**2. Results**

#### *2.1. Identification and Characterization of the S1fa Family Genes in Chinese Cabbage*

To identify and characterize the S1fa transcription factor genes in Chinese cabbage, we performed BLASTP searches against the Chinese cabbage genome database (http: //brassicadb.cn (accessed on 22 March 2022)) using three *Arabidopsis* S1fa protein sequences (AT2G37120, AT3G53370 and AT3G09735) as query sequences, and confirmed four candidate genes of the S1fa family in Chinese cabbage, including *Bra003132*, *Bra034084*, *Bra006994* and *Bra029784*. The S1fa family genes are distributed on different chromosomes of Chinese cabbage; *Bra034084, Bra029784, Bra003132* and *Bra006994* are located on A01, A05, A07 and A09, respectively (Figure 1A). The protein 3D structure of S1fa gene showed similar structural homology in Chinese cabbage (Figure 1B). The length of the S1fa genes is between 70—88 aa, with a molecular weight ranging from 7.8 to 9.3 kDa (Table 1). The isoelectric point of Chinese cabbage S1fa proteins is relatively high (pI > 10.38), indicating that they are rich in alkaline amino acids (Table 1). Subcellular location analysis showed that all S1fa genes were localized in the nucleus.

**Table 1.** The characteristics of the S1fa genes in Chinese cabbage.


**Figure 1.** *Cont*.

**Figure 1.** Chromosomal localization of the S1fa genes in Chinese cabbage and protein 3D structures. (**A**) Chinese cabbage has ten chromosomes, and the chromosome number (A01 to A10) is shown on the top of each chromosome. The position marked on the chromosome represents the location of the S1fa genes. (**B**) Protein 3D structure of Chinese cabbage S1fa genes.

#### *2.2. Phylogenetic Analysis of the S1fa Genes in Chinese Cabbage*

The phylogenetic analysis was used to investigate the evolution of the S1fa genes in Chinese cabbage. The S1fa proteins were compared with those in other species, including tomato, pepper, cotton, rice, *Arabidopsis*, cucumber, watermelon and rice, to investigate and explore the evolutionary relationships. A total of 27 S1fa proteins were clustered into three groups (I, II and III), which consisted of 6, 9 and 12 members, respectively. *Bra003132* was clustered into group I, while *Bra034084*, *Bra006994* and *Bra029784* were clustered into group II, indicating that Chinese cabbage S1fa genes have high homology with those in rice, cucumber and pepper (Figure 2A). Additionally, low bootstrap values in the phylogenetic tree are due to the divergence of the protein sequences that occur between Chinese cabbage and *Arabidopsis*, cotton and tomato during the evaluation. Multiple sequence alignments show that the amino acid sequences of the S1fa genes are highly conserved between Chinese cabbage and *Arabidopsis* (Figure 2B). The conserved domain of S1fa is highlighted in Figure 2B.

#### *2.3. Cis-Element Analysis of S1fa*

Cis-elements are the regions of non-coding DNA that regulate the transcription of the neighboring genes. The cis-elements of Chinese cabbage S1fa genes were identified in the promoter region as presented in Figure 3. The results show that the *Bra034084* gene is located on chromosome A07, which has 1 GATA-motif, 1 LTR, 1 TC-rich repeat, 1 TCA-element, 1 CGTCA-motif, 1 GT1-motif, 1 TGACG-motif, 2 AE-box, 3 AREs and 3 TCT-motifs. *Bra003132* has 1 LTR, 1 MBS, 1 AE-box, 1 CAT-box, 1 TCCC-motif, 1 TCTmotif, 1 LAMP-element, 2 CGTCA-motifs, 2 TGACG-motifs, 2 TGA-elements, 3 AREs, 3 MBSs, 4 ABREs and 6 G-boxes (Supplementary Table S1). *Bra006994* has 1 LTR, 1 TCAelement, 1 ARE, 1 Box II, 1 CAT-box, 1 TC-rich repeats, 2 TCT-motifs, 2 ABREs, 2 TCT-motifs and 3 O2-sites. *Bra029784* has 1 TCA-element, 1 ARE, 1 AE-box, 1 G-box, 1 Box-II, 1 *chs*-Unit 1 m1, 2 TC-rich repeats, 2 MBSs, 2 ABREs and 2 TCT-motifs, which are involved in facilitating a plant's physiological and biochemical mechanisms under abiotic stresses.

**B** 

**Figure 2.** Phylogenetic analysis and multiple sequence alignment of the S1fa proteins. (**A**) Phylogenetic tree of the S1fa genes in Chinese cabbage, tomato, pepper, cotton, rice, *Arabidopsis*, cucumber, watermelon and rice. The neighbor-joining tree was generated using the MEGA7 software with 100 bootstrap replicates. The different colored dots represent different plant species. (**B**) Multiple sequence alignment of S1fa proteins from Chinese cabbage and *Arabidopsis*. Multiple sequence alignment was performed using the MEGA7 software. The highlighted amino acids are highly conserved.

#### *2.4. Structure and Motif Analysis of the S1fa Genes*

To explore the features of the S1fa genes, the conserved motifs of the genes in Chinese cabbage were analyzed. The results show that S1fa consists of three common motifs, namely, motif 1, 2 and 3, as presented in Figure 4. Motif 1 is the largest motif with a length of 55 aa, which is localized in the middle of the S1fa gene, followed by motif 2 and motif 3, respectively. Similarly, the exon–intron structures of the S1fa genes were analyzed (Figure 4). *Bra006994*, *Bra003132* and *Bra029784* have the same structure, while *Bra034084* has a different structure. The coding sequence of *Bra006994*, *Bra003132* and *Bra029784* is localized on the left and right borders of the UTR, and shares a similar gene structure, but *Bra034084* does not have a UTR.

**Figure 3.** Cis-element analysis of the S1fa genes in Chinese cabbage. Different colors represent different cis-elements in the promoter region of the S1fa family genes in Chinese cabbage.

**Figure 4.** Analysis of motifs and gene structures of S1fa proteins in Chinese cabbage. (**A**) Protein motifs, location and phylogenetic trees in the S1fa family members. (**B**) The sequence of three identified motifs in Chinese cabbage. (**C**) Gene structure of the S1fa family member in Chinese cabbage.

## *2.5. Expression Profiles of the S1fa Genes in Different Tissues*

To explore the potential functions of the S1fa genes in growth and development, the tissue-specific characteristics were obtained from the Chinese cabbage database (http://brassicadb.cn/#/ (accessed on 22 March 2022)). The results show that the TPM (Transcript per million) values of the S1fa genes varied in different plant tissues. The S1fa genes were highly expressed in the silique of Chinese cabbage (Figure 5), while the expression level was the lowest in the leaf tissue. Comparative analyses of the S1fa genes show that *Bra034084* had the highest expression, followed by *Bra003132* and *Bra006994*, respectively. *Bra029784* had the least expression in all tissues except the silique tissues compared with other members of the S1fa genes. Moreover, the expression of the S1fa genes was downregulated in the leaf and flower tissues, while *Bra006994* showed no expression in the leaf and flower tissues. Taken together, these findings indicate that the S1fa genes are actively expressed in Chinese cabbage, which could play vital roles in Chinese cabbage growth and developmental process. Thus, it is necessary to investigate the functions of the S1fa genes in abiotic stress tolerance.

**Figure 5.** The transcription levels of the S1fa genes in different tissues, including root, leaves, flower, stem, callus and silique of Chinese cabbage. Data were obtained from Chinese cabbage database.

#### *2.6. Expression Patterns of the S1fa Genes under Abiotic Stress*

The S1fa transcription factor plays an important role in regulating plant growth and development, and abiotic stress tolerance. However, the involvement of the S1fa genes in response to abiotic stresses is not clear. To confirm the molecular mechanism of the S1fa genes in response to abiotic stresses, their transcript abundance was investigated under Hg, Cd and NaCl stresses (Figure 6). The expression levels of all four S1fa genes were investigated 24 h after the stress treatments. The S1fa genes were significantly expressed under abiotic stresses. Under Hg stress, the expressions of the *Bra034084* and *Bra029784* genes were significantly elevated compared with the control (CK) treatment, followed by *Bra003132*. Likewise, under Cd stress, the *Bra003132* and *Bra006994* genes showed high expression levels compared with CK, while NaCl stress significantly reduced the expression of the S1fa genes (Figure 6). These findings suggest that the S1fa genes are involved in and positively induced by various abiotic stresses in Chinese cabbage.

**Figure 6.** The expression levels of the S1fa genes under NaCl, Cd and Hg stresses. Different colors indicate different stresses and the letters above error bars represent significant differences at *p* > 0.05.
