Introduction: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential key enzyme in the glycolytic pathway and plays an important role in stress responses. Although GAPDH family genes have been found in different plant species, the determination of their gene family analysis and their functional roles in soybean are still unknown.

Methods: In this study, gene sequence and expression data were obtained using online tools, and systematic evolution, expression profile analysis, and qRT-PCR analysis were conducted.

Results and Discussion: Here a total of 16 GmGAPDH genes were identified on nine chromosomes, which were classified into three clusters. Additionally, all GmGAPDH genes harbor two highly conserved domains, including Gp_dh_N (PF00044) and Gp_dh_C (PF02800). The qRTPCR analysis also showed that most GmGAPDH genes significantly responded to multiple abiotic stresses, including NaHCO3, polyethylene glycol, cold, and salt. Among them, GmGAPDH14 was extraordinarily induced by salt stress. The GmGAPDH14 gene was cloned and overexpressed through soybean hair roots. The overexpressed transgenic soybean plants of the GmGAPDH14 gene have also shown better growth than that of control plants. Moreover, the overexpressed transgenic plants of GmGAPDH14 gene had higher activities of superoxide dismutase but lower malonaldehyde (MDA) content than those of control plants under salt stress. Meanwhile, a total of four haplotypes were found for the GmGAPDH14 gene, and haplotypes 2, 3, and 4 were beneficial for the tolerance of soybean to salt stress. These results suggest that the GmGAPDH14 gene might be involved in the process of soybean tolerance to salt stress. The results of this study will be valuable in understanding the role of GAPDH genes in the abiotic stress response of soybean.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolytic metabolic pathway, which widely exists in biological cells (Zhang et al., 2019). GAPDHs catalyzed glyceraldehyde-3-phosphate to form 1,3-biphosphoglycerate in the presence of NAD+ and inorganic phosphate (Sirover, 2011). The major functions of the GAPDH gene refer to immune response (Henry et al., 2015), expression regulation (Zhang et al., 2017), and autophagy (Colell et al., 2007).

In plants, GAPDH genes are involved in glycolytic or photosynthetic pathways (Plaxton, 1996). Meanwhile, GAPDH genes can be divided into three categories according to their functions in cells (Guo et al., 2012; Guo et al., 2014). In chloroplasts, NADP-specific GAPDHs (GAPA/B) were involved in photosynthetic CO2 fixation. In the cytoplasm, NAD-dependent GAPDH (GAPC) converted glyceraldehyde-3-P (Ga3P) to 1,3-bisphosphoglycerate. In plastids, GAPCp isoforms may be involved in glycolytic energy production. Moreover, all GAPDH proteins contained highly conserved domains, including the Gp_dh_N (PF00044) and Gp_dh_C (PF02800) domains (Jiao et al., 2011; Zeng et al., 2016; Miao et al., 2019). It was also found that some GAPDHs also contained CP12 (PF02672) domain.

To date, a series of GAPDH genes have been cloned and characterized, including Arabidopsis thaliana (Guo et al., 2014), Oryza sativa (Lim et al., 2021), Zea may (Bustos et al., 2007), and Cucumis sativus (Chaturvedi et al., 2016). Based on subcellular localization, it has been proven that GAPDH was divided into cytosolic (Cy) and plastic (P) isoforms (Miao et al., 2019; Wei et al., 2022). In A. thaliana, GAPDH genes distributed in different subcellular compartments: GAPC1 and GAPC2 were located in the cytosol, and the rest of the GAPDH genes were located in plastids (Rius et al., 2008; Anoman et al., 2015). Some researchers have revealed that GAPCs can regulate the accumulation of oil content in seeds (Guo et al., 2014). The seed oil content was reduced by 3% when GAPDH was knocked out of the cytoplasm in A. thaliana, suggesting that cytosolic GAPDH was vital for regulating the content of seed oil (Guo et al., 2014). Furthermore, the plastidic GAPCp has been shown to be involved in starch metabolism (Muñoz-Bertomeu et al., 2009). In soybean, the knockdown of GAPC1 decreased the nodule nitrogenase activity without affecting the nodule weight (Ke et al., 2022). Moreover, the key role of GAPDH genes in plant growth and development and responses to abiotic stresses has been extensively confirmed, including heat (Kim et al., 2020), cold (Liu et al., 2017), salinity (Cho et al., 2014), and drought (Li et al., 2019). Previous studies have shown that the overexpression of PsGAPDH can increased salt tolerance in potato (Jeong et al., 2001). In Arabidopsis, the overexpression of TaGApC gene from Chinese spring Triticum aestivum displayed improved drought tolerance by decreasing the reactive oxygen species (ROS) levels (Zhang et al., 2020). Furthermore, it was also found that salicylic acid restrains the GAPDH activity in vitro (Pokotylo et al., 2020).

Soybean was the main oil crop in the world (Holle and Damme, 2015). However, the yield and the quality of soybean were often affected by abiotic stresses such as low temperature, drought, and salinization (Feng et al., 2020). Therefore, it was significant to study the salt resistance mechanism of soybean and excavate the stress-resistant genes for improving the yield and quality of soybean. Although GAPDHs have been characterized and analyzed in many plant species, the characterization of the GAPDH gene family in soybean is still limited, and it is unknown how GAPDH regulates the molecular mechanism of salt stress in soybean.

Although most studies have described the biological and physiological functions of the GAPDH gene, few research were known in terms of the functional divergence of the GAPDH gene family in soybean. In this study, 16 of the GAPDH gene members in soybean were identified, and their phylogenetic relationships, gene structure, chromosomal localization, and stress responses were analyzed. Furthermore, the function of GmGAPDH14 gene in soybean tolerance to salt stress was tentatively verified, indicating the important role of GmGAPDH14 gene in salt stress.

To identify the GAPDH gene sequence of soybean, systematic BLASTP was conducted against the soybean reference genome database (https://www.soybase.org/) and the Phytozome database (https://phytozome-next.jgi.doe.gov/) using the published Arabidopsis GAPDH as alignment sequence. The screening threshold was set to E-value (