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. 2026 Mar 6;139(3):89. doi: 10.1007/s00122-026-05189-7

Genome-wide characterization of soybean alcohol dehydrogenase (ADH) genes identifies GmADH13 as a positive regulator of the salt stress response

Sihui Wang 2, Zhiyuan Xu 2, Peng Cheng 3, Jun Yang 2, Qiang Hao 2, Jinfang Wang 2, Ziqian Cheng 2, Lingshi Xia 2, Zhenbang Hu 2,✉, Xin Li 3,✉, Anyu Su 1,✉
PMCID: PMC12966252  PMID: 41790283

Abstract

Alcohol dehydrogenase (ADH) is a zinc-binding enzyme responsible for catalyzing the interconversion between ethanol and acetaldehyde, as well as other alcohol and aldehyde pairs, within the ethanol fermentation pathway. This enzyme plays a crucial role in plant adaptation to environmental stressors. However, knowledge regarding the ADH gene family in soybean remains limited. Here, a genome-wide analysis was conducted, leading to the identification of 22 ADH genes in soybean and their classification into five subfamilies based upon phylogenetic relationships. Cis-regulatory element analysis combined with qRT-PCR experiments demonstrated that GmADH genes exhibit significant upregulation on exposure to various abiotic stresses, such as drought, alkaline conditions, and salt stress, as well as to hormonal stimuli. Furthermore, GmADHs display distinct tissue-specific expression patterns. Notably, GmADH13 showed consistent upregulation across multiple stress conditions, suggesting its pivotal role in soybean’s salt stress response. Functional analyses revealed that GmADH13 enhances salt tolerance by modulating the reactive oxygen species scavenging system, maintaining redox homeostasis, and stabilizing Na⁺/K⁺ levels within cells, thereby reducing oxidative damage induced by salt stress. The results from transgenic hairy root experiments further support the role of GmADH13 in improving salt tolerance. Collectively, this study expands the current understanding of the ADH gene family's involvement in soybean stress responses and highlights potential genetic targets for enhancing soybean salt tolerance during cultivation.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00122-026-05189-7.

Introduction

Alcohol dehydrogenases (ADHs), also referred to as alcohol: NAD + oxidoreductases (EC 1.1.1.1), are extensively distributed across diverse organisms and are encoded by multigene families in both eukaryotes and prokaryotes. These enzymes facilitate the reversible oxidation of alcohols to aldehydes, playing a crucial role in metabolic and stress response pathways (Dixon and Hewett 2000). ADHs have been identified in a variety of species, including animals, plants, yeast, and bacteria (Alka et al. 2013; Plapp et al. 2013; Kumar et al. 2012). Structurally, plant ADHs are zinc-binding enzymes characterized by two conserved domains: a GroES-like domain (ADH_N) responsible for catalysis and a zinc-binding domain (ADH_zinc_N) essential for enzyme function (Murzin 1996; Taneja and Mande 1999). The ADH gene family exhibits considerable diversity and is primarily categorized into three subfamilies based on amino acid sequence length: short-chain dehydrogenase/reductases (SDR-ADH; ~ 250 amino acids), medium-chain dehydrogenase/reductases (MDR-ADH; ~ 350 amino acids), and long-chain dehydrogenase/reductases (LDR-ADH; 600–750 or 385–900 amino acids) (Alka et al. 2013; Jörnvall et al. 2013). Plant ADHs typically belong to the MDR subfamily and contain zinc-binding motifs essential for enzymatic activity (Nordling et al. 2002).

Prior studies have identified and isolated ADH genes from Arabidopsis thaliana (Koch et al. 2000), Nicotiana tabacum L. (Wang et al. 2024), Cucumis melo (Manríquez et al. 2006), Brassica napus (Zhang et al. 2024), and Solanum lycopersicum (Speirs et al. 1998), where they contribute to various physiological and developmental processes. In tobacco (N. tabacum L.), 53 ADH genes were classified into six subfamilies through phylogenetic analysis, with promoter cis-elements linked to cell development, hormone signaling, and stress responses (Wang et al. 2024). In crops including wheat, barley, and tomato, ADHs display tissue-specific expression patterns, suggesting roles in tissue development and stress adaptation (Komatsu et al. 2011; Pathuri et al. 2011; Yamauchi et al. 2014). For instance, barley (Hordeum vulgare) ADH1 modulates both anaerobic stress tolerance and susceptibility to the fungal pathogen Blumeria graminis, thus establishing a link between abiotic and biotic stress responses (Pathuri et al. 2011); oilseed rape (Brassica napus) BnADH36 enhances salt tolerance through reactive oxygen species (ROS) scavenging (Zhang et al. 2024); rice (Oryza sativa) ADHs are primarily induced by low temperature and hypoxia, which promotes fermentation to maintain energy supply under adverse conditions (Kato-Noguchi and Yasuda 2007). ADHs are also involved in fruit ripening and aroma synthesis (Echeverra et al. 2004; Straeten et al. 1991). For instance, overexpression of Le-ADH2 in tomato alters alcohol-aldehyde balance, influencing flavor profiles such that they are more “ripe.” In white pear, three genes have been closely linked to aromatic compound levels in the course of fruit development (Pbr013912.1, Pbr026289.1, and Pbr01252.1), while PbrADH6 in pear is associated with volatile ester production and total ADH activity (Zeng et al. 2020). ADH genes have also been implicated in primary and secondary metabolic pathways in species such as persimmon (DkADH), Artemisia annua (AaADH2), and Sedum sarmentosum (SsADH) (Polichuk et al. 2010; Tonfack et al. 2011; Sung et al. 2012).

Beyond metabolic functions, ADH genes are crucial in plant stress responses, including waterlogging, cold stress, and drought tolerance. Under stress conditions, ADHs help regulate ROS homeostasis by modulating the expression of ROS-related genes (Su et al. 2020). For example, drought stress significantly induces ADH expression in tobacco roots, and ADH gene silencing leads to wilting, highlighting its protective role (Senthil-Kumar et al. 2010). In rice seedlings, low-temperature stress activates ADH and PDC enzymes, enhancing ethanol fermentation and ADH gene expression (Kato-Noguchi and Yasuda 2007). A cold-responsive ADH gene identified in forest strawberry has also been identified as holding promise as a molecular marker for cold tolerance (Davik et al. 2013). Similarly, in maize seedlings, ADH activity increases under cold stress, while ADH1-ADH2 mutants exhibit severe lipid peroxidation and cellular injury. Under hypoxic conditions, maize ADH1 and ADH2 show peak expression at 6 h before declining, reflecting their dynamic role in stress adaptation (Kato-Noguchi 2000). In Arabidopsis, AtADH is mainly expressed in lateral roots and other roots but absent in shoots, resembling the expression pattern of ADH1 in maize. In barley, ADH genes are significantly induced and highly active under anaerobic stress, and in Panax ginseng, PgADH expression patterns in response to abiotic stimulation indicated that the involvement of this short-chain ADH in hormone-mediated stress responses (Kim et al. 2009). In addition, ADH expression is upregulated in soybean, grass bean, and Arabidopsis under salt stress (Manak et al. 2002; Sobhanian et al. 2010; Chattopadhyay et al. 2011).

Despite the identification of multiple ADH genes across various plant species and the advancement of transgenic technologies that enable the functional characterization of these genes, emphasizing their important roles in cells, a thorough assessment of the soybean ADH gene family is still lacking. Understanding their genetic architecture and functional roles is essential for crop improvement. Soybean is a globally important oilseed crop, functioning as a primary source of edible oils and biofuel.

Soil salinity poses a significant challenge to soybean growth, yield, and quality (Zhao et al. 2020). Identifying salt-tolerance-associated genes and elucidating their molecular mechanisms is crucial for breeding salt-tolerant soybean varieties. In this study, bioinformatics approaches were employed to identify 22 ADH genes in the soybean genome and performed detailed analyses of their phylogenetic relationships, cis-regulatory elements, gene structures, and expression profiles across various tissues and under abiotic stress conditions. Notably, GmADH13 exhibited significant upregulation under NaCl treatment, suggesting its critical role in salt stress response. Enzyme kinetics assays confirmed that the purified GmADH13 protein possesses ethanol dehydrogenase activity. Functional validation in transgenic soybean hairy roots demonstrated that GmADH13 overexpression enhances salt tolerance by stabilizing cellular redox homeostasis, increasing antioxidant enzyme activity, and maintaining intracellular Na⁺/K⁺ balance, thereby mitigating salt-induced oxidative damage. These findings highlight GmADH13 as a promising candidate for enhancing salt tolerance in soybean through genetic improvement strategies.

Materials and methods

Soybean ADH family genes identification and analysis

The complete genome and protein sequences of soybean were obtained via accessing the Phytozome v12.1 database (https://phytozome.jgi.doe.gov/pz/portal.html). To identify ADH family genes in soybean, ADH protein sequences from Arabidopsis thaliana were access through the Arabidopsis Information Resource (TAIR; https://www.arabidopsis.org/) and employed to conduct a BLASTP search against the soybean genome, applying an e-value threshold of 1.0 (Jacob et al. 2008). Candidate ADH genes were selected based on the presence of conserved ADH domains (PF08240 and PF00107). Subcellular localization predictions of the identified ADH proteins were conducted using WoLF PSORT (https://www.genscript.com/wolf-psort.html). ADH protein amino acid sequence lengths, isoelectric points, and molecular weights were computed via the ExPASy server (https://web.expasy.org/compute_pi/).

A phylogenetic tree was developed using homologous ADH amino acid sequences from A. thaliana, Medicago truncatula, Phaseolus vulgaris, Oryza sativa, Glycine max, and Sorghum bicolor, employing MEGA 5.0 with the neighbor-joining method (Tamura et al. 2011). Exon–intron structures of the identified genes were visualized using the Gene Structure Display Server v2.0 (https://gsds.gao-lab.org). Gene synteny relationships between soybean ADHs and those from other plant species were analyzed via the Plant Genome Duplication Database (PGDD; https://chibba.agtec.uga.edu/duplication/). Predictions of cis-regulatory elements within 2000 bp upstream of the translation initiation sites of GmADH genes were made with PlantCARE (http://bioinformatics.psb.ugent) (Passricha et al. 2017), with classification and visualization performed in TBtools (v 2.056) (Chen et al. 2020).

Evaluation of the effects of hormones and abiotic stress on GmADH expression

To investigate how GmADH genes respond to abiotic and hormonal stress, soybean seedlings (cultivar DN50) were cultivated to the second trifoliolate stage and then exposed to a range of stress conditions, including NaCl (150 mmol/L), jasmonic acid (JA; 100 μmol/L), NaHCO₃ (100 mmol/L), abscisic acid (ABA; 100 μmol/L), mannitol (200 mmol/L), PEG6000 (20%), salicylic acid (SA; 100 μmol/L), and brassinolide (BR; 100 μmol/L). Samples were collected at 0, 1, 3, 6, 12, and 24 h post-treatment, and total RNA was extracted and stored at −80°C for subsequent analysis.

RNA extraction was performed using a Quick Total RNA Isolation Kit, and RNA integrity was assessed via 1% agarose gel electrophoresis. PrimeScript® RT Master Mix was used to prepare cDNA, which was diluted to 500 ng/L and utilized for qRT-PCR analyses conducted with a Roche LightCycler® 96 system with SYBR Green detection (Bustin et al. 2009). GmACTIN4 served as the internal reference gene, and relative expression levels were calculated using the 2−∆∆CT method (Livak and Schmittgen 2001). Statistical analyses were conducted using Student’s t tests or ANOVAs, with p < 0.05 defining significance. All experiments were performed with at least three biological replicates, and data are presented as mean ± SD.

Plasmid preparation and subcellular localization analyses

The roots of the ‘DN50’ soybean cultivar were used to amplify the full-length coding sequence (CDS) of GmADH13. To generate the GmADH13::GFP fusion construct, the GmADH13 gene was inserted into the pBI121 vector, which includes a GFP tag, using gene-specific primers (Table S2). Recombinant GmADH13::GFP plasmid was transiently expressed in Arabidopsis mesophyll protoplasts via polyethylene glycol (PEG)-calcium-mediated transformation for 16 h (Yoo et al. 2007). An empty vector served as a control. Subcellular localization of GFP-tagged GmADH13 protein was assessed via LSM710 confocal laser scanning microscopy (Zeiss, Oberkochen, Germany).

Prokaryotic protein expression and enzyme kinetics

The GmADH13 CDS was cloned into the BamH I site of the pGEX-4T-3 vector, generating the recombinant plasmid pGEX-4T-3-GmADH13. This construct was transformed into Escherichia coli strain DE3 for heterologous protein expression. A positive clone was chosen and cultured at 37 °C in the presence of 1 mmol/L IPTG for 4 h to induce expression of the GST-GmADH13 fusion protein. The expressed protein was purified using glutathione Sepharose high-performance affinity chromatography and detected via Western blotting using anti-GST. Enzyme kinetics of the purified GmADH13 protein were assessed via an Eadie–Hofstee plot.

Heterologous expression

The GmADH13 CDS was cloned into the Kpn I/Xba I sites of the pYES2 vector, generating the recombinant plasmid pYES2-GmADH13. Both pYES2 (empty vector control) and pYES2-GmADH13 constructs were introduced into Saccharomyces cerevisiae strain INVSC1 via lithium acetate-mediated transformation (Gietz and Schiestl 2007). Transformed yeast cells were initially grown in SD (-Ura) liquid medium at 30°C for 24 h until reaching an OD600 of 0.5, after which they were transferred to SG (-Ura, 2% galactose) medium and diluted to an OD600 of 0.4 for subsequent stress treatments (Ibrahim et al. 2001).

GmADH13 overexpression and knockdown

To construct the overexpression vector pSOY1-GmADH13, the GmADH13 CDS was amplified and inserted into the pSOY1 vector under the control of the CaMV35S promoter. A guide RNA targeting GmADH13 was designed for CRISPR/Cas9-mediated knockdown, and the pGES401 vector was modified to incorporate the target sequence, generating pGES401-GmADH13 (Bai et al. 2022). Agrobacterium strain K599 was used for the transformation of soybean hypocotyls following a previously established hairy root induction protocol (Tóth et al. 2016). The hairy roots were transformed and confirmed by PCR, enzyme activity assays, and RT-PCR to obtain GmADH13 overexpressing hairy roots (OHR) and knockout hairy roots (KHR). Salt stress response was evaluated 3 days after NaCl treatment (0 and 120 mmol/L) by measuring plant height, maximum root length, fresh and dry weights of leaves and roots in independent transgenic lines.

Biochemical and physiological analyses

The effects of salt stress on photosynthesis in transformed hairy root plants were analyzed using the high-throughput phenotyping platform (Phenotrait) by measuring photosynthetic parameters (F0, Fm, and Fv/Fm) following appropriate stress treatments. Measurements of plant height and root length were taken following treatment with NaCl (0 or 120 mmol L−1) for 3 days. The upper portions of seedling roots were isolated, incubated for 15 min at 105 °C, and dried to a constant weight at 80 °C. Aboveground and belowground portions of seedlings were then weighed once dry.

Spectrophotometry was employed to quantify superoxide (O2−) and hydrogen peroxide (H2O2) levels (Fryer et al. 2002; Velikova et al. 2000). A thiobarbituric acid reaction was utilized to quantify levels of malondialdehyde (MDA) to assess lipid peroxidation (Hodges et al. 1999). Reactive oxygen species (ROS) scavenging was examined by preparing soybean extracts (0.5 g) in buffer (15 mL) with PVP (1%: w/v), K2HPO4-KH2PO4 (50 mmol/L; pH 7.0), EDTA (1.5 mmol/L), and ASA (0.5 mmol/L). Supernatants were then processed to quantify superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activity (Giannopolitis and Ries 1977; Nakano and Asada 1981). All measurements were performed with three biological replicates.

Histochemistry

Leaf samples from CHR, OHR, and KHR plants were collected both before and two hours after stress treatment and placed in 1.5-mL centrifuge tubes containing NBT, DAB, or Evans blue staining solutions. The samples were incubated overnight in darkness at room temperature to ensure optimal staining. Following incubation, the leaves were subjected to decolorization in a boiling water bath with 75% ethanol and 5% glycerin (Zhao et al. 2021). To investigate stomatal responses, the lower epidermis was carefully excised from CHR, OHR, and KHR leaves and immersed in a stomatal opening solution that contained either 0 or 120 mmol/L NaCl. After one hour of light exposure, the stomata were stained with DCFH-DA for 30 min, followed by three washes with distilled water. The stained samples were then visualized using a confocal microscope (Rajneesh et al. 2017). For root analysis, root tips from both stressed and unstressed hairy root plants were selected after 12 h of treatment. These root samples were immersed in PI staining solution (20 μg/mL) and CoroNa™ Green Sodium Indicator solution for 30 min. After three rinses with distilled water, confocal microscopy was used to capture images of the stained root tips (You et al. 2022; Jabeen et al. 2021).

Results

Identification and evaluation of the soybean ADH gene family

A total of 22 GmADH genes were identified in the soybean genome through a BLAST search using known AtADHs as queries. These genes were designated GmADH1 to GmADH22. The encoded proteins varied in length from 363 to 401 amino acids, with molecular weights between 38.64 and 43.57 kDa, as well as isoelectric points between 5.52 and 6.75 (Table S1). Sequence analyses confirmed that all GmADH proteins contain two conserved domains: a GroES-like domain (ADH_N) and a zinc-binding domain (ADH_zinc_N) (Figures S1-S2). Phylogenetic analysis of the GmADH family, along with homologous sequences from A. thaliana, G. max, O. sativa, M. truncatula, P. vulgaris, and S. bicolor, classified these genes into five distinct subfamilies (Fig. 1A). Subfamily V contained the largest number of soybean ADH genes (nine), while subfamilies I, II, and III each included four genes. Subfamily II comprised only a single gene.

Fig. 1.

Fig. 1

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Phylogenetic, gene structure, and collinearity analyses of GmADH genes. A Analysis of the evolution of ADH family proteins in Arabidopsis thaliana, Glycine max, Oryza sativa, Medicago truncatula, Phaseolus vulgaris, and Sorghum bicolor. B ADH gene structural characteristics. The CDS is marked with a yellow box, while UTR sequences are denoted with a green box. C Collinearity analyses of ADH family genes from A. thaliana, G. max, O. sativa, M. truncatula, P. vulgaris, and S. bicolor. D Collinearity analyses of GmADH genes from soybean. Circles represent chromosomes, with the curves of varying colors denoting syntenic ADH gene areas. Exon and intron sizes are marked with the provided scale

To better understand the evolution and structural organization of the GmADH genes (Kolkman and Stemmer 2001; Liu et al. 2005), their exon–intron structures were analyzed (Fig. 1B). The number of exons ranged from nine to ten, with members of the same subfamily exhibiting similar exon–intron structural patterns. This conservation suggests that exon–intron arrangements are closely linked to the evolutionary history of the ADH gene family and that gene family expansion may have been influenced by intron insertions or deletions.

Comparative genomic analysis of ADH genes among multiple plant species provided insight into their evolutionary trajectories. The 22 soybean ADH genes were distributed across 20 chromosomes, sharing 32 orthologous gene pairs with A. thaliana, O. sativa, M. truncatula, P. vulgaris, and S. bicolor. Among these, seven orthologous pairs were identified between G. max and A. thaliana, while three pairs were shared between G. max and O. sativa (Fig. 1C). Additionally, 14 paralogous gene pairs were detected within the soybean genome, indicating that duplication events played a role in expanding this gene family (Fig. 1D). The observed synteny indicates that the diversification of ADH genes occurred prior to soybean speciation.

Characterization of GmADH responses to hormones and abiotic stressors

Cis-regulatory elements play a crucial role in coordinating gene transcription. The 2000-bp promoter regions upstream of these GmADH genes was thus analyzed for regulatory motifs to better elucidate the factors that shape the expression of these genes. Several cis-acting elements linked to hormone signaling (ABRE, TCA, TGA, TGACG, and CGTCA) and stress responses (MBS, TC-rich, ARE, and LTR) were identified (Figure S4). These findings suggest that GmADH genes may be actively involved in stress adaptation and plant development.

To examine how GmADH genes respond to abiotic stress, their expression profiles were assessed under salt (150 mM NaCl), drought (200 mM mannitol), alkaline (100 mM NaHCO₃), and osmotic (20% PEG) stress conditions (Fig. 2A). Expression patterns varied, with some genes showing significant upregulation or downregulation in response to specific stresses. In subgroup I, GmADH12, GmADH13, GmADH14, and GmADH15 exhibited a characteristic pattern of initial upregulation followed by downregulation. Notably, GmADH13 showed the most pronounced response, with transcript levels peaking 12 h after salt treatment. Additionally, GmADH6 was significantly upregulated under stress conditions, while GmADH14 exhibited the highest expression increase after three hours of exposure to 20% PEG (Fig. 2C).

Fig. 2.

Fig. 2

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Evaluation of GmADH responses to abiotic stress and hormone stimulation. A GmADH expression patterns were assessed in the roots of soybean plants that underwent treatment for 0, 1, 3, 6, 12, or 24 h with NaCl (150 mmol L−1), PEG (20%), mannitol (200 mmol L−1), NaHCO3 (100 mmol L−1), or water. B GmADH expression patterns in the roots of soybean plants treated for 0, 1, 3, 6, 12, or 24 h with SA, ABA, BR, or JA (100 μmol L−1) or water. Unstimulated controls served as the reference for these analyses. C, D Patterns of candidate gene expression during abiotic stress (C) and hormone stimulation (D). *p < 0.05; **p < 0.01; ***p < 0.001, Student’s t test

Since hormones are key messengers within plants that coordinate many stress responses and cis-acting elements associated with hormone responses were identified in GmADH promoters (ABRE, P-box, TCA, TGA, TGACG, CGTCA) (Figure S4), qRT-PCR analysis was conducted to assess transcriptional responses to JA, SA, BR, and ABA (Fig. 2B). GmADH genes displayed varying degrees of hormone sensitivity. For instance, GmADH6, GmADH12, GmADH13, GmADH14, and GmADH15 responded strongly to JA. GmADH6 exhibited more than a 280-fold increase in expression after 12 h of stress. Similarly, GmADH13 expression was upregulated approximately 30-fold in response to both SA and ABA. Furthermore, GmADH6 expression increased by nearly 100-fold under BR treatment (Fig. 2D). Interestingly, most GmADHs exhibited significant expression changes in response to all four hormones, suggesting a broad involvement in hormone-mediated regulatory pathways. Among them, GmADH13 displayed the most substantial upregulation across stress and hormone treatments. Consequently, GmADH13 was selected for further functional analysis to assess its role in salt tolerance.

GmADH13 encodes a cytosolic protein with ADH enzyme activity

To determine the subcellular localization of GmADH13, its coding sequence was fused to the N-terminus of a GFP reporter gene and transiently expressed in Arabidopsis protoplasts. The fluorescence analysis revealed that while free GFP, used as a control, was distributed throughout the cytoplasm and nucleus, the GmADH13-GFP fusion protein was exclusively localized in the cytoplasm (Fig. 3A). This specific cytoplasmic localization suggests that GmADH13 primarily functions within the cytoplasm, potentially engaging in cellular processes linked to stress response or metabolic regulation.

Fig. 3.

Fig. 3

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GmADH13 encodes a cytosolic protein exhibiting ADH activity. A GmADH13 subcellular localization analysis. pBI121-GmADH13::GFP or pBI121-GFP as observed via confocal microscopy in transgenic Arabidopsis mesophyll protoplasts. Bars = 10 μm. B Western blotting analysis of the purified GmADH13 protein. C Eadie–Hofstee plots were utilized to assess the kinetic properties of GmADH13

To further characterize the enzymatic properties of GmADH13, the recombinant protein was expressed in Escherichia coli Rosetta (DE3) using a GST-tagged expression vector. The fusion protein was purified and analyzed via SDS-PAGE and Western blotting to confirm its expression and purity (Fig. 3B). Enzyme kinetics were assessed using NAD and ethanol as substrates, and the kinetic parameters were determined through Eadie–Hofstee data plots. The estimated Km values for NAD and ethanol were calculated as 0.13 and 0.08 mmol L−1, respectively, while the Vmax values were 7.27 and 2.31 µmol min−1 mg−1 protein, respectively (Fig. 3C). These findings confirm that GmADH13 exhibits ADH enzyme activity, supporting its functional role in metabolic processes.

GmADH13 overexpression enhances NaCl resistance in yeast

Yeast, as a eukaryotic model system, is frequently used for functional validation of stress-related genes. To examine the impact of GmADH13 on yeast growth under stress conditions, the GmADH13 CDS was cloned into the pYES2 expression vector, which is often utilized when validating stress resistance-related genes (Esquivel et al. 2020; Li and Ljungdahl 1996), and introduced into yeast cells. Growth analysis under control conditions showed no significant differences between yeast cells harboring the GmADH13-pYES2 construct and those carrying the empty pYES2 vector. However, under high-salinity stress, yeast cells expressing GmADH13 exhibited enhanced growth compared to control strains (Fig. 4A). Additionally, in the YDR strain, which exhibits growth inhibition under NaCl stress, overexpression of GmADH13 was able to partially restore growth (Fig. 4B). These results suggest that GmADH13 enhances cellular resistance to salt stress in yeast, likely through its enzymatic function.

Fig. 4.

Fig. 4

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GmADH13 enhances yeast (INVSc1) NaCl tolerance. A Recombinant yeast INVSc1 (GmADH13-pYES2) and control INVSc1 (pYES2) growth characteristics under NaCl treatment. B YDR, pYES2, and GmADH13-pYES2 yeast strain growth characteristics when exposed to NaCl stress in YPD liquid medium, assessing absorbance (OD600) at 24 h and 48 h following treatment with 200 mM NaCl. *p < 0.05; **p < 0.01 vs. YDR; Student’s t tests

GmADH13 overexpression improves the salt tolerance of soybean plants

To further clarify the role of GmADH13 in salt stress responses, transgenic soybean hairy roots were generated with either overexpression (OHR) or knockout (KHR) of GmADH13. Expression analysis confirmed a significant upregulation of GmADH13 in OHR plants and a marked reduction in KHR plants relative to control hairy roots (CHR). Enzymatic assays demonstrated that ADH activity in transgenic hairy roots correlated with GmADH13 transcript levels, confirming the successful genetic modifications.

Under normal growth conditions, no noticeable differences were observed between transgenic and control plants. However, following salt stress treatment, OHR plants exhibited significantly improved growth compared to CHR plants, while KHR plants displayed severe growth inhibition, including pronounced leaf yellowing (Fig. 5A). Measurements taken on the third day of NaCl exposure revealed that in comparison with CHR plants, OHR plants had greater root elongation and fresh root weight, whereas KHR plants exhibited markedly reduced root growth (Fig. 5B). To further assess stress adaptation, key physiological indicators, including chlorophyll fluorescence parameters, chlorophyll index (Chl-Idx), and anthocyanin index (Ari-Idx), were analyzed. These parameters provide insights into photosynthetic efficiency and plant resilience under stress. Under salt stress, OHR plants exhibited higher values of Fm, Fv/Fm, Chl-Idx, and Ari-Idx relative to CHR plants, whereas KHR plants displayed significantly elevated F0 values, indicating greater stress susceptibility (Fig. 5C). Collectively, these findings confirm that GmADH13 positively regulates salt stress tolerance in soybean.

Fig. 5.

Fig. 5

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GmADH13 enhances salt stress tolerance in transgenic soybean hairy roots. A Phenotypic overview of seedlings. Scale bar: 5 cm B Soybean plant height, root length, fresh weight, and dry weight for seedlings expressing GmADH13-OHR, GmADH13-KHR, or CHR following exposure to 0 or 120 mmol L−1 NaCl for 3 days. C Chlorophyll fluorescence parameters, chlorophyll index, and anthocyanin index analyses. Data are means ± SE (n = 4). *p < 0.05; **p < 0.01 vs. CHR plants; Student’s t tests

Exposure to abiotic stress often triggers excessive accumulation of ROS in plants, necessitating precise ROS regulation to mitigate oxidative damage (Schieber and Chandel 2014). To assess ROS levels in soybean leaves under salt stress, NBT and DAB staining were used to visualize superoxide (O₂⁻) and hydrogen peroxide (H₂O₂) accumulation, respectively (Kim et al. 2003). Under non-stress conditions, CHR, OHR, and KHR plants exhibited minimal staining, indicating low ROS accumulation. However, under salt stress, varying degrees of staining were observed, with OHR plants exhibiting the lightest staining, suggesting reduced ROS accumulation and minimal leaf damage. In contrast, KHR plants displayed the darkest staining, indicative of excessive ROS buildup, severe leaf damage, and impaired stress resistance (Fig. 6A-B). ROS play a crucial role in stomatal dynamics, influencing plant water regulation and stress adaptation (Carmody et al. 2016; Murata et al. 2015). To further investigate this role, H₂O₂ production in guard cells was visualized using DCFH-DA staining, where the intensity of green fluorescence corresponds to ROS levels. Under normal conditions, stomatal guard cells exhibited weak fluorescence signals, indicative of low ROS levels. Upon salt stress, OHR plants maintained relatively low H₂O₂ accumulation, whereas KHR plants displayed substantially higher fluorescence intensity compared to CHR plants (Fig. 6C). This suggests that GmADH13 overexpression contributes to the regulation of ROS homeostasis, particularly in stomatal function, thereby enhancing salt stress tolerance.

Fig. 6.

Fig. 6

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GmADH13 enhances antioxidant in the context of salt stress. A NBT and B DAB staining were conducted for CHR, OHR, and KHR plants subjected to 0 or 120 mmol L−1 NaCl treatment. C ROS levels were detected via DCFH-DA staining in salt stress-exposed lines. D H2O2, O2−, aperture ratio, and antioxidant enzyme activity levels were assessed in leaves from OHR, KHR, and CHR plants following 0 or 120 mmol L−1 NaCl treatment for 3 days. Data are means ± SE (n = 3). *p < 0.05; **p < 0.01 vs. CHR; Student’s t tests

To further substantiate the staining results, H₂O₂ and O₂⁻, along with the activities of key antioxidant enzymes, were systematically analyzed (Noctor and Foyer 2016). Following NaCl treatment, an overall increase was observed in H₂O₂ and O₂⁻ levels as well as in the activities of antioxidant enzymes. Among the analyzed plant lines, OHR plants exhibited the lowest accumulation of H₂O₂ and O₂⁻, whereas KHR plants displayed significantly elevated levels compared to the other strains. Under salt stress conditions, the enzymatic activities of CAT, POD, and SOD increased across all plant lines. However, this increase was most pronounced in OHR plants compared to CHR plants, while KHR plants exhibited a decline in their reactive oxygen scavenging capacity (Fig. 6D). These findings suggest that GmADH13 may play a pivotal role in mitigating ROS-induced cellular damage by modulating redox balance and enhancing the plant's antioxidant defense system.

GmADH13 maintains the Na⁺/K⁺ balance to mitigate salt stress

To further investigate oxidative stress-induced cell damage, cell viability assays were performed using Evans blue and propidium iodide (PI) staining (Yin et al. 2017). Figure 7A and B, under normal growth conditions, all plant lines exhibited minimal staining with no significant differences. However, upon exposure to NaCl stress, KHR plants showed extensive cell death, CHR plants displayed moderate damage, while OHR plants exhibited relatively mild stress-induced damage. Malondialdehyde (MDA) content, a key indicator of lipid peroxidation and oxidative stress, was measured to further assess cellular damage. Under normal conditions, MDA levels remained comparable across all plant lines. However, under salt stress, MDA accumulation was highest in KHR plants, followed by CHR plants, whereas OHR plants exhibited the lowest levels (Fig. 7D).

Fig. 7.

Fig. 7

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GmADH13 preserves the Na⁺/K⁺ balance and mitigates stress-induced cell death and membrane damage. A Evans blue staining results for CHR, OHR, and KHR plants that underwent 0 or 120 mmol L−1 NaCl treatment. B PI staining of CHR, OHR, and KHR plant root tips following treatment with 0 or 120 mmol L−1 NaCl. C Fluorescence analyses of root Na+ accumulation in plants subjected to 0 or 120 mmol L−1 NaCl treatment. Scale bar: 10 μm. D The impact of 0 or 120 mmol L⁻1 NaCl treatment on MDA, Na⁺, and K⁺ levels in CHR, OHR, and KHR Plants. Data are means ± SE (n = 3). *p < 0.05; **p < 0.01 vs. CHR; Student’s t test

Salt stress disrupts cellular ion homeostasis, particularly by altering Na⁺ and K⁺ balance, which adversely affects plant physiological processes. To monitor intracellular Na⁺ concentration, CoroNa™ Green, a green fluorescence-based Na⁺ probe, was utilized. Under normal conditions, the fluorescence intensity in root tips of all plant lines was low, with no significant differences observed. However, under salt stress, intracellular Na⁺ levels increased across all plant lines, resulting in an enhancement in fluorescence intensity. Among them, KHR plants exhibited the highest fluorescence intensity, indicating excessive Na⁺ accumulation, whereas OHR plants displayed the lowest intensity, suggesting improved Na⁺ exclusion (Fig. 7C). Additionally, the Na⁺/K⁺ ratio increased under salt stress in all plants, with the most substantial rise detected in KHR plants (Fig. 7E). These findings indicate that GmADH13 enhances salt tolerance by maintaining ionic homeostasis, reducing excessive Na⁺ accumulation, and promoting cellular resilience under salt stress conditions.

GmADH13 influences redox homeostasis and ROS scavenging-related gene expression

To elucidate the role of GmADH13 in redox regulation and ROS detoxification, the transcriptional expression of genes encoding key antioxidant enzymes, including SOD1 and SOD2 (Tiew et al. 2015), as well as those involved in ascorbate (ASA) and glutathione (GSH) biosynthesis (Koussevitzky et al. 2008; Schimmeyer et al. 2016), was analyzed. No differences in gene expression were detected among the plant lines under normal conditions. However, following NaCl treatment, the transcriptional levels of genes associated with ROS-scavenging and redox homeostasis were significantly upregulated in response to salt stress. Notably, their expression levels were highest in OHR plants, moderate in CHR plants, and lowest in KHR plants (Fig. 8). These findings suggest that GmADH13 contributes to the regulation of cellular redox balance and enhances stress resilience by modulating the expression of ROS-scavenging and redox-associated genes.

Fig. 8.

Fig. 8

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The expression of key redox and ROS homeostasis-related genes in GmADH13 transgenic hairy roots. mRNA levels for the indicated genes were assessed in OHR, KHR, and CHR plants that underwent 0 or 120 mmol L−1 NaCl treatment. Data are means ± SEs (n = 3). *p < 0.05; **p < 0.01 vs. CHR plants; Student’s t tests

Discussion

GmADH genes control hormone and abiotic stress responses, with GmADH13 as the primary regulator of salt stress responses

Here, a total of 22 GmADH proteins were identified, all of which contain conserved protein domains (PF), characteristic of typical ADHs (Figure S1) (Wang et al. 2024). Phylogenetic analysis of 72 ADH protein sequences across six species revealed five major clusters, with Cluster Ⅴ containing the largest number of members. Gene structural analysis indicated that genes with fewer or no introns are evolutionarily conserved, with Cluster I displaying a relatively lower intron count, suggesting higher evolutionary stability. Collinearity analysis further revealed that GmADH genes share homologous relationships with ADHs from Oryza sativa and Arabidopsis thaliana, implying that GmADHs originated before these species diverged (Fig. 1). Expression profiling demonstrated that 55% of GmADHs exhibit high transcript abundance in seeds, while 32% show predominant expression in both seeds and nodules (Figure S3). Additionally, several GmADH genes display tissue-specific expression patterns across roots, stems, leaves, flowers, seeds, and pods, indicating their potential regulatory roles in plant growth and development.

Cis-regulatory elements are known to mediate abiotic stress responses, with dehydration-responsive element (DRE) motifs playing a key role in drought and low-temperature adaptation. Analysis of the 2000-bp promoter regions of the 22 identified GmADH genes using the PlantCARE database revealed multiple cis-regulatory elements, including ARE, ABRE, LTR, and TGA motifs (Figure S4). These elements are associated with stress response and hormone signaling pathways, suggesting that GmADHs may participate in stress adaptation and hormonal regulation. Further investigation of GmADH transcriptional responses to various environmental stresses, including salt, alkali, and osmotic stress, as well as hormone treatments, demonstrated differential expression patterns across conditions. These findings highlight the regulatory significance of GmADH genes in soybean stress responses, with expression modulated in a stress-specific manner. Notably, prior studies have shown that ADH genes in woodland strawberry enhance cold tolerance (Davik et al. 2013), while ADH genes in tobacco confer resistance to drought (Senthil-Kumar et al. 2010). Consistent with these observations, GmADH12, 13, 14, and 15 exhibited significant transcriptional induction in response to saline and alkaline stress, as well as JA (Fig. 2). This indicates that GmADHs may play key roles in stress resistance and JA-mediated signaling. The mechanisms underlying their involvement in salt and alkaline stress adaptation warrant further investigation.

GmADH13 is an essential regulator of redox homeostasis and ROS levels under salt stress

Among the identified ADH genes, GmADH13 exhibited the most pronounced response to salt stress, reaching peak expression at 12-h post-treatment. Further investigations were conducted to elucidate the role of GmADH13 in salt stress adaptation at the molecular level. The recombinant GmADH13 protein expressed in Escherichia coli demonstrated ethanol dehydrogenase activity, as confirmed by enzymatic assays (Fig. 3). Additionally, heterologous expression of GmADH13 in yeast significantly enhanced salt tolerance (Fig. 4). Compared to CHR plants, OHR plants displayed greater root elongation and fresh weight, while KHR plants exhibited significantly reduced root length and fresh weight. Chlorophyll fluorescence parameters further corroborated these findings, reinforcing the role of GmADH13 in promoting salt tolerance in soybean (Fig. 5).

Previous studies have shown that activation of the antioxidant defense system is essential for mitigating ROS accumulation during abiotic stress (Schieber and Chandel 2014). When plants encounter salinity stress, redox homeostasis is disrupted, leading to oxidative stress and lipid peroxidation. Maintaining redox equilibrium is therefore critical for plant stress adaptation (Hossain and Dietz 2016). To assess ROS accumulation, NBT and DAB staining were used to visualize superoxide and hydrogen peroxide levels, respectively, while DCFH-DA staining was employed to examine ROS distribution in stomatal guard cells. The results indicated that OHR plants exhibited the lightest staining, suggesting reduced ROS accumulation, whereas KHR plants displayed the darkest staining, indicating excessive ROS buildup. Further biochemical assays revealed that antioxidant enzyme activities, including those of SOD, POD, and CAT, were significantly elevated in OHR plants compared to CHR plants, whereas KHR plants exhibited diminished enzymatic activity (Fig. 6). These findings suggest that GmADH13 enhances salt tolerance by modulating redox homeostasis and scavenging ROS to mitigate oxidative stress.

Hormonal signaling converges on GmADH13-mediated salt tolerance

Beyond direct ROS detoxification, the rapid induction of GmADH13 by ABA, SA, JA and BR (Fig. 2B) provides a mechanistic link between systemic hormone cues and local stress execution. ABA-enhanced CAT, POD and SOD expression aligns exactly with the antioxidant gene module up-regulated in GmADH13-OHR lines (Fig. 8), explaining how the hormone signal is translated into a stronger ROS-buffering capacity. JA-activated SOS1–NHX1 ion-transport cascade cooperates with GmADH13-dependent Na⁺ exclusion (Fig. 7E), while SA/BR-tuned ROS amplitudes are kept below the damage threshold by the same ADH-driven redox buffering. Thus, the early transcriptional burst of GmADH13 triggered by multiple hormones functions as a feed-forward loop that integrates systemic stress cues into the local antioxidant and ion-homeostatic machinery, thereby reinforcing the observed salt-tolerance phenotype.

GmADH13 is involved in regulating intracellular ion balance under salt stress to reduce cell damage

One of the primary consequences of salt stress is cellular damage, particularly to the cell membrane. To evaluate the extent of cellular damage, Evans blue staining was performed on leaves, while PI staining was used to assess root tip viability. Under salt stress conditions, the least staining intensity was observed in OHR plants, suggesting reduced cell damage, whereas KHR plants exhibited the highest staining intensity, indicating extensive cellular injury. MDA content, a key marker of membrane lipid peroxidation and oxidative damage, was also measured. The results showed that KHR plants had the highest MDA levels under salt stress, while OHR plants exhibited significantly lower MDA accumulation (Fig. 7). These findings indicate that overexpression of GmADH13 reduces membrane damage and cell death under salinity stress.

High salinity disrupts intracellular ionic balance, leading to excessive Na⁺ accumulation, structural damage to the cell membrane, metabolic disturbances, and ion toxicity (Fang et al. 2021). To further examine the impact of GmADH13 on ion regulation, CoroNa™ Green Na⁺ staining was used to visualize intracellular Na⁺ distribution in root tips, and Na⁺ and K⁺ ion concentrations were quantified. Under normal conditions, fluorescence intensity was minimal across all plant lines, with no significant differences observed. However, under salt stress, Na⁺ fluorescence intensity markedly increased in all plants, with KHR plants exhibiting the highest fluorescence, indicative of excessive Na⁺ accumulation. In contrast, OHR plants displayed the weakest fluorescence signal, suggesting lower Na⁺ retention (Fig. 7). Quantitative measurements further revealed that the Na⁺/K⁺ ratio increased significantly under salt stress, with the most substantial increase observed in KHR plants. These findings suggest that GmADH13 promotes Na⁺ efflux from root cells, thereby maintaining intracellular ion balance and mitigating salt-induced cellular toxicity, ultimately enhancing salt tolerance in soybean.

Conclusion

Here, a comprehensive analysis of the soybean ADH gene family identified 22 GmADH genes, which were classified into five phylogenetic subgroups. Differential expression analysis revealed that various GmADH subtypes exhibit distinct transcriptional responses to salt stress, with GmADH13 displaying the most robust induction. Functional characterization demonstrated that GmADH13 plays a pivotal role in soybean salt tolerance by enhancing antioxidant activity, reducing ROS accumulation, and maintaining ionic homeostasis to mitigate cellular damage. These findings offer new insights into the regulatory functions of ADH genes in plant stress responses and lay the groundwork for further exploration of ADH-mediated stress tolerance mechanisms in soybean and other crop species.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 2895 KB) (2.8MB, docx)

Author contributions

Sihui Wang contributed to writing—original draft, methodology, investigation, and data curation; Zhiyuan Xu contributed to data curation and validation; Peng Cheng contributed to resources and methodology; Jun Yang contributed to data curation and methodology; Qiang Hao contributed to data curation and resources; Jinfang Wang contributed to resources and methodology; Ziqian Cheng, Anyu Su and Lingshi Xia contributed to validation and data curation; ; Zhenbang Hu contributed to visualization and data curation; Xin Li contributed to writing—reviewing and editing.. All authors read and approved the final manuscript.

Funding

This study was financially supported by the National Key R&D Program of China (2021YFD1201104-02, 2023YFD2300101); the National Science and Technology Major Projects (2023ZD040360305, 2023ZD0403604); the National Natural Science Foundation of China (U23A20192, 31971899, 32272093, 32272072); the Natural Science Foundation of Heilongjiang Province (TD2022C003, YQ2022C010); and the Innovation Project of Heilongjiang Academy of Agricultural Sciences (CX23ZD05).

Data availability

Data will be made available on request.

Declarations

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Sihui Wang, Zhiyuan Xu, and Peng Cheng contributed equally to this work.

Contributor Information

Zhenbang Hu, Email: zbhu@neau.edu.cn.

Xin Li, Email: maize_lee@163.com.

Anyu Su, Email: aysu@neau.edu.cn.

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Associated Data

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Supplementary Materials

Supplementary file1 (DOCX 2895 KB) (2.8MB, docx)

Data Availability Statement

Data will be made available on request.

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