**1. Introduction**

Chinese cabbage (*Brassica rapa*) is a winter vegetable crop that originated in China and is mainly cultivated in north China [1,2]. It is the largest vegetable crop that is produced throughout the year, and because of its excellent nutritional value, it is highly consumed [3]. As a leafy vegetable, Chinese cabbage is more sensitive to environmental influences, including NaCl, heavy metals, low and high temperature, etc., which cause a series of physiological, molecular and biochemical changes that negatively affect plant growth and production [4,5]. To cope with different environmental influences, molecular, cellular and biochemical responses are regulated through many series of pathways, inducing antioxidant enzymes, hormones and transcription factors (TFs), to reduce the detrimental effects of stresses [6]. Under abiotic stresses, the plant produces ROS (reactive oxygen species), which is highly toxic and reactive, and hence causes oxidative damage and cell death [7]. Plant cells exhibit high efficiencies in scavenging ROS through the well-established coordination of antioxidant enzymes (SOD, POD, CAT, GS, APX and GR) and non-enzymatic antioxidants (ascorbic acid and glutathione) [8]. The overproduction of ROS initially causes damaged cells, hormonal imbalance and decreased metabolic and

**Citation:** Anwar, A.; Zhang, S.; Wang, L.-X.; Wang, F.; He, L.; Gao, J. Genome-Wide Identification and Characterization of Chinese Cabbage S1fa Transcription Factors and Their Roles in Response to Salt Stress. *Antioxidants* **2022**, *11*, 1782. https:// doi.org/10.3390/antiox11091782

Academic Editor: Nafees A. Khan

Received: 5 July 2022 Accepted: 2 September 2022 Published: 9 September 2022

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enzymatic activities, and thus interferes with signaling pathways that are involved in carbohydrate, protein, lipid, chlorophyll and photosynthetic machinery [6–8].

Plants have established a wide range of physiological and biochemical mechanisms to avoid the harmful effects of environmental influences [9,10]. The overproduction of ROS is diminished through the regulation of gene and protein expression levels in the mitochondria, nucleus, chloroplast and cell wall [11]. The cell wall is a complex structure, which is the first and central protective barrier for abiotic stress [11]. A number of signaling cascades have been reported to be involved in the regulation of stress responses in cell walls, including cell wall integrity (CWI), high-osmolality glycerol (HOG) and protein kinase A (PKA) [11]. *AtMyb41* regulates osmotic and salinity stresses through the activation of cell wall biosynthesis genes [12]. Rice *R2R3-type* MYB transcription factor, *OsMPS*, negatively regulates the expression of hormone signaling genes, EXPANSIN and biosynthesis genes in the cell wall to enhance NaCl stress tolerance [13]. Wall-associated kinases (WAKs), a potential sensor for the cell wall, regulate the pectic signaling network of the cell wall under abiotic stresses [14].

Transcription factors are crucial regulators of plant abiotic stresses, and are involved in the regulation of the defense system to stabilize ROS production [9,15,16]. Among different transcription factors, the S1fa transcription factor is highly conserved and belongs to the smallest family of the plant kingdom [17]. The members in this family have a small molecular weight (7 to 9 KD) and an average length of 70 to 80 aa, and are mainly localized in the nucleus [17]. Although usually no more than five S1fa proteins are found in most plants, such as maize, rice, tomato, soybean, *Arabidopsis* and Chinese cabbage, *Arachis duranensis* has 126 S1fa proteins [18]. Chinese cabbage has four members of the S1fa proteins, including *Bra003132*, *Bra034084*, *Bra006994* and *Bra029784*, which bind to the cis-element of the site 1 binding site, one of the three highly conserved binding sites (site 1, 2 and 3) located in the promoter region. Previous studies have reported that spinach S1fa has a nuclear localization signal peptide and a DNA recognition motif, which may function as a transcription factor [19]. The S1fa genes are mainly expressed in roots and etiolated seedlings rather than leaves, indicating their involvement in growth and development [17,18]. The S1fa gene plays a key role in abiotic stress tolerance. Under abiotic stresses, the S1fa genes are the most downregulated genes in cotton [17]. Moreover, in Chinese cabbage, the S1fa gene shows significant responses to salinity stress, suggesting that it may act as the upstream gene for salt responsive genes. Two genes of the S1fa family, *PtS1Fa1* and *PtS1Fa2*, have been characterized in *Populous trichocarpa* [17]. The results show that the *PtS1Fa2* overexpression lines of *P. trichocarpa* increase in fresh weight, root length and chlorophyll accumulation under drought stresses. However, the overexpression of *PtS1Fa1* has no obvious effect on the drought stress response. These findings suggest that *PtS1Fa2* plays a key role in the activation of antioxidant enzymes such as SOD and POD to reduce the MDA, H2O2 and O2 − contents, and induces drought tolerance. The *OsS1fa* gene in rice confirms drought stress tolerance in *Arabidopsis* [18]. These results demonstrate that the *OsS1fa* gene is highly expressed in the leaf, culm and root. Drought tolerance-related genes, such as *LEA*, *GRF7*, *YODA*, *RD29A* and *CPK6*, are significantly expressed in the *OsS1fa* overexpression line under drought stresses, suggesting that *OsS1fa* plays a fundamental role in plant development and abiotic stress responses.

In this study, four members of the S1fa family were identified and characterized through the investigation of phylogeny, motif, gene structure, cis-element and miRNA in Chinese cabbage. Furthermore, the functions of the S1fa genes in response to abiotic stresses (Hg, Cd, Al, Co, Cu, mannitol (osmotic stress), salt and cold and heat stress) were investigated in yeast, which showed that two S1fa genes were highly sensitive to NaCl stress. The TPM value of the S1fa genes was measured in different plant tissues, such as the root, leaf, stem, flower, callus, silique and specific leaves of Chinese cabbage. The pRS416- GFP vector was used to test the subcellular localization of the S1fa genes under salinity stress in yeast. The significance of this study will be helpful for the understanding of the S1fa gene's function and boost the genetic modification of Chinese cabbage, which can

improve crop production and adaptation to environmental cues. It will be more interesting to explore the role of S1fa genes in the hormone signaling pathway, interaction and crosstalk to identify novel genes in Chinese cabbage. Future research on S1fa will offer the possibility of genetic engineering of crop varieties with enhanced crop production.
