**3. Discussion**

Environmental influence impairs plant growth and development through oxidative stresses, which may change genome stability [10,20]. Plants have evolved a number of pathways to cope with ROS production and reduce the detrimental effects of abiotic stresses, including antioxidant enzymes, hormonal responses, the activation of transcription factors [8] and regulation of downstream genes [9,21].

Transcription factors (such as MYB, WRKY, ERF and NAC) play a crucial role in abiotic stress tolerance, and are involved in the transcriptional regulation of plant genes [22–25]. The S1fa transcription factor belongs to the smallest family of plants that are involved in plant growth and development [18]. However, no study has reported on their regulatory effects on abiotic stresses in Chinese cabbage. The present study was designed to investigate the physiological and molecular mechanism of the S1fa genes in abiotic stress tolerance. We identified and characterized four S1fa proteins in Chinese cabbage at the whole genome level, which were also compared with three *Arabidopsis* S1fa proteins. Systematic analyses including phylogenetic trees, gene and protein structures, motifs, physiochemical properties, miRNAs and cis-elements were conducted in the promoter region of the S1fa genes, and the effects of the S1fa genes on abiotic stress tolerance were investigated in yeast models. These findings provide novel insights into the functional characterization of the S1fa genes, which can be used in molecular breeding to enhance crop production and abiotic stress tolerance.

Based on phylogenetic analyses, the S1fa proteins were divided into three groups. Chinese cabbage S1fa proteins were classified in groups I and II, which share a high similarity with those in rice, pepper, cucumber and watermelon (Figure 2). The difference between these groups demonstrates that these proteins underwent great genetic variation after divergence, probably due to environmental influences, and that some gene fragments might have been lost during the evolutionary process [26]. The analysis of gene structure and motif suggested that the S1fa genes shared a similar exon–intron and motif structure, indicating there is a closer evolutionary relationship among the members in the same group but a functionally diversified relationship among the other group members [27–30]. The structure and motif analysis of Chinese cabbage S1fa genes showed a similar structure, which has three common motifs (Figure 4). Interestingly, based on motif analysis, the S1fa genes in Chinese cabbage can be divided into two subgroups, with *Bra003132* and *Bra006994* in one group, and *Bra029784* and *Bra034084* in the other (Figure 4). Protein analysis shows that motif 1 is highly conserved, which may be involved in or required for recognizing abiotic stress responsive cis-elements in response to stresses [31–33].

A gene's expression pattern can provide important indications for its biological functions. In the current study, the expression patterns of the S1fa genes were analyzed under abiotic stresses in Chinese cabbage (Figure 6). The S1fa genes showed different expression patterns in different tissues. The S1fa genes had the lowest expression in the leaves and the highest expression in the silique (Figure 5). *Bra006994* showed a minimum expression level in the leaf, flower and callus. The expression level of the S1fa genes was significantly different under different abiotic stresses (Figure 6). Under NaCl stress, *Bra034084* and *Bra029784* were significantly expressed compared with the other two S1fa genes. However, their expressions were significantly downregulated under Cd and Hg stresses (Figure 6). Taken together, these results suggest that the S1fa transcription factors are potentially involved in salinity stress tolerance, as well as plant growth and development [17,18].

The cis-element is a specific sequence on the promoter region of a given gene, which influences the expression of protein-coding and long non-coding RNA genes. Activation of the expression of the gene by binding with the cis-element is a common way of regulating developmental and physiological processes [33]. In plants, miRNAs act as a positive regulator in regulating related genes. Many studies have shown that miRNAs are involved in response to abiotic stresses [34]. *miR398b* negatively regulates the defense system in cotton and causes an adverse effect on plant growth. *miR1885* regulates plant growth and tolerance to viral infection through targeting *BraTNL1* and *BraCP24* genes in *Brassica* [35]. *Bra034084* and *Bra029784* were targeted by miRNAs including miR398b and miR1885, which might be involved in salt stress responses (Figure 7). Under abiotic stresses, the cis-elements are involved in controlling the transcriptional regulation of the core gene network [31]. The S1fa genes of Chinese cabbage contain a number of cis-elements including light and abiotic stress responsive elements, ABA, GA, methyl jasmonate (MeJA), and low and high temperature responsive elements (Figure 3). Previous studies have reported that hormonal cis-elements, such as O2-site, TGA-element, TGACG-motif, CGTCA-motif, TCA-element and ABRE motifs, are the key cis-regularity modules that stimulate the hormone signaling pathways under abiotic stresses [31,33]. The cis-elements activate specific transcription factors and their downstream genes, acting as a key cellular regulator in response to abiotic stresses [33]. These cis-elements may also be involved in salinity stress tolerance by regulating the specific hormonal signal transduction pathways [33].

The S1fa transcription factors have been reported to be involved in photomorphogenesis and abiotic stress tolerance [17]. However, their function has yet to be fully understood. Here, four Chinese cabbage S1fa genes were identified and cloned to investigate their function in abiotic stress tolerance using a yeast model (Figure 7). The results suggest that the *S1fa* genes did not respond to any abiotic stress. However, *Bra034084* and *Bra029784* were highly sensitive to NaCl stress (Figure 7(H)), suggesting that the S1fa transcription factors are involved in salinity stress tolerance. These findings are in line with a previous study, which shows that *OsS1fa* improves drought stress tolerance in *Arabidopsis* and increases the expression of the drought stress-related genes [18].

Plants exposed to abiotic stresses generate an excessive amount of ROS (H2O2 and O2 −), which is highly toxic and detrimental to proteins, lipids, DNA and carbohydrates, eventually leading to cell death [6,36,37]. The plant possesses an antioxidant enzyme defense system to normalize the overproduction of ROS. In this study, the overexpression of *Bra034084* and *Bra029784* significantly decreased the activities of antioxidant enzymes including SOD, POD and CAT under salinity stresses in yeast (Figure 11). On the other hand, the overexpression of *Bra034084* and *Bra029784* enhanced the accumulation of H2O2, O2 − and MDA in the *S1fa* overexpressing yeast cells compared with the wild type (EV) (Figure 11). These findings suggest that *Bra034084* and *Bra029784* inhibit the antioxidant enzyme activities, thereby leading to a higher hypersensitivity to salt stresses. *PsS1Fa2* overexpression in *Populus trichocarp* enhanced drought stress tolerance by increasing the activities of antioxidant enzymes (SOD and POD) and reducing the accumulation of MDA, H2O2 and O2 − [17]. A similar report has been conducted, which shows *CaDHN4* can protect against cold and salt stresses by activating the antioxidant enzyme defense system [38].

In eukaryotic organisms, the cell wall plays a dominant role in the protection of the cell from environmental influences [11]. In yeast, transcriptional re-programing can alter the expression of key genes for cell wall biosynthesis, energy generation, signal transduction and stress [11,39]. Several signaling pathway cascades such as *MAPK*, *MAPKK1*, *MAPKK2* and *Slt2* have been reported to be involved in cell wall biosynthesis [40,41]. In this study, the overexpressed S1fa genes, *Bra034084* and *Bra029784*, were located in the cytoplasm, and were translocated under NaCl stresses (Figure 7), and they significantly enhanced the expression level of cell wall biosynthesis genes (Figure 11). The *PKC1* (protein kinase C) pathway plays an important role in cell wall biogenesis, maintenance and cell integrity [41], and is regulated by *Bck1p*, *Mkk1p*, *Slt2p* and *Rom2p* [11,41]. Our study showed that the expression of these factors was significantly increased in the S1fa overexpressing yeast cells under salinity stresses, as presented in Figure 10. Additionally, the overexpression of *Bra034084* and *Bra029784* enhanced the transcript level of *Bck1p*, *Ptc1p Ccw14p*, *Crh1p*, *Mkk1p*, *Mkk2p*, *Rlm2* and *Rom2*, which are involved in the signaling pathway regulating cell wall integrity [11,39,40]. Subcellular localization analysis suggests that the S1fa gene was translocated from the cytoplasm to the cell wall under salt stresses, as presented in Figure 9, which in turn might promote the expression of many cell wall biosynthesis genes. These findings are consistent with previous studies, which show that abiotic stress induces a significant reduction in the activities of antioxidant enzymes and increases the contents of

ROS and MDA [11,14], thereby regulating the expression of the genes controlling cell wall integrity [11]. In summary, it can be concluded that the S1fa transcription factors activate genes involved in cell wall integrity under salinity stresses. Future studies are required to investigate the linkage between cell wall integrity and S1fa genes under abiotic stresses, especially salinity stresses.

#### **4. Materials and Method**

Chinese cabbage (Cv. Guangdongzao) seeds were soaked with 1% sodium hypochlorite for 3 min and then washed at least five times with ddH2O to remove the excessive sodium hypochlorite. Then, the seeds were germinated in <sup>1</sup> <sup>2</sup>MS media in a controlled growth chamber as described previously [42]. The uniform seedlings were transferred to a hydroponic culture and incubated for five more days before treated with 75 μM Cd, 75 mM Hg and 1 M NaCl, respectively. The samples were collected and ground in liquid nitrogen to extract the total RNA [43].

#### *4.1. Identification of the S1fa Genes in Chinese Cabbage*

In order to identify the S1fa genes in Chinese cabbage, the protein sequences of three S1fa genes from TAIR (www.Arabidopsis.org (accessed on 22 March 2022)) were downloaded and used as queries in Chinese cabbage genome database (http://brassicadb .cn (accessed on 22 March 2022)) with the BLASTP program [42]. All predicted Chinese cabbage S1fa proteins were confirmed through Pfam (http://pfam.xfam.org/ (accessed on 22 March 2022)) and SMART database. The physicochemical parameters including protein isoelectric point (pl), molecular weight (kDa) and length were calculated using the tools on the ExPASy server (http://web.expasy.org/compute\_pi/ (accessed on 22 March 2022)). The chromosomal locations and strand directions were obtained from the BRAD database (http://brassicadb.cn (accessed on 22 March 2022)), and the subcellular location of each protein was investigated using CELLO 2.5 (http://cello.life.nctu.edu.tw/ (accessed on 22 March 2022)) [29].

#### *4.2. Phylogenetic Trees and Sequence Alignment*

The full length protein sequences from Chinese cabbage, tomato, pepper, cotton, *Arabidopsis*, cucumber, watermelon and rice were obtained from the genome database and aligned using MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle (accessed on 22 March 2022)), which were used to construct the evolutionary tree using the MAGA7 software by the neighbor-joining method, and protein sequence alignment was performed as described previously [29].

#### *4.3. S1fa Structure and Conserved Motif*

The S1fa gene structure was analyzed using the online MEME program (http://meme -suite.org/tools/meme (accessed on 22 March 2022)), and the maximum number of motifs was set at 10. The structures of the S1fa genes were designed by using the online program GSDS 2.0 (http://gsds.cbi.pku.edu.cn/ (accessed on 22 March 2022)) [29]. The online tool PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 22 March 2022)) was used to analyze the cis-elements as described previously [28,29,44].
