Molecular Regulation of CBF Signaling in Cold Acclimation

Review

Molecular Regulation of CBF Signaling in Cold Acclimation Yiting Shi,1,2 Yanglin Ding,1,2 and Shuhua Yang1,* Cold stress restricts plant growth, development, and distribution. Understanding how plants transduce and respond to cold signals has long been a topic of interest. Traditional genetic and molecular analyses have identified C-repeat/ DREB binding factors (CBFs) as key transcription factors that function in cold acclimation. Recent studies revealed the involvement of pivotal protein kinases and transcription factors in CBF-dependent signaling, expanding our knowledge of cold signal transduction from perception to downstream gene expression events. In this review, we summarize recent advances in our understanding of the molecular regulation of these core components of the CBF cold signaling pathway. Knowledge of the mechanism underlying the ability of plants to survive freezing temperatures will facilitate the development of crop plants with increased freezing tolerance.

Highlights Plants withstand freezing stress by triggering cold acclimation processes; the CBF-dependent pathway has a central role in cold acclimation in plants. A cold acclimation mechanism is proposed by which the cold signal is transduced from the membrane to the nucleus, leading to a series of biochemical and physiological changes in the cell and the induction of coldresponsive genes. A subset of transcriptional regulators is involved in CBF-dependent and -independent pathways that regulate coldregulated (COR) gene expression.

General Effects of Cold Stress on Plants Cold stress is a major environmental stress that limits the geographical location of plants in nature, and may significantly reduce crop production. When a plant encounters low temperatures, a series of cellular responses and molecular strategies can be activated that allow the plant 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]. Chilling stress causes the membrane to rigidify, destabilizes protein complexes, and impairs photosynthesis [3], whereas freezing stress causes more serious injuries to the plant. Freezing temperatures promote ice formation in the apoplast (intercellular spaces) of plant tissues [4]. The accumulation of intercellular ice physically disrupts the cell membrane [5], causing a decrease in the water potential outside the cell, and leading to severe cell dehydration [4,5]. In response to freezing stress, plants in temperate climates, such as winter wheat (Triticum aestivum), rye (Secale cereale L.), barley (Hordeum vulgare), and oat (Avena sativa), have evolved sophisticated cold acclimation mechanisms that improve plant freezing tolerance upon exposure to nonfreezing temperatures [2,6].

Several protein kinases are important regulators of the CBF signaling pathway, including SNF1-related protein kinases (SnRK2s), receptor-like protein kinases, and mitogen-activated protein kinases (MAPKs). The interplay of cold, light, and phytohormone signaling is important for balancing freezing tolerance and plant growth.

Understanding Cold Acclimation in Plants Cold acclimation involves an array of physiological and biochemical modifications. The field took a step forward with the discovery of cold-regulated (COR) genes, which function in the plant 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), responsive to desiccation (RD), and early dehydration-inducible (ERD) genes. Some of these genes encode key enzymes for osmolyte biosynthesis that increase freezing tolerance via the accumulation of cryoprotective proteins and soluble sugars, thus repairing cold-rigidified membranes and stabilizing cellular osmotic potential [6]. CBF transcription factors, also known as dehydration responsive element (DRE) binding factor1 (DREB1) proteins, are critical for cold acclimation in higher plants [8,9]. CBF proteins recognize the CRT/DRE cis-element,

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1

State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China 2 These authors contributed equally

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

https://doi.org/10.1016/j.tplants.2018.04.002 © 2018 Elsevier Ltd. All rights reserved.

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which contains the conserved CCGAC sequence and is found in the promoters of a subset of COR genes [9–11]. When plants are exposed to nonfreezing low temperatures, CBF genes are rapidly 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 of chromosome 4 of the arabidopsis genome [11,13]. The functions of CBF genes have been characterized 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-acclimated 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 and exhibit functional redundancy. However, some contradictory conclusions were reached in these studies: (i) one study reported that a T-DNA insertion mutation of CBF2 showed enhanced freezing tolerance [17], whereas another study found that cbf2 was slightly susceptible to freezing stress [15]; and (ii) both the cbf1,3 RNAi lines produced in [17] and the null allele cbf1,3 double mutant obtained in [16] were less tolerant to freezing stress than was the wildtype. However, the cbf1 cbf3 double mutant generated in [17] exhibited increased freezing tolerance. The reasons for these discrepancies remain unknown. It might be that truncated CBF1/3 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 be destroyed due to the deletion of a large DNA fragment, leading to misregulation of the remaining CBF. 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, and maize (Zea mays), suggesting that CBF is conserved in plants that both have and lack the capacity to acclimate to cold temperatures [20,21]. Overexpression of CBF genes results in the induction of COR gene expression and increased freezing tolerance in many plant species [8,22–26]. Phylogenetic analysis of the arabidopsis CBF proteins and their orthologs in various plants suggests that CBFs are phylogenetically conserved across these species (Figure 1). Although CBF family proteins are primarily involved in regulating COR gene expression, other functions have been noted for individual CBF members. For example, CBF2 has been shown to negatively regulate the expression of CBF1 and CBF3 in arabidopsis [17]. CBF4, a unique member of the CBF transcription factor family, has a role in plant drought stress tolerance [27]. Bioinformatic analysis comparing the transcriptional profiles of individual cbf single 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 stress responses would be a challenging topic for further research. Studies conducted in Arabidopsis thaliana populations provide direct evidence that natural variation at the CBF locus has an important 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 mutations or deletions in CBF coding regions alter the structure or transactivation of CBF proteins to affect COR expression in accessions from warm habitats [33–35]. Further insight into the natural selection of the CBF pathway would enhance our understanding of the importance of freezing tolerance in the evolution of arabidopsis ecotypes.

The Transcriptional Regulatory Network Underlying Cold Acclimation Over the past two decades, various transcription factors have been identified that regulate CBF expression 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 624

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Glossary C-repeat/DREB binding factor (CBF) transcription factors: also known as DREB1s, these AP2 family transcription factors mediate plant freezing tolerance by binding to CRT/ DRE cis-elements containing the conserved CCGAC sequence in COR genes and upregulating their expression. Cold acclimation: a process whereby plants acquire freezing tolerance after exposure to nonfreezing temperatures. The key roles of cold acclimation are to stabilize the membrane and to maintain the osmotic potential of plant cells to help them resist freezing injury. The acclimation mechanism is conducted through transcriptional reprogramming of stress-responsive genes, which alters metabolic homeostasis according to the severity of the cold stress and the plant species. Cold-regulated (COR) genes: genes that are regulated by low temperature. In Arabidopsis thaliana, COR genes are classified as low temperature-induced (LTI), coldinducible (KIN), responsive to desiccation (RD), and early dehydration-inducible (ERD) genes, some of which encode osmolyte and cryoprotective proteins that protect plant cells against freezing injury. Cold stress: stress caused by temperatures below normal growth conditions, which can be classified as chilling stress (0–15 C) or freezing stress (below 0 C). Chilling stress usually has deleterious effects on photosynthesis, membrane fluidity, reactive oxygen species (ROS) homeostasis, and energy metabolism in tropical and subtropical plants. Freezing stress results in intercellular ice formation, which leads to permanent membrane lesions and structural damage to the cell. Post-translational modifications (PTMs): the covalent and generally enzymatic modification of proteins during or after translation. PTMs have a fundamental role in proteomic changes by regulating protein structure, activity, and localization. Such modifications include phosphorylation, glycosylation, ubiquitination, sumoylation, Snitrosylation, methylation, and

46

AtCBF2 (Arabidopsis thaliana)

acetylation. PTMs influence almost all aspects of cellular action. Transcriptional regulation: a process whereby cells control the expression of genes in part through transcription factors, transcriptional regulatory proteins that bind to particular sites of the genome and thereby enhance or repress the transcription of genes. This process allows the cell to appropriately respond to environmental, developmental, or other signals.

AtCBF3 (Arabidopsis thaliana)

97

AtCBF1 (Arabidopsis thaliana)

39

BoCBF1b (Brassica oleracea) 95

99

BoCBF3 (Brassica oleracea) BoCBF1a (Brassica oleracea)

98

96

BoCBF2 (Brassica oleracea) AtCBF4 (Arabidopsis thaliana)

85

95 100

BoCBF4 (Brassica oleracea) MtCBF2 (Medicago truncatula) MtCBF3 (Medicago truncatula)

76

LeCBF3 (Solanum lycopersicum)

70

LeCBF1 (Solanum lycopersicum)

94 57

99

LeCBF2 (Solanum lycopersicum) MtCBF1 (Medicago truncatula) ZmDREB1.8 (Zea mays)

100

74

56

OsDERB1F (Oryza saƟva) ZmDREB1.10 (Zea mays) OsDREB1E (Oryza saƟva)

82

HvCBF11 (Hordeum vulgare) OsDREB1G (Oryza saƟva)

100

ZmDREB1.9 (Zea mays)

56 51

HvCBF1 (Hordeum vulgare) HvCBF5 (Hordeum vulgare) ZmDREB1.7 (Zea mays)

99 52

OsDREB1C (Oryza saƟva)

63

HvCBF4A (Hordeum vulgare) HvCBF9 (Hordeum vulgare)

62 85

HvCBF14 (Hordeum vulgare)

86

HvCBF2A (Hordeum vulgare) OsDREB1B (Oryza saƟva) 94

ZmDREB1.6 (Zea mays) OsDREB1D (Oryza saƟva)

92 65

94

OsDREB1A (Oryza saƟva) HvCBF6 (Hordeum vulgare)

72

ZmDREB1.3 (Zea mays)

93

ZmDREB1.5 (Zea mays)

70

75

ZmDREB1.4 (Zea mays)

99

ZmDREB1.1 (Zea mays)

42

OsDREB1H (Oryza saƟva)

ZmDREB1.21 (Zea mays) 68

HvCBF12 (Hordeum vulgare) HvCBF13 (Hordeum vulgare)

72

HvCBF3 (Hordeum vulgare)

95 82

HvCBF10A (Hordeum vulgare)

(See figure legend on the bottom of the next page.) Trends in Plant Science, July 2018, Vol. 23, No. 7

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Cold stress Protein kinase OST1

ICE1

MPK3/6

MYB15

Ca2+

Light

CaM

PhyB

CAMTA3

1PIF3,4,7

Hormone BR

CESTA

ETH

BZR1/ BES1

EIN3

Circadian

Flowering

?

?

CCA1/LHY

SOC1 PRRs

MYC

MYB

CM2

G-box/E-box

BRRE

EBS

CBS

TranscripƟonal regulaƟon

CarG-box

CBF genes

Figure 2. Transcriptional Regulation of C-Repeat/DREB Binding Factor (CBF) Genes during Cold Acclimation in Arabidopsis. CBFs act as master transcription 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 transcription activator 3 (CAMTA3), brassinazole-resistant 1/brassinosteroid insensitive 1-EMS-supressor 1 (BZR1/BES1), CESTA (CES), and circadian clock associated 1/late elongated hypocotyl (CCA1/LHY), or repressed by MYB15, phytochrome-interacting factors (PIFs), ethylene insensitive 3 (EIN3), and suppressor of constans overexpression 1 (SOC1). In addition, pseudo-response regulators (PRRs) act either directly or indirectly as negative regulators of CBFs. The cold-activated protein kinases open stomata 1 (OST1) and MPK3/6 phosphorylate and regulate the stability of ICE1, thus modulating CBF expression. MPK6 phosphorylates MYB15 to release 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 their expression. Transcription factors involved in hormone signaling pathway, including brassinosteroids (BRs) and ethylene (ETH), also modulate CBF expression. The CBF genes 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 factors regulating the circadian clock, directly activate CBF expression. The flowering time component SOC1 is required for the downregulation of CBFs. PRRs are suggested to 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 transcriptional activator to date is inducer of CBF expression1 (ICE1), a MYC-like basic helix-loop-helix (bHLH) transcription factor [37]. ICE1 binds to the MYC-binding sites (CANNTG) [38] in the promoters of CBF1–3, thereby promoting their expression under cold stress [287_TD$IF][37,40]. The ice1 mutant was originally isolated in a genetic screen for plants with abnormal regulation of CBF3–LUC expression in the cold [37]. The ice1 mutant exhibits impaired freezing tolerance and significant defects in the induction of CBF genes, whereas overexpression of ICE1 enhances the coldinduced 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 Other Species. 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 branches represented bootstrap values (from 1000 replicates). The arabidopsis CBFs are illustrated in red.

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Table 1. Proteins Functioning in the CBF Pathway in Arabidopsis Factor

Protein characteristics

Direct target(s)

Effect on freezing tolerance (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 nascent polypeptide-associated complex (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 (direct or indirect)

Negative

[65]

FVE

Homolog of the mammalian retinoblastoma-associated protein

COR15A, COR47 (direct or 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 of leaf tissues [41,42], prompting speculation that ICE1/2 are specific regulators of the CBF-COR pathway in stomata in land plants. A previous study showed that low temperatures reduce stomatal apertures by inducing apoplastic calcium uptake by guard cells in Commelina communis [43]. It would be interesting to test whether CBF signaling pathways participate in regulating stomatal development and movement during cold acclimation. Another group of transcriptional activators that regulates CBFs is calmodulin-binding transcription activator (CAMTA) proteins, which contain conserved calmodulin-binding sites [44]. CAMTA3 protein was initially found to bind to the CM2 motif (CGCG-box) of the CBF2 promoter and activate its expression [45,46]. CAMTA1 and CAMTA2 are positive regulators of the coldinduced expression of CBF1–3 [45,46]. Moreover, CAMTA proteins (CAMTA1–5) function as transcriptional 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)

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decreases in temperature [47]. Considering that low temperatures result in a rapid increase in cytosolic calcium levels, this finding links calcium signaling and cold induction of the CBF pathway. 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/or are cofactors of CAMTA at low temperature. Investigations into these questions would expand our 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 of the R2R3 family of MYB transcription factors, regulates CBF expression under low temperature conditions [48]. A recent study showed that MPK6 interacts with, and phosphorylates, MYB15 to reduce its binding to CBF promoters [50], thus releasing the inhibitory effect of this transcription factor on CBF gene expression. PIFs are a subset of bHLH transcription factors that have central roles in the phytochrome-mediated light-signaling network [49,51]. PIF4 and PIF7 bind to the G-box motifs in the CBF1 and CBF2 promoters and the E-box motif in the CBF3 promoter under long-day conditions [52]. Additionally, PIF3 associates with the G-box and E-box motifs in the promoters of all three CBF genes, repressing their transcription under both dark and low temperature conditions [53]. These observations suggest that negative transcription factors have important roles in maintaining the homeostasis of CBF-regulated transcriptional networks during cold acclimation. Plants have evolved sophisticated strategies in which hormone and cold signaling pathways are coordinated to better adapt to freezing stress. Ethylene insensitive 3 (EIN3) is a key transcription factor involved in ethylene signaling that negatively regulates freezing tolerance in arabidopsis, partially through affecting the expression of CBFs by binding to the EBS motifs in their promoters [54]. Two F-box proteins EBF1/2 of the ethylene-signaling pathway positively regulate CBF expression by mediating the degradation of the transcriptional repressors EIN3 and PIF3 [53,54]. Consistently, ethylene-insensitive plants grown on agar plates displayed enhanced freezing tolerance [53,54]. These results support the notion that ethylene negatively regulates freezing tolerance. By contrast, another study showed that an arabidopsis ACS octuple mutant grown in soil pots was deficient in ethylene biosynthesis, was hypersensitive to freezing, and exhibited reduced induction of CBF and COR genes under cold treatment [55]. Moreover, overexpression of tomato ethylene response factor 2 (TERF2/[28_TD$IF]LeERF2) enhances plant freezing tolerance through promoting ethylene biosynthesis and the ethylene signaling pathway [56], suggesting that ethylene has a positive role in regulating plant freezing tolerance. It appears that the function of ethylene in plant freezing tolerance is different when plants are grown in soil versus agar plates, implying that the humidity or the decreasing rates of the temperature may impact the freezing tolerance. Thus, the exact roles of ethylene in freezing tolerance in different growth conditions and plant species might be diverse and require further investigation. Jasmonic acid (JA) is a lipid-derived plant hormone that positively regulates CBF signaling by mediating the interaction between JAZ1/4 protein and ICE1/2, thus modulating the transcriptional activity of ICE proteins and CBF1–3 gene expression [57]. Brassinosteroids (BRs) are a class of plant steroid hormones that also have a role in promoting freezing tolerance [58]. The bin2-3 bil1 bil2 mutant, 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, which act as downstream transcription factors in the BR-signaling pathway, positively regulate freezing tolerance by promoting CBF1 and CBF2 expression through binding to the BRRE and E-box motifs in their promoters [60]. The BR-controlled bHLH transcription factor CESTA regulates CBF expression by recognizing G-box motifs in their promoters [59]. Further studies 628

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of the interaction between hormone signaling and cold signaling are likely to provide novel insights into how plants precisely coordinate growth and freezing tolerance. CBF1–3 expression is not only responsive to low temperatures, but is also gated by the circadian clock [61–63]. The cold-induced upregulation of CBF genes depends on the time of day [61]. Both clock regulatory elements and cold-responsive elements have been identified in CBF promoters [62,64]. Consistent with this observation, the morning-expressed MYB transcription 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 reduced in the cca1 lhy double mutant [62]. Moreover, mutations in PRR5, PRR7, and PRR9, encoding key components of the circadian oscillator, result in the upregulation of CBFs [65], implying that the circadian clock is an essential cue for the full induction of CBFs at low temperatures. The floral activators SOC1 and FVE mediate the direct link between cold signaling and flowering time [66]. SOC1 is a MADS-box transcription factor that negatively regulates CBF expression through directly binding to the CArG boxes in their promoters [67], while FVE, an arabidopsis homolog 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, such as low temperature, the circadian clock, light, and flowering time (Figure 2). Cold treatment induces massive reprogramming of the plant transcriptome [69,70]. Genomewide expression profiling of wild-type versus cbfs plants revealed that CBF proteins extensively regulate the expression of genes involved in stress responses, carbohydrate and lipid metabolism, cell wall modification, transcription factors, kinases, and calcium (Ca2+[286_TD$IF]) and hormonal signaling [15,16]. Analysis of CBF-overexpressing lines and a cbfs triple mutant revealed that few hundreds of the approximately 4000 COR genes are regulated by CBFs [14–16], indicating that 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 -independent pathways in addition [60]. Thus, CBF-independent pathways may have major roles in modulating COR gene expression. However, how different transcription factors fine-tune COR expression to establish cold acclimation remains to be fully elucidated.

Perception of the Cold Signal The current model of cold-sensing mechanisms in plants is that cold shock leads to changes in membrane fluidity and rearrangement of the cytoskeleton that trigger Ca2+ influx, which in turn activates the expression of downstream COR 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 is whether calcium channels are involved in cold signal perception. The identification of COLD1, a transmembrane protein that functions as a cold sensor in rice (Oryza sativa), provides support for this hypothesis [74]. The transmembrane protein COLD1 regulates G-protein signaling by interacting with rice G-protein a subunit1 (RGA1) to accelerate GTPase activity. Under chilling stress, 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 determine whether COLD1 itself functions as a temperature-regulated membrane ion channel, and whether 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) Trends in Plant Science, July 2018, Vol. 23, No. 7

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

Post-Translational Modifications Are Important for Cold Signaling PTMs 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-dependent signaling (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 that PTMs are important for the functioning of ICE1. Indeed, multiple PTMs have been shown to control the turnover and duration of ICE1 under low temperatures. The RING finger protein, high expression of osmotically responsive gene1 (HOS1), is required for the 26S proteasomemediated degradation of ICE1 [90]. HOS1 physically interacts with and ubiquitinates ICE1, thus reducing its stability under low temperatures. The overexpression of HOS1 decreases freezing tolerance in plants by repressing the expression of CBF3 [90]. Sumoylation might protect target proteins from 26S proteasomal degradation by preventing their ubiquitination [91]. SIZ1, a SUMO 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 polyubiquitination mediated by HOS1 [89]. The siz1 null mutant is hypersensitive to freezing stress and 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 protein kinases (SnRK2s), mitogen-activated protein kinases (MAPKs), Ca2+-dependent protein kinases (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 in our understanding of the function of a protein kinase in cold signaling [39]. Ding et al. initially found that OST1, a Ser/Thr protein kinase in the ABA core signaling pathway, positively regulates CBF-mediated freezing tolerance through phosphorylating ICE1 at Ser278 to prevent its 26S proteasome-mediated degradation by HOS1 [39]. The ost1 mutants are sensitive to freezing stress, whereas overexpression of OST1 results in increased freezing tolerance. It was reported more recently that OST1 phosphorylates b-subunits of NAC (a nascent polypeptideassociated complex), basic transcription factor 3 (BTF3), and BTF3-like (BTF3L) proteins in 630

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Cold stress

COLD1

Plasma membrane

Cytoplasm [Ca2+] COLD1

Nucleus

26S proteasome

26S proteasome

CBFs

CORs CRT/DRE

26S proteasome

Plant freezing tolerance

Figure 3. Cold Sensing in the Plant Cell. The plasma membrane is thought to be the primary target of cold sensing and the starting point for transmission of the cold signal into the nucleus. After exposure to cold stress, the plasma and endoplasmic reticulum (ER) membrane-located cold sensor chilling-tolerance divergence 1 (COLD1) interacts with rice Gprotein a subunit 1 (RGA1), activating the Ca2+ signaling pathway in plant cells, and stimulating downstream cold signal transduction and C-repeat/DREB binding factor (CBF) expression. Plasma membrane Ca2+-regulated receptor-like kinases calcium/calmodulin-regulated receptor-like kinase 1/2 (CRLK1/2) positively regulate cold-induced gene expression by activating the MEKK1-MKK2-MPK4 pathway. Cold induces the activity of open stomata 1 (OST1), a pivotal kinase in the ABA signaling pathway, which interacts with and phosphorylates inducer of CBF expression 1 (ICE1) as well as basic transcription factor 3/BTF3-like protein (BTF3/BTF3L). The cold signal activates the MKK4/5-MPK3/6 cascade to regulate freezing tolerance through phosphorylation and degradation of ICE1, thus attenuating the CBF signaling. A receptor-like cytoplasmic kinase cold-responsive protein kinase 1 (CRPK1) is activated by low temperature stress and phosphorylates (Figure legend continued on the bottom of the next page.) Trends in Plant Science, July 2018, Vol. 23, No. 7

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arabidopsis. Phosphorylated BTF3s enhance plant freezing tolerance by promoting CBF protein stability through interacting with CBF proteins and promoting CBF transcription [93]. Cold-induced OST1 activation is independent of ABA [39]. Recently, two companion papers indicated that the arabidopsis MAPK signaling cascade regulates freezing tolerance through the ICE1-CBF-COR transcriptional pathway [95,94]. MPK3/MPK6 interact with, and phosphorylate, 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 transcriptional activity, thus negatively regulating CBF expression and freezing tolerance in arabidopsis [289_TD$IF][95,94]. In addition, the MEKK1-MKK[290_TD$IF]2-MPK4 cascade promotes CBF expression and freezing tolerance by antagonizing the MKK4/5-MPK3/6 pathway [291_TD$IF][95]. Interestingly, MPK3/MPK6mediated phosphorylation of ICE1 does not affect the HOS1–ICE1 interaction [94]. Whether MPK3/MPK6 recruit other E3 ligases, such as COP1 (an E3 ligase that mediates dark-induced degradation of ICE1 during stomatal development [96]), remains to be explored. A recent study showed that OsMAPK3 phosphorylates and stabilizes OsICE1 in rice, which directly regulates the expression of OsTPP1, encoding a key enzyme for trehalose synthesis, thereby positively regulating chilling tolerance [97]. These findings indicate that MAPK3 has distinct and even opposite roles in response to low temperatures in different plant species. Taken together, these compelling pieces of evidence suggest that phosphorylation is a PTM that synergistically or antagonistically regulates the stability and abundance of ICE1 under coldstress conditions. RLKs also mediate cold acclimation in plants [98]. CRLK1, a plasma membrane-localized Ca2+[289_TD$IF]/ calmodulin-regulated receptor-like kinase, positively regulates COR expression and cold acclimation, likely by phosphorylating MEKK1 and enhancing its activity [99,100]. CRLK1 and its closest homolog, CRLK2, act upstream of the MEKK1-MKK2-MPK4 pathway to positively regulate CBF expression and freezing tolerance [291_TD$IF][95]. It was recently revealed that the receptor-like cytoplasmic kinase cold-responsive protein kinase1 (CRPK1) is a negative regulator of the CBF pathway in arabidopsis [101]. Cold treatment activates the kinase activity of CRPK1 without affecting its localization. CRPK1 then directly interacts with, and phosphorylates, 14-3-3 proteins on the plasma membrane. These phosphorylated 14-3-3s translocate into the nucleus to facilitate the proteasome-mediated degradation of CBF1 and CBF3, thereby attenuating an overwhelming cold acclimation in arabidopsis [101]. Another study showed that RCI1A, a 14-3-3w, negatively regulates freezing tolerance in arabidopsis [55], implying that 143-3 family proteins are extensively involved in plant freezing tolerance. Given that receptor-like cytoplasmic kinases often act together with a receptor kinase, it is possible that phosphorylation is a primary event in cold perception and that unknown receptor-like kinase(s) may be involved in transmitting the cold signal from the membrane into the nucleus.

Tradeoff between Cold Tolerance and Plant Growth The constitutive overexpression of CBFs adversely affects plant growth under normal growth conditions [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 negatively regulate CBF signaling. The key components of CBF-dependent signaling, ICE1 and CBF, are modulated by posttranslational modifications (PTMs). As for ICE1, phosphorylation of ICE1 by OST1 and MPK3/6 promotes and reduces the stability of ICE1 under cold stress. ICE1 is degraded by high expression of osmotically responsive gene1 (HOS1) through the 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 mediate the phosphorylation and degradation of ICE1 by recruiting unknown E3 ligases. 14-3-3 proteins interact with CBF1/3 and facilitate the ubiquitin-mediated degradation of CBF1/3. Cold induces the accumulation of CBF proteins, which bind to the CRT/DRE motif in the promoters of cold-regulated (COR) genes and induce their expression.

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elusive. In plants overexpressing CBF1, the GA-inactivating GA2-oxidase gene and RGL3 are upregulated, leading to a decrease in bioactive GA levels and the accumulation of DELLA protein [102]. Consistently, the dwarf and late-flowering phenotypes of CBF1-overexpressing plants are rescued by either GA treatment or crosses with a della double mutant [102]. These findings suggest that CBF1 represses plant growth via a DELLA-dependent mechanism. At normal temperatures, cbfs triple mutants grow more slowly and have shorter roots, fewer and smaller rosette leaves, and less biomass compared with wild-type plants [15]. However, when grown under chilling stress (4 C), cbfs plants are larger than the wild-type [16]. Therefore, CBFs are master players in the trade-off between growth limitation and freezing tolerance, at least partially 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]. A recent breakthrough study revealed that the root stem cell niche adopts a novel ‘sacrifice-forsurvival’ mechanism under chilling stress [105]; chilling stress preferentially causes newly generated columella stem cell daughters to die, which results in the auxin maximum being re-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 stem cells to divide at a faster rate when optimal temperatures are restored, thereby improving the ability of the root to withstand chilling stress [105]. These findings shed light on a unique strategy of how sessile plants survive adverse conditions, and provide a potential strategy for engineering cold-tolerant crops.

Concluding Remarks and Future Perspectives The transcriptional network of the CBF signaling pathway has been extensively studied over the past two decades. Although genome-wide expression profiling has identified many COR genes, only approximately 10–25% of these genes are regulated by CBFs, implying that more early cold-regulated transcription factors are involved in improving freezing tolerance. Several elegant strategies have been used to isolate various transcription factors that function in parallel with CBFs. However, more studies are needed to identify additional transcriptional regulators and to explore their underlying relationships to increase our understanding of the cold-related transcriptional network.

Outstanding Questions How are CBF-independent COR genes regulated by cold stress at the transcriptional level? Which protein kinase(s) or E3 ligase(s) participate in the turnover of CBF transcription factors at the post-translational level? How does cold activate protein kinases at the early stage of the response of plants to cold stress? Is cold-activated protein kinase activity dependent on ROS or calcium signaling? Do plants have additional protein kinases or phosphatases that regulate the functions of OST1, CRPK1, and MAPKs? How do the positive and negative regulators of CBF signaling coordinate their function to fine-tune the response of plants to cold stress? How do CBF signaling components interact with plant hormones to balance plant growth and freezing tolerance?

Emerging evidence suggests that PTMs also have an important role in controlling the CBF pathway, with most studies to date focusing on the regulation of ICE1. An interesting phenomenon was recently reported, namely that CBF proteins are subject to 26S proteasomemediated degradation [101]. This suggests that an unidentified E3 ligase participates in the turnover of these proteins. Thus, additional studies are needed to investigate the PTMmediated regulation of CBF proteins. These studies will extend our understanding of the ICE1-CBF-COR cascade at the posttranslational level. Several protein kinases involved in CBF signaling have been successfully isolated. However, how cold activates these protein kinases during the early stages of low temperature perception remains unclear. CRPK1 is a cold-activated plasma membrane-localized protein kinase, supporting the hypothesis that the cold signal is initially transmitted from the plasma membrane [101]. Notably, CRPK1 lacks a classic transmembrane domain, suggesting 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 is dependent on reactive oxygen species (ROS) or Ca2+ signaling. Given that COLD1 might regulate Ca2+ levels in the cytoplasm, the next challenge is to identify the link between Ca2+ Trends in Plant Science, July 2018, Vol. 23, No. 7

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signaling and kinase activity. In particular, since phosphatases modulate protein phosphorylation levels, it would be interesting to identify phosphatases involved in cold-triggered kinase activity. Protein kinase and phosphatase pairs are important for signal perception and transduction in eukaryotes and prokaryotes [106]. In Bacillus subtilis, cold signals are perceived by plasma membrane-localized histidine kinase DesK, which has dual roles as a protein kinase and protein phosphatase [107]. It is possible that plasma membrane-localized protein kinases and their phosphatases are involved in low temperature sensing and signal transduction. Thus, it will be interesting to identify these putative proteins using quantitative phosphoproteomics assays. Cold stress activates negative regulatory pathways, such as the CRPK1-14-3-3s [101] and MPK3/6-ICE1 modules [289_TD$IF][95,94], to attenuate CBF-dependent signaling, but the physiological significance of these repressive pathways is 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 to prevent excessive growth retardation. Investigating the links between the positive and negative regulators of CBFs would shed light on the mechanisms underlying the balance between plant growth and freezing tolerance. Plants have evolved precise mechanisms to perceive fluctuating temperatures to improve their chance of survival. An important step in understanding the thermosensory mechanism in plants was the identification of phytochrome as a thermosensor [77,78]. Moreover, a blue-light receptor was found to function as a cold sensor in M. polymorpha, suggesting that 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 temperatures awaits further study. It would be interesting to investigate whether photoreceptors monitor cold signals to trigger CBF signaling and the expression of COR genes (see also Outstanding Questions). Acknowledgments This work was 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

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

2.

Thomashow, M.F. (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 571–599

3.

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

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

4.

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

5.

Steponkus, P.L. (1984) Role of the plasma membrane in freezing injury and cold acclimation. Annu. Rev. Plant Physiol. Plant Mol. Biol. 35, 543–584

6.

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

7.

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

8.

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

9.

Liu, Q. et al. (1998) Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and

634

Trends in Plant Science, July 2018, Vol. 23, No. 7

11. Gilmour, S.J. et al. (1998) Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early 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 transcription factors in configuring the low temperature transcriptome of Arabidopsis. Plant J. 41, 195–211 13. Medina, J. et al. (1999) The Arabidopsis CBF gene family is composed of three genes encoding AP2 domain-containing proteins whose expression Is regulated by low temperature but not by abscisic acid or dehydration. Plant Physiol. 119, 463–470 14. Park, S. et al. (2015) Regulation of the Arabidopsis CBF regulon by a complex low-temperature regulatory network. Plant J. 82, 193–207 15. Zhao, C.Z. et al. (2016) Mutational evidence for the critical role of CBF transcription factors in cold acclimation in Arabidopsis. Plant Physiol. 171, 2744–2759

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

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

17. Novillo, F. et al. (2004) CBF2/DREB1C is a negative regulator of CBF1/DREB1B and CBF3/DREB1A expression and plays a central role in stress tolerance in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 101, 3985–3990

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 tolerance by enhancing ICE1 stability in Arabidopsis. Dev. Cell 32, 278– 289

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

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

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

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

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

42. Kanaoka, M.M. et al. (2008) SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to arabidopsis stomatal differentiation. Plant Cell 20, 1775–1785

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

43. Wilkinson, S. et al. (2001) Rapid low temperature-induced stomatal closure occurs in cold-tolerant Commelina communis leaves but not in cold-sensitive tobacco leaves, via a mechanism that involves apoplastic calcium but not abscisic acid. Plant Physiol. 126, 1566–1578

22. Gilmour, S.J. et al. (2000) Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiol. 124, 1854–1865 23. Qin, F. et al. (2004) Cloning and functional analysis of a novel DREB1/CBF transcription factor involved in cold-responsive gene expression in Zea mays L. Plant Cell Physiol. 45, 1042– 1052

44. Bouche, N. et al. (2002) A novel family of calmodulin-binding transcription activators in multicellular organisms. J. Biol. Chem. 277, 21851–21861 45. Doherty, C.J. et al. (2009) Roles for Arabidopsis CAMTA transcription factors in cold-regulated gene expression and freezing tolerance. Plant Cell 21, 972–984

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

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

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

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

26. Ito, Y. et al. (2006) Functional analysis of rice DREB1/CBF-type transcription factors involved in cold-responsive gene expression in transgenic rice. Plant Cell Physiol. 47, 141–153 27. Haake, V. et al. (2002) Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis. Plant Physiol. 130, 639–648 28. Shi, Y. et al. (2017) The precise regulation of different COR genes by individual CBF transcription factors in Arabidopsis thaliana. J. Integr. Plant Biol. 59, 118–133 29. Koornneef, M. et al. (2004) Naturally occurring genetic variation in Arabidopsis thaliana. Annu. Rev. Plant Biol. 55, 141–172

48. Agarwal, M. et al. (2006) A R2R3 type MYB transcription factor is involved in the cold regulation of CBF genes and in acquired freezing tolerance. J. Biol. Chem. 281, 37636–37645 49. Leivar, P. et al. (2008) Multiple phytochrome-interacting bHLH transcription factors repress premature seedling photomorphogenesis in darkness. Curr. Biol. 18, 1815–1823 50. Kim, S.H. et al. (2017) Phosphorylation of the transcriptional repressor MYB15 by mitogen-activated protein kinase 6 is required for freezing tolerance in Arabidopsis. Nucleic Acids Res. 45, 6613–6627

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

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

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

52. Kidokoro, S. et al. (2009) The phytochrome-interacting factor PIF7 negatively regulates DREB1 expression under circadian control in Arabidopsis. Plant Physiol. 151, 2046–2057

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

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

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

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

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

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

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

56. Zhang, Z. and Huang, R. (2010) Enhanced tolerance to freezing in tobacco and tomato overexpressing transcription factor TERF2/LeERF2 is modulated by ethylene biosynthesis. Plant Mol. Biol. 73, 241–249

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

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

Trends in Plant Science, July 2018, Vol. 23, No. 7

635

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

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

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

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

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

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

61. Fowler, S.G. et al. (2005) Low temperature induction of Arabidopsis CBF1, 2, and 3 is gated by the circadian clock. Plant Physiol. 137, 961–968 62. Dong, M.A. et al. (2011) Circadian clock-associated 1 and late elongated hypocotyl regulate expression of the C-repeat binding factor (CBF) pathway in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 108, 7241–7246 63. Harmer, S.L. et al. (2000) Orchestrated transcription of key pathways in Arabidopsis by the circadian clock. Science 290, 2110–2113 64. Mikkelsen, M.D. and Thomashow, M.F. (2009) A role for circadian evening elements in cold-regulated gene expression in Arabidopsis. Plant J. 60, 328–339 65. Nakamichi, N. et al. (2009) Transcript profiling of an Arabidopsis PSEUDO RESPONSE REGULATOR arrhythmic triple mutant reveals 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 to flower: enabling, promoting, and resetting. Plant Cell 16, S18– S31 67. Seo, E. et al. (2009) Crosstalk between cold response and flowering in Arabidopsis is mediated through the flowering-time gene SOC1 and its upstream negative regulator FLC. Plant Cell 21, 3185–3197 68. Kim, H.J. et al. (2004) A genetic link between cold responses and flowering time through FVE in Arabidopsis thaliana. Nat. Genet. 36, 167–171 69. Fowler, S. and Thomashow, M.F. (2002) Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. Plant Cell 14, 1675–1690 70. Kreps, J.A. et al. (2002) Transcriptome changes for Arabidopsis in response to salt, osmotic, and cold stress. Plant Physiol. 130, 2129–2141 71. Catala, R. et al. (2011) Integration of low temperature and light signaling 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. Cell 160, 1209–1221 75. Manishankar, P. and Kudla, J. (2015) Cold tolerance encoded in one SNP. Cell 160, 1045–1046 76. Casal, J.J. and Questa, J.I. (2017) Light and temperature cues: multitasking receptors and transcriptional integrators. New Phytol. 217, 1029–1034 77. Legris, M. et al. (2016) Phytochrome B integrates light and temperature signals in Arabidopsis. Science 354, 897–900 78. Jung, J.H. et al. (2016) Phytochromes function as thermosensors in Arabidopsis. Science 354, 886–889 79. Fujii, Y. et al. (2017) Phototropin perceives temperature based on 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-red light receptor phytochrome B alter cell elongation and physiological responses throughout Arabidopsis development. Plant Cell 5, 147–157

636

Trends in Plant Science, July 2018, Vol. 23, No. 7

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 posttranslational modifications. Nat. Biotechnol. 21, 255–261 86. Barrero-Gil, J. and Salinas, J. (2013) Post-translational regulation of cold acclimation response. Plant Sci. 205–206, 48–54 87. Catala, R. and Salinas, J. (2008) Regulatory mechanisms involved in cold acclimation response. Span. J. Agric. Res. 6, 211–220 88. Lee, B.H. et al. (2005) The Arabidopsis cold-responsive transcriptome and its regulation by ICE1. Plant Cell 17, 3155–3175 89. Miura, K. et al. (2007) SIZ1-mediated sumoylation of ICE1 controls CBF3/DREB1A expression and freezing tolerance in Arabidopsis. Plant Cell 19, 1403–1414 90. Dong, C.H. et al. (2006) The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination 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, classification and 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 phosphorylation positively regulates CBFs during plant cold responses. EMBO J. 37, e98228 94. Li, H. et al. (2017) MPK3- and MPK6-mediated ICE1 phosphorylation negatively regulates ICE1 stability and freezing tolerance in Arabidopsis. Dev. Cell 43, 630–642 e634 95. Zhao, C. et al. (2017) MAP kinase cascades regulate the cold response by modulating ICE1 protein stability. Dev. Cell 43, 618–629 e615 96. Lee, J.H. et al. (2017) Light inhibits COP1-mediated degradation of ICE transcription factors to induce stomatal development in Arabidopsis. Plant Cell 29, 2817–2830 97. Zhang, Z. et al. (2017) OsMAPK3 phosphorylates OsbHLH002/ OsICE1 and inhibits its ubiquitination to activate OsTPP1 and enhances rice chilling tolerance. Dev. Cell 43, 731–743 e735 98. Shiu, S.H. and Bleecker, A.B. (2001) Plant receptor-like kinase gene family: diversity, function, and signaling. Sci. STKE 2001, re22 99. Yang, T. et al. (2010) A calcium/calmodulin-regulated member of the receptor-like kinase family confers cold tolerance in plants. J. Biol. Chem. 285, 7119–7126 100. Yang, T. et al. (2010) Calcium/calmodulin-regulated receptorlike kinase CRLK1 interacts with MEKK1 in plants. Plant Signal. Behav. 5, 991–994 101. Liu, Z. et al. (2017) Plasma membrane CRPK1-mediated phosphorylation of 14-3-3 proteins induces their nuclear import to fine-tune CBF signaling during cold response. Mol. Cell 66, 117– 128 102. Achard, P. et al. (2008) The cold-inducible CBF1 factor-dependent signaling pathway modulates the accumulation of the growth-repressing DELLA proteins via its effect on gibberellin metabolism. Plant Cell 20, 2117–2129 103. Ning, S.B. et al. (2002) Characterization of the early stages of programmed cell death in maize root cells by using comet assay and the combination of cell electrophoresis with annexin binding. Electrophoresis 23, 2096–2102

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

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

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

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

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: a two-component signal transduction thermometer in Bacillus subtilis. EMBO J. 20, 1681–1691

Trends in Plant Science, July 2018, Vol. 23, No. 7

637