*2.7. Statistical Analysis*

Three biological and six analytical replicates were used to run all reactions. Gene expression differences were assessed using normalized expression (Cq) in the Bio-Rad CFX MaestroTM/Software version 2.3 and were found to be significant for *<sup>p</sup>* ≤ 0.05 (\*), *<sup>p</sup>* ≤ 0.01 (\*\*), *p* ≤ 0.001 (\*\*\*) after ANOVA and Shapiro–Wilk Normality tests. The standard errors of the mean are shown as vertical bars (n = 6).

#### **3. Results**

#### *3.1. Characterization and Phylogenetic Analysis of DsDBF1*

A *DBF1* gene was identified based on the metatranscriptome data for the moss *D. scoparium* downloaded from the Sequence Read Archive in the NCBI. To verify the *DBF1* gene identified from *D. scoparium*, specific primers were designed and the PCR product (717 bp) was cloned into the pAL2-T vector (Evrogen, Moscow, Russia) and then sequenced. Blasting the *DBF1* sequence from *D. scoparium* after cloning and sequencing revealed high homology with *ERF/DREBs* of other mosses and vascular plants in the NCBI database. Further analyses of the protein sequence using PFAM [37], NCBI CDD [38], InterProScan [39] and HMMER [40] databases revealed that this protein had a classic AP2 domain structure (Figure 1) and was named DsDBF1. In addition, coding domain sequence (CDS) length (bp), subcellular localization, and physico-chemical properties such as protein length (aa), molecular weight (MW, kDa), isoelectric point (pI), instability index, and GRAVY were predicted (Table 1). The results showed that the cloned *DsDBF1* encoded a 238 amino acid polypeptide (Figure S1) with a predicted molecular weight of 26 kDa and isoelectric point of 5.98. Calculation of the instability index classified the protein as unstable with a value greater than 40. A negative value of GRAVY indicated that DsDBF1 was hydrophilic and subcellular prediction showed that the protein was localized within the nucleus and cytoplasm (Table 1).


**Figure 1.** Sequence alignments of DsDBF1 and other known ERF/DREB proteins showing the classical AP2 domains from mosses and vascular plants such as *Syntrichia caninervis* (DREBP5\_AMT92109.1), *Physcomitrium patens* (ERF RAP2-1-like\_XP 024372564.1; PpDBF1 ABA43687.2; ERF\_TINY-like\_XP 024390306.1), *Gossypium hirsutum* (GhDREBP1\_AAO43165.1; GhDBP\_RAP2-4-like\_NP 001314591.1), *Glycine max* (GmDREBP3\_ABB36646.1; GmDREBP\_NP 001345276.1; GmDREBP\_NP 001345278.1), *Oryza sativa* (OsRAP\_XP 468111.1; OsDREBP2A\_XP 025878770.1; DREBP1A\_XP 015610912.1; Os-DREB1F\_NP 001359120.1; ERF038\_XP 015614793.1), *Triticum aestivum* (TaDREBP1\_AAL01124.1; CRT/DREBP\_XP 044398325.1), *Zea mays* (ZmDBF1\_AAM80486.1; ZmDBF2\_AAM80485.1), *Capsicum annuum* (CaCBF1B\_AAQ88400.1), *Capsella bursa-pastoris* (CbCBF\_AAR26658.1), *Brassica napus* (BnCBF\_AAL38243.1), *Prunus avium* (PaDREB1F\_XP 021803652.1), *Arabidopsis thaliana* (TINY2\_NP 196720.1), *Bryum argenteum* (ERF\_QDB64575.1), *Pohlia nutans* (ERF\_QCF46602.1), *Selaginella moellendorffii* (ERF039-like\_XP 024530345.1), *Citrus sinensis* (ERF016\_XP 006474696.1), *Apostasia shenzhenica* (ERF034\_PKA61103.1), and *Theobroma cacao* (ERF016\_XP 007012585.2). Amino acid sequences are highlighted with different colors. Sequences marked by (\*) show conserved amino acid residues. Two conserved elements (YRG and RAYD) are marked by black horizontal lines. The differences in the moss conserved elements are shown in black frames. The red line shows the classical AP2 domain.

**Table 1.** Physico-chemical properties and subcellular localization of DsDBF1.


Sequence alignment analysis indicated that DsDBF1 shared high homology and a conserved AP2/ERF domain with other DREBs (Figure 1), but with low similarity in their overall amino acid sequences (Figure S2). Additionally, two conserved elements (YRG and RAYD) were found in the AP2/ERF domain after sequence analysis, although arginine (R) is replaced by lysine (K) in both the first YRG and second RAYD elements in DsDBF1

and some DREBs of other mosses (Figure 1). The homologous protein sequences obtained after BLASTP search of the DsDBF1 sequence and other known ERF/DREB proteins from GenBank were used to construct a phylogenetic tree demonstrating the evolutionary relationship between DsDBF1 and other similar sequences from mosses and vascular plants. The evolutionary tree showed that DsDBF1 belongs to the A-5 group of the DREB subfamily as it shared a common ancestry and homology with other known A-5 DREBs from mosses such as *S. caninervis*, *P. patens, Bryum argenteum*, *Pohlia nuntans,* and vascular plants such as *Selaginella moellendorffii*, *G. max*, *O. sativa*, *Citrus sinensis*, *Theobroma cacao,* and *Gossypium hirsutum* (Figure 2). Furthermore, it was found that group A-5 was divided into seven subgroups, with *S. moellendorffii* positioning between the protein subgroups of mosses and the vascular plants. As shown in Figure 2, all other known DREBs from vascular plants were clustered into different DREB subfamilies such as A-1, A-2, A-4, and A-6.

**Figure 2.** Phylogenetic analyses of DsDBF1 and other ERF/DREB proteins from mosses and vascular plant constructed using the neighbor-joining method with 1500 bootstrap test showing the relationship between the amino acid sequences. Evolutionary distances were calculated using the Poisson correction method and all ambiguous positions were removed by pairwise deletion. Amino acid sequences used for phylogenetic tree construction were retrieved, in part, from GenBank and after blast analysis from GenBank: *Syntrichia caninervis* (DREBP5\_AMT92109.1), *Physcomitrium patens* (ERF RAP2-1-like\_XP 024372564.1; PpDBF1 ABA43687.2; ERF\_TINY-like\_XP 024390306.1), *Gossypium hirsutum* (GhDREBP1\_AAO43165.1; GhDBP\_RAP2-4-like\_NP 001314591.1), *Glycine max* (GmDREBP3\_ABB36646.1; GmDREBP\_NP 001345276.1; GmDREBP\_NP 001345278.1), *Oryza sativa* (OsRAP\_XP 468111.1; OsDREBP2A\_XP 025878770.1; DREBP1A\_XP 015610912.1; OsDREB1F\_NP

001359120.1; ERF038\_XP 015614793.1), *Triticum aestivum* (TaDREBP1\_AAL01124.1; CRT/DREBP\_XP 044398325.1), *Zea mays* (ZmDBF1\_AAM80486.1; ZmDBF2\_AAM80485.1), *Capsicum annuum* (CaCBF1B\_AAQ88400.1), *Capsella bursa-pastoris* (CbCBF\_AAR26658.1), *Brassica napus* (BnCBF\_AAL38243.1), *Prunus avium* (PaDREB1F\_XP 021803652.1), *Arabidopsis thaliana* (TINY2\_NP 196720.1), *Bryum argenteum* (ERF\_QDB64575.1), *Pohlia nutans* (ERF\_QCF46602.1), *Selaginella moellendorffii* (ERF039-like\_XP 024530345.1), *Citrus sinensis* (ERF016\_XP 006474696.1), *Apostasia shenzhenica* (ERF034\_PKA61103.1), and *Theobroma cacao* (ERF016\_XP 007012585.2).

Additionally, the results of MEME analyses showed that DsDBF1 contained a total of six motifs, among them, motifs 1–3 represented the basic conserved motifs that made up the AP2 domain (Figure 3). Motif 4 was absent only in the DREB protein of *Triticum aestivum* (TaDREBP1\_AAL01124.1), while motif 5 was absent in the DREB proteins of *Gossypium hirsutum* (GhDREBP1 AAO43165.1 and GhDBP RAP2-4-like\_NP 001314591.1), *G. max* (GmDREBP3 ABB36646.1 and GmDREBP\_NP 001345278.1), *O. sativa* (OsRAP\_XP 468111.1 and DREBP1A\_XP 015610912.1), *Z. mays* (ZmDBF1\_AAM80486.1) including *T. aestivum* (TaDREBP1\_AAL01124.1). However, an additional motif 9 was detected in DsDBF1, which was only conserved in *S. caninervis* (DREBP5\_AMT92109.1) and two in *P. patens* (ERF RAP2-1-like\_XP 024372564.1 and PpDBF1\_ABA43687.2) (Figure 3).

\_XP 024372564.1; PpDBF1 ABA43687.2; ERF\_TINY-like\_XP 024390306.1), *Gossypium hirsutum* (Gh-DREBP1\_AAO43165.1; GhDBP\_RAP2-4-like\_NP 001314591.1), *Glycine max* (GmDREBP3\_ABB36646.1; GmDREBP\_NP 001345276.1; GmDREBP\_NP 001345278.1), *Oryza sativa* (OsRAP\_XP 468111.1; Os-DREBP2A\_XP 025878770.1; DREBP1A\_XP 015610912.1; OsDREB1F\_NP 001359120.1; ERF038\_XP 015614793.1), *Triticum aestivum* (TaDREBP1\_AAL01124.1; CRT/DREBP\_XP 044398325.1), *Zea mays* (ZmDBF1\_AAM80486.1; ZmDBF2\_AAM80485.1), *Capsicum annuum* (CaCBF1B\_AAQ88400.1), *Capsella bursa-pastoris* (CbCBF\_AAR26658.1), *Brassica napus* (BnCBF\_AAL38243.1), *Prunus avium* (PaDREB1F\_XP 021803652.1), *Arabidopsis thaliana* (TINY2\_NP 196720.1), *Bryum argenteum* (ERF\_QDB64575.1), *Pohlia nutans* (ERF\_QCF46602.1), *Selaginella moellendorffii* (ERF039-like\_XP 024530345.1), *Citrus sinensis* (ERF016\_XP 006474696.1), *Apostasia shenzhenica* (ERF034\_PKA61103.1), and *Theobroma cacao* (ERF016\_XP 007012585.2). Distribution of 10 putative conserved motifs in DREB proteins is shown. Conserved motifs are represented by different colored boxes numbered 1–10.

#### *3.2. Expression Patterns of DsDBF1 in Response to Abiotic Stress Treatments*

The expression pattern of *DsDBF1* was studied after application of abiotic stresses such as desiccation/rehydration, exposure to DCMU, CdCl2, paraquat, high and freezing temperatures to moss apical segments. Desiccation of the hydrated moss for 2, 24, and 72 h over silica gel resulted in almost up to 94% loss of RWC in the moss samples, accompanied by downregulation of *DsDBF1*, with the lowest expression observed after 24 h of dehydration (Figure 4A). Rehydration of the mosses after 72 h of desiccation showed a gradual increase in *DsDBF1* expression after 0.5 h and further 2 h with the expression of *DsDBF1* 2-fold higher compared to that in the hydrated mosses before desiccation (Figure 4A). Treatment of moss segments with an inhibitor of photosynthesis DCMU downregulated *DsDBF1* expression after 1 and 12 h (Figure 4B). Subjecting the mosses to heavy metal CdCl2 and prooxidant paraquat significantly increased the expression of *DsDBF1* after 1 h (Figure 4B); however, further treatment for 12 h downregulated *DsDBF1* expression. No significant changes in *DsDBF1* expression were observed after exposing moss to +30 ◦C for 1 and 12 h. Exposure of mosses to a freezing temperature of −20 ◦C reduced the level of *DsDBF1* expression after 1 h (Figure 4B); however, further exposure for 12 h at −20 ◦C upregulated gene expression almost 10-fold compared to a 1 h treatment (Figure 4B).

**Figure 4.** Expression patterns of *DsDBF1* under abiotic stress treatments analyzed using RT-qPCR. (**A**) Relative expression of *DsBF1* during desiccation over silica gel and rehydration. Shaded bars

represent the hydrated and rehydrated moss, and solid bars represent the desiccated moss. (**B**) Relative expression of *DsDBF1* exposed to DCMU, CdCl2, paraquat and high/low temperature for 1 and 12 h. Open bars correspond to control samples of mosses kept at room temperature. Red bars with white dots represent mosses treated with 100 μM DCMU, bars with orange horizontal stripes show moss treated with 100 μM CdCl2, bars with green stripes correspond to samples subjected to 100 μM paraquat, yellow and blue bars correspond to mosses exposed to +30 ◦C and −20 ◦C, respectively. *p* ≤ 0.05 (\*), *p* ≤ 0.01 (\*\*), *p* ≤ 0.001 (\*\*\*). The vertical bars indicate the standard errors of the mean (n = 6).

### **4. Discussion**

Members of the AP2/ERF family of TFs are among the most important key regulators of genes responsible for stress tolerance and developmental transitions of plants. These TFs regulate transcriptional networks to activate or repress gene expression in response to biotic and abiotic factors through the modulation of several signaling pathways [7,8,17,50]. In the last few years, many DREBs have been identified and characterized in several angiosperms, including *A. thaliana* [11], rice (*O. sativa*) [13,51], soybean (*G. max*) [52], maize (*Z. mays*) [15,53], cotton (*G. hirsutum*) [54,55], barley (*H. vulgare*) [16,56,57], wheat (*T. aestivum*) [58], *Populus euphratica* [59], *Caragana korshinskii* [60], and others. Surprisingly, the AP2/ERF gene family has been rarely studied in stress-tolerant moss species [19–21]. Several recent studies have shown that AP2/ERF TFs play an important role in the developmental processes and stress responses in some moss species, such *P. patens* [22,23,61], *S. caninervis* [24,62–65], *B. argenteum* [21], and *P. nutans* [66]. *Dicranum scoparium* is a desiccation-tolerant moss [25] whose genome has not been fully sequenced, and no TF families of this species have been reported to date. In this present study, we first identified in silico a cDNA of the *DBF1* gene in the moss *D. scoparium*. Then, the identified gene was verified by cloning and sequencing. In addition, we performed molecular characterization of the protein, including analysis of the conserved domain, physico-chemical properties, subcellular localization, phylogenetic relationship, and motif analyses of identified Ds-DBF1 and DREBs of other plants, and finally, we examined the expression patterns of this gene in response to abiotic stresses. Our results demonstrate that the expression of *DsDBF1* is strongly induced by rehydration after desiccation, and treatments with CdCl2, paraquat, and freezing temperature, providing insights into the roles of *DBF1* in response of *D. scoparium* to abiotic stresses.

Analyses of the physico-chemical properties and the subcellular localization showed that *DsDBF1* encodes a 238-amino acid polypeptide with a molecular weight of 25 kDa and a pI of 5.98 and the protein is localized within the nucleus and cytoplasm (Table 1). While the majority of TFs are nuclear localized, some are not when initially synthesized [67]. Some of these TFs are kept inactive in the cytoplasm when synthesized or expressed as membrane proteins, but when stimulated, they are activated by proteolytic cleavage, releasing the active form, which enters the nucleus and activates target genes [67,68].

Furthermore, the BLASTP search of the NCBI database revealed that DsDBF1 shares high sequence similarities with some DREBs from angiosperms and mosses. In addition, some uncharacterized proteins from mosses such as *Ceratodon purpureus* and *S. fallax* also show very high similarities to DsDBF1. The amino acid composition of the AP2 domain of DsDBF1 revealed that it contains 65 amino acid residues (Figure 1), which approximately corresponds to the conserved 60 amino acids of the AP2/ERF domain found in all DREBs [11]. Amino acid alignments of DREB proteins from different plants show high sequence similarity in the middle of AP2/ERF domain of these proteins (Figure 1), which is a significant feature of plant DREBs [64,69]. However, in general, outside the domain box, low similarity is observed in their overall amino acid sequences (Figure S2).

Analysis of the AP2/ERF domain after multiple sequence alignments revealed the presence of two conserved YRG and RYAD elements (Figure 1), although only glycine (G) is conserved in the YRG element among all the DREBs, while alanine (A) and aspartic acid (D) are conserved in the RYAD elements (Figure 1). Furthermore, in the first YRG

element, tyrosine (Y) and arginine (R) are replaced by phenylalanine (F) and lysine (K), respectively, whereas in the second RYAD element, R is substituted by K, leucine (L), and histidine (H), and the Y is substituted by F and H. The AP2/ERF domain is a type of DNA-binding module that contains two known conserved elements (YRG and RAYD), and these two elements can bind with the promoter sequence or some other interacting proteins [69,70]. Studies have shown that YRG is involved in DNA binding activity and is the basic hydrophilic N-terminal side of the AP2/ERF domain. The N-terminal region is approximately 19 to 22 amino acids in length [50,69,70]. In addition, the second element, RAYD, is located in the acidic C-terminal region of the AP2/EREBP domain with a length of 42 to 43 amino acids. It is suggested that the RAYD element plays a crucial role in mediating protein–protein interactions [50,70]. However, in this study, the substitution of amino acids observed at various positions within the conserved elements in the AP2/ERF domain after multiple sequence alignments (Figure 1) of DREB proteins, may imply their functional divergence within DREB subfamilies.

To understand the evolutionary relationship between DsDBF1 and other well-known DREBs from other plants, a neighbor-joining tree was constructed using the deduced amino acid residues of these DREB proteins (Figure 2). In this analysis, DsDBF1 was found to belong to the A-5 group of the DREB subfamily as it shares a common ancestor with other known A-5 DREBs from mosses such as *S. caninervis*, *P. patens*, *B. argenteum*, *P. nuntans*, a lycophyte, for example, *S. moellendorffii*, and the angiosperms, such as *G. max*, *O. sativa*, *C. sinensis*, *T. cacao,* and *G. hirsutum* (Figure 2). In the A-5 subgroup, *S. moellendorffii* branches from the moss subgroup, positioning itself between the mosses and the angiosperms. This supports the report of early divergence of vascular plants from the ancient non-vascular plants [71]. It has been proposed that *PpDBF1*, an A-5 type DREB from *P. patens*, is an ancestor of DREB proteins and plays a general role in various stresses in non-vascular moss, which has diverged into different subclasses with different functions in the higher plants [23]. Consequently, the grouping of DsDBF1 and some other A-5 DREB proteins from mosses, lycophytes, and angiosperms in one clade suggests that they were established in the early stages of land plant evolution. Additionally, it was found that all other known DREBs from vascular plants diverged into different DREB subfamilies such as A-1, A-2, A-4, and A-6 (Figure 2). The DREB gene subfamily may have evolved and assumed new roles as a result of the divergence of the AP2 genes. The functional diversity and divergence of DREB genes during the adaptive evolution of stress signaling pathways in plants is most likely the result of subsequent duplication and transposition events [23].

Moreover, an investigation of the conserved motifs in DsDBF1 and other selected DREB proteins was carried out using MEME software. From the results, DsDBF1 contains a total of six motifs. Motifs 1–3 represent the conserved motifs of the AP2 domain (Figure 3). Furthermore, an additional motif 9 was detected in DsDBF1. This motif is present in *S. caninervis* (DREBP5\_AMT92109.1) and *P. patens* (ERF RAP2-1-like\_XP 024372564.1 and PpDBF1\_ABA43687.2) (Figure 3), suggesting their common origin. Genome-wide sequence analysis of AP2/ERF family TFs in numerous plants revealed conserved regions and motifs on both sides of the AP2/ERF domain with important roles in transcriptional activity, protein–protein interactions, and nuclear localization. These conserved motifs can serve as an evidence for further classification of subgroups [50,72].

Plant DREB TFs play critical roles in the response to dehydration, salinity, and cold stresses [73,74]. To further understand the role of *DsDBF1* in response to stresses, we examined the expression profile of *DBF1* gene by RT-qPCR in the *D. scoparium* subjected to desiccation/rehydration, exposure to DCMU, CdCl2, paraquat, heat and freezing temperature. Our results indicate that *DsDBF1* gene is upregulated by most of these stresses, suggesting that this gene is involved in *D. scoparium* response to abiotic stresses (Figure 4A,B). Surprisingly, *DsDBF1* gene is downregulated following exposure of the moss to DCMU (Figure 4B). It has been reported that genes assigned to different groups within the same gene family show diverse stress response patterns and stress tolerance [64,75]. To date, most reports on DREB and Cold binding factors (CBFs) have mainly focused on DREBA1

and DREBA2, the largest among the subgroups [69,76]. The A-1 type DREBs (DREB1) are induced by cold and improve plant stress tolerance to low temperatures [73,77], whereas A-2 type DREBs (DREB2) play a major role in response to dehydration and heat stress, and improve drought and salt tolerance in plants [78]. A-5 DREBs have rarely been studied, and the functional and stress response mechanisms are still unclear [63].

Interestingly, desiccation of the hydrated mosses for 2, 24, and 72 h decreased *DsDBF1* expression (Figure 4A). However, rehydrating moss thalli after 72 h of desiccation progressively increased *DsDBF1* expression to 2-fold higher compared to the hydrated mosses before desiccation (Figure 4A). *PpDBF1*, a homolog of *DsDBF1,* was weakly induced by dehydration stress but strongly induced by ABA [23]. Out of ten A-5 type DREBs from *S. caninervis*, *ScDREB5* was downregulated under rapid desiccation stress over silica gel, while *ScDREB3*, *ScDREB9,* and *ScDREB10* were poorly induced by desiccation [63]. However, these four DREBs were significantly induced by cold stress, while *ScDREB3* and *ScDREB5* were upregulated during heat stress [63]. The rapid desiccation used in our experiment, in which moss thalli were dried over silica gel and reached an RWC of 6% [28] after 24 h, never occurs in boreal forests, where drying rates of mosses are much slower [27,79]. Other A-5-type DREBs such as *GhDBP1* and *GmDREB3* have been shown to improve plant stress tolerance [23,55,77]. Moreover, photosynthesis inhibitor DCMU decreased *DsDBF1* expression (Figure 4B). Meanwhile, CdCl2 and paraquat significantly altered the expression of *DsDBF1*, as short-term exposure increased *DsDBF1* expression after 1 h (Figure 4B). Longterm treatment of moss samples with paraquat and CdCl2 resulted in downregulation of *DsDBF1* expression after 12 h. Furthermore, exposure of moss to +30 ◦C had little effect on *DsDBF1* expression, although a freezing temperature of −20 ◦C for 12 h upregulated gene expression almost 10-fold compared to a 1 h cold treatment (Figure 4B). It has been reported that *PpDBF1*, *GmDREB2*, *StDREB2, ScDREB1*, *ScDREB2*, *ScDREB4*, *ScDREB6*, *ScDREB7,* and *ScDREB8* responded to drought, salt, and cold treatment among members of the A-5 subgroups [23,63,80,81]. Taken together, the upregulation of *DsDBF1* during rehydration after desiccation, exposure to CdCl2, paraquat, and freezing-temperature stress suggests that *DsDBF1,* like other A-5 DREBs, plays important roles in *D. scoparium* stress tolerance.

### **5. Conclusions**

An A-5 type gene, *DsDBF1*, encoding DRE-binding transcription factor TF was identified and cloned in the moss *D. scoparium*. *DsDBF1* protein was predicted to be localized within the nucleus and cytoplasm. Furthermore, RT-qPCR analysis showed that *DsDBF1* expression was significantly induced in response to abiotic stresses such as desiccation/rehydration, exposure to paraquat, CdCl2, high and freezing temperatures. *D. scoparium* is a desiccation tolerant moss species. Based on our results, we believe that *DsDBF1* could be a promising gene candidate to improve stress tolerance in various crop plants, and characterization of transcription factors of a stress-tolerant moss such as *D. scoparium* provides a better understanding of plant response and adaptation mechanisms.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/life13010090/s1, Figure S1: *DsDBF1* coding domain sequence and protein sequence; Figure S2: Sequence alignments of DsDBF1 and other known ERF/DREB proteins from mosses and vascular plants such as *Syntrichia caninervis* (DREBP5\_AMT92109.1), *Physcomitrium patens* (ERF RAP2-1-like\_XP 024372564.1; PpDBF1 ABA43687.2; ERF\_TINY-like\_XP 024390306.1), *Gossypium hirsutum* (GhDREBP1\_AAO43165.1; GhDBP\_RAP2-4-like\_NP 001314591.1), *Glycine max* (GmDREBP3\_ABB36646.1; GmDREBP\_NP 001345276.1; GmDREBP\_NP 001345278.1), *Oryza sativa* (OsRAP\_XP 468111.1; OsDREBP2A\_XP 025878770.1; DREBP1A\_XP 015610912.1; Os-DREB1F\_NP 001359120.1; ERF038\_XP 015614793.1), *Triticum aestivum* (TaDREBP1\_AAL01124.1; CRT/DREBP\_XP 044398325.1), *Zea mays* (ZmDBF1\_AAM80486.1; ZmDBF2\_AAM80485.1), *Capsicum annuum* (CaCBF1B\_AAQ88400.1), *Capsella bursa-pastoris* (CbCBF\_AAR26658.1), *Brassica napus* (BnCBF\_AAL38243.1), *Prunus avium* (PaDREB1F\_XP 021803652.1), *Arabidopsis thaliana* (TINY2\_NP 196720.1), *Bryum argenteum* (ERF\_QDB64575.1), *Pohlia nutans* (ERF\_QCF46602.1), *Selaginella moellendorffii* (ERF039-like\_XP 024530345.1), *Citrus sinensis* (ERF016\_XP 006474696.1), *Apostasia shenzhenica*

(ERF034\_PKA61103.1) and *Theobroma cacao* (ERF016\_XP 007012585.2). Multiple alignment was performed using Clustal Omega. Amino acid sequences are highlighted with different colors. Sequences marked by (\*) show conserved amino acid residues, Table S1: Primers of RT-qPCR.

**Author Contributions:** Conceptualization, A.B.M. and F.V.M.; methodology, A.B.M. and A.O.O.; software, A.O.O., A.B.M. and I.Y.L.; formal analysis, A.O.O. and I.Y.L.; investigation, A.B.M.; writing original draft preparation, A.O.O.; writing—review and editing, F.V.M.; supervision, F.V.M.; project administration, F.V.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the state assignment of the FRC KazSC RAS. F.V.M. thanks Russian Foundation for Basic Research [grant number 20-04-00721] for partial financial support (gene cloning and sequencing).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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