD1, atransmembrane protein that functions as a cold sensor in rice (Oryza sativa), provides supportfor this hypothesis [74]. The transmembrane protein COLD1 regulates G-protein signaling byinteracting with rice G-protein a subunit1 (RGA1) to accelerate GTPase activity. Under chillingstress, COLD1-RGA1 form a complex that mediates the cold-induced influx of intracellular Ca2+, subsequently leading to COR gene expression [74]. It would be interesting to determinewhether COLD1 itself functions as a temperature-regulated membrane ion channel, andwhether cold-induced Ca2+ influx is sufficient for enhancing cold tolerance in plants [75].

Light and temperature are environmental cues that provide seasonal information to plants [76].Recent studies have shown that the photoreceptors phyB [77,78] and phototropins (PHOTs)



[79] act as thermosensory molecules that perceive fluctuating temperatures in plants. Thephytochrome phyB has a major role in regulating photomorphogenesis [80]. Two companionpapers demonstrate that changes in ambient temperature could affect the reversion of phyBbetween the Pfr-Pr forms in arabidopsis [77,78], suggesting that phyB functions as a thermo-sensor. Interestingly, the expression of the CBF regulon is upregulated in phyB and phyDmutants at 16�C [81], suggesting that phytochrome also has a role in cold acclimation bymodulating CBF signaling. A recent study showed that the blue-light (BL) photoreceptor PHOTis responsible for the cold-avoidance response in the fern Adiantum capillus-veneris andliverwort Marchantia polymorpha [82–84]. In the liverwort, low temperatures are perceivedby the light/oxygen/voltage (LOV) domain of PHOT, thereby inducing chloroplast relocation andthe cold-avoidance response [79]. These findings imply that photoreceptors perceive both lightand temperature to optimize photosynthesis under different temperatures. It would be inter-esting to investigate the underlying mechanism mediated by photoreceptors that functions inthe perception of low temperatures in plants.

Post-Translational Modifications Are Important for Cold SignalingPTMs have profound effects on the functioning of proteins, including their activity, localization,and stability [85]. Accumulating evidence suggests that major PTMs, such as phosphorylation,ubiquitination, and sumoylation, have important roles in CBF-dependent signaling in plants[86,87]. Here, we summarize the regulatory roles of PTMs in ICE-CBF-COR-dependentsignaling (Figure 3 and Table 1).

As described above, the transcription factor ICE1 activates CBF and COR gene expression[37,88]. However, ICE1 expression is not responsive to cold treatment [89], suggesting thatPTMs are important for the functioning of ICE1. Indeed, multiple PTMs have been shown tocontrol the turnover and duration of ICE1 under low temperatures. The RING finger protein, highexpression of osmotically responsive gene1 (HOS1), is required for the 26S proteasome-mediated degradation of ICE1 [90]. HOS1 physically interacts with and ubiquitinates ICE1, thusreducing its stability under low temperatures. The overexpression of HOS1 decreases freezingtolerance in plants by repressing the expression ofCBF3 [90]. Sumoylation might protect targetproteins from 26S proteasomal degradation by preventing their ubiquitination [91]. SIZ1, aSUMO E3 ligase, is required for the accumulation of SUMO conjugates during cold treatment.SIZ1-mediated sumoylation of ICE1 at Lys393 increases its stability and reduces its poly-ubiquitination mediated by HOS1 [89]. The siz1 null mutant is hypersensitive to freezing stressand exhibits impaired cold-induced CBF expression [89].

Phosphorylation is the most common PTM that leads to signal transduction and amplification[85]. Several protein kinases belonging to diverse families, including SNF1-related proteinkinases (SnRK2s), mitogen-activated protein kinases (MAPKs), Ca2+-dependent protein kin-ases (CDPKs), calcineurin-B-like interacting protein kinases (CIPKs), and receptor-like kinases(RLKs), are involved in regulating cold acclimation [92].

The identification of cold-activated OST1/SnRK2.6 represents a remarkable advancement inour understanding of the function of a protein kinase in cold signaling [39]. Ding et al. initiallyfound that OST1, a Ser/Thr protein kinase in the ABA core signaling pathway, positivelyregulates CBF-mediated freezing tolerance through phosphorylating ICE1 at Ser278 to preventits 26S proteasome-mediated degradation by HOS1 [39]. The ost1 mutants are sensitive tofreezing stress, whereas overexpression of OST1 results in increased freezing tolerance. It wasreported more recently that OST1 phosphorylates b-subunits of NAC (a nascent polypeptide-associated complex), basic transcription factor 3 (BTF3), and BTF3-like (BTF3L) proteins in



Cold stress

COLD1

[Ca2+]

COLD1

Plasma membrane

Cytoplasm

Nucleus

26S proteasome26S proteasome

26S proteasome

Plant freezing tolerance

CRT/DRE

CBFs

CORs

Figure 3.

(Figure legend continued on the bottom of the next page.)

Cold Sensing in the Plant Cell. The plasma membrane is thought to be the primary target of cold sensingand the starting point for transmission of the cold signal into the nucleus. After exposure to cold stress, the plasma andendoplasmic reticulum (ER) membrane-located cold sensor chilling-tolerance divergence 1 (COLD1) interacts with rice G-protein a subunit 1 (RGA1), activating the Ca2+ signaling pathway in plant cells, and stimulating downstream cold signaltransduction and C-repeat/DREB binding factor (CBF) expression. Plasma membrane Ca2+-regulated receptor-likekinases calcium/calmodulin-regulated receptor-like kinase 1/2 (CRLK1/2) positively regulate cold-induced gene expres-sion by activating the MEKK1-MKK2-MPK4 pathway. Cold induces the activity of open stomata 1 (OST1), a pivotal kinasein the ABA signaling pathway, which interacts with and phosphorylates inducer of CBF expression 1 (ICE1) as well as basictranscription factor 3/BTF3-like protein (BTF3/BTF3L). The cold signal activates the MKK4/5-MPK3/6 cascade to regulatefreezing tolerance through phosphorylation and degradation of ICE1, thus attenuating the CBF signaling. A receptor-likecytoplasmic kinase cold-responsive protein kinase 1 (CRPK1) is activated by low temperature stress and phosphorylates



arabidopsis. Phosphorylated BTF3s enhance plant freezing tolerance by promoting CBFprotein stability through interacting with CBF proteins and promoting CBF transcription[93]. Cold-induced OST1 activation is independent of ABA [39]. Recently, two companionpapers indicated that the arabidopsis MAPK signaling cascade regulates freezing tolerancethrough the ICE1-CBF-COR transcriptional pathway [95,94]. MPK3/MPK6 interact with, andphosphorylate, ICE1 at conserved Ser/Thr residues (Ser94/403; Thr366 in [95]; Ser94/203/403; Thr366/382/384 in [94]), which reduces the stability of ICE1 as well as its transcriptionalactivity, thus negatively regulating CBF expression and freezing tolerance in arabidopsis[289_TD$DIFF][95,94]. In addition, the MEKK1-MKK[290_TD$DIFF]2-MPK4 cascade promotesCBF expression and freezingtolerance by antagonizing the MKK4/5-MPK3/6 pathway [291_TD$DIFF][95]. Interestingly, MPK3/MPK6-mediated phosphorylation of ICE1 does not affect the HOS1–ICE1 interaction [94].Whether MPK3/MPK6 recruit other E3 ligases, such as COP1 (an E3 ligase that mediatesdark-induced degradation of ICE1 during stomatal development [96]), remains to be explored.A recent study showed that OsMAPK3 phosphorylates and stabilizes OsICE1 in rice, whichdirectly regulates the expression of OsTPP1, encoding a key enzyme for trehalose synthesis,thereby positively regulating chilling tolerance [97]. These findings indicate that MAPK3 hasdistinct and even opposite roles in response to low temperatures in different plant species.Taken together, these compelling pieces of evidence suggest that phosphorylation is a PTMthat synergistically or antagonistically regulates the stability and abundance of ICE1 under cold-stress conditions.

RLKs alsomediate cold acclimation in plants [98]. CRLK1, a plasmamembrane-localized Ca2+[289_TD$DIFF]/calmodulin-regulated receptor-like kinase, positively regulates COR expression and coldacclimation, likely by phosphorylating MEKK1 and enhancing its activity [99,100]. CRLK1and its closest homolog, CRLK2, act upstream of the MEKK1-MKK2-MPK4 pathway topositively regulate CBF expression and freezing tolerance [291_TD$DIFF][95]. It was recently revealed thatthe receptor-like cytoplasmic kinase cold-responsive protein kinase1 (CRPK1) is a negativeregulator of the CBF pathway in arabidopsis [101]. Cold treatment activates the kinase activityof CRPK1 without affecting its localization. CRPK1 then directly interacts with, and phosphor-ylates, 14-3-3 proteins on the plasma membrane. These phosphorylated 14-3-3s translocateinto the nucleus to facilitate the proteasome-mediated degradation of CBF1 and CBF3, therebyattenuating an overwhelming cold acclimation in arabidopsis [101]. Another study showed thatRCI1A, a 14-3-3w, negatively regulates freezing tolerance in arabidopsis [55], implying that 14-3-3 family proteins are extensively involved in plant freezing tolerance. Given that receptor-likecytoplasmic kinases often act together with a receptor kinase, it is possible that phosphor-ylation is a primary event in cold perception and that unknown receptor-like kinase(s) may beinvolved in transmitting the cold signal from the membrane into the nucleus.

Tradeoff between Cold Tolerance and Plant GrowthThe constitutive overexpression of CBFs adversely affects plant growth under normal growthconditions [8,22]; although this has been known for years, the underlying mechanism remains

14-3-3 proteins. Phosphorylated 14-3-3s are translocated from the cytoplasm into the nucleus, where they negativelyregulate CBF signaling. The key components of CBF-dependent signaling, ICE1 and CBF, are modulated by post-translational modifications (PTMs). As for ICE1, phosphorylation of ICE1 by OST1 and MPK3/6 promotes and reduces thestability of ICE1 under cold stress. ICE1 is degraded by high expression of osmotically responsive gene1 (HOS1) throughthe 26S proteasome pathway. SUMO E3 ligase SIZ1-mediated sumoylation of ICE1 reduces polyubiquitylation of ICE1.The OST1-mediated phosphorylation of ICE1 interrupts the HOS1-mediated degradation of ICE1. MPK3/MPK6 mediatethe phosphorylation and degradation of ICE1 by recruiting unknown E3 ligases. 14-3-3 proteins interact with CBF1/3 andfacilitate the ubiquitin-mediated degradation of CBF1/3. Cold induces the accumulation of CBF proteins, which bind to theCRT/DRE motif in the promoters of cold-regulated (COR) genes and induce their expression.



Outstanding QuestionsHow are CBF-independent CORgenes regulated by cold stress at thetranscriptional level?

Which protein kinase(s) or E3 ligase(s)participate in the turnover of CBF tran-scription factors at the post-transla-tional level?

How does cold activate protein kin-ases at the early stage of the responseof plants to cold stress? Is cold-acti-vated protein kinase activity depen-dent on ROS or calcium signaling?

Do plants have additional protein kin-ases or phosphatases that regulate thefunctions of OST1, CRPK1, andMAPKs?

How do the positive and negative reg-ulators of CBF signaling coordinatetheir function to fine-tune the responseof plants to cold stress?

How do CBF signaling componentsinteract with plant hormones to bal-ance plant growth and freezingtolerance?

elusive. In plants overexpressing CBF1, the GA-inactivating GA2-oxidase gene and RGL3 areupregulated, leading to a decrease in bioactive GA levels and the accumulation of DELLAprotein [102]. Consistently, the dwarf and late-flowering phenotypes of CBF1-overexpressingplants are rescued by either GA treatment or crosses with a della double mutant [102]. Thesefindings suggest that CBF1 represses plant growth via a DELLA-dependent mechanism. Atnormal temperatures, cbfs triple mutants grow more slowly and have shorter roots, fewer andsmaller rosette leaves, and less biomass compared with wild-type plants [15]. However, whengrown under chilling stress (4�C), cbfs plants are larger than the wild-type [16]. Therefore, CBFsare master players in the trade-off between growth limitation and freezing tolerance, at leastpartially by integrating hormone-related pathways.

The effect of chilling stress on root development and growth has not been extensively studied.Studies have shown that chilling stress leads to DNA damage in the root stem cells [103,104]. Arecent breakthrough study revealed that the root stem cell niche adopts a novel ‘sacrifice-for-survival’ mechanism under chilling stress [105]; chilling stress preferentially causes newlygenerated columella stem cell daughters to die, which results in the auxin maximum beingre-established in the quiescent center, and functional stem cell niche activity being maintained,thus preventing further division of columella stem cells. This mechanism allows the plant stemcells to divide at a faster rate when optimal temperatures are restored, thereby improving theability of the root to withstand chilling stress [105]. These findings shed light on a uniquestrategy of how sessile plants survive adverse conditions, and provide a potential strategy forengineering cold-tolerant crops.

Concluding Remarks and Future PerspectivesThe transcriptional network of the CBF signaling pathway has been extensively studied over thepast two decades. Although genome-wide expression profiling has identified many CORgenes, only approximately 10–25% of these genes are regulated by CBFs, implying that moreearly cold-regulated transcription factors are involved in improving freezing tolerance. Severalelegant strategies have been used to isolate various transcription factors that function in parallelwith CBFs. However, more studies are needed to identify additional transcriptional regulatorsand to explore their underlying relationships to increase our understanding of the cold-relatedtranscriptional network.

Emerging evidence suggests that PTMs also have an important role in controlling the CBFpathway, with most studies to date focusing on the regulation of ICE1. An interesting phe-nomenon was recently reported, namely that CBF proteins are subject to 26S proteasome-mediated degradation [101]. This suggests that an unidentified E3 ligase participates in theturnover of these proteins. Thus, additional studies are needed to investigate the PTM-mediated regulation of CBF proteins. These studies will extend our understanding of theICE1-CBF-COR cascade at the posttranslational level.

Several protein kinases involved in CBF signaling have been successfully isolated. How-ever, how cold activates these protein kinases during the early stages of low temperatureperception remains unclear. CRPK1 is a cold-activated plasma membrane-localized pro-tein kinase, supporting the hypothesis that the cold signal is initially transmitted from theplasma membrane [101]. Notably, CRPK1 lacks a classic transmembrane domain, sug-gesting it is recruited to the membrane and activated by an unidentified membrane kinase(such as a RLK). Future studies should investigate whether cold-induced kinase activity isdependent on reactive oxygen species (ROS) or Ca2+ signaling. Given that COLD1 mightregulate Ca2+ levels in the cytoplasm, the next challenge is to identify the link between Ca2+



signaling and kinase activity. In particular, since phosphatases modulate protein phos-phorylation levels, it would be interesting to identify phosphatases involved in cold-triggeredkinase activity. Protein kinase and phosphatase pairs are important for signal perceptionand transduction in eukaryotes and prokaryotes [106]. In Bacillus subtilis, cold signals areperceived by plasma membrane-localized histidine kinase DesK, which has dual roles as aprotein kinase and protein phosphatase [107]. It is possible that plasma membrane-local-ized protein kinases and their phosphatases are involved in low temperature sensing andsignal transduction. Thus, it will be interesting to identify these putative proteins usingquantitative phosphoproteomics assays. Cold stress activates negative regulatory path-ways, such as the CRPK1-14-3-3s [101] and MPK3/6-ICE1 modules [289_TD$DIFF][95,94], to attenuateCBF-dependent signaling, but the physiological significance of these repressive pathwaysis unknown. High levels of CBF expression could have deleterious effects on the plant.Thus, a braking system for CBF signaling is important for fine-tuning the CBF pathway toprevent excessive growth retardation. Investigating the links between the positive andnegative regulators of CBFs would shed light on the mechanisms underlying the balancebetween plant growth and freezing tolerance.

Plants have evolved precise mechanisms to perceive fluctuating temperatures to improvetheir chance of survival. An important step in understanding the thermosensory mechanismin plants was the identification of phytochrome as a thermosensor [77,78]. Moreover, ablue-light receptor was found to function as a cold sensor in M. polymorpha, suggestingthat low temperature and light cues are integrated immediately at the sensor level [79].Nevertheless, the exact role of phytochromes in the response of plants to low temperaturesawaits further study. It would be interesting to investigate whether photoreceptors monitorcold signals to trigger CBF signaling and the expression of COR genes (see also Outstand-ing Questions).

AcknowledgmentsThis workwas supported by grants from the National Natural Science Foundation of China (No. 31730011 and 31670261).

We apologize to all colleagues whose relevant work could not be cited owing to space limitations.

References
1. Yadav, S.K. (2010) Cold stress tolerance mechanisms in plants.
Agron. Sustain. Dev. 30, 515–527

2. Thomashow, M.F. (1999) Plant cold acclimation: freezing toler-ance genes and regulatorymechanisms. Annu. Rev. Plant Phys-iol. Plant Mol. Biol. 50, 571–599

3. Ruelland, V. et al. (2009) Cold signalling and cold acclimation inplants. Adv. Bot. Res. 35–150

4. Pearce, R.S. (2001) Plant freezing and damage. Ann. Bot. 87,417–424

5. Steponkus, P.L. (1984) Role of the plasmamembrane in freezinginjury and cold acclimation. Annu. Rev. Plant Physiol. Plant Mol.Biol. 35, 543–584

6. Chinnusamy, V. et al. (2007) Cold stress regulation of geneexpression in plants. Trends Plant Sci. 12, 444–451

7. Guy, C.L. et al. (1985) Altered gene-expression during cold-acclimation of spinach. Proc. Natl. Acad. Sci. U. S. A. 82, 3673–3677

8. Jaglo-Ottosen, K.R. et al. (1998) Arabidopsis CBF1 overexpres-sion induces COR genes and enhances freezing tolerance.Science 280, 104–106

9. Liu, Q. et al. (1998) Two transcription factors, DREB1 andDREB2, with an EREBP/AP2 DNA binding domain separatetwo cellular signal transduction pathways in drought- and


low-temperature-responsive gene expression, respectively, inArabidopsis. Plant Cell 10, 1391–1406

10. Stockinger, E.J. et al. (1997) Arabidopsis thaliana CBF1 enc-odes an AP2 domain-containing transcriptional activator thatbinds to the C-repeat/DRE, a cis-acting DNA regulatory elementthat stimulates transcription in response to low temperature andwater deficit. Proc. Natl. Acad. Sci. U. S. A. 94, 1035–1040

11. Gilmour, S.J. et al. (1998) Low temperature regulation of theArabidopsis CBF family of AP2 transcriptional activators as anearly step in cold-induced COR gene expression. Plant J. 16,433–442

12. Vogel, J.T. et al. (2005) Roles of the CBF2 and ZAT12 transcrip-tion factors in configuring the low temperature transcriptome ofArabidopsis. Plant J. 41, 195–211

13. Medina, J. et al. (1999) The Arabidopsis CBF gene family iscomposed of three genes encoding AP2 domain-containingproteins whose expression Is regulated by low temperaturebut not by abscisic acid or dehydration. Plant Physiol. 119,463–470

14. Park, S. et al. (2015) Regulation of the Arabidopsis CBF regulonby a complex low-temperature regulatory network. Plant J. 82,193–207

15. Zhao, C.Z. et al. (2016) Mutational evidence for the critical role ofCBF transcription factors in cold acclimation in Arabidopsis.Plant Physiol. 171, 2744–2759

http://refhub.elsevier.com/S1360-1385(18)30086-4/sbref0005

















































16. Jia, Y. et al. (2016) The cbfs triple mutants reveal the essentialfunctions of CBFs in cold acclimation and allow the definition ofCBF regulons in Arabidopsis. New Phytol. 212, 345–353

17. Novillo, F. et al. (2004) CBF2/DREB1C is a negative regulator ofCBF1/DREB1B and CBF3/DREB1A expression and plays acentral role in stress tolerance in Arabidopsis. Proc. Natl. Acad.Sci. U. S. A. 101, 3985–3990

18. Novillo, F. et al. (2007) Arabidopsis CBF1 and CBF3 have adifferent function than CBF2 in cold acclimation and definedifferent gene classes in the CBF regulon. Proc. Natl. Acad.Sci. U. S. A. 104, 21002–21007

19. Hua, J. (2016) Defining roles of tandemly arrayed CBF genes infreezing tolerance with new genome editing tools. New Phytol.212, 301–302

20. Agarwal, P.K. et al. (2006) Role of DREB transcription factors inabiotic and biotic stress tolerance in plants. Plant Cell Rep. 25,1263–1274

21. Mizoi, J. et al. (2012) AP2/ERF family transcription factors inplant abiotic stress responses. Biochim. Biophys. Acta 1819,86–96

22. Gilmour, S.J. et al. (2000) Overexpression of the ArabidopsisCBF3 transcriptional activator mimics multiple biochemicalchanges associated with cold acclimation. Plant Physiol. 124,1854–1865

23. Qin, F. et al. (2004) Cloning and functional analysis of a novelDREB1/CBF transcription factor involved in cold-responsivegene expression in Zea mays L. Plant Cell Physiol. 45, 1042–1052

24. Zhang, X. et al. (2004) Freezing-sensitive tomato has a functionalCBF cold response pathway, but a CBF regulon that differs fromthat of freezing-tolerant Arabidopsis. Plant J. 39, 905–919

25. Savitch, L.V. et al. (2005) The effect of overexpression of twoBrassica CBF/DREB1-like transcription factors on photosyn-thetic capacity and freezing tolerance in Brassica napus. PlantCell Physiol. 46, 1525–1539

26. Ito, Y. et al. (2006) Functional analysis of rice DREB1/CBF-typetranscription factors involved in cold-responsive gene expres-sion in transgenic rice. Plant Cell Physiol. 47, 141–153

27. Haake, V. et al. (2002) Transcription factor CBF4 is a regulator ofdrought adaptation in Arabidopsis. Plant Physiol. 130, 639–648

28. Shi, Y. et al. (2017) The precise regulation of different CORgenes by individual CBF transcription factors in Arabidopsisthaliana. J. Integr. Plant Biol. 59, 118–133

29. Koornneef, M. et al. (2004) Naturally occurring genetic variationin Arabidopsis thaliana. Annu. Rev. Plant Biol. 55, 141–172

30. Hannah, M.A. et al. (2006) Natural genetic variation of freezingtolerance in Arabidopsis. Plant Physiol. 142, 98–112

31. Zhen, Y. and Ungerer, M.C. (2008) Clinal variation in freezingtolerance among natural accessions of Arabidopsis thaliana.New Phytol. 177, 419–427

32. Zuther, E. et al. (2012) Clinal variation in the non-acclimated andcold-acclimated freezing tolerance of Arabidopsis thalianaaccessions. Plant Cell Environ. 35, 1860–1878

33. McKhann, H.I. et al. (2008) Natural variation in CBF genesequence, gene expression and freezing tolerance in the Ver-sailles core collection of Arabidopsis thaliana. BMC Plant Biol. 8,105

34. Kang, J. et al. (2013) Natural variation ofC-repeat-binding factor(CBFs) genes is a major cause of divergence in freezing toler-ance among a group of Arabidopsis thaliana populations alongthe Yangtze River in China. New Phytol. 199, 1069–1080

35. Gehan, M.A. et al. (2015) Natural variation in the C-repeatbinding factor cold response pathway correlates with localadaptation of Arabidopsis ecotypes. Plant J. 84, 682–693

36. Zarka, D.G. et al. (2003) Cold induction of Arabidopsis CBFgenes involves multiple ICE (inducer of CBF expression) pro-moter elements and a cold-regulatory circuit that is desensitizedby low temperature. Plant Physiol. 133, 910–918

37. Chinnusamy, V. et al. (2003) ICE1: a regulator of cold-inducedtranscriptome and freezing tolerance in Arabidopsis. GenesDev. 17, 1043–1054

38. Meshi, T. and Iwabuchi, M. (1995) Plant transcription factors.Plant Cell Physiol. 36, 1405–1420

39. Ding, Y. et al. (2015) OST1 kinase modulates freezing toleranceby enhancing ICE1 stability in Arabidopsis. Dev. Cell 32, 278–289

40. Kim, Y.S. et al. (2015) The unified ICE-CBF pathway provides atranscriptional feedback control of freezing tolerance during coldacclimation in Arabidopsis. Plant Mol. Biol. 89, 187–201

41. Fursova, O.V. et al. (2009) Identification of ICE2, a gene involvedin cold acclimation which determines freezing tolerance in Ara-bidopsis thaliana. Gene 429, 98–103

42. Kanaoka, M.M. et al. (2008) SCREAM/ICE1 and SCREAM2specify three cell-state transitional steps leading to arabidopsisstomatal differentiation. Plant Cell 20, 1775–1785

43. Wilkinson, S. et al. (2001) Rapid low temperature-induced sto-matal closure occurs in cold-tolerant Commelina communisleaves but not in cold-sensitive tobacco leaves, via amechanismthat involves apoplastic calcium but not abscisic acid. PlantPhysiol. 126, 1566–1578

44. Bouche, N. et al. (2002) A novel family of calmodulin-bindingtranscription activators in multicellular organisms. J. Biol. Chem.277, 21851–21861

45. Doherty, C.J. et al. (2009) Roles for Arabidopsis CAMTA tran-scription factors in cold-regulated gene expression and freezingtolerance. Plant Cell 21, 972–984

46. Kim, Y. et al. (2013) Roles of CAMTA transcription factors andsalicylic acid in configuring the low-temperature transcriptomeand freezing tolerance of Arabidopsis. Plant J. 75, 364–376

47. Kidokoro, S. et al. (2017) Different cold-signaling pathwaysfunction in the responses to rapid and gradual decreases intemperature. Plant Cell 29, 760–774

48. Agarwal, M. et al. (2006) A R2R3 typeMYB transcription factor isinvolved in the cold regulation of CBF genes and in acquiredfreezing tolerance. J. Biol. Chem. 281, 37636–37645

49. Leivar, P. et al. (2008) Multiple phytochrome-interacting bHLHtranscription factors repress premature seedling photomorpho-genesis in darkness. Curr. Biol. 18, 1815–1823

50. Kim, S.H. et al. (2017) Phosphorylation of the transcriptionalrepressor MYB15 by mitogen-activated protein kinase 6 isrequired for freezing tolerance in Arabidopsis. Nucleic AcidsRes. 45, 6613–6627

51. Castillon, A. et al. (2007) Phytochrome Interacting Factors:central players in phytochrome-mediated light signaling net-works. Trends Plant Sci. 12, 514–521

52. Kidokoro, S. et al. (2009) The phytochrome-interacting factorPIF7 negatively regulates DREB1 expression under circadiancontrol in Arabidopsis. Plant Physiol. 151, 2046–2057

53. Jiang, B. et al. (2017) PIF3 is a negative regulator of the CBFpathway and freezing tolerance inArabidopsis.Proc. Natl. Acad.Sci. U. S. A. 114, E6695–E6702

54. Shi, Y. et al. (2012) Ethylene signaling negatively regulatesfreezing tolerance by repressing expression of CBF and type-A ARR genes in Arabidopsis. Plant Cell 24, 2578–2595

55. Catala, R. et al. (2014) The Arabidopsis 14-3-3 protein RARECOLD INDUCIBLE 1A links low-temperature response and eth-ylene biosynthesis to regulate freezing tolerance and cold accli-mation. Plant Cell 26, 3326–3342

56. Zhang, Z. and Huang, R. (2010) Enhanced tolerance to freezingin tobacco and tomato overexpressing transcription factorTERF2/LeERF2 is modulated by ethylene biosynthesis. PlantMol. Biol. 73, 241–249

57. Hu, Y. et al. (2013) Jasmonate regulates the inducer of CBFexpression-C-repeat binding factor/DRE binding factor1 cas-cade and freezing tolerance in Arabidopsis. Plant Cell 25, 2907–2924











































































































































58. Kagale, S. et al. (2007) Brassinosteroid confers tolerance inArabidopsis thaliana and Brassica napus to a range of abioticstresses. Planta 225, 353–364

59. Eremina, M. et al. (2016) Brassinosteroids participate in thecontrol of basal and acquired freezing tolerance of plants. Proc.Natl. Acad. Sci. U. S. A. 113, E5982–E5991

60. Li, H. et al. (2017) BZR1 positively regulates freezing tolerancevia CBF-dependent and CBF-independent pathways in Arabi-dopsis. Mol. Plant 10, 545–559

61. Fowler, S.G. et al. (2005) Low temperature induction of Arabi-dopsis CBF1, 2, and 3 is gated by the circadian clock. PlantPhysiol. 137, 961–968

62. Dong, M.A. et al. (2011) Circadian clock-associated 1 and lateelongated hypocotyl regulate expression of the C-repeat bindingfactor (CBF) pathway in Arabidopsis. Proc. Natl. Acad. Sci. U. S.A. 108, 7241–7246

63. Harmer, S.L. et al. (2000) Orchestrated transcription of keypathways in Arabidopsis by the circadian clock. Science 290,2110–2113

64. Mikkelsen, M.D. and Thomashow, M.F. (2009) A role for circa-dian evening elements in cold-regulated gene expression inArabidopsis. Plant J. 60, 328–339

65. Nakamichi, N. et al. (2009) Transcript profiling of an ArabidopsisPSEUDO RESPONSE REGULATOR arrhythmic triple mutantreveals a role for the circadian clock in cold stress response.Plant Cell Physiol. 50, 447–462

66. Boss, P.K. et al. (2004) Multiple pathways in the decision toflower: enabling, promoting, and resetting. Plant Cell 16, S18–S31

67. Seo, E. et al. (2009) Crosstalk between cold response andflowering in Arabidopsis is mediated through the flowering-timegene SOC1 and its upstream negative regulator FLC. Plant Cell21, 3185–3197

68. Kim, H.J. et al. (2004) A genetic link between cold responsesand flowering time through FVE in Arabidopsis thaliana. Nat.Genet. 36, 167–171

69. Fowler, S. and Thomashow, M.F. (2002) Arabidopsis transcrip-tome profiling indicates that multiple regulatory pathways areactivated during cold acclimation in addition to the CBF coldresponse pathway. Plant Cell 14, 1675–1690

70. Kreps, J.A. et al. (2002) Transcriptome changes for Arabidopsisin response to salt, osmotic, and cold stress. Plant Physiol. 130,2129–2141

71. Catala, R. et al. (2011) Integration of low temperature and lightsignaling during cold acclimation response in Arabidopsis. Proc.Natl. Acad. Sci. U. S. A. 108, 16475–16480

72. Zhu, J.K. (2016) Abiotic stress signaling and responses in plants.Cell 167, 313–324

73. Venkatachalam, K. and Montell, C. (2007) TRP channels. Annu.Rev. Biochem. 76, 387–417

74. Ma, Y. et al. (2015) COLD1 confers chilling tolerance in rice. Cell160, 1209–1221

75. Manishankar, P. and Kudla, J. (2015) Cold tolerance encoded inone SNP. Cell 160, 1045–1046

76. Casal, J.J. and Questa, J.I. (2017) Light and temperature cues:multitasking receptors and transcriptional integrators.New Phy-tol. 217, 1029–1034

77. Legris, M. et al. (2016) Phytochrome B integrates light andtemperature signals in Arabidopsis. Science 354, 897–900

78. Jung, J.H. et al. (2016) Phytochromes function as thermosen-sors in Arabidopsis. Science 354, 886–889

79. Fujii, Y. et al. (2017) Phototropin perceives temperature basedon the lifetime of its photoactivated state. Proc. Natl. Acad. Sci.U. S. A. 114, 9206–9211

80. Reed, J.W. et al. (1993) Mutations in the gene for the red/far-redlight receptor phytochrome B alter cell elongation and physio-logical responses throughout Arabidopsis development. PlantCell 5, 147–157


81. Franklin, K.A. andWhitelam, G.C. (2007) Light-quality regulationof freezing tolerance in Arabidopsis thaliana. Nat. Genet. 39,1410–1413

82. Kodama, Y. et al. (2008) Low temperature-induced chloroplastrelocation mediated by a blue light receptor, phototropin 2, infern gametophytes. J. Plant Res. 121, 441–448

83. Huala, E. et al. (1997) Arabidopsis NPH1: a protein kinase with aputative redox-sensing domain. Science 278, 2120–2123

84. Christie, J.M. (2007) Phototropin blue-light receptors. Annu.Rev. Plant Biol. 58, 21–45

85. Mann, M. and Jensen, O.N. (2003) Proteomic analysis of post-translational modifications. Nat. Biotechnol. 21, 255–261

86. Barrero-Gil, J. and Salinas, J. (2013) Post-translational regula-tion of cold acclimation response. Plant Sci. 205–206, 48–54

87. Catala, R. and Salinas, J. (2008) Regulatory mechanismsinvolved in cold acclimation response. Span. J. Agric. Res. 6,211–220

88. Lee, B.H. et al. (2005) The Arabidopsis cold-responsive tran-scriptome and its regulation by ICE1. Plant Cell 17, 3155–3175

89. Miura, K. et al. (2007) SIZ1-mediated sumoylation of ICE1 con-trols CBF3/DREB1A expression and freezing tolerance in Ara-bidopsis. Plant Cell 19, 1403–1414

90. Dong, C.H. et al. (2006) The negative regulator of plant coldresponses, HOS1, is a RING E3 ligase that mediates the ubiq-uitination and degradation of ICE1. Proc. Natl. Acad. Sci. U. S.A. 103, 8281–8286

91. Johnson, E.S. (2004) Protein modification by SUMO. Annu. Rev.Biochem. 73, 355–382

92. Lehti-Shiu, M.D. and Shiu, S.H. (2012) Diversity, classificationand function of the plant protein kinase superfamily. Philos.Trans. R. Soc. Lond. B Biol. Sci. 367, 2619–2639

93. Ding, Y. et al. (2018) OST1-mediated BTF3L phosphorylationpositively regulates CBFs during plant cold responses. EMBO J.37, e98228

94. Li, H. et al. (2017) MPK3- and MPK6-mediated ICE1 phosphor-ylation negatively regulates ICE1 stability and freezing tolerancein Arabidopsis. Dev. Cell 43, 630–642 e634

95. Zhao, C. et al. (2017) MAP kinase cascades regulate the coldresponse by modulating ICE1 protein stability. Dev. Cell 43,618–629 e615

96. Lee, J.H. et al. (2017) Light inhibits COP1-mediated degradationof ICE transcription factors to induce stomatal development inArabidopsis. Plant Cell 29, 2817–2830

97. Zhang, Z. et al. (2017) OsMAPK3 phosphorylates OsbHLH002/OsICE1 and inhibits its ubiquitination to activate OsTPP1 andenhances rice chilling tolerance. Dev. Cell 43, 731–743 e735

98. Shiu, S.H. and Bleecker, A.B. (2001) Plant receptor-like kinasegene family: diversity, function, and signaling. Sci. STKE 2001,re22

99. Yang, T. et al. (2010) A calcium/calmodulin-regulated memberof the receptor-like kinase family confers cold tolerance inplants. J. Biol. Chem. 285, 7119–7126

100. Yang, T. et al. (2010) Calcium/calmodulin-regulated receptor-like kinase CRLK1 interacts with MEKK1 in plants. Plant Signal.Behav. 5, 991–994

101. Liu, Z. et al. (2017) Plasma membrane CRPK1-mediated phos-phorylation of 14-3-3 proteins induces their nuclear import tofine-tune CBF signaling during cold response.Mol. Cell 66, 117–128

102. Achard, P. et al. (2008) The cold-inducible CBF1 factor-depen-dent signaling pathway modulates the accumulation of thegrowth-repressing DELLA proteins via its effect on gibberellinmetabolism. Plant Cell 20, 2117–2129

103. Ning, S.B. et al. (2002) Characterization of the early stages ofprogrammed cell death in maize root cells by using comet assayand the combination of cell electrophoresis with annexin bind-ing. Electrophoresis 23, 2096–2102









































































































































104. Koukalova, B. et al. (1997) Chromatin fragmentation associatedwith apoptotic changes in tobacco cells exposed to cold stress.FEBS Lett. 414, 289–292

105. Hong, J.H. et al. (2017) A sacrifice-for-survival mechanism pro-tects root stem cell niche from chilling stress. Cell 170, 102–113

106. Fuchs, S. et al. (2013) Type 2C protein phosphatases in plants.FEBS J. 280, 681–693

107. Aguilar, P.S. et al. (2001) Molecular basis of thermosensing: atwo-component signal transduction thermometer in Bacillussubtilis. EMBO J. 20, 1681–1691

108. Miura, K. et al. (2011) ICE1 Ser403 is necessary for proteinstabilization and regulation of cold signaling and tolerance. PlantJ. 67, 269–279

109. Lee, C.M. and Thomashow, M.F. (2012) Photoperiodic regula-tion of the C-repeat binding factor (CBF) cold acclimation path-way and freezing tolerance in Arabidopsis thaliana. Proc. Natl.Acad. Sci. U. S. A. 109, 15054–15059



















Molecular Regulation of CBF Signaling in Cold Acclimation

Documents

Transcript of Molecular Regulation of CBF Signaling in Cold Acclimation