Comprehensive analysis of cucumber C-repeat/dehydration-responsive element binding factor family genes and their potential roles in cold tolerance of cucumber

Cold stress is one of the main abiotic stresses limiting cucumber (Cucumis sativus L.) growth and production. C-repeat binding factor/Dehydration responsive element-binding 1 protein (CBF/DREB1), containing conserved APETALA2 (AP2) DNA binding domains and two characteristic sequences, are key signaling genes that can be rapidly induced and play vital roles in plant response to low temperature. However, the CBF family has not been systematically elucidated in cucumber, and the expression pattern of this family genes under cold stress remains unclear. In this study, three CsCBF family genes were identified in cucumber genome and their protein conserved domain, protein physicochemical properties, gene structure and phylogenetic analysis were further comprehensively analyzed. Subcellular localization showed that all three CsCBFs were localized in the nucleus. Cis-element analysis of the promoters indicated that CsCBFs might be involved in plant hormone response and abiotic stress response. Expression analysis showed that the three CsCBFs could be significantly induced by cold stress, salt and ABA. The overexpression of CsCBFs in cucumber seedlings enhanced the tolerance to cold stress, and importantly, the transcript levels of CsCOR genes were significantly upregulated in 35S:CsCBFs transgenic plants after cold stress treatment. Biochemical analyses ascertained that CsCBFs directly activated CsCOR genes expression by binding to its promoter, thereby enhancing plant resistance to cold stress. This study provided a foundation for further research on the function of CsCBF genes in cold stress resistance and elucidating its mechanism.


Introduction
Low temperature is a major environmental factor affecting the growth and seasonal distribution of field crops and horticultural crops. When plants are exposed to freezing temperatures, their cold tolerance and freezing resistance are increased, that is, this adaptation process is called cold adaptation [1]. Many plants, such as Arabidopsis and oilseed rape, subjected to low temperature stress, have evolved a complex set of cold-adapted mechanisms involving gene transcriptional regulation and a wide range of physiological, biochemical and metabolic changes [2][3][4]. In recent years, the molecular mechanism of cold adaptation has been studied extensively and the Open Access *Correspondence: 374141645@qq.com; 163.hwx@163.com 1 School of Biological Science and Technology, University of Jinan, Jinan 250022, China 2 Shandong Institute of Pomology, Tai'an, Shandong 271000, China key regulatory factors of this complex network have been explored. In the Arabidopsis genome, 4-20% of the genes are regulated by cold signaling. Many transcription factors including MYB, NAC, MYC, AP2/ERF (APETALA2/ ethylene response factor) and bZIP can induce the expression of stress-related genes to protect cells from injury at low temperature [5][6][7]. Studies have shown that the cold signal regulation pathway dependent on CBF (CRT/DRE binding factor), namely ICE1 (Inducer of CBF/DREB1 expression 1)-CBF-COR (cold-responsive) pathway, is the most clearly studied cold response pathway in plants [4,8,9]. In this pathway, ICE1 is rapidly expressed after low temperature induction and promotes the expression of CBF genes and CBFs bind to the CRT/ DRE (C-repeat/Dehydration responsive element) cis-element on the COR gene promoters to activate COR gene expression. Thus, plant resistance to low temperature can be improved [10][11][12].
CBF transcription factors belong to the DREB subfamily of APETALA2/Ethylene-Responsive Factor (AP2/ERF) family, so all CBF proteins have a highly conserved AP2 DNA binding domain, and there are also two conserved characteristic sequences (PKK/RPAGRxKFxETRHP and DSAWR) on both sides of the AP2 domain [13]. The former sequence is directly located in the upstream of AP2 DNA binding domain and may be related to protein transport [13]. Arabidopsis thaliana contains six CBF genes, namely AtCBF1 (DREB1B), AtCBF2 (DREB1C) and AtCBF3 (DREB1A), AtCBF4, AtDDF1 and AtDDF2, among which AtCBF1, AtCBF2 and AtCBF3 are extensively induced by low temperature and can all improve the cold tolerance of Arabidopsis thaliana. These three AtCBF proteins have high homology and are arranged in tandem array as AtCBF1-AtCBF3-AtCBF2 on the short arm of chromosome 4 [14][15][16]. The expression of AtCBF4 is induced by drought and ABA, but not by low temperature, while overexpression of AtCBF4 gene can enhance drought tolerance and cold tolerance of Arabidopsis thaliana [17]. AtDDF1 and AtDDF2 are related to salt tolerance of Arabidopsis thaliana [18].
Currently, CBF genes have been isolated from many plant species, such as apple, soybean, rice, tomato, wheat, barley and maize, and have been shown to play important roles in plant cold response [19]. Overexpression of AtCBFs in other species can enhance the cold resistance of each species, and the heterogeneous expression of CBFs from other plants in Arabidopsis can also enhance the cold tolerance of Arabidopsis [20][21][22]. When TaCBF14 and TaCBF15 genes from wheat were transferred into barley, the frost tolerance of transgenic plants was higher than that of wild-type barley [23]. Overexpression of OsDREB1A could enhance cold resistance of rice [24]. Transgenic potato with AtCBF1 gene enhanced cold tolerance and induced physiological changes related to adaptation to cold environment [20]. Overexpression of CsGG3.2 could enhance cold resistance via positively regulated the expression of CBF genes in cucumber [25]. Moreover, exogenous melatonin could increase the expression of CBF1 and enhanced the cold tolerance of cucumber seedlings [26]. CBFs are important regulators of plant growth and low temperature response, and the biological function of CBFs in regulating cold tolerance is highly conserved among plants, but also species-specific [27].
Low temperature rapidly activates the expression of CBF in plants, which then binds specifically to the DRE/ CRT cis-element of the COR gene promoters to induce its expression [28,29]. Many COR genes have been isolated from plants and are known as KIN (cold-inducible), ERD (early engend-inducible), LTI (low temperature induced) and RD (response to dehydration). These genes include LTI78, COR78 (RD29A), COR47, COR15A and KIN1 [30][31][32][33]. In addition to Arabidopsis, many COR homologues have been cloned from other plants [34]. The expression of CsCOR1 gene in tea was significantly induced by low temperature and drought, and the heterologous expression of CsCOR1 in tobacco enhanced its salt tolerance and dehydration tolerance [35]. Under cold stress, two cucumber CBF-inducible COR genes, CsCOR15b and CsKIN1, were higher expressed in CsGG3.2 overexpression plants [25].
The discovery of CBF transcriptional activators provide a new way to improve plant cold resistance, and lay a theoretical foundation for further discovery of key genes in plant cold resistance mechanism, which has a wide application prospect and important application value in crop and vegetable quality improvement. Cucumber (Cucumis sativus L.) is an economically important crop cultivated worldwide [36]. The functions of CsCBFs have not been systematically identified in cucumber. In this study, three CsCBF genes were identified in cucumber and the comprehensive analyses including the gene structures, conserved domains, phylogenetic analysis and cis-elements in promoters were further performed. In addition, the expression patterns of CsCBFs under different abiotic stresses were analyzed. Furthermore, overexpressed CsCBFs cucumber seedlings increased their cold tolerance by activating CsCOR genes expression. These results provide a basis for further research on cold tolerance mechanism of cucumber.

Identification and analysis of CBF genes in Cucumber
To identify CsCBF family genes in cucumber genome, the six Arabidopsis CBF protein sequences and the AP2/ ERF conserved domain and characteristic motif of CBF protein were employed as queries to search against the cucumber genome database using BlastP programme, respectively. Finally, three cucumber CBF family genes were identified, and named CsCBF1 to CsCBF3 according to their sequence similarity and phylogenies with individual AtCBF proteins. Multi-sequence alignment (MSA) analysis of the three cucumber CBF protein sequences and six Arabidopsis CBF protein sequences showed that the AP2/ERF domain and the characteristic motifs of CBF family were highly conserved, and the C-terminal and N-terminal of CsCBF proteins were significantly different (Fig. 1). The information of the CsCBFs, including the gene ID, gene name, chromosomal locations, isoelectric points (pI), and amino acid length was shown in Table 1. These three CsCBF genes were mapped on chromosome 3, 5 and 5 of cucumber, respectively, and the isoelectric points of CsCBF1, CsCBF2, CsCBF3 protein were 5.10, 5.16 and 4.85, respectively ( Table 1). The isoelectric points of all three CsCBF proteins were less than 7, indicating that the three proteins were acidic.

Phylogenetic analysis, gene structure and conserved motif analysis of CBF genes in cucumber, tomato and Arabidopsis
To analyze orthologous or paralogous relationships of the CBF genes from nine plant species, a phylogenetic tree of these genes was constructed from amino acid sequences (Fig. 2). As shown in Fig. 2, the 70 CBF proteins could be roughly divided into 18 groups, of which CsCBF1 and CsCBF2 belonged to group 5 and CsCBF3 belonged to group 8 ( Fig. 2; Table S1). To better evaluate the evolutionary relationships of the CsCBF proteins, we further analyzed the phylogenetic tree, gene structures and conserved motifs of six AtCBFs, seven SlCBFs and three CsCBFs (Fig. 3). As shown in Fig. 3A, the resulting tree categorized these CBF proteins into three clades, designated CladeI, CladeII, and Clad-eIII, and cucumber CBF members were classified into Clade I and III. Phylogenetic analysis also revealed that CsCBF1 and CsCBF2 had the highest homology with SlCBF1, SlCBF2 and SlCBF3, and all clustered in Clade I (Fig. 3A). CsCBF3 was classified into CladeIII, which included four SlCBF and two AtCBF proteins, and Cla-deII only composed of four AtCBF proteins.
The gene structure of CBFs from cucumber, Arabidopsis and tomato were also analyzed, which was consistent with the results of phylogenetic analysis (Fig. 3B). The number of exons in AtCBF, SlCBF and CsCBF genes was conserved, ranging from one to two exons. We found that the two clades, CladeI and Cla-deII, had same gene structures, which all contained only one exon and no intron (Fig. 3B). The six proteins, AtDDF1, AtDDF2 and SlCBF4-SlCBF7, in CladeIII  contained one exon, while CsCBF3 in CladeIII had two exons and one intron (Fig. 3B).
To further analyze the structural diversity and predict the function of the CBF proteins, the motif analysis of them was carried out by MEME ( Fig. 3C; Fig. S1). A total of ten distinct motifs, named Motif1-Motif10, were identified (Fig. S1). Motif1, which was representative AP2/ERF domain, and motif 2 and 4, which were the characteristic domains of CBF family, were identified in all CBFs. Some of the specific motifs were absent in certain clades. For example, motif 6, 7 and 9 existed only in Clade II, but were absent in all the members of the CladeI Collectively, CBF proteins with close evolutionary relationships in the phylogenetic tree generally had similar gene structures and conserved motifs, indicating that evolution of each subfamily in the three different species was relatively conserved.

Cis-elements identification of CsCBF gene promoters in Cucumber
Previous studies have indicated that most of CBF proteins regulate plant growth and tolerance to various abiotic stresses [37]. To investigate the biological function of CsCBF genes in cucumber, the potential cis-elements were identified on the 2-kb promoter regions of the CsCBF genes by PlantCARE (Table S2). As shown in Fig. 4, cis-elements responding to hormones such as abscisic acid (ABA), salicylic acid (SA), jasmonate acid (MeJA), auxin and gibberellin (GA) were presented on the promoters of CsCBF genes. Moreover, the CsCBF promoters also contained cis-elements in response to abiotic stresses, such as low temperature, defense and stress. In addition, the three CsCBF gene promoters all had light response signal elements (Fig. 4), suggesting that CsCBF genes might also be involved in the regulation of cucumber growth and development by light signal. In conclusion, the three CsCBF genes in cucumber may be involved in the response to multiple plant hormones and abiotic stresses.

Expression patterns of CsCBFs under different abiotic stresses
To investigate whether CsCBF genes respond to abiotic stress in cucumber, qRT-PCR was used to detect the expression levels of CsCBF genes under different stress conditions including low temperature (4°C), salt (100 mM NaCl) and ABA (100 μΜ ABA) (Fig. 5). Under low temperature treatment, the expression patterns of CsCBF1 and CsCBF2 showed a similar trend, which was firstly rapidly increased, and reached maximum values at 3 h, decreasing thereafter (Fig. 5A). However, the expression levels of CsCBF3 reached the maximum at 9 h, suggesting that CsCBF3 was less sensitive to low temperature than CsCBF1 and CsCBF2. Similar to the expression pattern after low temperature treatment, the expression levels of CsCBF1 and CsCBF2 achieved the maximum after 3 h of salt stress, while the expression level of CsCBF3 was highest after just 1 h of NaCl treatment, then rapidly decreased to the initial level and remained at a low level all the time (Fig. 5B). Different from the above treatments, CsCBF genes responded to ABA more rapidly, and all significantly increased at 0.5 h. The expression levels of CsCBF2 and CsCBF3 increased rapidly at 0.5h and reached the maximum at 1 h, then dropped to the initial level at 3h and increased again at 6h, decreasing  B 100 mM NaCl treatment. C 100 μΜ ABA treatment. The extracted RNA samples were collected at 0.5h, 1h, 3h, 6h, 9h, 12h and 24h after the corresponding treatment, respectively. The cucumber β-actin gene was used as an internal control, and three biological replicates were used for gene expression analyses. Error bars were the standard errors (SE). Different lowercase letters represented significant differences (P < 0.05) thereafter (Fig. 5C). Unlike CsCBF2 and CsCBF3, the expression level of CsCBF1 reached its highest value at 6h of ABA treatment (Fig. 5C). These results suggested that CsCBFs were involved in response to low temperature, salt and ABA.

Subcellular Localization of CsCBFs
PredictProtein software was used to predict the subcellular localization of CsCBF1, CsCBF2 and CsCBF3 proteins [38], and the prediction result showed that CsCBF1, CsCBF2 and CsCBF3 proteins were all in the nucleus (Fig.  S2). To verify the predicted results of CsCBF proteins, the fusion protein vectors 35S:CsCBF1-GFP, 35S:CsCBF2-GFP and 35S:CsCBF3-GFP were constructed, respectively (Fig. 6A). The GFP emited a green fluorescent signal under a laser-scanning confocal microscopy to determine where the gene is expressed. Microscopically, epidermal cells from tobacco leaves expressing the different CsCBF fusion proteins all only showed a fluorescence signal in the nucleus. As a control, the 35S:GFP fluorescence was observed throughout the whole cell (Fig. 6B). These data indicated that CsCBF proteins were all nuclear localization proteins, which were consistent with the previous prediction. The three CsCBF transcription factors may play roles in transcriptional regulation.

Overexpression of CsCBFs enhanced the tolerance of transgenic cucumber seedlings to cold stress
The expression levels of CsCBFs were significantly induced by low temperature (Fig. 5). In order to further investigate the response of CsCBF1, CsCBF2 and CsCBF3 to cold stress and their biological functions, the agrobacterium-mediated transient transformation experiments were conducted in cucumber cotyledons to clarify CsCBFs tolerance to cold stress. Cucumber seedlings overexpressing CsCBFs with GFP fluorescence signal in cotyledons were selected for subsequent experiments (Fig. S3A). The qRT-PCR analysis showed that the expression of CsCBF genes in their transgenic cucumbers were significantly higher than those in WT  (Fig. S3B), and different stress treatments all promoted the expression of CsCBF genes in transgenic cucumber (Fig. S3C-E). To test whether CsCBFs can enhance cold resistance of cucumber cotyledons, transgenic cucumber seedlings and WT were treated at 0°C, respectively. Before treatment, the 35S:CsCBFs transgenic plants and WT all grew well, while after 3 h of cold treatment, slight wilting appeared in cotyledons of WT compared with transgenic seedling overexpressing CsCBF1, CsCBF2 and CsCBF3, and serious wilting in WT showed more obvious difference from all transgenic seedlings after 24 h (Fig. 7A). After 48 h, the survival rate of WT was only 27%, while the survival rates of the 35S:CsCBF1, 35S:CsCBF2 and 35S:CsCBF3 transgenic plants were 60%, 67% and 56% ( Fig. 7A-B).
The contents of proline and MDA are important physiological indexes to measure cold resistance of plant [39]. Compared with WT plants, the 35S:CsCBFs transgenic plants all showed a significant increase in proline and great decrease in MDA content (Fig. 7C-D). In addition, to verify that overexpression of CsCBFs enhanced cold tolerance of cucumber seedlings by regulating COR genes, the expression levels of two CsCOR genes (CsCOR15A and CsKIN1) in CsCBFs overexpressed plants and WT were detected under cold treatment, respectively. As shown in Fig. 7E-F, the transcriptional levels of the two genes in transgenic plants were significantly higher than those in WT plants after cold treatment. Based on survival rate, physiological indexes and CsCOR genes expression, CsCBF1 and CsCBF2 played stronger roles in cold stress response than CsCBF3 (Fig. 7). Taken all together, these results suggested that overexpression of CsCBF genes in cucumber could significantly enhance cold tolerance of cucumber.

CsCBFs directly activate CsCORs expression by binding to their promoters
Overexpression of CsCBFs greatly induced the expression of CsCORs (Fig. 7E-F), and previous reports have shown that CBFs can directly bind to the promoter regions of COR genes to activate their expression [12]. To verify whether CsCBFs can bind to the promoters of CsCORs, the 2-kb promoter fragments of CsCOR15A and CsKIN1 were selected and inserted into the pHIS2 plasmid, respectively. The CDSs of CsCBFs were separately cloned into the pGADT7 vector. The yeast one-hybrid (Y1H) assays were carried out and the results showed that CsCBF1, CsCBF2, and CsCBF3 proteins all could specifically directly bind to the promoters of CsCOR15A and CsKIN1, but not empty pHIS2 vector (Fig. 8A). The transient GUS activity assays were carried out in tobacco leaves to verify the above results. The above DNA fragments were separately inserted into pCAMBIA1300-GUS vector containing GUS reporter gene, and the CDSs of CsCBFs were cloned into the pCAMBIA1300 plasmid to obtain 35S:CsCBFs recombinant plasmids. The results showed that all three CsCBFs could activate the expression of CsCOR15A and CsKIN1 in vivo (Fig. 8B). These data revealed that CsCBFs could directly activate CsCORs expression by binding to their promoters.

Discussion
Cucumber (Cucumis sativus L.) is an economically important crop cultivated worldwide and one of the main vegetables grown in the facility [36]. Low temperature is a major environmental factor affecting the growth and seasonal distribution of cucumber. The transcription factor CBFs (C-repeat Binding Factor) are key "molecular switch" for plant to sense low temperature signals and regulate their adaptive responses. These genes can activate the expression of several downstream cold-tolerance related functional genes to improve plant resistance to low temperature [40]. Therefore, bioinformatics analysis of CBF gene family in cucumber was conducted to provide theoretical basis for analyzing the mechanism of CBFs regulating cucumber cold stress response.
Studying the differences in gene structure and conserved motifs is an important reference for analyzing the evolutionary relationships of gene families [33]. In our study, multi-sequence alignment analysis of CsCBFs showed that the three CsCBF proteins all contained conserved AP2 domains and their flanks (Fig. 1). By analyzing the gene structures of CsCBFs, it was found that the three CsCBF genes in cucumber did not contain UTR (Fig. 3). Phylogenetic tree analysis showed that 70 CBF proteins from cucumber and other species were divided into 18 subgroups. Among them, the three CsCBF proteins are closely related to CBF proteins in apple and pomegranate (Fig. 2). Previous studies have shown that CBF genes play important roles in plant growth and development. Overexpression of AtCBFs results in plant growth retardation and delayed flowering [16,41].
Here, we showed that light response signal elements were found on the promoters of the three CsCBF genes (Fig. 4), suggesting that CsCBFs may be involved in the regulation of light signal in the growth and development of cucumber.
Studies have shown that light is also an important environment, and photochrome-interacting factors (PIFs) play a key role in regulating plant development [42]. Part PIFs can bind to G-box and E-box cis-elements in AtCBF promoters to regulate their transcription, and the PIF3/4/7 are negatively involved in the low temperature response pathways of plants and can negatively regulate CBF expression [43]. In our study, cis-elements that respond to abiotic stresses and hormones such as low temperature, defense and stress, abscisic acid, salicylic acid, jasmonate acid, auxin and gibberellin were simultaneously screened on promoters of CsCBF genes (Fig. 4). The expression analysis showed that CsCBFs were indeed regulated by low temperature (Fig. 5A), which will help further elucidate the molecular regulation mechanism of cold signaling.
The functions of CBF genes in apple, soybean, rice, tomato, wheat, barley and maize have been widely reported [19,[44][45][46]. Overexpression of DREB can enhance plant resistance to stress, and 35S:PpDBF1 transgenic tobacco has higher salt tolerance, drought tolerance and cold tolerance [47]. The heterologous expression of ZmCBF3 and CsCBF3 (tea) in Arabidopsis can significantly enhance the frost resistance of Arabidopsis thaliana [48,49]. Overexpression of CsCBFs in the cotyledons of cucumber seedlings could markedly enhance its cold resistance and the changes of Pro and MDA in 35S:CsCBFs transgenic cucumber seedlings indicated that CsCBFs could enhance cold tolerance of cucumber (Fig. 7). Low temperature induces the expression of CBFs, thereby activating the expression of downstream target genes. Heterologous expression of VvCBF1 in Arabidopsis can enhance the expression of AtCOR15A, AtRD29B and AtRD29A [50]. The expression of AtCOR15A, AtCOR47, AtKIN1 and AtRD29A in 35S:SmCBFs transgenic Arabidopsis thaliana were up-regulated, thus enhancing its cold resistance [51]. In this study, the transcript level of CsCOR genes was significantly upregulated in 35S:CsCBFs transgenic plants after cold stress treatment (Fig. 7). Y1H and GUS experiments demonstrated that CsCBFs, as transcription factors, could directly bind to promoters of COR genes and activate their expression (Fig. 8). These results provided a foundation for further research on the function of CsCBFs gene in cold stress resistance and elucidating its mechanism.

Conclusions
In this study, we comprehensively analyzed the cucumber CBF family genes. The expression patterns of CsCBF genes under different stress treatments were also investigated, and the roles of CsCBFs in cucumber cold tolerance were analyzed in detail by transient transgenic method. This study provided a foundation for further research on the function of CsCBF genes in cold stress resistance and elucidating its mechanism.

Genome-wide identification of CsCBFs in cucumber
To identify the CsCBF genes from cucumber genome database (http:// cucur bitge nomics. org/ organ ism/ 20), six Arabidopsis AtCBF proteins were used as query sequences and Blastp was used to search for the predicted proteins. All candidate genes were further confirmed by the existence of conserved characteristic sequences (PKK/RPAGRxKFxETRHP and DSAWR) and AP2 (PF00847.20) domains using the Pfam (available online: http:// pfam. janel ia. org) and Simple Modular Architecture Research Tool (SMART) datebase (http:// smart. embl-heide lberg. de).

Physicochemical properties of CsCBF proteins
ExPASy software (http:// web. expasy. org/ protp aram/) was used to analyze protein sequences of CsCBFs to predict amino acid length and isoelectric point (pI). The location of CsCBF genes on cucumber chromosome was determined according to the physical location information in cucumber genome database.

Phylogenetic analysis, gene structure and conserved motif analysis
Multiple sequence alignments of these proteins were performed using ClustalW with default parameters. An un-rooted phylogenetic tree was constructed with the full-length amino acid sequences of the 70 CBFs using MEGA 7.0, and the neighbour-joining (NJ) method was used with the following parameters: Poisson correction, pairwise deletion, and bootstrap (1000 replicates; random seed) [52]. The corresponding DNA and cDNA sequences of each predicted gene were downloaded from genomes, and the gene structures were analyzed as described by [53]. The conserved motifs in CsCBFs were identified using Multiple Expectation Maximization for Motif Elicitation (MEME) online program (http:// memesuite. org/ index. html) [53].

Identification of cis-elements on CsCBFs promoter in cucumber
The entire cucumber genome data were downloaded from the cucumber genome database (Chinese Long 9930: http:// cucur bitge nomics. org/), and the 2 kb sequences upstream of the transcription start site of of CsCBFs were extracted by TBtools. The cis-elements on the promoter regions of CsCBF genes were analysed by PlantCARE website (http:// bioin forma tics. psb. ugent. be/ webto ols/ plant care/ html/) [52].

Plasmid construction and transient transformation of cucumber cotyledons
To generate 35S:CsCBFs-GFP, the full-length coding sequence of CsCBF1, CsCBF2 and CsCBF3 were amplified and cloned into pCAMBIA1300 vector with a GFP tag, respectively. The recombinant plasmids were transformed into Agrobacterium tumefaciens LBA4404, and then transferred into 8-d-old cucumber cotyledons for subsequent cold tolerance tests [54]. The primers used are listed in Table S3.

Subcellular localization of CsCBFs
To determine the subcellular localization of CsCBFs, the empty GFP vector and the recombinant plasmid of 35S:CsCBF1-GFP, 35S:CsCBF2-GFP and 35S:CsCBF3-GFP were injected into tobacco leaf epidermal cells, respectively. The injected tobaccos were grown under normal conditions for about 48 hours. The fluorescent signal was observed by a fluorescence microscope.

Expression pattern of CsCBF genes under different abiotic stresses
The cucumber inbred line Xintaimici was used for transient genetic transformation and stress treatments, and all plants were cultured in a light incubator under 28 °C with 16 h light /20 °C with 8 h dark cycle conditions. The two-week-old cucumber seedlings were placed in a 4 °C incubator for low temperature treatment until the leaves were collected, and the leaves of two-week-old cucumber seedlings with consistent growth were sprayed with 100 μM ABA and 100 mM NaCl, respectively. The leaves were selected at 0, 0.5, 1, 3, 6, 9, 12 and 24h for subsequent quantitative analysis. The mixed leaves of five plants were one replicate, and each sample contained three biological replicates. Each treatment was repeated at least three times.

Yeast one-hybrid assays
The 2-kb promoter fragments of CsCOR15A and CsKIN1 were selected and inserted into the pHIS2 plasmid, respectively. The coding sequences (CDSs) of CsCBFs were separately cloned into the pGADT7 vector to obtain the constructs AD-CsCBFs. The optimal 3-AT concentration which could inhibit the growth of background histidine of pHIS2 vector was screened. Then the recombinant pHIS2 vector and AD-CBFs were co-transferred to yeast strain Y187 and grown on medium SD/-Trp-Leu-His with optimal 3-AT concentration. Empty vector pGADT7 was used as the control. Y1H was conducted according to [55]. The primers used are listed in Table S3.

Transient GUS activity assays
The ORFs of CsCBFs were separately inserted into the pCAMBIA1300 vector. The 2kb promoter fragments of CsCOR15A and CsKIN1 were separately inserted into pCAMBIA1300-GUS plasmids to obtain proCsCOR15A:GUS and proCsKIN1:GUS recombinant plasmids. The tobacco leaves were used to conduct GUS activity assays. The different combinations were injected into 5-week-old tobacco leaves by agrobacterium-mediated method. Under normal conditions, the injected tobacco grew 2-3 days for subsequent experiments. The transient activity assays were measured as described previously [54].