Genome-Wide Identification of the CBF Gene Family and ICE Transcription Factors in Walnuts and Expression Profiles under Cold Conditions

Cold stress impacts woody tree growth and perennial production, especially when the temperature rapidly changes in late spring. To address this issue, we conducted the genome-wide identification of two important transcription factors (TFs), CBF (C-repeat binding factors) and ICE (inducers of CBF expression), in three walnut (Juglans) genomes. Although the CBF and ICE gene families have been identified in many crops, very little systematic analysis of these genes has been carried out in J. regia and J. sigillata. In this study, we identified a total of 16 CBF and 12 ICE genes in three Juglans genomes using bioinformatics analysis. Both CBF and ICE had conserved domains, motifs, and gene structures, which suggests that these two TFs were evolutionarily conserved. Most ICE genes are located at both ends of the chromosomes. The promoter cis-regulatory elements of CBF and ICE genes are largely involved in light and phytohormone responses. Based on 36 RNA sequencing of leaves from four walnut cultivars (‘Zijing’, ‘Lvling’, ‘Hongren’, and ‘Liao1’) under three temperature conditions (8 °C, 22 °C, and 5 °C) conditions in late spring, we found that the ICE genes were expressed more highly than CBFs. Both CBF and ICE proteins interacted with cold-related proteins, and many putative miRNAs had interactions with these two TFs. These results determined that CBF1 and ICE1 play important roles in the tolerance of walnut leaves to rapid temperature changes. Our results provide a useful resource on the function of the CBF and ICE genes related to cold tolerance in walnuts.


Introduction
Cold stress (low temperature) is an important environmental factor significantly constraining plant growth, development, and geographical distribution [1,2].Cold stress has effects on crop productivity, such as rice and wheat yield [2].To acclimate to the environmental temperature changes, plants have evolved a cascade of physiological and biochemical regulatory strategies to avoid the limitations [3,4].There are two different cold signals, rapid and gradual temperature decreases, referring to a complex gene regulation pathway involving transcriptional upregulation or downregulation.It is crucial that C-repeat binding factors/drought response element binding factor (CBFs/DREBs) pathways are upregulated for cold tolerance [5][6][7].The CBF pathway responding to cold stress is found in numerous plants, such as Acer species [8], soybean [9], Gossypium [10], Malus baccata [11], tomato [12], and Betula platyphylla [13], etc.In plants, the ICE1-CBF-COR transcriptional cascade is the key regulation pathway for cold temperature [14,15].Furthermore, the CBFs/DREBs genes are regulated by the inducer of CBF expression (ICEs, inducer of CBF expression) to enhance cold tolerance in plants [16][17][18].The conserved

Genome-Wide Identification and Phylogenetic Analysis of the CBF and ICE Gene Family in Three Walnut Genomes
The whole-genome data from three walnut genomes and those from another five species (Table S1) were used to identify the CBF and ICE1 gene family.A total of 16 CBFs (4 JrCBF, 7 JreCBF, and 5 JsCBF) and 12 ICEs (6 JrICE, 4 JreICE, and 2 JsICE) were identified in the three walnut cultivars, respectively (Table 1).These CBFs were divided into three subgroups (subgroup I, subgroup II, and subgroup III) and four subgroups of ICE1 genes (subgroup I, subgroup II, subgroup III, and subgroup IV) (Figure 1).The largest subgroup included 25 members of the CBF gene family (green branch, Figure 1A).The results indicated that ATCBFs and ATDDFs were divided into different groups.The largest subgroup included 13 members of the ICE gene family (green branch), followed by another 10 members of the ICE gene family (orange branch, Figure 1B).The subgroup with the most members of the ICE1 gene family in A. thaliana was represented by a blue branch (Figure 1B).
Table 1.Information of CBF and ICE proteins from three walnut genomes.

Chromosomal Distribution and Duplication Patterns of Juglans regia and Juglans sigillata
The chromosomal distribution of CBF and ICE1 genes was determined using the genome annotation data(*.gff).For the three cultivars (Juglans regia), the CBF distribution was different.The CBF genes of 'Chandler' were located on four distinct chromosomal: Chr01, Chr02, Chr03, and Chr10.Only one CBF gene was located on each chromosome (Figure 2A).For 'Zhongmucha-1', there were seven CBF genes distributed on six chromosomes, with two CBF genes on Chr3 (JreCBF3 and JreCBF6, Figure 2A).In addition, the five CBF genes from 'Yangbi' walnut were dispersed on five different chromosomes (Figure 2A).According to the ICE1 distribution analysis, there were two fewer members in 'Yangbi' walnut than the two other walnut types (six ICE1 genes in 'chandler' and four in 'Zhongmucha-1', respectively; Figure 2B).Two JrICE1 genes were located on Chr01 and Chr10, separately (Figure 2B).
To further explain the complexity and diversification of CBFs and ICE1s, we conducted a duplication mode analysis (Table S2).A total of three duplication patterns in Juglans regia ('Chandler', 'Zhongmucha-1', and 'Yangbi') were observed in which the main mode was whole-genome duplication (WGD) event, which occurred at the maximum proportion.In total, 15 out of 16 CBFs (93.75%) and 10 out of 12 ICE1s (83.33%) experienced the WGD event.Otherwise, only JreCBF6 exhibited tandem duplication (TD), and two ICE1 genes (JrICE1c and JrICE1c) experienced dispersed duplication (DSD) (Figure 2; Table S2).The result of the duplication analysis indicated that WGD induced the expansion of the CBF and ICE1 gene family in Juglans regia (Figure 2; Table S2).

Chromosomal Distribution and Duplication Patterns of Juglans regia and Juglans sigillata
The chromosomal distribution of CBF and ICE1 genes was determined using the genome annotation data(*.gff).For the three cultivars (Juglans regia), the CBF distribution was different.The CBF genes of 'Chandler' were located on four distinct chromosomal: Chr01, Chr02, Chr03, and Chr10.Only one CBF gene was located on each chromosome (Figure 2A).For 'Zhongmucha-1', there were seven CBF genes distributed on six chromosomes, with two CBF genes on Chr3 (JreCBF3 and JreCBF6, Figure 2A).In addition, the five CBF genes from 'Yangbi' walnut were dispersed on five different chromosomes (Figure 2A).According to the ICE1 distribution analysis, there were two fewer members in 'Yangbi' walnut than the two other walnut types (six ICE1 genes in 'chandler' and four in 'Zhongmucha-1', respectively; Figure 2B).Two JrICE1 genes were located on Chr01 and Chr10, separately (Figure 2B).

Protein Domain and Gene Structure Analysis
To exhibit the protein domain, motifs, and gene structure of CBF and ICE1 with respect to similarity, the phylogenetic tree (Neighbor-Joining tree) was devised based on the protein sequences (Figure 3).The results showed that all CBF proteins contained only one conserved domain named AP2, and ICE proteins had five conserved domains (named bHLH_AtAMS_like, bHLH_SF superfamily, HLH, ACT_UUR-ACR-like, and ACT superfamily, respectively).The five domains overlapped in the ICE protein of bHLH_At-AMS_like and bHLH_SF superfamily, HLH, ACT_UUR-ACR-like, and ACT superfamily (Figure 3B).The motifs of the CBF proteins were similar in terms of size and type (six motifs) but were different in terms of locations (Figure 3A).The same methods were found for the ICE protein, including similar motifs; however, some ICEs had variations, such as JrICE1 that lacked motif9 and JrICE1e that lacked motif6, respectively (Figure 3B).For gene structures, overall, the CBF genes generally had one CDS of long length of one mainly CDS (~600 bp) in three walnut genomes, while there were at least four CDSs (one longer CDS and other shorter CDSs) in walnut ICE genes (Figure 3).In addition, JrCBF1 and JrCBF2 had UTRs of long length, indicating that they play regulatory functions.[20], J. regia ('Zhongmucha-1') [43], and J. sigillata (iron walnut) [23], respectively.(A) Chromosomal localization position and duplication types of the CBF genes in Juglans regia ('Chandler') [20], J. regia ('Zhongmucha-1') [43], and J. sigillata (iron walnut) [23], respectively.(B) Chromosomal localization position and duplication types of the CBF genes in Juglans regia ('Chandler') [20], J. regia ('Zhongmucha-1') [43], and Juglans sigillata (iron walnut) [23], respectively.The genome information of the three walnuts is shown at the bottom of each figure of chromosomal locations.The red dot and green dot indicate Tandem duplication and dispersed duplication.

Protein Domain and Gene Structure Analysis
To exhibit the protein domain, motifs, and gene structure of CBF and ICE1 with respect to similarity, the phylogenetic tree (Neighbor-Joining tree) was devised based on the protein sequences (Figure 3).The results showed that all CBF proteins contained only one conserved domain named AP2, and ICE proteins had five conserved domains (named bHLH_AtAMS_like, bHLH_SF superfamily, HLH, ACT_UUR-ACR-like, and ACT superfamily, respectively).The five domains overlapped in the ICE protein of bHLH_AtAMS_like and bHLH_SF superfamily, HLH, ACT_UUR-ACR-like, and ACT superfamily (Figure 3B).The motifs of the CBF proteins were similar in terms of size and type (six motifs) but were different in terms of locations (Figure 3A).The same methods were found for the ICE protein, including similar motifs; however, some ICEs had variations, such as JrICE1 that lacked motif9 and JrICE1e that lacked motif6, respectively (Figure 3B).For gene structures, overall, the CBF genes generally had one CDS of long length of one mainly CDS (~600 bp) in three walnut genomes, while there were at least four CDSs (one longer CDS and other shorter CDSs) in walnut ICE genes (Figure 3).In addition, JrCBF1 and JrCBF2 had UTRs of long length, indicating that they play regulatory functions.Although the ICEs had conserved motifs and domains, three ICE genes (JrICE1c, JrICE1b, JreICE1c) contained introns of long length (Figure 3).

Collinearity Analysis and Selective Pressure
Among the walnut genomes, all CBF and ICE genes had collinearity relationships (Figure 4).The collinearity predictions showed that there were eight CBF and eight ICE paralogous gene pairs and 44 CBF and 26 ICE orthologous gene pairs between the three Juglans genomes, respectively (Figures 4 and S1).Among these genes, four CBFs (JreCBF4, JreCBF1, JreCBF5, and JreCBF2) had higher collinearity with other CBF genes (Figure 4 and S1A).For ICEs, the analysis showed that each ICE gene had at least two collinearity relationships with other ICEs, suggesting that all ICE genes were very similar and conserved.Both JsICE1a and JsICEb had three collations with ICE genes of J. regia (Figure S1B).Although there were different members of CBF and ICE groups in the three walnut genomes, these two gene families were highly correlated (Figure 4).To further reveal the selection pressure of the homologous gene pairs of CBFs and ICEs in the three walnut genomes, we determined the Ka/Ks values of these homologous CBF and ICE gene pairs (Table S3).The Ka/Ks ratios of eight homologous gene pairs were more than 1, while the other CBF and ICE homologous gene pairs were less than 1 (Table S3).The Ka/Ks value was more than 1 for eight orthologous gene pairs (JsCBF1 vs. JreCBF5, JrCBF1 vs. JsCBF1, JrCBF1 vs. JreCBF4, JrICE1b vs. JsICE1a, JsICE1a vs. JreICE1c, JrCBF2 vs. JsCBF2, JrICE1b vs. JreICE1b,  [20], J. regia ('Zhongmucha-1') [43], and J. sigillata ('Yangbi') [23], respectively.The Neighbor-Joining (NJ) phylogenetic tree of two genes in three Juglans assemblies is shown on the left.The motifs and protein domains are represented by different colored boxes.Green boxes and gray lines indicated exons and introns, respectively.

Collinearity Analysis and Selective Pressure
Among the walnut genomes, all CBF and ICE genes had collinearity relationships (Figure 4).The collinearity predictions showed that there were eight CBF and eight ICE paralogous gene pairs and 44 CBF and 26 ICE orthologous gene pairs between the three Juglans genomes, respectively (Figures 4 and S1).Among these genes, four CBFs (JreCBF4, JreCBF1, JreCBF5, and JreCBF2) had higher collinearity with other CBF genes (Figures 4  and S1A).For ICEs, the analysis showed that each ICE gene had at least two collinearity relationships with other ICEs, suggesting that all ICE genes were very similar and conserved.Both JsICE1a and JsICEb had three collations with ICE genes of J. regia (Figure S1B).Although there were different members of CBF and ICE groups in the three walnut genomes, these two gene families were highly correlated (Figure 4).To further reveal the selection pressure of the homologous gene pairs of CBFs and ICEs in the three walnut genomes, we determined the Ka/Ks values of these homologous CBF and ICE gene pairs (Table S3).The Ka/Ks ratios of eight homologous gene pairs were more than 1, while the other CBF and ICE homologous gene pairs were less than 1 (Table S3).The Ka/Ks value was more than 1 for eight orthologous gene pairs (JsCBF1 vs. JreCBF5, JrCBF1 vs. JsCBF1, JrCBF1 vs. JreCBF4, JrICE1b vs. JsICE1a, JsICE1a vs. JreICE1c, JrCBF2 vs. JsCBF2, JrICE1b vs. JreICE1b, and JrICE1f vs. JrICE1a), suggesting that they were occurred positively selections and may be experienced a rapidly evolutionary rate.Within the eight gene pairs, only one in the same genome (JrICE1f and JrICE1a).In addition, the Ka/Ks ratios were less than 1, indicating that these gene pairs experienced relatively purify selection during evolution (Table S3).and JrICE1f vs. JrICE1a), suggesting that they were occurred positively selections and may be experienced a rapidly evolutionary rate.Within the eight gene pairs, only one in the same genome (JrICE1f and JrICE1a).In addition, the Ka/Ks ratios were less than 1, indicating that these gene pairs experienced relatively purify selection during evolution (Table S3).[20], J. regia ('Zhongmucha-1') [43], and J. sigillata (iron walnut) [23], respectively.Red lines indicate orthologous gene pairs.

Cis-Acting Elements Analysis of Promoters
To investigate the potential functions of the CBF and ICE genes in the three walnut genome assemblies, we conducted a cis-acting element analysis of promoter regions of these two genes.The cis-acting elements were divided into four major classes, namely those with a light-responsive element, phytohormones responsive, stress resistance, and those associated with plant development and growth (Figure 5).The upstream promoter regions of the main CBF and ICE genes contained cis-acting elements associated with stress resistance, indicating that these CBF and ICE genes may play an essential role in light, abiotic stress, and resistance in the walnuts.In addition, the promoter CBF regions of the 'Zhongmucha-1' walnut genome contained a larger number of cis-acting elements than those of CBFs in the other two walnut genomes ('Chandler' and 'Yangbi'), suggesting that CBFs in walnuts may be involved in genetic variations in the cold stress signaling and pathways.We also found that the ICEs of three walnuts contained a large number of cisacting elements in the promoter regions of LTR and ARE related to stress resistance.Furthermore, most of the CBF and ICE genes contained the cis-acting elements associated with  [20], J. regia ('Zhongmucha-1') [43], and J. sigillata (iron walnut) [23], respectively.Red lines indicate orthologous gene pairs.

Cis-Acting Elements Analysis of Promoters
To investigate the potential functions of the CBF and ICE genes in the three walnut genome assemblies, we conducted a cis-acting element analysis of promoter regions of these two genes.The cis-acting elements were divided into four major classes, namely those with a light-responsive element, phytohormones responsive, stress resistance, and those associated with plant development and growth (Figure 5).The upstream promoter regions of the main CBF and ICE genes contained cis-acting elements associated with stress resistance, indicating that these CBF and ICE genes may play an essential role in light, abiotic stress, and resistance in the walnuts.In addition, the promoter CBF regions of the 'Zhongmucha-1' walnut genome contained a larger number of cis-acting elements than those of CBFs in the other two walnut genomes ('Chandler' and 'Yangbi'), suggesting that CBFs in walnuts may be involved in genetic variations in the cold stress signaling and pathways.We also found that the ICEs of three walnuts contained a large number of cis-acting elements in the promoter regions of LTR and ARE related to stress resistance.Furthermore, most of the CBF and ICE genes contained the cis-acting elements associated with three light responsive, namely, G-box and box4, while namely the ABRE (related to the abscisic acid hormone response), CGTCA-motif and TGACG-motif (related to the methyl jasmonate hormone response) genes associated with phytohormones response were found in all three walnut varieties (Figure 5).three light responsive, namely, G-box and box4, while namely the ABRE (related to the abscisic acid hormone response), CGTCA-motif and TGACG-motif (related to the methyl jasmonate hormone response) genes associated with phytohormones response were found in all three walnut varieties (Figure 5).

Gene Expression Patterns Analysis
To investigate the CBF and ICE expression patterns in four walnut cultivars ('Zijing', 'Lvling', 'Hongren', and 'Liao1') under different temperature conditions, transcriptomic data from leaves under three levels of temperature (8 • C, 22 • C, and 5 • C) were analyzed (Figures 6 and S3; Tables S4-S6).Based on the transcriptome data, we evaluated the DEGs (differentially expressed genes) between the three different temperature conditions (8 • C, 22 • C, and 5 • C) in the four walnut cultivars (Figure S3; Table S4).A total of 18,740 DEGs were identified between the three different temperature conditions, including 465 shared DEGs in three comparison pairs (Figure S4; Table S5).Some of the annotated genes relating to plant growth, development, and stress in those overlapped in 465 DEGs (Table S5), such as four Auxilin-like protein (AUL), two AP2/ERF, three Ethylene-responsive transcription factor (ERF4), three Gibberellin 2-beta-dioxygenase (GA2OX), five E3 ubiquitin-protein ligase (ATL), two F-box protein, four Calcium-binding protein (CML), two Caffeoyl-CoA O-methyltransferase (CCOAOMT), five Cytochrome P450 (CYP), one Dehydrin (COR), four NAC domain-containing protein (NAC), Transcription factor bHLH96, six transcription factor (MYB), four WRKY transcription factor (WRKY), three Omega-3 fatty acid desaturase (FAD), and four Pentatricopeptide repeat-containing protein (PCMP).We performed the statistical analysis of the different expression levels of CBF genes and ICE genes under three different temperature conditions (8 • C, 22 • C, and 5 • C) among four walnut cultivars (Figure S5).The results showed that CBF genes were expressed relatively significantly differently between the walnut cultivars at 22 • C and 5 • C (Figure S5A), while there were no significant differences in the ICE genes (Figure S5B).In addition, we found that the ICE genes were expressed significantly higher than CBF genes under the three different temperature conditions (8 • C, 22 • C, and 5 • C) in the four walnut cultivars (Figure S5C).Similarly, the linear relative regression analysis of the expression levels of the four cultivars was different for the CBFs and ICEs (Figure S5D).We performed an analysis of variance (ANOVA) for the three temperatures and four cultivars.The results showed that there was a significant difference in the expression level among the three temperatures (p = 2.7 × 10 −8 ***), among the four cultivars (p = 0.027 *), and between the temperature and cultivars (p = 0.00045 ***), respectively.For the CBF gene, there was a significantly different expression level among the four cultivars (p = 0.00289 **), but not among the temperatures or between the temperature and cultivars (Table S7).In addition, the ANOVA of the CBF and ICE genes showed that 14 of 28 (50%) were expressed significantly differently among the three temperatures, among the four cultivars, and between the temperature and cultivars, including JreCBF1, JreCBF2, JreCBF4, JreCBF5, JrCBF1, JrCBF2, JrCBF4, JsCBF1, JsCBF2, JreICE1a, JreICE1b, JrICE1a, JrICE1d, and JsICE1b (Table S8).Furthermore, we found that only one of sixteen CBF genes (6.25%) was expressed significantly at 5 • C for the walnut cultivars 'Hongren' and 'Liao1', but the CBF gene was expressed slightly less at 5 • C for the cultivars of 'Zijing' and 'Lvling' (Figure 6B).We only found three ICE genes that were expressed to a lesser degree in all of the walnut samples, while three ICE1 genes (JsICE1b, JrICE1b, and JreICE1d) were overexpressed at 5 • C for all four cultivars (Figures 6  and S5).Based on the three sets of ANOVA results, for the temperatures, there were seven genes (JreCBF4, JreCBF5, JreCBF6, JrCBF2, JrCBF4, JsCBF1, and JrICE1e) were expressed significantly differently among the four cultivars at 8 • C, four genes (JreCBF1, JrCBF1, JsCBF2, and JreICE1e) at 22 • C, and three genes (JrCBF2, JsCBF2, and JreICE1e) at 5 • C, respectively (Table S8).For the cultivars, there were seven genes (JreCBF1, JreCBF4, JreCBF5, JrCBF1, JrCBF2, JsCBF1, and JsCBF2) in 'Liao1' were expressed significantly differently under the three temperature conditions, three DEGs (JreCBF5, JsCBF3, and JreICE1a) in 'Lvling', three DEGs (JrCBF1, JsCBF1, and JreICE1) in 'Hongren', and one DEG (JsCBF5) in 'Zijing' under the three temperature conditions (Table S9).In general, the ICE gene expression was significantly higher than for CBF genes (Figure S5C).All of the statistical analyses of gene expression suggested that most ICE genes regulated the expression in walnut leaves after the temperature decreased, while CBF1 might be the most important transcription factor in walnut in terms of cold stress.Both CBF and ICE genes had different expression profiles in the four walnut cultivars under the three temperature conditions in the same environment, suggesting that different walnut varieties may have different cold stress resistance mechanisms.
In plants, miRNAs play many biological functions by regulating the expression of target genes, such as in clod tolerance [44][45][46].There is no published miRNA database in psRNATarget for walnut species, so we predicted the interaction network between the CBF and ICE proteins in walnuts and the putative miRNA in Arabidopsis for target prediction.All CBFs and ICEs had at least one interaction with miRNA in our analysis.The ICE genes had more putative miRNA interactions (124) compared to CBF (39 miRNAs) in walnuts (Figure 8; Table S11).For the CBF genes, JreCBF7 had the highest number of nine putative miRNA interactions, while JrCBF1 had only one putative miRNA (ath-miR170-3p) interaction.A total of 12 CBF genes had at least three miRNA interactions.For the ICE genes, JrICE1c had the highest number of putative miRNA interactions at 47, while JrICE1a had 9 putative miRNA interactions.All 12 ICE genes had at least 9 miRNA interactions with an average interaction of 23 (Figure 8).On the other hand, ath-miR164b-5p and ath-miR5021 targeted the most CBF genes (JreCBF2, JsCBF3, JrCBF4, and JsCBF4) and ICE genes (JrICE1d,

Evolution of CBF1 and ICE1 in Angiosperms
The CBF1 and ICE1 genes have been identified as major regulatory TFs of cold-related proteins in many angiosperm plants [5][6][7]14,15].To better understand the evolution and variations of CBF1 and ICE1 proteins in the walnut family and other plants, we selected a total of 19 plant genomes, including six Juglans varieties (J.regia, 'Chandler'; J. regia, 'Zhongmucha-1'; J. sigillata, 'Yangbi'; J. nigra, J. microcarpa, and J. mandshurica), three monocotyledons (Oryza sativa, Zea mays, and Triticum aestivum), and another ten plants (Carya cathayensis, Castanea mollissima, Quercus robur, Sesamum indicum, Malus pumila, Populus trichocarpa, Vitis vinifera, Olea europaea, Arabidopsis thaliana, and Theobroma cacao).The phylogenetic tree of CBF1 and ICE1 showed that the species have converged into the same group with close systematic relationships; for instance, all of the CBFs and ICEs of Juglans are clustered into one group, except for JmCBF1 (Figure 9).Three CBFs and three ICEs of the monocotyledons were clustered into one group of more than 85, all CBFs and ICEs of Arabidopsis were clustered into a single group, and the CBF1 and ICE1proteins of Fagales were also clustered into one group in the phylogenetic tree.These results suggest the convergent evolution of CBF1 and ICE1 in angiosperms, which is also supported by the conserved domains in these two proteins.The CBF1 proteins had only one conserved domain, named AP2, while the ICE1 proteins had two conserved domains, named bHLH_At-AMS_like and ACT_UUR-ACR-like, in all nineteen plant genomes.The same patterns of gene structure and motifs were found for CBF1 and ICE1, which suggests that all CBF1 and ICE1 had conserved protein domains, motifs, and structures.However, CmCBF1 had one more domain, named AP2 superfamily, and CmICE1 had one more domain, named PRK13855 superfamily, respectively.In addition, we found that CmCBF1 and JmCBF1 had long introns between two CDSs, which suggests that there is some genetic variation among the CBF genes in plants.In our study, we found some long UTRs in many ICEs from the 19 plant species (Figure 9).The ICE genes were long in length, and the UTRs differed between species.The UTRs were also differently located among the plant species.This phenomenon was also observed in the CBF genes, such as CcCBF1 and OsCBF1 had long UTRs.Furthermore, we found that one long CDS in all ICE1 genes and at similar

Evolution of CBF1 and ICE1 in Angiosperms
The CBF1 and ICE1 genes have been identified as major regulatory TFs of coldrelated proteins in many angiosperm plants [5][6][7]14,15].To better understand the evolution and variations of CBF1 and ICE1 proteins in the walnut family and other plants, we selected a total of 19 plant genomes, including six Juglans varieties (J.regia, 'Chandler'; J. regia, 'Zhongmucha-1'; J. sigillata, 'Yangbi'; J. nigra, J. microcarpa, and J. mandshurica), three monocotyledons (Oryza sativa, Zea mays, and Triticum aestivum), and another ten plants (Carya cathayensis, Castanea mollissima, Quercus robur, Sesamum indicum, Malus pumila, Populus trichocarpa, Vitis vinifera, Olea europaea, Arabidopsis thaliana, and Theobroma cacao).The phylogenetic tree of CBF1 and ICE1 showed that the species have converged into the same group with close systematic relationships; for instance, all of the CBFs and ICEs of Juglans are clustered into one group, except for JmCBF1 (Figure 9).Three CBFs and three ICEs of the monocotyledons were clustered into one group of more than 85, all CBFs and ICEs of Arabidopsis were clustered into a single group, and the CBF1 and ICE1proteins of Fagales were also clustered into one group in the phylogenetic tree.These results suggest the convergent evolution of CBF1 and ICE1 in angiosperms, which is also supported by the conserved domains in these two proteins.The CBF1 proteins had only one conserved domain, named AP2, while the ICE1 proteins had two conserved domains, named bHLH_AtAMS_like and ACT_UUR-ACR-like, in all nineteen plant genomes.The same patterns of gene structure and motifs were found for CBF1 and ICE1, which suggests that all CBF1 and ICE1 had conserved protein domains, motifs, and structures.However, CmCBF1 had one more domain, named AP2 superfamily, and CmICE1 had one more domain, named PRK13855 superfamily, respectively.In addition, we found that CmCBF1 and JmCBF1 had long introns between two CDSs, which suggests that there is some genetic variation among the CBF genes in plants.In our study, we found some long UTRs in many ICEs from the 19 plant species (Figure 9).The ICE genes were long in length, and the UTRs differed between species.The UTRs were also differently located among the plant species.This phenomenon was also observed in the CBF genes, such as CcCBF1 and OsCBF1 had long UTRs.Furthermore, we found that one long CDS in all ICE1 genes and at similar locations in all of the plant species, while shorter CDSs were distributed differently among the species (Figure 9).locations in all of the plant species, while shorter CDSs were distributed differently among the species (Figure 9).

Discussion
The C-repeat binding factors (CBFs) and ICE transcription factors are well known to play essential roles in plant development, growth, and stress responses, particularly in regulating cold and chilling responses in plants [1][2][3]47].They are important candidate genes for abiotic stress, such as in the cold acclimation processes [47], while 12 CBFs were found to be responsive to low temperature (4 °C) in lettuce [44], LpCBFs were expressed significantly under cold treatment conditions in Lolium perenne [48], and GthICE2 has been shown to respond to cold and drought stress [18].Gene function experiments have been carried out in model plants; such as AtCBF1 has been found to be involved in response to low temperatures and abscisic acid in A. thaliana [49,50], OsCBF3 is responsible for lowtemperatures-induced expression in rice [51], and SlCBF1 enhances the resistance to cold tolerance and water deficit stress in tomato (Solanum lycopersicum) and Brassica napus [52].Thus, a genome-wide investigation of the CBF and ICE gene family will be useful to elucidate plant cold stress.However, there is little information available about the CBF and ICE gene family information in the economic perennial tree, walnut.In this study, the two most important two TFs gene families of CBF and ICE were identified and analyzed in three walnut genomes using bioinformatic methods, including the examination of phylogenetic trees, gene chromosome locations, gene structure, motifs, conserved domains, expression levels, and protein interaction networks, as well as evolutionary relationships.

Characteristics of CBF and ICE1 in Three Walnut Genomes
Walnut is an important food nut and oil tree species that is widely cultivated across the world for its valuable nuts and wood [20][21][22].It is cultivated extensively for nut and wood production [53].J. regia is widely grown in diverse temperature regions of Asia, Europe, America, Australia, South Africa, and New Zealand [53].As its sister taxon, J. sigillata (iron walnut), is only grown in the southwest of China, these two walnuts are growing sympatric and exhibit strong gene introgressions [23][24][25].A rapid change in the climate can damage the buds, flowers, young stems, and even the entire plant [31][32][33].Climate change also affects the growth of tree species in their distribution region, and in particular, low temperatures affect plant development and growth [23,30].The

Discussion
The C-repeat binding factors (CBFs) and ICE transcription factors are well known to play essential roles in plant development, growth, and stress responses, particularly in regulating cold and chilling responses in plants [1][2][3]47].They are important candidate genes for abiotic stress, such as in the cold acclimation processes [47], while 12 CBFs were found to be responsive to low temperature (4 • C) in lettuce [44], LpCBFs were expressed significantly under cold treatment conditions in Lolium perenne [48], and GthICE2 has been shown to respond to cold and drought stress [18].Gene function experiments have been carried out in model plants; such as AtCBF1 has been found to be involved in response to low temperatures and abscisic acid in A. thaliana [49,50], OsCBF3 is responsible for lowtemperatures-induced expression in rice [51], and SlCBF1 enhances the resistance to cold tolerance and water deficit stress in tomato (Solanum lycopersicum) and Brassica napus [52].Thus, a genome-wide investigation of the CBF and ICE gene family will be useful to elucidate plant cold stress.However, there is little information available about the CBF and ICE gene family information in the economic perennial tree, walnut.In this study, the two most important two TFs gene families of CBF and ICE were identified and analyzed in three walnut genomes using bioinformatic methods, including the examination of phylogenetic trees, gene chromosome locations, gene structure, motifs, conserved domains, expression levels, and protein interaction networks, as well as evolutionary relationships.

Characteristics of CBF and ICE1 in Three Walnut Genomes
Walnut is an important food nut and oil tree species that is widely cultivated across the world for its valuable nuts and wood [20][21][22].It is cultivated extensively for nut and wood production [53].J. regia is widely grown in diverse temperature regions of Asia, Europe, America, Australia, South Africa, and New Zealand [53].As its sister taxon, J. sigillata (iron walnut), is only grown in the southwest of China, these two walnuts are growing sympatric and exhibit strong gene introgressions [23][24][25].A rapid change in the climate can damage the buds, flowers, young stems, and even the entire plant [31][32][33].Climate change also affects the growth of tree species in their distribution region, and in particular, low temperatures affect plant development and growth [23,30].The transcription factors CBF and ICE have evolved into an important aspect of functional genomics research; thus, the genome-wide identification and gene functional research of the essential TFs in woody plants becomes increasingly more significant for genome sequencing and technology development [20][21][22][23][24][25].Here, we identified a total of 16 CBFs and 12 ICEs in three walnut genomes.All putative CBF genes were divided into three groups, while the ICE genes were clustered into four groups.The number of CBF and ICE genes varied in the three walnut varieties, indicating the diversity of these three walnut genomes.Among the CBF and ICE genes in the three walnuts, most of the genes (25/28, 89.3%) exhibited whole genome duplication events (WGDs), while JrCBF6 exhibited tandem duplication and JrICE1e and JreICE1 showed dispersed duplication.The members of the CBF and ICE gene families are associated with the size of the genome and the effects of plant evolution [28].This is in contrast to the number of CBF family members in annual plants; for example, there are 4 in A. thaliana, 12 in Lolium perenne [48], 14 in lettuce [47], 32 in the biennial herb Brassica oleracea [54], and 24 in the perennial herbs Taraxacum kok [55].In the perennial woody plants, there are some reports of the presence of CBF members in whole genomes; for instance, in three Acer species, a total of five, four, and seven CBFs were identified in Acer truncatum, Acer pseudosieboldianum, and Acer yangbiense, respectively [8].There were few reports of ICE genes in the genome-wide identification of plants.The use of domain query revealed that there were five, four, six, seven, eight, seven, eight, and eight genes coding the ICE domain in cotton (Gossypium) of G. arboteum, G. raimondii, G. thurberi, G. hirsutum, G. barbadense, G. tomentosum, G. mustelinum, and G. darwinii, respectively [10].Differences in the number of CBFs and ICEs among the angiosperms can be explained by gene duplication, genetic diversity, and evolution.Differences in the molecular weight (16.68-90.91kDa), number of amino acids (150-648), and isoelectric point (4.98-10.00) of walnut CBFs and ICEs indicate putative differences in the walnut genomes.All CBF and ICE TFs are primarily localized to the nucleus according to the subcellular localization prediction analysis, and similar results have been reported in other plants [8][9][10].

Conserved Domain and Cis-Acting Elements of CBF and ICE1 in Three Walnut Genomes
The previous studies showed that the conserved domains in transcription factors (TFs) might be related to gene functions, and the cis-acting elements play roles in the regulation of genes and post-transcriptional modification; similar results have also been found in other plant studies [28,37,39].All CBF proteins contained only one domain, AP2, indicating that they play key roles in plant growth, development, and stress, which is consistent with the previous studies [54,55].The AP2 domain is presented specifically in plants but not animals and important TFs belong to the superfamily AP2/ERF [28,[56][57][58][59].We found the consistent and conserved CBF proteins in three walnut genomes, which have also been found in other plants [9][10][11][12].The similar pattern and conserved domains of CBF and ICE proteins in different plant species suggest that it is useful for CBFs and ICEs to be identified as diverse in terms of plant evolution and environmental adaption, which further demonstrates the essential nature of these two families for plant growth, development, and stresses response [16][17][18]60].Although the identified CBF and ICE protein domains and motifs were conserved, their gene structure was different in the three walnut genomes, particularly ICEs (Figure 2).The CBF genes were structurally conserved in Arabidopsis and other plants (with one conserved exon), and the length and sequence of the CDS were also highly conserved, while a few species had two exons, such as Castanea mollissima and Juglans mandshurica [28].For the ICE genes, there was high diversity in the gene structure in Juglans species and other plants (Figure 9).In the present study, we found that the number of exons in many ICEs of the 19 plant species was greater than 3, and all members had long exons (Figure 9).The ICE genes had long lengths and conserved exons between species, and the exons were also differently located among the plant species, which suggests that ICE genes play important roles and varieties during plant evolution and keep the conserved domains for their functions [61,62].This phenomenon was not observed in the CBF genes of other plant species, so it might be unique to ICE genes.Furthermore, we found that the introns were very short in the CBF1 genes but very long introns in the ICE1 genes, which suggests that they have different benefits and regulation mechanisms in plants [63].Both UTR and introns could lead to an increase in protein diversity and are involved in gene expressions [63] Cis-acting elements are well-known participants in the control of gene expression [37,38,48].In our study, many cis-acting elements associated with responsiveness to light and phytohormones were identified in the upstream CBF and ICE promoter regions (Figure 4), which suggests that both CBF and ICE proteins might be associated with photosynthetic and hormone responses and regulations.As a light-preferring tree species, walnuts mainly grow in temperate forest areas [21][22][23].Light is an important element that regulates plant growth, development, and stress.Many studies have shown that light can affect the cold stress in plants via CBF and ICE genes [2][3][4][5][6].Plant hormones are well known to regulate plant stresses, and the hormones play very important roles in plant adaptions and growth in diverse environments [64,65].Thus, these results revealed that CBF and ICE might play a critical role in the stress response of walnuts.In addition, we found some cis-acting elements associated with stress resistance, suggesting that CBF and ICE genes are also involved in some kinds of stress responses.

Gene Expression Profiles of CBF and ICE1 in Response to Cold Stress
Currently, cold stress has become one of the essential factors limiting the flower and fruit productivity of walnuts, and the exploration of important salt and drought tolerance genes is key for the advancement of local walnut adaption breeding programs [1][2][3]20,[27][28][29][30].To reveal the CBF and ICE gene expression profiles, we detected the RNA sequencing data to calculate the FPKM values in different cold conditions and tissues.Most CBF and ICE genes (9 out of 10) were expressed more highly in the leaves compared to the flowers, hulls, and other tissues; however, five out of six ICE genes (JrICE1a, JrICE1b, JrICE1c, JrICE1d, and JrICE1e) were highly expressed in J. regia ('Chandler') female flowers, while all four CBF genes were less expressed in female flowers (Figure S2).Two ICE genes, JrICE1d and JrICE1e, were expressed highly in all tissues.These results indicated that both CBF and ICE genes play specific roles in different tissues and organs in walnuts.Among these CBF and ICE genes, JreCBF4, JrCBF1, JsCBF1, JreICE1e, JsICE1a, and JrICE1b were expressed highly in leaves at 5 • C after the temperature decreased quickly in two walnut cultivars, 'Hongren' and 'Liao', which suggests that both CBF and ICE played an important role during rapidly changes in the temperature in spring (Figure 6).In addition, four walnut varieties had different gene expression patterns of CBF genes and ICE genes under three temperature conditions (Figures 5 and S5, Tables S6 and S9), suggesting that walnut species have experienced diverse environmental adaption through natural selection and domestication [20][21][22].This is possibly related to the temperate climate of the natural habitat of iron walnuts in southwest China, mainly in Yunnan province [23,24].Walnut species are distributed and cultivated in widely diverse ecologically regions in different climate areas in the world [20][21][22][25][26][27].Previous reports showed that CBFs activate many downstream genes, and ICE1 acts upstream to regulate CBFs, which increases cold stress in plants [66].Our analysis of CBF and ICE gene expression patterns under rapid temperature changes in the leaves of four walnut cultivars also suggested that CBF and ICE genes might play critical roles in the cold stress response of J. regia and J. sigillata (Figure 6), a finding that is also supported by those from other woody plant species [11,13,23].Due to the potential role that the identified CBF genes and ICE genes play in walnut varieties with increased cold tolerance, CBFs and ICEs will be candidate genes to improve the resilience of walnut and woody crops to climate change.

Protein-Protein and Protein Interactions, and the Putative microRNA Interaction Predictions
In this study, we investigated the interactions between CBF and ICE proteins and other proteins (Figure 7).Our results showed that there were a total of eleven shared interaction proteins for both CBF and ICE proteins, including CAMTA3, FAMA, HOS1, MYB15, SCE1, SIZ1, SPCH, SRK2E, SUMO1, TIFY10A, and TIFY10A.The proteins had potential functions related to cold tolerance.The Arabidopsis HOS-1 mutation regulated the genes RD29A, COR47, COR15A, KIN1, and ADH (ALCOHOL DEHYDROGENASE), and it mediated the FLC (FLOWERING LOCUS) and degradation of ICE1 as an important negative regulator of cold signal transduction in plant cells [67][68][69].The important transcription factor MYB15 positively regulated cold stress by activating the gene expression of CBFs (CBF1, CBF2, and CBF3), targeted by Syl-miR156e-3p and ABA-mediation in tomato, but MYB15 interacts with the ICE1 and negatively expression of CBFs in Arabidopsis [70][71][72][73].In addition, PUB25 (PLANT U-BOX) and PUB26 negatively regulate MYB15 and positively regulate CBF expression in Arabidopsis [72,73].SIZ1 is a small ubiquitin-related modifier (SUMO) E3 ligase that mediates ICE1 and CBF3 to enhance cold tolerance in Arabidopsis [74].Within the unique interaction proteins in CBFs and ICEs, there were 7 and 16 proteins relative to cold response, respectively (Table S10).The AP2/ERF is an important transcript factor that positively enhances cold tolerance in plants, has been identified in Juglans mandshurica [28], and positively regulates cold stress in Arabidopsis, rice, and grape [75,76].These shared and unique interactions among proteins will be useful resources and could be very important candidates in walnut cold tolerance studies.
MicroRNAs (miRNAs) play an important role in regulating target gene expression and are key regulators in plant development and stress responses [45].In woody species, some miRNAs have been identified, but little is known about these in walnuts.We found that a total of 14 miRNAs interacted with both CBFs and ICEs.Both ath-miR472-3p and ath-miR5024-3p have been reported to respond to cold tolerance by regulating their target genes in wheat and Arabidopsis [77,78].A total of four miRNAs are related to salt, and four miRNAs are related to drought tolerance, suggesting that miRNAs play important roles in plant responses to abiotic stresses.Low temperature is a major environmental factor affecting the productivity of woody plants.These findings related to CBFs and ICEs interaction proteins and miRNAs indicate that protein-protein, and protein-microRNA interaction predictions could provide useful analysis and gene function studies in walnut cold tolerance breeding.

Plant Materials and RNA Sequencing
In this study, we used four cultivars of Persian walnut (Juglans regia), including 'Zijing', 'Lvling', 'Hongren', and 'Liao1', respectively.All trees were grown in Xi'an Botanical Garden, Shaanxi Province, China.We collected the leaf samples from four cultivars after a rapid decrease in the temperature in cold late spring.In April 2023, we collected the plant materials fresh leaves from four cultivars under three low temperatures: 8 • C (4 April), 22 • C (9 April), and 5 • C (23 April), respectively (Figure S5A; Table S2).We collected three biological duplications for each temperature condition and each cultivar.Then, the tissue samples were flash-frozen in liquid nitrogen and stored at −80 • C prior to utilization.The total RNA was extracted to analyze further the genes that responded to the rapid decrease in temperature.All thirty-six leaf samples were sequenced using the Illumina HiSeq X Ten platform (Illumina, San Diego, CA, USA).All of the transcriptome clean reads were then mapped to the Juglans regia ('Chandler') reference genome using HISAT version 2.2.0 [20,79] with default parameters.The numbers of fragments per kb of transcript sequence per million bp sequenced (FPKM) were used to calculate the gene expression levels [80].We identified the DEGs (differentially expressed genes) between the three temperature conditions (8 • C, 22 • C, and 5 • C) and combined the four walnut cultivars ('Zijing', 'Lvling', 'Hongren', and 'Liao1') using DESeq2 (https://support.bioconductor.org/tag/DeSeq2/,accessed on 2 May 2023) [81,82] with fold change (FC) > 1 and FDR < 0.05.The volcano plot was employed to show the different expression patterns of upregulated and downregulated genes using the R package ggplot2 [83,84].We analyzed the shared and unique DEGs between the three temperatures using the online program jvenn (https://jvenn.toulouse.inrae.fr/app/index.html,accessed on 2 December 2023) [85].We further annotated the overlapped DEGs using the Juglans regia ('Chandler') reference genome [20].

Construction of Phylogenetic Tree and Analysis of Gene Structure, Conserved Motif
The phylogenetic relationships among the CBF and ICE gene families were analyzed using the software MEGA version 7.0 using the Neighbor-Joining (NJ) tree with a bootstrap of 1000.Then, the Neighbor-Joining tree was embellished using the online tool iTOL (https://itol.embl.de/,accessed on 12 April 2023).Based on the CBF protein sequences belonging to Arabidopsis thaliana, Juglans regia, J. nigra, J. mandshurica, and Cyclocarya paliurus, we performed gene structure analysis using the website GSDS (http://gsds.gao-lab.org/,accessed on 12 July 2023).To visualize the candidate genes, the conserved motifs were predicted with a maximum of 10 motifs using the MEME program and the software TBtools version 2.008 [88].The gene domain was explored using Batch CD-Search in NCBI (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi,accessed on 12 July 2023).Additionally, the detected domain was visualized using software TBtools version 2.008 [88].

Chromosomal Location, Collinearity of Transcription Factors, and Selective Pressure Analysis in Juglans Regia
To identify the location of the genes on each chromosome, the target gene annotation information was retrieved from the Juglans regia whole genome annotation files.According to the physical position of the candidate CBF and ICE genes, the chromosomal locations of the CBF genes and ICE genes of J. regia ('Chandler'), J. regia ('Zhongmucha-1') and J. sigillata ('Yangbi') were determined using the software TBtools version 2.008 [88].The collinearity and duplication analyses were performed using McScanX software (https: //github.com/wyp1125/MCScanx,accessed on 12 May 2023) [89].All of the results above were visualized using the software TBtools version 2.008 [88].In addition, we calculated the Ka/Ks values to estimate the selective pressure among the identified gene pairs using KAKS_CALCULATOR version 2.0 software [90].The values of nonsynonymous (Ka), synonymous (Ks), and Ka/Ks were estimated in each CBF and ICE gene pair in three walnut genomes using the method of Yang and Nielsen (YN) [91].Following the screening, several gene pairs were not listed if either the Ka or Ks values were unavailable for analysis.The Ka/Ks values more than 1 and less than 1 indicate positive selection and negative selection, respectively.

Analysis of Subcellular Localization and Protein Physicochemical Properties
The physicochemical properties of identified candidates were predicted by the online tool Expasy (https://www.expasy.org/,accessed on 13 July 2023).The subcellular localization of all genes was predicted using Protein Subcellular Localization Prediction: WoLF PSORT tools (https://wolfpsort.hgc.jp/,accessed on 12 July 2023).

Cis-Acting Elements Analysis of Promoter and Gene Expression
Based on the upstream 2000 bp sequences of the candidates extracted from the wholegenome files, the promoter sequences were uploaded to the online website PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/,accessed on 12 July 2023).For the transcriptome gene expression analysis, we first filtered all RNA raw sequencing data using the software FASTP (https://github.com/OpenGene/fastp,accessed on 1 May 2023) [92].We utilized FASTP to ensure quality control and perform adapter trimming, quality filtering (limit the percentage of low-quality bases, default 40%), and per-read quality pruning, among other operations, to attain clean data from raw data for further analysis [92], and then the clean reads data were mapped to the reference genome of J. regia ('Chandler') using the software HISAT version 2.2.0 [79].Then, the gene expression level was calculated using the FPKM values via the program featureCounts [84].Subsequently, a heatmap was displayed using the software TBtools version 2.008 [88].For the statistical analysis of the expression levels of CBFs and ICEs in the four walnut cultivars under three temperatures, we first performed the Bartlett test of expressions for these studied genes.Then, the ANOVA and Kruskal-Wallis test were employed to compare the differences amongst the different cultivar groups and temperature conditions, respectively.All statistical analysis was conducted using R version 4.2.3 software.The MANOVA (multivariate analysis of variance) and MRA (multiple regression analysis) were executed using R software to estimate the influence of temperature and cultivar on the gene expression level, respectively.

Protein-Protein Interactions and microRNA Targeting Analysis
We annotated all CBF and ICE protein sequences using the eggnog-mapper tool (http://eggnog-mapper.embl.de/(accessed on 31 October 2023) [93].The CBF and ICE protein sequences were uploaded to the STRING online website (https://cn.string-db.org,accessed on 31 October 2023) to analyze the protein-protein interaction predictions using default parameters [94].Since there is no Juglans proteins-proteins and miRNAs interaction database, we mapped the representative CBF and ICE protein sequences with the A. thaliana genome using default parameters [93,95].We analyzed the PPI prediction in STRING (https://cn.string-db.org,accessed on 31 October 2023) and performed the CBF and ICE genes with the putative miRNA interactions.We submitted all identified CBF and ICE proteins as candidates to predict the putative targeting of Arabidopsis miRNAs using the online program psRNATarget using default parameters [94].Both proteinprotein interactions and protein-microRNA interactions were visualized using the software Cytoscape version 3.10.0with default parameters [96].We analyzed the shared and unique proteins and miRNAs between the CBFs and ICEs using the online program jvenn (https: //jvenn.toulouse.inrae.fr/app/index.html,accessed on 2 December 2023) [85].We searched for the potential gene functions of all 14 overlapping interactive miRNAs on the NCBI website.We further annotated the overlapped proteins using the Juglans regia ('Chandler') reference genome [20].

Conclusions
In the present study, we identified a total of sixteen CBF and eleven ICE genes in three walnut genomes.The phylogenetic tree analysis showed that the CBF genes could be divided into three groups and that the ICE genes could be clustered into four groups, respectively.The phylogenetic, synchronic, and collinearity analyses indicated that both the CBF and ICE gene families were evolutionarily conservative.The CBFs had only one conserved domain, AP2, while the ICEs had bHLH and ACT conserved domains.Although the CBF genes were not evenly located across the chromosomes in the three walnut genomes, they exhibited consistently high collinearity, which revealed that the CBFs were relatively conserved proteins.Most of the ICE genes were distributed at the ends of the chromosomes in the three walnut genomes.The transcriptome expression profile analysis indicated that the ICE genes were expressed more highly than the CBF genes under cold conditions in four walnut cultivars ('Zijing', 'Lvling', 'Hongren', and 'Liao1'), indicating that CBF1 and ICE1 have important functions under rapidly temperature changes in walnut leaves.

Figure 5 .
Figure 5. Cis-acting element analysis in promoter regions of CBF and ICE1 genes in two to three walnut genomes.The cis-acting elements are shown on the top of the functional annotations, and they are divided into four major categories: light responsiveness cis-acting elements, phytohormone responsiveness, stress resistance, and plant development and growth.The gene names are provided on the left, and the number of cis-acting elements is given in the colored green box.

Figure 5 .
Figure 5. Cis-acting element analysis in promoter regions of CBF and ICE1 genes in two to three walnut genomes.The cis-acting elements are shown on the top of the functional annotations, and they are divided into four major categories: light responsiveness cis-acting elements, phytohormone responsiveness, stress resistance, and plant development and growth.The gene names are provided on the left, and the number of cis-acting elements is given in the colored green box.

Figure 7 .
Figure 7. Protein-protein interaction networks of CBF and ICE with other proteins.The color line indicates protein interactions.The darker color indicate that the more numbers of protein-protein interaction relationships.

Figure 7 .
Figure 7. Protein-protein interaction networks of CBF and ICE with other proteins.The color line indicates protein interactions.The darker color indicate that the more numbers of protein-protein interaction relationships.

Figure 8 .
Figure 8. Representation of the regulatory networks between the targeted CBF (left) and ICE (right) genes and putative miRNAs.The bigger circles represent proteins, and the small circles represent miRNAs.The colored line indicates interaction relationships.

Figure 8 .
Figure 8. Representation of the regulatory networks between the targeted CBF (left) and ICE (right) genes and putative miRNAs.The bigger circles represent proteins, and the small circles represent miRNAs.The colored line indicates interaction relationships.

Figure 9 .
Figure 9. Phylogenetic analysis, gene structure, conserved domains, and motif of CBF1 and ICE1 in 19 angiosperms.The numbers on the NJ tree indicate that the bootstrap rate.

Figure 9 .
Figure 9. Phylogenetic analysis, gene structure, conserved domains, and motif of CBF1 and ICE1 in 19 angiosperms.The numbers on the NJ tree indicate that the bootstrap rate.