**1. Introduction**

Abiotic stresses, such as drought, salinity, heavy metals, high and low temperatures, can disrupt cellular homeostasis, resulting in redox imbalances and the accumulation of reactive oxygen species (ROS), which can limit plant development and crop productivity [1,2]. To react and adapt to these environmental challenges, plants have developed complex mechanisms including physiological, biochemical, and molecular processes [3–5]. Significant progress has been made over the past two decades regarding the identification and characterization of stress-responsive genes and proteins that directly protect plants against stresses [5,6]. Numerous genes are regulated by transcription factors (TF) in response to various abiotic stimuli [7]. Transcription factors play important roles in controlling the expression of genes in various signaling pathways due to their DNA-binding specificity [8,9].

Dehydration-responsive element binding (DREB) TFs play critical roles in plant tolerance by regulating the expression of stress-inducible genes during abiotic stresses [10]. They have been extensively identified in a wide variety of higher plants (angiosperms), such as *Arabidopsis thaliana* [11], soybean (*Glycine max*) [12], rice (*Oryza sativa*) [13,14], maize (*Zea mays*) [15], barley (*Hordeum vulgare*) [16], and others. The dehydration-responsive element (DRE)-binding factor, also known as DBF1, belongs to the APETALA2/Ethylene-Responsive Factor (AP2/ERF) TF family, which has been demonstrated to be involved in various biological processes in plants, including metabolism, development, and stress

**Citation:** Onele, A.O.; Mazina, A.B.; Leksin, I.Y.; Minibayeva, F.V. *DsDBF1*, a Type A-5 DREB Gene, Identified and Characterized in the Moss *Dicranum scoparium*. *Life* **2023**, *13*, 90. https://doi.org/10.3390/ life13010090

Academic Editors: Wajid Zaman and Hakim Manghwar

Received: 5 December 2022 Revised: 23 December 2022 Accepted: 25 December 2022 Published: 28 December 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

response [12,17,18]. So far, AP2/ERF genes annotated in the mosses *Physcomitrium patens* and *Sphagnum fallax* are the largest TF families found in the plant TF databases (TFDB), although the AP2/ERF gene family has been rarely studied in the moss species [19–21]. Additionally, it has been shown that AP2/ERFs are regulated in response to numerous stresses, such as salinity and UV in *P. patens* [22] and the gene *PpDBF1* played a role in drought, salt, and cold tolerance in transgenic tobacco [23]. Furthermore, in the desiccation tolerant moss *Syntrichia caninervis*, AP2/ERFs were found to be the most abundant TFs [24].

*Dicranum scoparium* is a category "A" moss, one of the most desiccation tolerant moss species [25]. It is a widely distributed Holarctic moss that grows in various habitats and is one of the most polymorphic species in their genus [26]. Our preliminary analysis of class III peroxidase activity in three feather mosses such as *D. scoparium*, *Hylocomium splendens* and *Pleurozium schreberi* growing together in Aisha forest, Tatarstan, Russia revealed that they have high peroxidase activity and diverse peroxidase isoforms [27]. However, *D. scoparium* had the highest activity, approximately double that of *H. splendens* and *P. schreberi*, and this activity was stimulated by the desiccation/rehydration cycle. Therefore, *D. scoparium* was chosen for further investigation of desiccation tolerance mechanisms. Although to date the full genome of *D. scoparium* has not been sequenced and no reports of DREB families are available for this species, we have previously described in *D. scoparium* how temperature and desiccation/rehydration stresses change the expression of genes encoding Class I ascorbate peroxidase (*DsAPX*) and Class III peroxidases (*DsPODs*) [28]. We hypothesized that in Dicranum, abiotic stress will also influence the expression of *DBF1*. In this study, we report the isolation of a cDNA from *D. scoparium* that encodes a new DNA-binding TF, designated as DsDBF1. Furthermore, we analyzed the physico-chemical properties and subcellular localization of protein and gene expression patterns after desiccation/rehydration, high and low temperature, paraquat, DCMU, and CdCl2 stresses. Our study showed that *DsDBF1* was significantly upregulated after exposure of *D. scoparium* to abiotic stresses, especially desiccation/rehydration, freezing temperature, paraquat, and CdCl2, suggesting that this TF plays multiple roles in the tolerance of the moss to abiotic stresses.

#### **2. Materials and Methods**

#### *2.1. Plant Material*

*Dicranum scoparium* Hedw. was collected in the Aisha Forest in Tatarstan, Russia (55◦53 21.3 N 48◦38 14.3 E). Plant material was placed between sheets of paper and left to dry slowly in the open air for 2 days before being stored in the refrigerator at +4 ◦C in the dark until usage [28].

### *2.2. Identification and Retrieval of DsDBF1protein*

Metatranscriptome data for the moss *D. scoparium* deposited to the Sequence Read Archive in the NCBI under accession numbers: PRJEB21674, ID: 393814 and PRJNA499105, ID: 499105 were extracted from the database [29,30]. The files were downloaded using the SRA Toolkit [31] and then converted to fastq format. FastQC software [32] was used to evaluate library quality control (QC). Adapter removal and trimming was done using Trimmomatic software version 0.39 [33]. After trimming, the reads were reassessed using the FastQC software [32]. Library assembly was performed using Trinity software [34]. All contaminants and foreign fungal and bacterial sequences were removed from the original data [29,30].

BLASTX [35] of representative sequences from mosses and other predicted taxa was used to determine the taxonomic classification of the identified transcripts (E-value < 1 × <sup>10</sup><sup>−</sup>6). To reduce transcript redundancy, moss transcripts containing the top hits were isolated to a separate file and filtered using the EvidentialGene package (https://sourceforge.net/ projects/evidentialgene/, accessed on 10 October 2022) with the default parameters. The DBF1 amino acid sequence from *P. patens* was used as a query in a TBLASTN search [36] for a similar protein among the filtered transcripts from *D. scoparium*.

A DBF1 transcript was found after using TBLASTN and open reading frames (ORFs) were detected using the Augustus gene prediction and AssemblyPostProcessor tools in Galaxy version 1.0.3.0 (https://usegalaxy.org/, accessed on 10 October 2022). To confirm the domain-identifying members of the *DBF1* gene family, the predicted sequence was submitted against PFAM [37], NCBI Conserved Domains Database (CDD) [38], Inter-ProScan [39], and HMMER [40].

#### *2.3. Cloning and Sequencing of DBF1 Gene*

Total RNA was extracted using the GeneJET Plant RNA Purification Mini Kit (Thermo Scientific, Vilnius, Lithuania). RNA concentration and purity were assessed using a NanoDrop® ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and integrity was confirmed using 1% (*m/v*) agarose gel electrophoresis. First strand and double strand cDNA were synthesized using Evrogen Mint 2 synthesis kit according to manufacturer's protocols.

To verify the *DBF1* from *D. scoparium* identified in silico, the *DBF1* sequence with the highest homology to *DBF1* from *P. patens* was cloned into the pAL2-T vector (Evrogen, Moscow, Russia) using primers: F TGGGTTCACACGATGCGGA; R ACGCTTTGAATC-CACTGACGG and then sequenced.

#### *2.4. Sequence Analysis*

BLASTN software available online at (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 12 October 2022) was used to perform a homology search to compare our sequenced *DsDBF1* with other genes in the database. Files in Fasta format were downloaded from the NCBI database after BLAST search and then subjected to multiple sequence alignments using Clustal Omega [41] and ClustalW [42] in MEGA X [43]. The Expasy ProtParam tool [44] was used to predict the physico-chemical properties of the DsDBF1 protein, including molecular weight, isoelectric point, instability index, and grand average of hydropathicity (GRAVY). Subcellular localization was predicted by MULocDeep [45].

The homologous sequences of DsDBF1 proteins obtained after BLASTX and other known DREB proteins from the NCBI database were aligned by ClustalW [42] in MEGA X [43]. A phylogenetic tree was constructed in MEGA X [43] using the neighbor joining method for 1500 bootstraps [46]. Evolutionary distances were calculated using the Poisson correction method [47] and all ambiguous positions were removed by pairwise deletion.

The MEME suite (http://meme-suite.org/index.html, accessed on 12 October 2022) was used to analyze DREB protein sequences to find conserved motifs with the following parameters: zero or one site per sequence, number of motifs (1–10), motif width (6–50) [48]. After MEME, the motif map was rebuilt using the TBtools software [49].

#### *2.5. Stress Treatments*

For stress treatment in this study, we followed the protocol developed in our early studies [28]. Before the experiment, 2 cm apical stem segments of dry mosses were prehydrated at +4 ◦C for 24 h on wet filter paper. For stress treatments, 0.2 g moss segments were incubated in 20 mL of 100 μM paraquat (1,1-dimethyl-4,4-bipyridylium dichloride), 100 μM DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea), or 100 μM CdCl2 for 1 and 12 h. Hydrated apical stem segments were also thermally stressed by their exposure to −20 ◦C or +30 ◦C for 1 or 12 h in a dark temperature-controlled chamber (Thermostat LOIP, St. Petersburg, Russia). In all treatments, hydrated mosses kept at room temperature served as controls.

For desiccation stress, three biological replicates per treatment were used, each containing 0.17 g dry mass from 2 cm apical stem segments. Initially, air-dry mosses were fully hydrated by immersing them in a 20 mL volume of distilled water for 1 h while slowly shaking them on an orbital shaker. Then, the hydrated moss was gently blotted with filter paper and placed in the desiccator above silica gel. Here, moss samples were taken at time 0 (after 1 h of hydration), and after 2, 24, and 72 h of desiccation. After 72 h of desiccation,

moss samples were rehydrated for 0.5 and 2 h. The change in relative water content (RWC) was monitored according to the protocol previously described in [28].

#### *2.6. RNA Extraction, cDNA Synthesis and RT-qPCR*

Samples exposed to stresses were immersed in liquid nitrogen, then, each sample was ground into a fine powder. For RT-qPCR, 0.1 g of material from each replicate was immediately frozen in liquid nitrogen and stored at −80 ◦C until use. Extraction of total RNA from *D. scoparium* thalli was performed using the RNeasy Plant Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. RNA concentration and purity were measured with NanoDrop® ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA), and the integrity was further evaluated by gel electrophoresis in a 1% (*w/v*) agarose gel. First strand cDNA was synthesized using protocols from the Evrogen Mint 2 synthesis kit.

The vector NTI Suite 9 software was used to design RT-qPCR primers with the following parameters: amplicon length from 60 to 300 bp and a Tm range of 55 to 65 ◦C. RT-qPCR was performed on CFX Connect™ Real-Time System (Bio-Rad Laboratories, Singapore) with qPCRmix-HS SYBR (Evrogen). The templates were amplified three times at 95 ◦C for 3 min followed by 40 cycles of amplification (94 ◦C for 10 s and 55/60 ◦C for 40 s). Melting curve analysis after RT-qPCR and gel electrophoresis examination of the amplified products were used to assess the specificity of the primers. The gene-specific primers used for RT-qPCR are listed in Table S1. Ribosomal RNA (*18S*), glyceraldehyde-3-phosphate dehydrogenase (*GAPDH2*) and α-tubulin (*α-TUB1* and *α-TUB2*) were used as internal controls for RT-qPCR normalization [28].
