Functional Characterisation of the Transcription Factor GsWRKY23 Gene from Glycine soja in Overexpressed Soybean Composite Plants and Arabidopsis under Salt Stress

WRKY proteins are a superfamily of transcription factors (TFs) that play multiple roles in plants’ growth, development, and environmental stress response. In this study, a novel WRKY gene called GsWRKY23 that is specifically upregulated in salt-tolerant Glycine soja accession BB52 seedlings was identified by transcriptomic analysis under salt stress. How the physiological functions and mechanisms of the GsWRKY23 gene affect salt tolerance was investigated using transformations of soybean hairy roots and Arabidopsis, including wild-type (WT) and atwrky23-mutant plants. The results showed that GsWRKY23 in the roots, stems, and leaves of BB52, along with its promoter in the cotyledons and root tips of GsWRKY23pro::GUS Arabidopsis seedlings, displayed enhanced induction under salt stress. GsWRKY23 localises to the nucleus and shows transcriptional activation ability in yeast cells. Compared to GsWRKY23-RNAi wild soybean hairy-root composite plants under salt stress, obvious improvements, such as superior growth appearance, plant height and fresh weight (FW), and leaf chlorophyll and relative water content (RWC), were displayed by GsWRKY23-overexpressing (OE) composite plants. Moreover, their relative electrolytic leakage (REL) values and malondialdehyde (MDA) contents in the roots and leaves declined significantly. Most of the contents of Na+ and Cl− in the roots, stems, and leaves of GsWRKY23-OE plants decreased significantly, while the content of K+ in the roots increased, and the content of NO3− displayed no obvious change. Ultimately, the Na+/K+ ratios of roots, stems, and leaves, along with the Cl−/NO3− ratios of roots and stems, decreased significantly. In the transgenic WT-GsWRKY23 and atwrky23-GsWRKY23 Arabidopsis seedlings, the salt-induced reduction in seed germination rate and seedling growth was markedly ameliorated; plant FW, leaf chlorophyll content, and RWC increased, and the REL value and MDA content in shoots decreased significantly. In addition, the accumulation of Na+ and Cl− decreased, and the K+ and NO3− levels increased markedly to maintain lower Na+/K+ and Cl−/NO3− ratios in the roots and shoots. Taken together, these results highlight the role of GsWRKY23 in regulating ionic homeostasis in NaCl-stressed overexpressed soybean composite plants and Arabidopsis seedlings to maintain lower Na+/K+ and Cl−/NO3− ratios in the roots and shoots, thus conferring improved salt tolerance.


Introduction
Soil salinization is an increasingly serious problem worldwide, and the global salinized land area has reached approximately 1 billion hectares, accounting for 7% of the total land area [1].In addition to natural soil salinity, climate change and human irrigation have also exacerbated soil salinization [2].Soil salinity is one of the major environmental stresses that restrict plants' growth and development, as well as crop yield.NaCl is the most soluble salt, with the highest content in soil [3].When plants are exposed to a high-salt environment, plants will first be subjected to osmotic stress, and the lower water potential in the external soil will lead to a decrease in the water absorption capacity of the plants' roots [4].Second, the high concentration of Na + will reduce the absorption of K + and stimulate the outflow of K + , resulting in insufficient intracellular K + concentration and inhibition of the enzyme catalysis controlled by K + , thus affecting normal metabolism in plants [5,6].As anions such as Cl − , NO 3 − , and SO 4 2− can be transported through nonselective anion channels or transports, excessive accumulation of Cl − will lead to reduced absorption of anions such as NO 3 − and SO 4 2− , resulting in the lack of nutrient elements such as N and S [7].Therefore, Na + and Cl − constitute the main components of ionic toxicity for plants under salt-stressed environments.During their long evolutionary process, plants have formed complex and effective signal networks that can transform extracellular stress factors into intracellular signals and mediate plants' response processes and adaptation mechanisms to various stresses by activating or inhibiting stress-related gene expression to improve adaptability to adverse environments.In this process, various transcription factors (TFs), which can be regulated as cascade switches, often play the role of upstream connection and coordination [8].
Transcription factors (TFs), also known as trans-acting factors, can activate or inhibit the transcription of downstream target genes by recognising cis-acting elements in the promoter of target genes, and they participate in a variety of biological processes in plants, including cell division, growth, development, ageing, and various abiotic or biotic stress responses [9].When plants suffer from environmental stress, TFs, such as bZIP, NAC, MYB, DREB, WRKY, and other families, can activate or suppress the expression of target genes by binding to cis-acting elements, and then regulate related biochemical reactions and physiological metabolic processes through the expressed substances to reduce or avoid greater damage to plants.Regulating or suppressing the expression of genes encoding these transcription factors will generally improve the adaptability of plants to various stresses, such as salt, drought, low temperature, pathogens, and insects [10,11].As one of the largest and most important TF families, the WRKY family derives its name from a highly conserved WRKY domain containing approximately 60 amino acid residues and a highly conserved WRKYGQK heptapeptide in its N-terminus, which is associated with DNA-binding activity.The C-terminus has a typical zinc-finger motif (C 2 H 2 or C 2 HC type), which is involved in protein interaction and assisted DNA binding [12,13].WRKY proteins can activate or suppress the transcription of target genes by specifically binding their domain to the T/CTGACC/T (W-box) cis-acting element in the target gene promoter.According to the number of WRKY domains and the type of zinc-finger motifs, WRKY proteins can be divided into three groups: group I proteins contain two WRKY domains and a C 2 H 2 -type (or C 2 HC-type) zinc-finger motif; group II proteins contain only one WRKY domain with a C 2 H 2 -type zinc-finger motif, which can be subdivided into five subgroups (IIa-IIe) according to the difference in amino acid sequence; group III proteins consist of a WRKY domain and a zinc finger of the C 2 HC type [13].Since SPF1, the first member of the WRKY gene family, was isolated from sweet potatoes [14], an increasing number of WRKY gene family members with multiple roles in plants have been identified.Many studies have shown that these WRKY genes mainly respond to biotic or abiotic stress [15,16] and play regulatory roles in leaf senescence [17], seed development [18], plant dormancy [19], and biosynthesis of secondary metabolites [20].For example, the expression of AtWRKY23 is controlled by auxin through the ARF7/19 transcriptional response pathway, which can stimulate flavonoid biosynthesis and is needed for proper root growth and embryo development [20].WRKY23 from cucumber (Cucumis sativus L.), which is homologous to AtWRKY23, may be involved in the positive regulation of fruit peel yellowing [21].
Cultivated soybeans (Glycine max), as the main source of plant-derived protein, edible oil, and animal feed, play an important role in global agricultural production, sustainable development, and the human diet [22].G. max was domesticated from its wild relative G. soja in China, and generally, G. soja displays relatively higher salt tolerance than G. max [23,24].During long-term domestication and improvement, some key genes in the G. soja species for adaptation to stressful environments have been lost, resulting in obviously decreased genetic diversity in G. max cultivars [25,26].Nevertheless, due to the lack of a reproductive barrier between G. soja and G. max, it is possible for these stress-tolerance-related genes in G. soja to be reintroduced into the cultivated soybeans through breeding approaches [27].Therefore, it is of great importance to discover and identify the key salt-tolerance genes in G. soja for molecular design breeding of salt-tolerant G. max cultivars.In our previous study, transcriptomic analysis was carried out on the roots of halophytic G. soja BB52 seedlings under NaCl treatment to explore the molecular mechanisms of its strong salt tolerance.A large number of differentially expressed genes (DEGs) were enriched in Ca 2+ -signalling-related pathways in the plant-pathogen interaction pathway [25].On this basis, according to the Ca 2+ signal action on the MPK4 protein, the transcription factor WRKY gene family was enriched downstream of the MPK4 regulation pathway, and the transcription factor GsWRKY23 gene, which was significantly upregulated in the roots of BB52 seedlings under salt stress, was identified.In this work, the function of the GsWRKY23 gene in the salt tolerance of G. soja accession BB52 was investigated through multiple experimental techniques, including tobacco leaf transient expression, protein transcriptional activation assay and subcellular localisation, overexpression or RNA interference (RNAi) transformation of G. soja hairy roots mediated by Agrobacterium rhizogenes, and transgenic Arabidopsis (including WT and atwrky23 mutant) mediated by Agrobacterium tumefaciens.The changes in the phenotype and physiological parameters of GsWRKY23-overexpressing/RNAi hairy-root soybean composite plants and transgenic Arabidopsis exposed to NaCl treatment were determined.The aims of this study were to reveal the physiological mechanisms of GsWRKY23 mediating the alleviation of ionic toxicity in G. soja plants under salt stress, and to provide a crucial scientific basis and gene resources for molecular design breeding and salt-tolerance improvement in cultivated soybeans or other crops in the future.

Discovery of the GsWRKY23 Gene and Amino Acid Sequence Analysis of Its Encoding Protein
In a previous study, Pi et al. [25] conducted a transcriptomic analysis of the roots of halophytic G. soja accession BB52 seedlings under NaCl treatment.On this basis, we screened 11 WRKY family members that play a role in regulating downstream gene expression involved in Ca 2+ -signalling-related pathways of the plant-pathogen interaction pathway, among which GsWRKY23 was the member with the most salt-induced upregulation (Table 1 and Figure 1A).The coding DNA sequence (CDS) of the GsWRKY23 protein contained 320 amino acids, and a DNA-binding domain was identified at amino acid positions 170~211, including a WRKY domain and a C 2 H 2 zinc-finger motif; thus, GsWRKY23 belonged to the WRKY IIc subfamily.Amino acid sequence alignment showed that GsWRKY23 shared 99.6%, 93.1%, 91.2%, 74.8%, 67.9%, 63.7%, and 55.7% sequence identity with GmWRKY3, GmWRKY54, VuWRKY23, AhWRKY23, MtWRKY23, JrWRKY23, and AtWRKY23, respectively (Figure 1B).Further analysis using conserved domain alignments revealed that the WRKY domain was highly conserved, indicating that these proteins may actually have functions similar to GsWRKY23 (Figure 1C).

Response of GsWRKY23 and Its Promoter to Salt Stress
When 2-week-old wild soybean BB52 seedlings were exposed to 120 mM NaCl for 24 h, the expression levels of GsWRKY23 in different tissues were analysed using qRT-PCR.At 0, 3, 6, 12, and 24 h of salt treatment, the expression patterns of GsWRKY23 in roots, stems, and leaves first increased and then decreased.The upregulation multiples in roots, stems, and leaves reached maxima at 6 h of salt treatment, which were 5.71, 4.97, and 27.49 times, respectively (Figure 2A).
When 2-week-old wild soybean BB52 seedlings were exposed to 120 mM NaCl for 24 h, the expression levels of GsWRKY23 in different tissues were analysed using qRT-PCR.At 0, 3, 6, 12, and 24 h of salt treatment, the expression patterns of GsWRKY23 in roots, stems, and leaves first increased and then decreased.The upregulation multiples in roots, stems, and leaves reached maxima at 6 h of salt treatment, which were 5.71, 4.97, and 27.49 times, respectively (Figure 2A).
The promoter region (1632 bp) of GsWRKY23 was inserted upstream of the uidA (GUS) gene of the PBI101 vector to obtain the recombinant reporter vector GsWRKY23pro::GUS, and GsWRKY23pro::GUS-transgenic Arabidopsis was then obtained by A. tumefaciens GV3101-mediated transformation (Figure S1A).When treated with 120 mM NaCl for 24 h, obvious GUS staining (blue colour) was observed in both the cotyledons and root tips of transgenic Arabidopsis seedlings when compared to the untreated seedlings (Figure 2B).Thus, both the GsWRKY23 gene and its promoter can respond to salt stress with an enhanced effect in G. soja.The promoter region (1632 bp) of GsWRKY23 was inserted upstream of the uidA (GUS) gene of the PBI101 vector to obtain the recombinant reporter vector GsWRKY23pro::GUS, and GsWRKY23pro::GUS-transgenic Arabidopsis was then obtained by A. tumefaciens GV3101mediated transformation (Figure S1A).When treated with 120 mM NaCl for 24 h, obvious GUS staining (blue colour) was observed in both the cotyledons and root tips of transgenic Arabidopsis seedlings when compared to the untreated seedlings (Figure 2B).Thus, both the GsWRKY23 gene and its promoter can respond to salt stress with an enhanced effect in G. soja.

Transcriptional Activation Ability and Subcellular Localisation of the GsWRKY23 Protein
The CDS of GsWRKY23 was cloned into the pGBKT7 to construct a pGBKT7-GsWRKY23 plasmid, and then pGBKT7 and pGBKT7-GsWRKY23 were introduced into Y2HGold yeast cells to detect the transcriptional activation ability of the GsWRKY23 protein.As shown in Figure 3A, the yeastY2HGold strain and Y2HGold transformed with pGBKT7 or pGBKT7-GsWRKY23 plasmids grew well on YPDA medium, but only the yeast strain transformed with the pGBKT7-GsWRKY23 grew on SD/-Trp/-His medium and turned blue when Xα-Gal was added.This indicates that GsWRKY23 activated the expression of the reporter genes (HIS and MEL1) in yeast cells and showed transcriptional activation activity.
and turned blue when X-α-Gal was added.This indicates that GsWRKY23 activated the expression of the reporter genes (HIS and MEL1) in yeast cells and showed transcriptional activation activity.
To explore the subcellular localisation of the GsWRKY23 protein, the CDS of GsWRKY23 without a termination codon was cloned into the pCAMBIA1300-eGFP vector to obtain the 35S:: GsWRKY23::eGFP recombinant plasmid, and then the empty vector pCAMBIA1300-GFP (35S::eGFP) and the fusion vector 35S::GsWRKY23::eGFP were transferred into the young leaves of tobacco plants by A. tumefaciens GV3101-mediated transformation.As shown in Figure 3B by laser confocal microscopy, compared with the leaf cells transformed with 35S::eGFP, obvious green fluorescence signals were observed only in the cell nuclei of tobacco leaves injected with 35S::GsWRKY23::eGFP.These results may indicate that the GsWRKY23 protein was mainly located in the nucleus and functioned as a transcription factor.To explore the subcellular localisation of the GsWRKY23 protein, the CDS of GsWRKY23 without a termination codon was cloned into the pCAMBIA1300-eGFP vector to obtain the 35S:: GsWRKY23::eGFP recombinant plasmid, and then the empty vector pCAMBIA1300-GFP (35S::eGFP) and the fusion vector 35S::GsWRKY23::eGFP were transferred into the young leaves of tobacco plants by A. tumefaciens GV3101-mediated transformation.As shown in Figure 3B by laser confocal microscopy, compared with the leaf cells transformed with 35S::eGFP, obvious green fluorescence signals were observed only in the cell nuclei of tobacco leaves injected with 35S::GsWRKY23::eGFP.These results may indi-cate that the GsWRKY23 protein was mainly located in the nucleus and functioned as a transcription factor.

Analysis of Salt Tolerance in GsWRKY23-Overexpressing and GsWRKY23-RNAi Wild Soybean Hairy-Root Composite Plants
Using the GsWRKY23-overexpressed vectors (GsWRKY23-OE) and RNA interference (GsWRKY23-RNAi) (Figure S2A) and a hairy-root transformation system mediated by A. rhizogenes, empty vector (EV), GsWRKY23-OE, and GsWRKY23-RNAi soybean hairyroot composite plants were obtained (Figures 4A and S2B-D).Under normal conditions, there were no apparent differences in the growth phenotype, FW per plant, plant height, leaf chlorophyll content and RWC, REL values, and MDA contents in roots and leaves among the EV, GsWRKY23-OE, and GsWRKY23-RNAi soybean plants (Figure 5).When exposed to 120 mM NaCl solution for 3 d, the growth of all soybean plants was found to be significantly inhibited, while the growth of GsWRKY23-OE was observed to be relatively good.Compared with EV plants, FW per plant, plant height, leaf chlorophyll content, and RWC of GsWRKY23-OE plants were obviously increased, and the changes in plant height, REL value, and MDA content of GsWRKY23-OE plants reached significant levels (p ≤ 0.05).However, the salt damage to GsWRKY23-RNAi plants was seriously aggravated, with more stunted plant height, clearly decreased leaf chlorophyll content, and increased root and leaf REL values and MDA contents, especially in the aspect of leaf-related physiological indices, e.g., leaf chlorophyll content decreased by 22.22%, and leaf REL value and MDA content increased by 62.62% and 42.94%, respectively, which all reached significant levels in comparison with EV (p ≤ 0.05) (Figure 5).
Furthermore, when compared with the control, the contents of Na + and Cl − significantly increased in the roots, stems, and leaves of EV, GsWRKY23-OE, and GsWRKY23-RNAi wild soybean plants under NaCl stress, while the K + content was significantly decreased and the NO 3 − content showed no visible change.When compared with GsWRKY23-RNAi plants, the Na + contents in the roots, stems, and leaves of OE plants decreased by 28.44%, 13.53%, and 4.86%, respectively, while the K + contents increased by 8.35%, 21.73%, and 3.12%, respectively; correspondingly, the Na + /K + ratios of roots, stems, and leaves were markedly decreased, by 33.89%, 28.87%, and 7.68%, respectively (p ≤ 0.05) (Figure 6A-C).Under NaCl treatment, and when compared with RNAi plants, the Cl − contents in the roots and stems of OE plants decreased by 24.61% and 19.33%, respectively, which resulted in remarkable decreases in the Cl − /NO 3 − ratio of roots and stems, by 21.52% and 26.88%, respectively (p ≤ 0.05) (Figure 6D-F).
Considering the ion-transporter-related genes as the pointcut, when GsWRKY23-OE and GsWRKY23-RNAi soybean composite seedlings were subjected to 120 mM NaCl solution for 6 h, compared with the control conditions, and except for the obvious downregulated expression of GsNRT2, the expression levels of GsHKT1-1, GsSOS1, GsNHX2, GsNRT2, GsCLC1, GsCLC-b2, and GsCLC-c2 in roots were significantly upregulated under salt treatment.Among them, the expression levels of GsHKT1-1 and GsSOS1 were significantly upregulated in GsWRKY23-OE plants in contrast to EV plants but were clearly downregulated in GsWRKY23-RNAi plants, and GsCLC-c2 and GsCLC-b2 exhibited the opposite trend.In addition, there were no remarkable differences in the expression levels of GsNHX2, GsNRT2, and GsCLC1 among the three soybean materials (Figure 4).Furthermore, when compared with the control, the contents of Na + and Cl − significantly increased in the roots, stems, and leaves of EV, GsWRKY23-OE, and GsWRKY23-RNAi wild soybean plants under NaCl stress, while the K + content was significantly decreased and the NO3 − content showed no visible change.When compared with GsWRKY23-RNAi plants, the Na + contents in the roots, stems, and leaves of OE plants decreased by 28.44%, 13.53%, and 4.86%, respectively, while the K + contents increased by 8.35%, 21.73%, and 3.12%, respectively; correspondingly, the Na + /K + ratios of roots, stems, Considering the ion-transporter-related genes as the pointcut, when GsWRKY23and GsWRKY23-RNAi soybean composite seedlings were subjected to 120 mM NaCl lution for 6 h, compared with the control conditions, and except for the obvious downr ulated expression of GsNRT2, the expression levels of GsHKT1-1, GsSOS1, GsNH GsNRT2, GsCLC1, GsCLC-b2, and GsCLC-c2 in roots were significantly upregulated un salt treatment.Among them, the expression levels of GsHKT1-1 and GsSOS1 were sign cantly upregulated in GsWRKY23-OE plants in contrast to EV plants but were clea downregulated in GsWRKY23-RNAi plants, and GsCLC-c2 and GsCLC-b2 exhibited opposite trend.In addition, there were no remarkable differences in the expression lev of GsNHX2, GsNRT2, and GsCLC1 among the three soybean materials (Figure 4).

GsWRKY23-Transgenic Arabidopsis Exhibits Enhanced Salt Tolerance
After the Arabidopsis atwrky23 mutant was confirmed by PCR identification (Figure S1B), transgenic WT-GsWRKY23 and atwrky23-GsWRKY23 homozygous lines (T 3 ) were generated in this study (Figure S1C).When cultured on 1 /2 MS medium, there was no visible variation in the seed germination rates of WT, atwrky23, WT-GsWRKY23, and atwrky23-GsWRKY23, all of which reached over 98% on the 7th day.When exposed to 1 /2 MS medium plus 75 mM or 150 mM NaCl, the seed germination rates of the four Arabidopsis materials were all reduced, but WT-GsWRKY23 and atwrky23-GsWRKY23 displayed relatively higher germination rates (96.81% and 93.72%, respectively) than WT and atwrky23 (81.92% and 75.0%, respectively) under 150 mM NaCl treatment on the 7th day (Figure 7A,B).nificantly increased.When the GsWRKY23 gene was transformed into Arabidopsis WT and the atwrky23 mutant, the salt tolerance of WT-GsWRKY23 and atwrky23-GsWRKY23 was enhanced, and this was simultaneously accompanied by the obvious recovery of FW per plant, leaf chlorophyll content, and RWC, as well as a significant decrease in REL value and MDA content in the shoots (p ≤ 0.05), and the mitigation effect of salt damage on atwrky23-GsWRKY23 was more obvious (Figure 7D-H).When the abovementioned 3-week-old Arabidopsis plants were treated with 120 mM NaCl for 7 d, their growth was markedly inhibited, and the WT and atwrky23-especially the mutant-suffered the most damage, while the WT-GsWRKY23 and atwrky23-GsWRKY23 showed a relatively better growth phenotype (Figure 7C).Compared with WT plants under salt stress, the FW per plant, leaf chlorophyll content, and RWC of atwrky23 were significantly decreased, while the REL value and MDA content in shoots were significantly increased.When the GsWRKY23 gene was transformed into Arabidopsis WT and the atwrky23 mutant, the salt tolerance of WT-GsWRKY23 and atwrky23-GsWRKY23 was enhanced, and this was simultaneously accompanied by the obvious recovery of FW per plant, leaf chlorophyll content, and RWC, as well as a significant decrease in REL value and MDA content in the shoots (p ≤ 0.05), and the mitigation effect of salt damage on atwrky23-GsWRKY23 was more obvious (Figure 7D-H).
Further analysis of the contents of Na + , K + , Cl − , and NO 3 − , as well as the ratios of Na + /K + and Cl − /NO 3 − , in the roots and shoots of WT, atwrky23, WT-GsWRKY23, and atwrky23-GsWRKY23 plants under salt stress showed that, compared with the control, salt treatment significantly increased the contents of Na + and Cl − in the roots and shoots of the above four Arabidopsis plants, especially in the shoots; for example, the contents of Na + and Cl − in WT increased by 10.71 and 5.47 times, respectively, and those of atwrky23 increased by 16.33 and 6.76 times, respectively.However, the contents of Na + and Cl − in the shoots of WT-GsWRKY23 and atwrky23-GsWRKY23 only increased by 4.29, 2.54 and 4.97, 3.07 times, respectively (Figure 8A,D).

Identification and Characteristics of the Transcription Factor GsWRKY23 Gene from t Salt-Tolerant G. soja Accession BB52
Under saline environments, a great amount of Na + and Cl -enters plants' cell the cellular absorption of K + and NO3 − clearly declines, leading to sharp rises in the N Under salt stress, the contents of K + and NO 3 − in the roots and shoots of WT and atwrky23 declined significantly, but the decrease in K + content in the roots and shoots of WT-GsWRKY23 and atwrky23-GsWRKY23 was obviously lower.Furthermore, the NO 3 − contents in the roots and shoots increased significantly compared with those of WT and atwrky23.Among them, the increase in NO 3 − content in the shoots was larger, increasing by 46.12% and 77.80% compared with WT and atwrky23, respectively, which already exceeded the control level (p ≤ 0.05) (Figure 8B,E).
The above changes resulted in the ratios of Na + /K + and Cl − /NO 3 − in the roots and shoots of WT and atwrky23 plants under salt stress being significantly higher than the control levels, and the increases in atwrky23 plants were greater.The ratios of Na + /K + and Cl − /NO 3 − in roots increased by 8.16 and 12.41 times, respectively, while those in shoots increased by 30.02 and 10.85 times, respectively.Under the transformation of the GsWRKY23 gene into Arabidopsis WT and the atwrky23 mutant, the ratios of Na + /K + and Cl − /NO 3 − in the roots and shoots of WT-GsWRKY23 and atwrky23-GsWRKY23 plants decreased significantly (p ≤ 0.05), especially in atwrky23-GsWRKY23 plants, whose ratios of Na + /K + and Cl − /NO 3 − in roots decreased by 69.42% and 72.88%, respectively, while those in the shoots decreased by 80.89% and 75.10%, respectively (Figure 8C,F).

Identification and Characteristics of the Transcription Factor GsWRKY23 Gene from the Salt-Tolerant G. soja Accession BB52
Under saline environments, a great amount of Na + and Cl -enters plants' cells, and the cellular absorption of K + and NO 3 − clearly declines, leading to sharp rises in the Na + /K + and Cl − /NO 3 − ratios in plants, disrupted ionic homeostasis, and a series of physiological metabolism disorders and final plant growth inhibition [28].The salt-borne G. soja accession BB52 was discovered and collected from the coastal area in Shandong Province, China, and many years of research have suggested that the BB52 accession used in this work possesses a higher salt tolerance than other G. soja species (such as N23227) and G. max cultivars (such as Jackson and N23674) [23,24,29].Compared with the G. max cv.N23674, G. soja BB52 materials display stronger salt tolerance, which is mainly related to their superior ability to accumulate the absorbed Na + and Cl − in the roots and reduce the transport and distribution to the stems and leaves under salt stress [23][24][25].Furthermore, maintaining anion homeostasis by maintaining relatively high NO 3 − levels in the stems and leaves of soybean plants to reduce the Cl − /NO 3 − ratio under salt treatment is also attributed to their stronger salt tolerance [24,29].
In the present study, previous transcriptomic data were used to analyse the differentially expressed genes (DEGs) in the roots of salt-stressed BB52 seedlings, and the significantly upregulated GsWRKY23 was screened downstream of the MPK4 regulation pathway (Figure 1A).This is consistent with the tissue expression patterns of the GsWRKY23 gene in BB52 seedlings observed during the 24 h of 120 mM NaCl treatment (Figure 2A), and it may also indicate enhanced induction of the GsWRKY23 gene under NaCl treatment.Meng et al. found that specifically upregulated genes in salt-tolerant plant species may play more positive roles in salt stress response and adaptation [30].The deeper GUS staining of the transgenic GsWRKY23pro::GUS Arabidopsis seedlings (Figure 2B) may suggest that the promoter of the GsWRKY23 gene also displayed an enhanced response to NaCl stress.Moreover, obvious transcriptional activation activity of GsWRKY23 was verified using a yeast system (Figure 3A), and transient transformation of tobacco indicated that GsWRKY23 was expressed in the nucleus (Figure 3B), which indicates that GsWRKY23 is a nucleus-localised protein and functions as a transcription factor.

Mechanisms of the GsWRKY23 Gene Conferred Salt Tolerance to Overexpressed Soybean Composite Plants and Arabidopsis
To date, the WRKY23 gene and its homologues have been reported in plants such as Arabidopsis thaliana, Cucumis sativus, Populus tremula, Oryza sativa, and G. max, and this suggests biological functions involved in the responses of plants to stressful environments.For example, overexpression of AtWRKY30 enhanced the seed germination rate of transgenic Arabidopsis thaliana under salt and oxidative stresses [31].OsWRKY42, as a negative regulator of jasmonic acid (JA)-mediated plant stress responses, conferred enhanced salt tolerance to OsWRKY42-transgenic Arabidopsis, which was mainly related to inducing anthocyanin production and reducing ROS levels [32].In G. max, Li et al. found that GmWRKY45-transgenic Arabidopsis plants displayed a significantly higher survival rate and soluble sugar content and, thus, improved salt tolerance compared to WT plants [33].Zhou et al. found that GmWRKY21-transgenic Arabidopsis plants were tolerant to cold stress, whereas GmWRKY54 conferred salt and drought tolerance, and GmWRKY13-transgenic Arabidopsis seedlings were more sensitive to NaCl and mannitol stress [34].Bao et al. found that overexpression of PcWRKY33 from Polygonum cuspidatum increased salt sensitivity in transgenic Arabidopsis seedlings by inhibiting the increased expression of AtSOS1, AtSOS2, AtSOS3, and AtNHX1 under salt treatment, accumulating excessive Na + and thereafter increasing the Na + /K + ratio [35].Song et al. reported that SbWRKY50, a negative transcription factor of the salt stress response in sweet sorghum, enhanced salt sensitivity in transgenic Arabidopsis by binding to the W-box in the upstream promoter of AtSOS1 and, accordingly, significantly increased the Na + content and Na + /K + ratio in roots [36].Currently, there are few reports on the G. soja transcription factor WRKY gene family, such as GsWRKY15a, which is involved in the control of seed size in wild soybeans and was significantly more weakly expressed than its orthologous GmWRKY15a in G. max during pod development [37].Compared to WT, GsWRKY20-transgenic Arabidopsis displayed enhanced drought tolerance, which was associated with increased susceptibility to ABA and facilitation of ABA-induced stomatal closure [38].Furthermore, GsWRKY20-overexpressing alfalfa showed increased salt tolerance as a result of accumulating less Na + and more K + in both leaves and roots [39], and GsWRKY20-overexpressing soybeans displayed strongly increased drought tolerance, with lower stomatal density and faster stomatal closure [40].
However, there have been no reports on the GsWRKY23 gene of wild soybeans and its related functions under salt stress or other stressful conditions.Therefore, the physiological functions of GsWRKY23 in the responses of overexpressed hairy-root composite soybean plants and transgenic Arabidopsis to NaCl treatment were investigated for the first time in this study, to further decipher the molecular mechanisms of the G. soja BB52 accession with strong salt tolerance in terms of transcription factors regulating functional gene expression.In this work, the Na + /K + ratios in the roots, stems, and leaves of GsWRKY23-OE soybean hairy-root composite plants, as well as Cl − /NO 3 − in roots and stems, were significantly lower than those of GsWRKY23-RNAi plants under NaCl treatment (Figure 6C,F).These findings are also consistent with the enhanced salt tolerance phenotype (Figure 5A-C) or mitigated salt injury indices (Figure 5D-G) of GsWRKY23-OE plants compared to GsWRKY23-RNAi plants.Du et al. reported that RtWRKY1-transgenic Arabidopsis displayed enhanced salt stress tolerance in addition to exhibiting lower MDA content, Na + content, and Na + /K + ratio than WT plants under NaCl treatment, resulting in upregulated AtSOS1 expression, but no effect on the expression level of AtNHX1 was observed in response to NaCl stress [41].OsWRKY50-overexpressing rice plants displayed increased tolerance to salt stress compared to WT plants by improving the expression of OsLEA3, OsRAB21, OsHKT1;5, and OsP5CS1 under NaCl stress [16].In our study, we also found that the expression levels of GsHKT1-1 and GsSOS1 were significantly upregulated in the roots of NaCl-treated GsWRKY23-OE plants in contrast to EV plants, but they were clearly downregulated in GsWRKY23-RNAi plants (Figure 4B,C), which may be causally related to the significantly lower Na + /K + ratios in the roots, stems, and leaves of saltstressed GsWRKY23-OE plants compared to those of GsWRKY23-RNAi plants.However, no similar situation was found for the other ion-transporter-related coding genes, such as GsNHX2, GsNRT2, GsCLC1, GsCLC-b2, and GsCLC-c2; in particular, the performance of GsCLC-b2 and GsCLC-c2 (their encoded proteins are generally thought to be related to the accumulation of anions such as Cl − and NO 3 − in the cell vacuoles of plants under salt stress [24,29]) was opposite to that of GsHKT1-1 and GsSOS1 (Figure 4D-H).When the GsWRKY23 gene was transferred into Arabidopsis WT plants and atwrky23 mutants, both transgenic plants displayed alleviated salt injury, especially atwrky23-GsWRKY23 (Figure 7C).Correspondingly, the ratios of Na + /K + and Cl − /NO 3 − in the shoots and roots of WT-GsWRKY23 and atwrky23-GsWRKY23 were significantly lower than those of WT and atwrky23 (Figure 8C,F).Overexpression of GsWRKY23 also enhanced the expression of AtSOS1 and AtHKT1 in the shoots of transgenic Arabidopsis WT and atwrky23 seedlings under salt treatment (Figure S3).These results indicate that GsWRKY23 is involved in alleviating the ionic toxicity of overexpressed soybean and Arabidopsis plants under salt stress by regulating ionic uptake and transport in plants to adjust the ratios of Na + /K + and Cl − /NO 3 − for ionic homeostasis.Alabd et al. reported that PpABF3 could target the G-box cis-element in the promoter of PpWRKY44, and PpWRKY44 directly bound to a W-box on the promoter of PpALMT9 to activate its expression and, thus, was positively involved in salinity-induced malate accumulation and pear fruit quality [42].However, little is known about the downstream target genes and/or interacting proteins of GsWRKY23.Further studies are needed to identify these genes and/or interacting proteins, and to better understand how GsWRKY23 (alone or together with its interacting proteins) regulates target gene expression and functions for maintaining ionic homeostasis and enhancing antioxidant capacity to alleviate salt damage or attribute strong salt tolerance to G. soja BB52 plants.In addition, considering that GmWRKY3 and GsWRKY23 possess highly similar amino acid sequences (Figure 1B,C), joint exploration of the role of GmWRKY3 may better assist us in fully revealing the salt tolerance function of GsWRKY23.
In conclusion, the transcription factor GsWRKY23 gene, which was significantly upregulated under NaCl stress, was screened for the first time from the genome of the salt-tolerant G. soja BB52 accession.Both the GsWRKY23 gene and its promoter displayed an enhanced response to NaCl stress.GsWRKY23 is a nucleus-localised protein that functions as a transcription factor.Analysis of phenotypic and physiological indices of EV, GsWRKY23-OE, and GsWRKY23-RNAi hairy-root composite soybean plants under salt stress indicated that GsWRKY23 played a positive role in the process of salt stress response and adaptation, which could reduce the contents of Na + and Cl − in plants and increase their K + content to maintain lower Na + /K + and Cl − /NO 3 − ratios, along with ionic homeostasis for contributing to stronger salt tolerance.These similar effects were further verified in GsWRKY23-transgenic Arabidopsis.These findings not only provide an important research basis for further revealing the molecular functions of the GsWRKY23 gene and the mechanisms underlying the strong salt tolerance of G. soja BB52 materials, but also provide a valuable theoretical basis and gene resources for molecular genetic improvement and germplasm innovation of salt-tolerant soybean cultivars and other crops.

Phylogenetic Analysis and Multiple Alignment of Amino Acid Sequences of the WRKY Family
The amino acid sequence of GsWRKY23 (Glysoja.02G002639)was obtained from the wild soybean database (http://www.wildsoydb.org/Gsoja_W05/(accessed on 5 June 2023)), and amino acid sequences of the WRKY23 family members with high homology to GsWRKY23 were retrieved and downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/(accessed on 6 June 2023)).The phylogenetic tree of the WRKY family was constructed using the neighbour-jointing (NJ) method with MEGA X software, and the amino acid sequence alignment of WRKY family members was performed using DNAMAN 8 software.Seeds of G. soja BB52 were surface-sterilised with 75% ethanol, soaked with deionised water for 12 h, and germinated in the dark at 25 • C.Then, the germinated seeds were grown in plastic pots containing quartz sand fertigated with 1 /2-strength Hoagland nutrient solution, and then maintained in a greenhouse at 22~25 • C with a 16 h light/8 h dark photoperiod, light irradiation of approximately 100 µmol m −2 s −1 , and 60~70% relative humidity.When the first trifoliate leaves were expanded, the seedlings were cultured with 1 /2-strength Hoagland solution containing 120 mM NaCl for 0, 3, 6, 12, and 24 h, and then the roots, stems, and leaves were sampled, immediately frozen in liquid nitrogen, and stored at −80 • C. Total RNA was isolated from plants using the Spin Column Plant Total RNA Purification Kit (Sangon Biotech, Shanghai, China).The reverse transcription was performed using Hifair II 1st Strand cDNA Synthesis SuperMix (Yeasen, Shanghai, China).Quantitative real-time PCR (qRT-PCR) was performed on an ABI 7500 fluorescence quantitative PCR instrument (Thermo Fisher Scientific China, Inc., Shanghai, China) using the 2 × Hieff qPCR SYBR Green Master Mix (Low Rox Plus) (Yeasen).Three independent biological replicates and three independent technical replicates were included for each sample.The soybean UBI3 gene (LOC114401261) was used as an internal control, and the relative expression was calculated using the 2 −∆∆Ct method [43].All of the primers used for qRT-PCR are listed in Table S1.

Expression Vector Construction
The promoter (1632 bp upstream of ATG) of GsWRKY23 was inserted upstream of the GUS (uidA) gene of the PBI101 vector to obtain the recombinant vector GsWRKY23pro::GUS.The CDS of GsWRKY23 was amplified by PCR using the specific primers (Table S1) and inserted into the yeast expression vectors pGBKT7, plant binary vectors pCAMBIA1300-GFP, and pFGC5941, to generate the fusion constructs pGBKT7-GsWRKY23, 35S::GsWRKY23::eGFP (without a stop codon), and 35S::GsWRKY23 overexpression (OE) vector.A 200 bp fragment was amplified from the CDS of GsWRKY23 as the RNAi target and inserted upstream and downstream of the CHSA intron of the pFGC5941 vector in forward and reverse orientations, respectively, to obtain the GsWRKY23-RNAi vector.The CDS of OsMADS (encoding a nuclear locator protein as a label) without a stop codon was inserted into the pCX-DR-RFP vector to obtaine the RFP fusion construct 35S::OsMADS::RFP.

GUS Staining of Transgenic Arabidopsis
The fusion construct GsWRKY23pro::GUS was transformed into Arabidopsis Col-0 plants using the A. tumefaciens strain GV3101-mediated floral dip method [44].Ten-day-old transgenic Arabidopsis seedlings planted on 1 /2 MS agar medium were stressed with 120 mM NaCl solution for 24 h, and the untreated seedlings were used as a control.Then, these seedlings were sampled for the GUS staining assay as described by Jefferson et al. [45].

Transcriptional Activation Assay of GsWRKY23 in Yeast
A transcriptional activation assay of GsWRKY23 was conducted as described by Liu et al. [46].Briefly, the fusion plasmid pGBKT7-GsWRKY23 was introduced into the yeast strain Y2HGold, and the empty vector pGBKT7 was used as a negative control.The strains carrying pGBKT7 or pGBKT7-GsWRKY23 and the Y2H strain were inoculated on YPDA, SD/-Trp/-His, and SD/-Trp/-His/X-α-Gal media to observe the growth of the strains and identify the transcriptional activation of GsWRKY23.

Subcellular Localisation Analysis of GsWRKY23
The fusion constructs 35S::GsWRKY23::GFP and 35S::OsMADS::RFP were transformed into the A. tumefaciens strain GV3101.Leaves of four-week-old tobacco plants were cotransformed with the fusion expression vector 35S::GsWRKY23::eGFP or 35S::OsMADS::RFP as described by He et al. [11], and the empty vector 35S::eGFP was used as a control.Then, the injected tobacco plants were cultured normally in a greenhouse for 24~48 h.Then, the GFP and RFP signals were visualised under a laser confocal fluorescence microscope (Ultra VIEW VoX, PerkinElmer, Waltham, MA, USA).

Salt-Tolerance Assays of GsWRKY23-OE and -RNAi Soybean Hairy-Root Composite Plants
The 35S::GsWRKY23-OE vector, -RNAi vector, and empty vector (pFGC5941) were transformed into A. rhizogenes K599, and soybean hairy-root transformation was mediated according to the methods described in our previous work [47].Positive transgenic soybean hairy-root composite plants were identified by PCR, and then they were transferred to 1 /2-strength Hoagland solution with or without 120 mM NaCl, and the plants transformed with an empty vector (EV) were used as negative controls.After 6 h, total RNA was extracted from the hairy roots of plants under control and NaCl treatments.Using the same method as previously described, qRT-PCR was used to detect the expression level changes of GsWRKY23 and the ion-transporter-related genes (such as GsHKT1-1, GsSOS1, GsNHX2, GsNRT2, GsCLC1, GsCLC-b2, and GsCLC-c2) in EV, GsWRKY23-OE, and GsWRKY23-RNAi plants.Three independent biological replicates and three independent technical replicates were included for each sample.The specific primers used for qRT-PCR are also listed in Table S1.After 3 d, phenotypic analysis of EV, GsWRKY23-OE, and GsWRKY23-RNAi plants was performed.Physiological parameters such as FW per plant, plant height, leaf chlorophyll content and RWC, REL values, and MDA contents in the roots and leaves were measured as described in our previous work [48].The roots, stems, and leaves were sampled, dried, and ground into powder for the assays of the contents of Na + , K + , Cl − , and NO 3 − according to the method described by Zhou and Yu [49].

Salt-Tolerance Analysis of GsWRKY23-Transgenic Arabidopsis WT and Mutant Plants
The Arabidopsis atwrky23 mutant was obtained from the Arashare platform, and homozygous mutants were screened using the three-primer method.The vector GsWRKY23-OE was transformed into Arabidopsis WT and mutant plants using the floral dip method [44].Homozygous T 3 lines of WT-GsWRKY23 (such as L1, L2, L3) and atwrky23-GsWRKY23 (such as l1, l2, and l3) were obtained.The seeds of WT, atwrky23, WT-GsWRKY23, and atwrky23-GsWRKY23 were sterilised with 75% ethanol and sown on 1 / 2 MS containing 0 (control), 75, and 150 mM NaCl, kept at 4 • C for 2 d and cultured horizontally in a greenhouse.Germination rates were calculated daily and photographed on the 7th day.Furthermore, seeds of the WT, atwrky23, and transgenic lines were sown in pots filled with peat moss and vermiculite (1:1).Three-week-old Arabidopsis plants were stressed with 120 mM NaCl for 7 d for phenotypic analysis, and the untreated plants were used as controls.Measurements of FW per plant, leaf chlorophyll content and RWC, REL values, and MDA contents in the shoots were also conducted.Finally, the shoots and roots were sampled, dried, and ground into powder for the assays of the contents of Na + , K + , Cl − , and NO 3 − .

Data Analysis
Data were analysed and presented as the mean ± SD for each treatment (n = 3, except for FW per plant and plant height, where n = 6, and seed germination of Arabidopsis where n = 32) using SPSS software (version 20.0).Significant differences among means were determined by Duncan's test at the p ≤ 0.05 significance level.

Figure 1 .
Figure 1.Screening of GsWRKY23 and its encoding protein amino acid sequence analysis: (A) Screening the Ca 2+ -signal-related pathway of plant-pathogen interaction and the expression change heatmap of enriched WRKY transcription factors in the transcriptome data.(B) Phylogenetic analysis of GsWRKY23 and the closely related WRKY protein in other plant species.The selected WRKYs are GmWRKY3 (AJB84597.1)and GmWRKY54 (NP_001237438.1) from Glycine max, VuWRKY23 (XP_047168319.1) from Vigna umbellate, AhWRKY23 (XP_029144762.1) from Arachis hypogaea, JrWRKY23 (XP_018824247.2) from Juglans regia, MtWRKY23 (XP_003625994.2) from Medicago truncatula, and AtWRKY23 (NP_182248.1)from Arabidopsis thaliana.(C) Alignment of the amino acid sequence of GsWRKY23 with its homologous proteins.Identical amino acids are indicated with black shadowing and GsWRKY23 is indicated with red star.Features of the sequence include a

Figure 1 .
Figure 1.Screening of GsWRKY23 and its encoding protein amino acid sequence analysis: (A) Screening the Ca 2+ -signal-related pathway of plant-pathogen interaction and the expression change heatmap of enriched WRKY transcription factors in the transcriptome data.(B) Phylogenetic analysis of GsWRKY23 and the closely related WRKY protein in other plant species.The selected WRKYs are GmWRKY3 (AJB84597.1)and GmWRKY54 (NP_001237438.1) from Glycine max, VuWRKY23 (XP_047168319.1) from Vigna umbellate, AhWRKY23 (XP_029144762.1) from Arachis hypogaea, Jr-WRKY23 (XP_018824247.2) from Juglans regia, MtWRKY23 (XP_003625994.2) from Medicago truncatula, and AtWRKY23 (NP_182248.1)from Arabidopsis thaliana.(C) Alignment of the amino acid sequence of GsWRKY23 with its homologous proteins.Identical amino acids are indicated with black shadowing and GsWRKY23 is indicated with red star.Features of the sequence include a WRKY domain (WRKYGQK) and a C 2 H 2 zinc-finger domain (both highlighted with the bold black lines above the alignment).

Figure 2 .
Figure 2. Tissue expression patterns of GsWRKY23 gene in wild BB52 soybean seedlings during 24 h of 120 mM NaCl treatment (A), and GUS staining of wild-type and GsWRKY23pro::GUS Arabidopsis seedlings under 120 mM NaCl for 24 h; bar = 3 mm (B); different letters indicate groups with statistically significant differences using Duncan's test (p ≤ 0.05).

Figure 2 .
Figure 2. Tissue expression patterns of GsWRKY23 gene in wild BB52 soybean seedlings during 24 h of 120 mM NaCl treatment (A), and GUS staining of wild-type and GsWRKY23pro::GUS Arabidopsis seedlings under 120 mM NaCl for 24 h; bar = 3 mm (B); different letters indicate groups with statistically significant differences using Duncan's test (p ≤ 0.05).

Figure 5 .Figure 6 .
Figure 5. Analysis of salt tolerance of GsWRKY23−OE and GsWRKY23−RNAi soybean hairy−root composite plants: (A) Growth phenotype under 120 mM NaCl solutions for 3 d; bar = 5 cm.(B) Fresh weight (FW) per plant.(C) Plant height.(D) Leaf chlorophyll content.(E) Leaf RWC.(F) REL value.(G) MDA content.Means ± SD are shown (n = 3, except for the measurement of plant height and FW per plant, where n = 6); different letters indicate groups with statistically significant differences using Duncan's test (p ≤ 0.05).

Figure 7 .
Figure 7. Salt-tolerance assays of GsWRKY23-transgenic Arabidopsis at the seed germination and seedling stages: (A) Pictorial representation of seed germination tests of WT, atwrky23, WT-GsWRKY23, and atwrky23-GsWRKY23 in the presence of 0, 75, or 150 mM NaCl for 7 d.(B) Graphical representation of seeds' germination rate in the presence of different NaCl concentrations.(C) Growth phenotype of the 3-week-old plants under 120 mM NaCl for 7 d; bar = 5 cm.(D) Fresh weight per plant.(E) Leaf chlorophyll content.(F) Leaf RWC.(G) REL value.(H) MDA content in

Figure 7 .
Figure 7. Salt-tolerance assays of GsWRKY23-transgenic Arabidopsis at the seed germination and seedling stages: (A) Pictorial representation of seed germination tests of WT, atwrky23, WT-GsWRKY23, and atwrky23-GsWRKY23 in the presence of 0, 75, or 150 mM NaCl for 7 d.(B) Graphical representation of seeds' germination rate in the presence of different NaCl concentrations.(C) Growth phenotype of the 3-week-old plants under 120 mM NaCl for 7 d; bar = 5 cm.(D) Fresh weight per plant.(E) Leaf chlorophyll content.(F) Leaf RWC.(G) REL value.(H) MDA content in the shoots.Means ± SD are shown (n = 3, except for fresh weight per plant, where n = 6, for seed germination, n = 32); different letters indicate groups with statistically significant differences using Duncan's test (p ≤ 0.05).

Table 1 .
Differentially expressed transcription factor WRKY gene family enriched downstream of the MAPK4 pathway in the roots of G. soja BB52 seedlings under control and NaCl treatments.

Table 1 .
Differentially expressed transcription factor WRKY gene family enriched downstream of the MAPK4 pathway in the roots of G. soja BB52 seedlings under control and NaCl treatments.