**3. Discussion**

The WRKY transcription factor superfamily, as a recently described member of the TF family, has been studied by many researchers due to its numerous and diverse biological functions. Since the first reports of WRKY TFs [72], research conducted in different species [4,52,57,73,74] has shown that WRKY TFs play significant roles in plant development and stress responses. Recently, many studies of GmWRKY TFs have been based on biotechnological and RNA-Seq approaches [30,57]. However, these studies mainly reported genome-wide annotation of the WRKYs and structure analysis of some genes involved in response to abiotic and biotic stresses. Although these genes have been identified through biochemistry and bioinformatics approaches, knowledge about soybean stress tolerance was limited. In this study, based on qRT-PCR and RNA-Seq data, *GmWRKY12* was selected for investigation of stress tolerance in soybean (Figure S1).

According to classifications in the WRKY family [18,28,75], WRKY12 belongs to Group IIc and contains a single WRKY domain and a CX4-5CX22-23HXH zinc-finger motif. Recent studies have shown that the WRKYGQK heptapeptide, which can specifically recognize and bind to the W-box consensus sequence (TTGACY) in the promoters of target genes, can be replaced by WRKYGKK, WRKYGEK, WKKYEDK, or WKKYCEDK; variations of the WRKYGQK motif might change the DNA binding specificities to downstream target genes [75]. However, multiple sequence alignment results showed that WRKY12 in different species only harbor the same WRKYGQK heptapeptide, demonstrating that WRKY12 protein is evolutionarily conserved and can recognize and bind to downstream target genes (Figure 4A). The result was consistent with the results observed in other species [54,57,65,76,77]. Structural conservation determines functional specificity: in rice, *OsWRKY12* was related to normal plant growth and expression of *OsWRKY12* was low at the seedling stage but increased gradually with growth [78]; similar results were found in specific tissues in our study. *GmWRKY12* has low expression in young leaf, flower, one cm pod, pod shell 10 DAF, seed 10 DAF, seed 14 DAF, seed 21 DAF, seed 25 DAF, seed 28 DAF, seed 35 DAF, seed 42 DAF and root under normal conditions. At the pod shell 14 DAF and nodule stages, the expression levels gradually increase (Table S2), which may be because genes are differentially expressed at different growth stages, or may perform different activities, such as metabolism, nutrient absorption or material transformation. For example, at the nodule stage, plants are primarily vegetative, while at seed 42 DAF, plants are accumulating nutrients [57]. In addition, WRKY12 was related to plant flowering time: *Arabidopsis* plants overexpressing *MlWRKY12* showed early flowering phenotype [79]. WRKY12 and WRKY13 have opposite effects on flowering time in the action of GA [80]. Overexpression of three *Triticum* genes, *TaWRKY12*, *TaWRKY18* and *TaZFP2* induced the expression of some genes related to Pi absorption and transportation, enhancing the abilities of Pi uptake and Pi use efficiency in plants under low-Pi stress conditions [81]. Thus, *GmWRKY12*, like other WRKYs, is involved in plant growth and development.

There are many *cis*-acting elements in the *GmWRKY12* promoter region, such as MYC (ABA and wound responsive element), W-box (SA responsive element), ABER4 (ABA responsive element), MYB (drought responsive element), CCAATB (heat-responsive element), GT-1 (salt stress responsive element), DPBF (dehydration-responsive element) and GARE (GA-responsive element) (Table 3). The presence of these elements indicates that *GmWRKY12* may take part in various biotic and abiotic responses except for growth and development of plants. Research of tobacco transcription factors *NtWRKY12* and *TGA2.2* found that *NtWRKY12* alone was able to induce PR-1a::GUS expression to high levels, the PR-1a gene was salicylic acid-inducible to activate the expression of SA-inducible genes [82]. SA is an important endogenous molecule that activates plant hypersensitive response and systemic acquired resistance, which are often involved in disease resistance of plants [83]. As the closest orthologue of *AtWRKY12*, *BrWRKY1*2 from Chinese cabbage conferred enhanced resistance to *Pectobacterium carotovorum ssp. carotovorum* (*Pcc*) through transcriptional activation of defense-related genes [84]. Furthermore, *LrWRKY12* were responsive to SA and methyl jasmonate (MeJA) treatments and conferred more resistance to *B. cinerea* than in wild-type plants [85]. These results show that

WRKY12 plays an important role in disease defense of plants, mainly because WRKYGQK specifically binds to the W-box to induce expression of downstream target genes.

In addition to the significant roles of WRKY12 identified in development and disease defense of plants, WRKY12 also functions in plant stress responses. Under treatment with NaCl and PEG, the expression level of *THWRKY12* in *Tamarix* tissues was increased, the expression pattern of *THWRKY12* after ABA treatment was approximately the same as the expression level changes under NaCl and PEG treatment, showing that the gene may participate in regulating salt and drought tolerance through the signaling pathway regulated by ABA [86]. In our study, *GmWRKY12* was first screened following both drought and salt treatment using RNA-Seq. In order to confirm whether it was responsive to salt and drought stress, qRT-PCR was conducted and further showed that *GmWRKY12* was highly expressed under drought and salt treatment, which indicated that the gene was related to drought and salt tolerance (Figure 3). *Cis*-acting elements and expression pattern analysis of *GmWRKY12* also showed that it may participate in the ABA signaling pathway (Table 3 and Figure 5). However, compared to the high expression level under drought and salt treatment, on the condition of ABA, *GmWRKY12* had low expression. Resistance identification of *GmWRKY12* using a soybean hairy root assay further showed that *GmWRKY12* may be involved in regulating salt and drought tolerance by promoting the combination of *cis*-acting elements with drought and salt-related genes, thereby enhancing plant resistance (Figure 7). Similar results were also found in other studies [87–89].
