Genome-Wide Analysis of DREB Family Genes and Characterization of Cold Stress Responses in the Woody Plant Prunus nana

Dehydration response element binding factor (DREB) is a family of plant-specific transcription factors, whose members participate in the regulation of plant responses to various abiotic stresses. Prunus nana, also known as the wild almond, is a member of the Rosaceae family that is rare and found to grow in the wild in China. These wild almond trees are found in hilly regions in northern Xinjiang, and exhibit greater drought and cold stress resistance than cultivated almond varieties. However, the response of P. nana DREBs (PnaDREBs) under low temperature stress is still unclear. In this study, 46 DREB genes were identified in the wild almond genome, with this number being slightly lower than that in the sweet almond (Prunus dulcis cultivar ‘Nonpareil’). These DREB genes in wild almond were separated into two classes. All PnaDREB genes were located on six chromosomes. PnaDREB proteins that were classified in the same groups contained specific shared motifs, and promoter analyses revealed that PnaDREB genes harbored a range of stress-responsive elements associated with drought, low-temperature stress, light responsivity, and hormone-responsive cis-regulatory elements within their promoter regions. MicroRNA target site prediction analyses also suggested that 79 miRNAs may regulate the expression of 40 of these PnaDREB genes, with PnaDREB2. To examine if these identified PnaDREB genes responded to low temperature stress, 15 of these genes were selected including seven homologous to Arabidopsis C-repeat binding factor (CBFs), and their expression was assessed following incubation for 2 h at 25 °C, 5 °C, 0 °C, −5 °C, or −10 °C. In summary, this analysis provides an overview of the P. nana PnaDREB gene family and provides a foundation for further studies of the ability of different PnaDREB genes to regulate cold stress responses in almond plants.


Introduction
Plant growth can be profoundly regulated by adverse environmental factors including drought and cold stress [1,2]. Adverse environmental conditions drive plants to upregulate specific TFs and other genes that help mitigate these stressors or their impacts on the host plant. The AP2/ERF (APETALA2/ethylene response factor) superfamily is the largest group of TFs in plants, regulating their development and ability to tolerate a wide array of abiotic and biotic stressors [3,4]. According to amino acid sequence similarity and the number of conserved domains, the AP2/ERF family is classified into AP2, ERF, DREB, RAV, and five other subgroups. The DREB and ERF subgroups only encode one AP2 conserved domain [5][6][7][8]. Dehydration response element binding factor (DREB) proteins are members of the ERF subfamily [9], each of which harbors conserved AP2/ERF domains that bind to the dehydration-responsive element (DRE) regions of the DNA with a core motif of A/GCC GAC [10][11][12]. The domain is composed of 60~70 amino acids, and the encoded protein is mainly composed of α-spiral, and β-folding is primary [13,14]. DREB associated with cold tolerance in this species. Therefore, understanding the cold response mechanism of P. nana is an important way to develop the P. nana industrial chain, screen high-quality genotypes of cold tolerant plants, and breeding.
In this paper, we excavated 46 DREB genes from the P. nana genome. Their conserved motifs, intron/exon structures, domains, interaction analyses, and cis-acting regulatory elements were systematically analyzed. Meanwhile, their evolutionary relationships with dicotyledonous A. thaliana, P. dulcis cultivar 'Nonpareil', P. avium, P. mume, P. persica, and P. armeniaca were compared, and further analyses were performed to gauge their possible roles in the regulation of environmental stress responses. Overall, these results provide a basis for further studies of the role of DREB family genes in the genetic improvement of stress resistance in cultivated almonds, and aimed to provide convenience for further analysis of the functions of the DREB genes in P. nana.

DREB Family Transcription Factor Identification
A combined search strategy was employed to detect DREB transcription factors within the P. nana genome. Initially, a hidden Markov model (HMM) file corresponding to AP2 (PF00847) was utilized as a query to search the genomes of cultivated and wild almond plants, with the later dataset having been unpublished. Of the identified proteins harboring AP2/ERF domains, proteins in the DREB subfamily were selected based upon the detection of conserved amino acids at positions 14 and 19 within this AP2/ERF domain. These protein sequences were further assessed to validate these AP2/ERF domains through an NCBI CD-search query [34]. DREB protein classification was achieved in other plants (A. thaliana, P. dulcis cultivar 'Nonpareil', P. avium, P. mume, P. persica, and P. armeniaca) by using this same approach with P. nana. After which, MEGA6 was used to construct a maximum likelihood (ML) phylogenetic tree based on full-length DREB protein sequences that had been aligned using Clustalx2.1, with 500 bootstrap replicates.
Duplicate PnaDREB sequences were identified by collinearity analysis with MCScanX software [35]. Nonsynonymous (Ka) and synonymous (Ks) PnaDREB sequence substitutions were estimated with DnaSP in order to predict the evolutionary strain (Ka/Ks) and divergence time [36].

Plant Cultivation and Cold Stress Treatment
A total of 15 genes were selected for the plant culture and cold stress treatment experiments, of which PnaDREB22, PnaDREB31, PnaDREB36, PnaDREB38, PnaDREB40, PnaDREB41, PnaDREB42, and PnaDREB43 were homologous genes to CBFs, while PnaDREB1, PnaDREB3, PnaDREB9, PnaDREB12, PnaDREB16, PnaDREB18, PnaDREB19, and PnaDREB27 were selected as low temperature homeopathic response elements. Wild (P. dulcis) and sweet almond (P. dulcis cultivar 'Nonpareil') seeds were treated for 24 h with a solution containing gibberellin (200 mM) prior to planting in a culture chamber. Seeds were then cultivated under consistent conditions (22 • C, 16 h light/8 h dark). Gene expression changes in response to different levels of cold stress were evaluated by transferring 2-month-old P. nana seedlings into incubators and then cooling them at a rate of 3 • /h to the final target temperature (25 • C, 5 • C, 0 • C, −5 • C, and −10 • C), which was held for 2 h. Leaves were then harvested from these seedlings, snap-frozen with liquid nitrogen, and stored at −80 • C.

qPCR Analyses
Total RNA was extracted from stored leaf samples with the Plant RNA Extraction Kit (Tiangen, Beijing, China) based on provided directions, after which a TIANScript First Strand cDNA Synthesis Kit (Tiangen, Beijing, China) was used to prepare cDNA. A CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and SYBR Premix ExTaq (TaKaRa, Dalian, China) were then utilized for qPCR analyses using appropriate primers constructed with the RealTime qPCR Assay tool (https://sg.idtdna.com/scitools/ Applications/RealTimePCR/ accessed on 6 December 2022). PdPP2A genes served as the internal reference controls, and primers used for this study are compiled in Table S1. The 2∆∆Ct method was used to assess the relative gene expression, and samples were analyzed in triplicate. Results were compared with one-way ANOVAs.

Identification of DREB TF Genes
Whole-genome sequencing results from wild almond and sweet almond plants were used to identify DREB TF-encoding genes based on the presence of a conserved AP2/ERF domain, with the protein sequences for the identified DREBs being shown in Supplementary Materials. In total, 46 and 51 DREB genes were identified in the wild almond and sweet almond genomes, respectively. While these genes were distributed across all six chromosomes in wild almond, they were not present on chromosomes 4 or 7 in sweet almond. These genes were designated PnaDREB1-PnaDREB46 according to their chromosomal locations (Figure 1), which were chromosomes 1, 2, 3, 5, 6, and 8, respectively, containing 12 (26.1%), 5 (10.9%), 4 (8.7%), 6 (13.0%), 6 (13.0%), and 13 (28.3%) PnaDREB genes, some of which were present in clusters. These uneven PnaDREB chromosomal distribution patterns highlight the complexity and diversity of this DREB gene family.

PnaDREB Gene Phylogenetic Analyses
Phylogenetic relationships among the PnaDREB TF family members and the potential functional characteristics of these proteins were next explored by constructing a phylogenetic tree based upon the full-length DREB protein sequences from A. thaliana (54 sequences), wild P. nana (46 sequences), P. dulcis cultivar 'Nonpareil' (51 sequences), P. avium (57 sequences), P. mume (49 sequences), P. persica (53 sequences), and P. armeniaca (50 sequences). These DREB proteins exhibited high levels of sequence diversity, and were separated into two broad groups (A and B) as well as five subgroups (Aα, Aβ, Aγ, Bα, Bβ) ( Figure 2). Clear differences in the phylogenetic distributions of DREBs from different species were observed, with, for example, 15, five, and six DREBs from A. thaliana, sweet almond, and wild almond plants, respectively, being assigned to the Aβ group. Phylogenetic analyses also revealed a high degree of DREB sequence similarity between the sweet and wild almond plants, as confirmed through sequence alignment and collinearity analysis (Tables S2 and S3, Figure 2). The nonsynonymous to synonymous mutation rate (Ka/Ks) was assessed as a metric for selective pressure, driving the evolution of this DREB gene family (Table S3). For 38 orthologous gene pairs identified when comparing the sweet almond and wild almond genomes, the Ka/Ks of most gene pairs was <1, while the Ka/Ks for PnaDREB12-XP_034226506.1 was >1, suggesting that this gene evolved under purifying selection following the divergence between wild and sweet almond plants, consistent with the potential adaptive evolution of XP_034226506.1. These 46 PnaDREBs exhibited predicted sizes ranging from 166 (PnaDREB15) to 1273 (PnaDREB12) amino acids, with an average molecular weight of 32.14 kDa. The isoelectric point (pI) values for these PnaDREBs also varied widely from 4.57 (PnaDREB18) to 9.39 (PnaDREB5), and 20 and 27 of these PnaDREBs, respectively, exhibited cytosolic and nuclear localization patterns (Table S1).

PnaDREB Gene Phylogenetic Analyses
Phylogenetic relationships among the PnaDREB TF family members and the potential functional characteristics of these proteins were next explored by constructing a phylogenetic tree based upon the full-length DREB protein sequences from A. thaliana (54 sequences), wild P. nana (46 sequences), P. dulcis cultivar 'Nonpareil' (51 sequences), P. avium (57 sequences), P. mume (49 sequences), P. persica (53 sequences), and P. armeniaca (50 sequences). These DREB proteins exhibited high levels of sequence diversity, and were separated into two broad groups (A and B) as well as five subgroups (Aα, Aβ, Aγ, Bα, Bβ) ( Figure 2). Clear differences in the phylogenetic distributions of DREBs from different species were observed, with, for example, 15, five, and six DREBs from A. thaliana, sweet almond, and wild almond plants, respectively, being assigned to the Aβ group. Phylogenetic analyses also revealed a high degree of DREB sequence similarity between the sweet and wild almond plants, as confirmed through sequence alignment and collinearity analysis (Tables S2 and S3, Figure 2). The nonsynonymous to synonymous mutation rate (Ka/Ks) was assessed as a metric for selective pressure, driving the evolution of this DREB gene family (Table S3). For 38 orthologous gene pairs identified when comparing the sweet almond and wild almond genomes, the Ka/Ks of most gene pairs was <1, while the Ka/Ks for PnaDREB12-XP_034226506.1 was >1, suggesting that this gene evolved under purifying selection following the divergence between wild and sweet almond plants, consistent with the potential adaptive evolution of XP_034226506.1. DREB genes clustered within a single group largely exhibited similar functional roles due to co-adaptations and relationships among these genes. For group Aα, homologous genes in the Arabidopsis CBF subgroup were chosen to construct a phylogenetic tree, revealing that there were pronounced differences in the numbers and levels of the sequence similarity of CBF homologous genes across species. To better clarify the phylogenetic relationships among these CBF genes, homologous genes from four Prunus species were selected and used to incorporate a phylogenetic tree along with Arabidopsis, sweet almond, and wild almond homologs for these genes ( Figure 2). Clear differences in the CBF gene numbers were evident across species, with only five CBF genes in P_armeniaca, while P. mume and P. dulcis cultivar 'Nonpareil' both harbored eight CBF genes.

Structural Analyses of PnaDREB Genes and Proteins
To additionally assess the functions and characteristics of these PnaDREBs, conserved motifs and intron/exon positioning were next assessed. The majority of these genes either exhibited no introns (PnaDREB33) or a single intron (PnaDREB12), whereas PnaDREB44 and PnaDREB12 harbored 2 and 21 introns, respectively ( Figure 3). were subjected to a phylogenetic analysis. (B) Phylogenetic analysis of homologous CBF genes from A. thaliana, P. nana, P. dulcis cultivar 'Nonpareil', P. avium, P. mume, P. persica, and P. armeniaca. MEGA 6.0 was used to generate a maximum likelihood (ML) phylogenetic tree with γ-distributed rates and Jones-Taylor-Thornton. The resultant phylogenetic tree was separated into two large subgroups (A and B) and five subgroups are represented with different colors. Red indicates Dreb genes encoded by P. dulcis. The initial letter indicates different species such as XP_021: P. avium; XP_034: P. dulcis cultivar 'Nonpareil'; XP_008: P. mume; XP_007: P. persica; CAB: P_armeniaca.
DREB genes clustered within a single group largely exhibited similar functional roles due to co-adaptations and relationships among these genes. For group Aα, homologous genes in the Arabidopsis CBF subgroup were chosen to construct a phylogenetic tree, revealing that there were pronounced differences in the numbers and levels of the sequence similarity of CBF homologous genes across species. To better clarify the phylogenetic relationships among these CBF genes, homologous genes from four Prunus species were selected and used to incorporate a phylogenetic tree along with Arabidopsis, sweet almond, and wild almond homologs for these genes ( Figure 2). Clear differences in the CBF gene numbers were evident across species, with only five CBF genes in P_armeniaca, while P. mume and P. dulcis cultivar 'Nonpareil' both harbored eight CBF genes.

Structural Analyses of PnaDREB Genes and Proteins
To additionally assess the functions and characteristics of these PnaDREBs, conserved motifs and intron/exon positioning were next assessed. The majority of these genes either exhibited no introns (PnaDREB33) or a single intron (PnaDREB12), whereas PnaDREB44 and PnaDREB12 harbored 2 and 21 introns, respectively (Figure 3). To more fully characterize the diverse structural characteristics of proteins in the PnaDREB family, the MEME program was used to identify specific conserved motifs. In total, 10 such conserved motifs were identified among these 46 PnaDREBs (Figure 4). Similar motif patterns were observed within each of the established PnaDREB subgroups, consistent with their similar functional roles. Motifs 1-4 were conserved AP2 domain motifs that were present in all of these PnaDREBs, while motifs 6 and 5 were unique to the PnaDREB A and Aα subfamilies, respectively. These PnaDREBs thus harbor conserved characteristics consistent with those of the DREB family TFs, indicating that they are likely to play related functional roles. To more fully characterize the diverse structural characteristics of proteins in the PnaDREB family, the MEME program was used to identify specific conserved motifs. In total, 10 such conserved motifs were identified among these 46 PnaDREBs (Figure 4). Similar motif patterns were observed within each of the established PnaDREB subgroups, consistent with their similar functional roles. Motifs 1-4 were conserved AP2 domain motifs that were present in all of these PnaDREBs, while motifs 6 and 5 were unique to the PnaDREB A and Aα subfamilies, respectively. These PnaDREBs thus harbor conserved characteristics consistent with those of the DREB family TFs, indicating that they are likely to play related functional roles.

Identification of Cis-Regulatory Elements and miRNA Target Sites Associated with PnaDREB Genes
To better understand the mechanisms that may regulate the expression of genes in the PnaDREB family, the 2000 bp regions preceding the start codon for each member of this gene family were subjected to promoter analyses that ultimately identified cis-regulatory elements associated with light, low-temperature, and drought stress responsivity as well as hormone-responsive elements( Figure 5). These included regulatory elements responsive to methyl jasmonate, gibberellin, auxin, abscisic acid, and salicylic acid signaling. Based on orthologous gene analyses in sweet almonds, light-, ABA-, and MeJA-responsive elements were found to be the most abundant in these analyzed PnaDREB promoter regions. Of note, 17 of these PnaDREB genes (PnaDREB1/3/9/12/16/18/19/20/25/27/31/32/35/36/37/38/40) harbored low temperature-responsive elements, suggesting a need to further study these genes in the context of cold stress.
To evaluate the potential roles of microRNAs (miRNAs) as regulators of PnaDREB expression, a miRNA target site prediction analysis was performed. This approach suggested that 79 miRNAs may regulate these PnaDREBs (Table S4), with PnaDREB2, for example, being subject to regulation by miR-156, miR-774, miR-854, and miR-5638.

Identification of Cis-Regulatory Elements and miRNA Target Sites Associated with PnaDREB Genes
To better understand the mechanisms that may regulate the expression of genes in the PnaDREB family, the 2000 bp regions preceding the start codon for each member of this gene family were subjected to promoter analyses that ultimately identified cis-regulatory elements associated with light, low-temperature, and drought stress responsivity as well as hormone-responsive elements( Figure 5). These included regulatory elements responsive to methyl jasmonate, gibberellin, auxin, abscisic acid, and salicylic acid signaling. Based on orthologous gene analyses in sweet almonds, light-, ABA-, and MeJA-responsive elements were found to be the most abundant in these analyzed PnaDREB promoter regions. Of note, 17 of these PnaDREB genes (PnaDREB1/3/9/12/16/18/19/20/25/27/31/32/35/36/37/38/40) harbored low temperature-responsive elements, suggesting a need to further study these genes in the context of cold stress. To evaluate the potential roles of microRNAs (miRNAs) as regulators of PnaDREB expression, a miRNA target site prediction analysis was performed. This approach suggested that 79 miRNAs may regulate these PnaDREBs (Table S4), with PnaDREB2, for example, being subject to regulation by miR-156, miR-774, miR-854, and miR-5638.
The majority of the analyzed PnaDREBs exhibited varying degrees of induction response to cold treatment (Figure 7), with some exhibiting similar induction pattern both wild and sweet almond seedlings. PnaDREB3, PnaDREB27, PnaDREB PnaDREB41, PnaDREB42, and PnaDREB43 were all upregulated in response to cold st in both of these species, whereas PnaDREB9, PnaDREB18, and PnaDREB22 were dow regulated, suggesting that these genes may play roles in cold stress responses. Nota PnaDREB27, PnaDREB41, PnaDREB42, and PnaDREB43 were upregulated more t 10-fold relative to the control (25 °C) conditions. The expression levels of PnaDREB PnaDREB27, PnaDREB41, PnaDREB42, and PnaDREB43 were greater in sweet almo relative to wild almond at different tested temperatures, whereas PnaDREB PnaDREB31, PnaDREB38, and PnaDREB39 were expressed at significantly higher lev in wild almond. Strikingly, PnaDREB16 and PnaDREB38, which were respective m bers of subgroups Bα and Aα, were upregulated in wild almond plants but downre lated in sweet almond plants in response to cold treatment. These differential express patterns may belie differences in the cold stress tolerance of these two almond variet PnaDREB1 was downregulated in sweet almond plants, but was first downregulated then upregulated at −10 °C in wild almonds (Figure 7a). PnaDREB12 was significan upregulated in wild almond leaves at −5 °C and −10 °C, whereas it was downregulate sweet almond leaves at 5 °C and −10 °C (Figure 7b). PnaDREB19 expression levels in leaves of wild almond plants also declined in response to cold treatment but rose nificantly in sweet almond plants at both −5 °C and −10 °C (Figure 7).
The majority of the analyzed PnaDREBs exhibited varying degrees of induction in response to cold treatment (Figure 7), with some exhibiting similar induction patterns in both wild and sweet almond seedlings. PnaDREB3, PnaDREB27, PnaDREB31, PnaDREB38, PnaDREB41, PnaDREB42, and PnaDREB43 were all upregulated in response to cold stress in both of these species, whereas PnaDREB9, PnaDREB18, and PnaDREB22 were downregulated, suggesting that these genes may play roles in cold stress responses. Notably, PnaDREB27, PnaDREB41, PnaDREB42, and PnaDREB43 were upregulated more than 10-fold relative to the control (25 • C) conditions. The expression levels of PnaDREB27, PnaDREB41, and PnaDREB42 were greater in sweet almond relative to wild almond at different tested temperatures, whereas PnaDREB12, PnaDREB16, PnaDREB31, PnaDREB38, and PnaDREB39 were expressed at significantly higher levels in wild almond. Strikingly, PnaDREB16, which were respective members of subgroups Bα , was upregulated in wild almond plants but downregulated in sweet almond plants in response to cold treatment. These differential expression patterns may belie differences in the cold stress tolerance of these two almond varieties. PnaDREB1 was downregulated in sweet almond plants, but was first downregulated and then upregulated at −10 • C in wild almonds (Figure 7a). PnaDREB12 was significantly upregulated in wild almond leaves at −5 • C and −10 • C, whereas it was downregulated in sweet almond leaves at 5 • C and −10 • C (Figure 7b).
PnaDREB19 expression levels in the leaves of wild almond plants also declined in response to cold treatment but rose significantly in sweet almond plants at both −5 • C and −10 • C (Figure 7).

Discussion
P. nana is a wild species that is related to the more widely cultivated sweet almond species. P. nana trees have evolved a diverse range of adaptive genetic mechanisms that enable them to tolerate stressors to which they are exposed in their harsh habitats [37]. According to the different uses of almonds, almonds are divided into many varieties. Among them, sweet almonds are widely cultivated for their edible functions, but their distribution in the wild is limited due to their suitability for growing in warm and arid regions. Both drought and cold stress resistance-related genes are invaluable genetic resources that have the potential to aid in efforts to improve the hardiness of cultivated almond varieties. AP2/ERF family TFs are important regulators of plant growth and development [38,39]. DREB transcription factors play an important role in plant response to cold stress signaling pathways [40,41]. However, there are few studies on the response of P. nana to cold stress at the molecular level. This study is the first publicly published analysis and identification of DREB transcription factors through genomic data, providing a basis for a better understanding of the functions of the DREB gene family of P. nana under cold stress. Accordingly, this study conducted a whole genome analysis of P. nana in an effort to identify target genes with the potential to aid in the future breeding of almond cultivars exhibiting superior cold resistance.
In the present study, we named these 46 PnaDREB genes from PnaDREB1 to PnaDREB46 based on their order on the chromosomes and classified them into five subgroups according to the phylogenetic relationships with A. thaliana. The results showed that the number of DREB genes in A was similar to that of reported plants such as A. thaliana 57 DREBs; Triticum aestivum 57 DREBs [42]; Phyllostachys edulis 47 DREBs [43]; Sorghum bicolor 52 DREBs [44]; Cicer arietinum 43 DREBs [45]. The above data indicate that the DREB gene family of A is highly conserved, which may be related to gene duplication during speciation and evolution. According to the analysis results of the phylogenetic tree, among the five subgroups, the proportion of subgroups was the highest at 45.7% and 32.6%, respectively, in Aγ and Bβ. The subgroup had the least PnaDREB genes, with only one and four, respectively. ICE1 belongs to the bHLH transcription factor family. Under cold stress, it activates the expression of CBFs genes, ultimately enhancing the cold resistance of plants by activating cold resistant proteins or ROS scavenging systems [46]. CBFs are located in the DREB family Aα subgroup, and it has been reported that Aα family members are sensitive to cold stress, almost all of which can be induced by cold stress, and can regulate the expression of cold stress-related genes [47,48]. In summary, the DREB family of P. nana may have the ability to cope with cold stress, laying a foundation for subsequent experiments.
PnaDREB genes have different numbers of introns, and most of the identified PnaDREB genes contain 0 or 1 intron, although PnaDREB12 contains 21 introns. In the description of the presence of PnaDREB12 Aα, there were some mutations. It is worth noting some PnaDREB genes, especially B β. The PnaDREB gene in group B is generally deficient in introns, which may shorten the post transcriptional process to respond immediately to abiotic stresses [49]. In this study, in Aα, the large number of introns in group I suggests that the molecular structure of group I of the DREB gene may be relatively conservative during evolution, which is conducive to evolution caused by protein diversity [50,51]. It seems that the key function of the DREB gene family in A is similar to that of Aα. Group A is closely related, and similar results have been found in Solanum torvum L. [52]. With the exception of the 1273 amino acids in the PnaDREB12 protein, the other PnaDREBs identified herein were predicted to range from 166 (PnaDREB15) to 515 (PnaDREB16) amino acids in size, with an average length of 295 amino acids. Molecular weight (17.91-58.10 kDa) and pI (4.57-9.39) values for these proteins were also relatively similar to those for other species including pepper (Capsicum annuum L.; 12.13-59.27 kDa, 4.6-10.64 pI) [53], suggesting that the characteristics and functions of these TFs are conserved across species to some degree. These results thus provide a foundation for future efforts to explore the functions of P. nana DREBs.
According to the prediction of cis-acting regulatory elements, PnaDREBs may play a regulatory role in various biological processes in cotton. The low-temperature responsive (LTR) element (CCGAAA) may be an important mediator of the ability of PnaDREB proteins to respond to cold stress, given that it was present in the promoter regions of seven PnaDREBs. This LTR sequence has been shown to be an essential cis-regulatory element within heat-shock protein promoter regions in both Ulva prolifera and Hordeum vulgare [9,54]. Sweet almonds were the a control for P. nana, B α subgroup PnaDREB16, was both upregulated in wild almond plants and downregulated in sweet almond plants. PnaDREB3, PnaDREB12, PnaDREB16, PnaDREB27, PnaDREB31, and PnaDREB38 were expressed at significantly higher levels in wild almond seedlings exposed to cold stress, suggesting that they may be important regulators of cold stress responses worthy of further study. After cold treatment, the expression levels of PnaDREB9, PnaDREB18, and PnaDREB22 in the selected 15 putative PnaDREB genes were downregulated. During cold treatment, we found that the expression levels of PnaDREB27, PnaDREB41, and PnaDREB42 in sweet almonds were higher than those in wild almonds. This suggests that some PnaDREB transcription factors may play a negative regulatory role. In summary, our analysis adds evidence that DREB can assist plants in coping with cold stress.

Conclusions
This study is the first genome-level description of the DREB gene family of P. nana. We identified 46 DREB genes in P. nana, and all of them were located on six chromosomes. In addition, based on phylogenetic relationships, PnaDREBs were classified into three groups. The motifs were highly conserved in the same subgroup. Meanwhile, cis-acting regulatory elements indicate that PnaDREBs play an important role in coping with cold stress. PnaDREB interaction analyses showed that PnaDREB36 and PnaDREB40 exhibited a high degree of homology to Arabidopsis CBF1. The qPCR results demonstrated that among the 15 selected PnaDREB genes, seven genes were significantly upregulated in both plant species, and three genes were significantly downregulated, of which the Bα and Aα members of the subgroup showed the opposite behavior when the two plants responded to cold treatment.
In summary, the results of this study not only provide clues for future analysis of the mechanism of PnaDREB response to cold stress, but also in expanding our understanding of the DREB family in plants.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/genes14040811/s1, Table S1: Characteristics of DREB transcription factors in Prunus nana; Table S2: Collinearity analysis of DREB gene in wild and sweet almond; Table S3: The nonsynonymous and synonymous mutation rate analysis of DREB gene in wild and sweet almond; Table S4: Prediction of MicroRNA target sites in PnaDREB genes.  Data Availability Statement: Data supporting the present study are available from the corresponding author upon reasonable request.