C-repeat binding factors, or dehydration responsive element (CBF/DREB1), are a larger subfamily of transcription factors that belong to the APETALA2/ethylene-responsive element binding factor (AP2/ERF) protein family and are induced/activated in response to osmotic stresses such as cold or drought. The AP2/ERF domain binds to the C-repeat/dehydration responsive elements (CRT/DRE) in the promoter region of a variety of genes involved in the abiotic stress response, also known as “CBF’s regulon.” These genes protect against the adverse effects of losing water caused by frost and drought with the biosynthesis of osmoprotectant proteins, carbohydrate metabolism-related activity, and sugar transport [85,86,87]. Among these, cold-regulated genes (COR) are the most important family, including late embryo abundant proteins (LEA), low-temperature-induced (LTI), cold-inducible (KIN), responsive to desiccation (RD), early dehydration-inducible (ERD), and the dehydrin (DHN) genes [88,89,90,91]. The distinctive element of CBFs within the AP2/ERF family is the specific “CBF signature” flanking the AP2 domain [86,92].

CBF1 was the first CBF gene isolated and characterized by Stockinger and colleagues in Arabidopsis thaliana [93]. Subsequently, other important works discovered the CBF family and its role in the model plant [94,95] and then in other 54 genera: 31 dicotyledons, 23 monocotyledons, and 13 woody species [96,97,98,99,100]. In Poaceae, multiple elements of the family were isolated and characterized, either in chilling-sensitive (e.g., rice and maize) or frost-tolerant species (e.g., wheat, barley, and rye) [96,101,102,103]. The CBF genes are characterized by short, mono-exon coding sequences (average length 700 bp) with no introns [96,104,105]. Interestingly, Shi et al. [91] performed a phylogenetic analysis and found that the CBF gene structure is remarkably conserved across various species (monocots/dicots), independently of their degree of frost tolerance. As reported by Campoli et al. and Badawi et al. [106,107], CBF genes are classified into four phylogenetic groups, each with two or more sub-groups. Some elements of the CBF gene family are scattered along the genome, while others, more frequently, are organized in clusters of tandemly duplicated genes on the long arm of homoeologous chromosome group 5 of Triticeae [74,108,109,110]. The cluster of CBF genes has been shown to coincide with a QTL for frost tolerance, namely Frost Resistance 2 (FR-2) in barley (FR-H2), diploid (FR-A^m2) and polyploid wheats (FR-A2 and FR-B2), and rye (FR-R2) [74,110,111]. In Triticeae crops, beside FR-2, part of the phenotypic variation for frost tolerance is attributed to another QTL located about 25–30 cM apart from FR-2 on the long arm of homoeologous chromosome group 5: Frost Resistance 1 (FR-1). This locus was identified by Hayes et al. in 1993 and Galiba et al. in 1995 [109,112] in barley and wheat, respectively, and reported to co-segregate with VRN-1, the vernalization requirement gene [74], whose expression leads the plant to become competent for flowering [113].

Thirteen TmCBF were described in Triticum monococcum L.; eleven of them were mapped on FR-A^m2, while TmCBF15 and TmCBF18 were mapped on chromosomes 7A^m and 6A^m, respectively [118]. Vágújfalvi et al. [110] attributed the locus for FT to chromosome 5A, and subsequently Knox and colleagues [119] divided the FR-A2 locus into: proximal (CBF 2, 4, 9, and 17), central (CBF 12, 14, and 15), and distal (CBF 3, 10, 13, and 16).

The genome of hexaploid wheat encodes 65 TaCBFs [120], 27 of which are paralogs with 1–3 homoeologous A, B, and D copies [120]. As reported by The International Wheat Genome Sequencing Consortium (IWGSC) [115], 54 TaCBFs are located on chromosome Group 5: 17 genes on 5A, 19 on 5B, and 18 on 5D chromosomes. Other TaCBFs are located on homoloegous chromosomes 6 (A, B, and D).

In winter cereals, cold acclimation, also known as “hardening”, has the vital function of protecting the crown and young leaves from ice damage [125]. Even after a severe stress episode, if the crown and young leaves survive, the plant maintains the potential to restore from tillering nodes [126]. This peculiarity is linked to the ability of the meristematic tissue to survive thanks to the physiological phenomenon of cold acclimation [127]. Phenolic compounds, sugars, soluble proteins, new enzyme isoforms, proline and organic acids, modification of the fatty acid composition in the phospholipid membrane, and higher levels of antioxidants are all proactive compounds connected to the reduction of frost damage [87,125,128,129].

In winter barley, wheat, and rye, cold acclimation occurs only in the vegetative phase, and it has two different signaling pathways: abscisic acid or ABA-dependent (ABA pathway) and ABA-independent (also known as the ICE-CBF-COR pathway) [130]. Although the ABA and CBF signal transmissions were considered distinct from each other, recent studies suggest a cross-talk between these two pathways [131].

In short-day conditions, the ICE-CBF-COR pathway is promptly activated after a brief exposure to low, non-harmful temperatures [132,133], and the CBF gene has a pivotal role in the coordination of the acclimation processes [134]. In Arabidopsis, a marked increase in CBF transcript levels was observed 15 min after cold exposure, followed by up-regulation of the effector genes about 2 h later [32,135]. On the other hand, in wheat and barley, an increase in CBF transcript levels was observed 4–12 h later after the cold exposure [75,136,137]. The gene induction relies on a temperature threshold dependent on the species and occurs in a 10 °C to 12 °C range in winter barley, wheat, and rye [138,139]. The result of the ICE-CBF-COR pathway cascade is the activation of the effector genes that modify the plant metabolism, conferring frost tolerance [140]. The temperature must be below 10 °C for 4–6 weeks in short-day conditions to complete the adaptive response in Triticeae [141,142]; once the process is completed, crops can withstand freezing at −7/12 °C for barley, −9/18 °C for wheat, and −18/−30 °C for rye [132,143].

Interestingly, no receptors receiving the low temperature signal have been identified so far [128]. The ICE-CBF-COR pathway is activated by an increase in intracellular Ca²⁺ concentration by either rigidification of the plasma membrane or ligand-activated channels. After calcium influx into the cytosol and its binding by Ca-sensors (such as calmodulins), a signal cascade based on calcium-binding proteins (CBPs) is initiated to target the ICE (inducers of CBF-gene expression) transcription factors that up-regulate the CBF genes [144,145]. ICE transcription factors belong to the MYC family and MYC subfamily of bHLH (basic helix–loop–helix) [78] and are known as positive CBF expression regulators, considered to act upstream of the low-temperature signaling pathway [78,146,147].

In addition, temperature variation is not the only environmental stimulus influencing the expression of the CBFs; also, circadian rhythms and light characteristics (i.e., quality and quantity) have been reported to be involved in cold acclimation [133]. For example, recent studies showed that the expression of some barley HvCBF genes (HvCBF2A, HvCBF4B, HvCBF6, and HvCBF14) is regulated by the circadian rhythm and day length [133,148,149]. In warm conditions, CBF genes show high expression late in the afternoon and continue to decrease early in the night [148]. The peak of expression is 8–12 h after the dawn, either in short- or long-day conditions. However, the amplitude of the peaks is wider in short-day compared to long-day conditions [150]. This peak does not coincide with the coolest period of the day, but it may be functional for the preparation of the cell for the subsequent cold of the night [107]. The circadian clock regulates the expression of several genes. The G-Box-like motifs are necessary for transcriptional regulation by the circadian pseudo-response regulators binding basic helix–loop–helix transcription factors [151]. Other environmental stimuli are the light spectra and intensity; several works have elucidated that the variation of light spectra and light intensity might modulate the expression of CBF genes and also increase frost tolerance [90,152,153,154,155,156].

The vernalization process is controlled by three major genes: VRN-1, VRN-2, and VRN-3 [73,157]. VRN-1 is a flowering promoter that was shown to be an AP1-like MADS-box transcription factor, whose expression leads the plant to the transition from the vegetative to the reproductive phase [158,159]. Moreover, it was also proven to be involved in cold acclimation and frost tolerance [71]. VRN-2 is a dominant flowering repressor down-regulated by vernalization treatment and includes two tandem zinc finger-CCT domain genes (ZCCT1 and ZCCT2) [31,160]. VRN-3, the main integrator of the photoperiod and vernalization signals that lead to the transition of the apical meristem [161], is homologous to the flowering integrator FLOWERING LOCUS T gene in Arabidopsis [162,163]. Due to their diploid nature, WH barleys can be considered a model for vernalization in Triticeae crops [24]. VRN-H2 is expressed in long and neutral day conditions [164]. In autumn, when plants are still in the seedling stages, VRN-H2 is highly expressed and represses the VRN-H3, which is the flowering induction gene [161,165]. The repression of VRN-H3 also limits the expression of VRN-H1 [158,159]. Exposure to cold temperatures activates VRN-H1 and results in the down-regulation of VRN-H2 and, consequently, the release of VRN-H3 from repression [31,73]. After prolonged cold exposure, the expression level of VRN-H1 reaches a threshold necessary to induce the transition phase, up-regulating VRN-H3, and initiating the flowering process [165]. Exposure to long-day conditions mediated by the photoperiod genes PPD-H1 and PPD-H2 is also necessary [53].

The expression of VRN-H1 changes in function of the plant growth habit; as mentioned above, in winter genotypes, the expression of the recessive vrn-h1 allele is induced by prolonged periods of cold [166,167]. The quantity of time under cold and short-day conditions necessary to satisfy the vernalization requirements varies with the geographical origin of the genotype and the environmental condition, changing from 6 to 10 weeks of temperatures in a range between 6 °C and 2 °C under short-day conditions [31,141,142,168]. In spring genotype, the dominant Vrn-h1 allele has a constitutive high expression that rapidly induces the transition [169]. The vernalization in wheat is more complex compared to barley due to the presence of three homoeologous VRN-A1, VRN-B1, and VRN-D1 loci mapped on the long arm of chromosome group 5 [170], with the major effect of VRN-A1 in determining the growth habit [171].

The interaction between VRN-1/FR-1 and FR-2 (CBFs) has also been demonstrated [172]; VRN-H1 can bind promoter regions of the CBF genes, inducing a reduction of their transcription levels; nevertheless, the mechanism is still not fully understood [117,173].

However, a question remains: how does the ICE-CBF-COR pathway confer frost tolerance?

The efforts to identify the molecular mechanisms underlying FR-2 in Triticeae crops were based on integration studies on structural and functional aspects of the locus. Several barley genotypes have been sequenced, and a pan-genome has been assembled [174]. Thanks to this data, FR-H2 was studied in different frost-prone and tolerant genotypes to evaluate the CBFs position in the cluster, the variability in the structure, the CBF coding sequences, and the promoter regions [24,116,175,176,177].

Initially, four HvCBF genes (HvCBF3, HvCBF6, HvCBF9, and HvCBF14) have been selected as candidate genes due to the presence of homologs in other Triticeae already reported to be involved in cold resistance [107,178]. Then, HvCBF14 has emerged as the major candidate for the frost tolerance in barley in several works [90,96,117,155,178,179,180]. Two SNP linked to HvCBF14, associated with frost tolerance, were identified by Fricano and colleagues [178] in an association analysis of a panel of European cultivars, landraces, and H. spontaneum accessions. Later on, a correlation between frost tolerance and the same HvCBF14 gene in spring haplotypes was demonstrated by Guerra et al. [179], who investigated a panel of 403 accessions with exome sequencing-based allele mining.

Structural variation is recognized as a common feature and evolutionary force of genomes, where copy number variations (CNV) and resulting gene dosage effects determine a number of traits/phenotypes in plants [181,182,183,184,185]. One of the first clear associations between CNV and phenotype was reported for the boron-toxicity tolerance in barley [186]. The first indication of the involvement of CNV at the FR-H2 locus and frost tolerance in Triticeae was reported by Knox et al. [121]. Two HvCBF2 paralogs (HvCBF2A and HvCBF2B) and multiple copies of the HvCBF2A-HvCBF4B genomic segment were identified in the frost-tolerant genotypes ‘Dicktoo’ and ‘Nure’. On the other hand, genomic clones of ‘Morex’ and ‘Tremois’ showed only single paralogs of HvCBF4 and HvCBF2. Results on CNV were confirmed by sequencing the same physical region in the tolerant ‘Nure’ [117] and susceptible ‘Morex’ [116] genotypes, in successive, independent experiments. Francia et al. [122] and Rizza et al. [53] confirmed that frost-resistant varieties of barley were characterized by a high number of copies for the HvCBF2 and HvCBF4 genes and maintained two distinct HvCBF2 paralogs (HvCBF2A and HvCBF2B). In summary, the influence of structural variation on determining the FR-2 effect remains a long-standing conundrum and leaves an open question: is the phenotype influenced by the expression of the HvCBF14 gene alone, or are multiple copies of other CBFs involved? Is the number of copies at the HvCBF2A–HvCBF4B segment relevant for the modulation of the HvCBF14 expression level and the resulting phenotype?

The influence of the gene dosage (i.e., the pool of transcripts) of a specific CBF on the expression of other elements of the ICE-CBF-COR pathway was tested/evaluated in two elegant experiments. The overexpression of HvCBF2 in the spring susceptible cultivar ‘Golden Promise’ resulted in higher transcript levels of COR genes; HvCOR14B and HvDHN5, already at warm temperatures, were raised strongly at cold temperatures. Moreover, higher transcription levels of HvCBF12, HvCBF15, and HvCBF16 and greater frost tolerance were observed in overexpressed lines [187]. According to authors, HvCBF2 may activate target genes at warm temperatures, and transcript accumulation for some of them is greatly enhanced by cold temperatures.

The influence of CNV at HvCBF2A-HvCBF4B on the expression levels of HvCBF12, HvCBF14, and HvCBF16 was investigated using the high frost-tolerant variety ‘Admire’ and different descendent genotypes (namely, Missouri barley—MO B lines) by Dhillon and colleagues [149]. MO B lines harboring a higher number of copies of HvCBF2A-HvCBF4B had higher expression levels of all three genes under normal growth conditions.

While barley has a diploid genome (2n = 2x = 14, HH) of 5 giga base pairs (Gbp) [190], tetraploid durum wheat (2n = 4x = 28, AABB) has 12 Gbp [191], and hexaploid wheat (2n = 6x = 42, AABBDD) has approximately 17 Gbp [192]. Thereby, FR-2 organization in wheat is more complex compared to barley due to the contribution of one/multiple homoeologous chromosome regions and redundancy caused by the ploidy level [193,194,195]. Wheat exhibits high variability in frost tolerance traits, given that hexaploid wheat genotypes (AABBDD) exhibit greater frost tolerance than diploid (AA) and tetraploid genotypes (AABB) [196,197].

The first works on CBF/FR-2 in wheat were carried out in mapping populations of einkorn diploid wheat (Triticum monococcum L.), which is the ancestor of the A genome in hexaploid wheat and is considered a practical model for the functional genetics of wheat [75,110,118,119,170,198,199]. First expression studies showed the association of CBF genes at the FR-A^m2 with the expression of COR genes and frost tolerance [75,110].

TmCBF12, TmCBF14, TmCBF15, and TmCBF16 (central cluster) expression levels were significantly associated with frost tolerance, measured as regrowth capacity after stress. Moreover, a high-density mapping study confirmed that TmCBF12, TmCBF14, and TmCBF15 were the candidates for the observed differences [119].

Thanks to the works carried out on T. monococcum, the number and position of CBF genes in bread wheat were identified in different works. While in barley, a CNV has never been associated with a central cluster at FR-H2 (see above), in diploid and polyploid wheat, a lower copy number of CBF14 in the B genome compared to the A and D genomes was reported [200].

TaCBF14 and TaCBF15 were associated with increased frost tolerance in doubled haploid (DH) mapping populations of ‘Norstar’ × ‘Winter Manitou’ and ‘Norstar’ × ‘Cappelle-Desprez’ (all WH genotypes) [198]. Higher levels of TaCBF14 induced by temperature shift and blue light were reported in winter wheat ‘Cheyenne’ [180].

Recent studies expanded the investigation of ICE-CBF-COR interconnection with other environmental stimuli with high-throughput functional analysis [78,195,203,204,205,206]. Guo et al. [78] carried out RNAseq and qPCR analysis in wheat tissues under different stress conditions, observing the expression of 53 genes belonging to the ICE-CBF-COR signaling cascade that revealed tissue-specific expression patterns of the ICE, CBF, and COR genes under different stress conditions. Six genes related to the ICE-CBF-COR pathway (TaCBF11a, TaCBF16b, TaICE1a, TaICE1d, TaCOR5a, and TaCOR6d.1) were induced by all treatments (drought, heat, drought, and cold). Three genes, two CBFs and one COR (TaCBF1b, TaCBF4a, and TaCOR3b), were induced specifically by cold.

Zheng et al. [206] carried out an isoform sequencing experiment at four leaf stages under frost stress (at −6 °C), and expression levels of TaCBF8a and TaCBF14a decreased, while TaCBF6a, TaCBF9a, TaCBF10a, TaCBF13a, and TaCBF15a expression levels increased. Recently, Wang et al. [203] performed a transcriptome analysis during the vernalization (4 °C) time-course with sampling from one to six weeks. Six CBF genes of the III subgroup were highly expressed exclusively before vernalization (“steady state” at 22 °C), while 10 CBFs, mainly from the IV subgroup, were not expressed before and were highly induced by vernalization, reaching the highest level of expression after three weeks and decreasing after five/six weeks of treatment. Two different homologs of the MYC-like bHLH transcription factor ICE were identified in wheat as TaICE41 and TaICE87 [146], and their overexpression in Arabidopsis enhanced frost tolerance after hardening. The recent availability of the wheat genome allowed us to locate three TaICE1 genes on the long arm of homoeologous chromosome group 3; these genes were shown to be induced by drought and cold treatment [78]. In addition, Wang et al. [203] reported that TaICE41 was expressed at extremely high levels after five weeks of vernalization.

Compared to other Triticeae crops, rye is uniquely tolerant to biotic and abiotic stresses, showing high yield potential under marginal conditions [207,208,209]. However, it received little attention in terms of breeding efforts and genomic research due to its limited distribution worldwide. Likewise barley and rye have a diploid genome (2n = 2x = 14, RR); however, it has not become a reference crop for genomic analysis in the Triticeae tribe due to its elevated level of allogamy and the fact that the first chromosome-scale assembly of its large 7.9 Gbp genome was released only recently, in 2021 [114], showing 92% of repetitive elements [207,210,211,212,213].

Investigation of rye genome evolution and chromosome synteny [213] revealed, as expected, that the chromosome 5R harboring FR-2 and FR-1 loci is entirely collinear with wheat homoeologous chromosome group 5. Initially, eleven ScCBF genes were isolated in a winter rye genome, and nine of them were mapped on chromosome 5R with a cluster organization (FR-R2) [106]. Subsequently, Jung and Seo [207] identified 12 new CBF genes and five new CBF gene alleles. The genome assembly [114] reported CNV for 4 members of CBF Group IV between tolerant and resistant varieties.

Concerning the structure of the locus, FR-R2 haplotyped variation has been associated with different frost tolerance levels in different rye genotypes [114,214].

CBF genes are members of a large protein family of the C-repeat binding factor/Dehydration responsive element-binding 1 (CBF/DREB1), known to be involved in the growth and development processes and responses to different environmental stressors (cold, heat, drought, salt, etc.) [215]. CBF genes could thus have in Triticeae a role in a cross-talk between the cold and drought response pathways, as already reported for Arabidopsis [98,216].

The CBF/DREB1 regulon modifies the plant metabolism in conditions of water deficiency, and their activation might also be triggered by drought conditions in the seedling phase [78,217]. In several drought-responsive genes, such as AtRD29A (responsive to desiccation), HvDHN1–HvDHN11 (dehydrin), or AtCOR6.6, a DRE/CRT motif is present in the promoter regions. When drought conditions occur, the plant reduces its water uptake by closing the stomata, which also reduces its CO₂ uptake, which results in a reduction in the photosynthesis and physiological activities. To cope with drought stress, plants activate several morphological and physiological modifications to conserve water and reduce its loss. The molecular response to drought follows a similar pathway to cold acclimation due to the same trigger of water scarcity, which activates both responses. As already summarized, water deficit activates, like low temperature stress, two different signaling pathways: ABA-dependent and ABA-independent [131]. The interaction between CBF genes in hormone-mediated acid abscisic (ABA) pathways has been reported [218,219]. In A. thaliana, the ABA-independent pathway is regulated by AtCBF4, which increases the production of a class of small, highly expressed, and stress-inducible proteins called late embryogenesis abundant (LEA), protecting the cellular membranes and the cytoskeleton from desiccation [216,220]. Interestingly, it has been shown in A. thaliana that ABA-responsive genes contain in the promoter regions both the ABA-response cis-element ABRE/ABF and the CRT/DRE motif [221]. Overall, drought stress in barley and wheat can have a significant negative impact on plant growth, yield, and grain quality; however, plants have evolved mechanisms to cope with water scarcity and to survive in dry environments [222]. Nevertheless, which CBF genes and which pathways are activated have not been determined yet [88,148,215]. In a study of A. thaliana transgenic lines, the overexpression of AtCBF1 and AtCBF3 genes resulted in an increase in drought tolerance [223]. A review of the conservative role of CBF genes throughout the Poaceae family reported rice OsDREB1A localized in the cluster OsDREB1H, syntenic with the FR-2 locus on chromosome 5 of Triticeae involved in chilling tolerance [96]. However, few examples of studies of the role of CBF genes in drought tolerance are available for barley and wheat.

A common phenotypic response observed in transgenic lines overexpressing CBF genes in different crops can be identified as an increased tolerance to frost and/or drought and modified growth and development, as originally reported for Arabidopsis [215,223]. Overexpression of two CBF/DREBs (TaDREB3 and TaCBF5L) in wheat and barley was reported to lead to an increase in drought and frost tolerance in transgenic barley. Moreover, in transgenic wheat, the TaCBF5L gene significantly increased the grain yield under severe drought during flowering [77]. Javadi and colleagues mined available GeneChip microarray data [224] in order to detect key genes involved in drought tolerance in barley and identified hub genes from the AP2 and NAC families that might be among the key TFs that regulate drought-stress response in barley. What is interesting is that HvCBF6 (distal cluster of FR-H2) was included among the hub genes. In rye, the PEG treatment (drought condition simulation) revealed that there is a specific type of response to stress among ScCBF genes; most of them were highly responsive to cold stress, whereas ScCBF2 and ScCBF7b were induced by water deprivation and were almost insensitive to low temperature [207]. Guo and colleagues [78] characterized the expression profile of the ICE-CBF-COR pathway in different wheat tissues under different stress conditions. Authors showed that TaCBF11a, TaCBF16b, TaICE1a, TaICE1d, TaCOR5a, and TaCOR6d.1 were induced by drought, and the induction level was higher in tolerant genotypes [205].

The overlapping of cold/frost and drought conditions is a relatively unexpected new form of combination of abiotic stress, and it usually happens during the late autumn, after sowing, when winter genotypes are in the seedling phase. The drought stress in the seedling phase induces root architecture modification that might act as a constitutive resistance mechanism, useful when the stress re-occurs in other phenological phases [225,226,227]. As observed in numerous studies in the past years, CBF overexpression in the model plant Arabidopsis enhances abiotic stress tolerance but, on the other hand, reduces growth. CBF genes are known to interact with plant hormones [131], and the current model of CBF-GA (gibberellic acid) interplay proposes that overexpression of CBFs, either via cold induction or by transgenic means, stimulates the accumulation of DELLAs. Those growth-repressing proteins act downstream in the GA signaling pathway, leading to stunted growth. As far as the underlying molecular mechanism is concerned, in warm temperatures, DELLAs interact with JAZs to prevent JAZs binding to ICE1, leading to its inactivation. In cold temperatures, ICE1 is modified to gain the function for activation of CBF transcription [228]. Understanding the relationship between CBF genes, GA and DELLA proteins might help to get an overall picture of the role of CBFs in plant physiology. One of the new frontiers that regard CBF genes is to evaluate their contribution in the tillering phase, crucial in the growth and development of wheat and barley, as it directly influences the potential yield and overall productivity of cereal crops [229]. Moreover, in this phase, winter cereals reach the maximum of their stress tolerance [28,132,143]. The main actors in tillering formation are gibberellic and abscisic acids; moreover, the roles of VRN-1 and VRN-2 and the photoperiod response gene PPD-1 have been described [230,231,232]. All these components interact with CBF genes; however, mechanisms of interaction are still not clear.