Genetically optimizing soybean nodulation improves yield and protein content.

Symbiotic nitrogen fixation in legume nodules requires substantial energy investment from host plants, and soybean (Glycine max (L.) supernodulation mutants show stunting and yield penalties due to overconsumption of carbon sources. We obtained soybean mutants differing in their nodulation ability, among which rhizobially induced cle1a/2a (ric1a/2a) has a moderate increase in nodule number, balanced carbon allocation, and enhanced carbon and nitrogen acquisition. In multi-year and multi-site field trials in China, two ric1a/2a lines had improved grain yield, protein content and sustained oil content, demonstrating that gene editing towards optimal nodulation improves soybean yield and quality.

miRNA Expression Analysis of IPEC-J2 Cells Damaged by Soybean 7S Globulin Reveals ssc-miR-221-5p as the Factor Alleviating Cell Damage.

The most significant and sensitive antigen protein that causes diarrhea in weaned pigs is soybean 7S globulin. Therefore, identifying the primary target for minimizing intestinal damage brought on by soybean 7S globulin is crucial. MicroRNA (miRNA) is closely related to intestinal epithelium's homeostasis and integrity. However, the change of miRNAs' expression and the function of miRNAs in Soybean 7S globulin injured-IPEC-J2 cells are still unclear. In this study, the miRNAs' expression profile in soybean 7S globulin-treated IPEC-J2 cells was investigated. Fifteen miRNAs were expressed differently. The differentially expressed miRNA target genes are mainly concentrated in signal release, cell connectivity, transcriptional inhibition, and Hedgehog signaling pathway. Notably, we noticed that the most significantly decreased miRNA was ssc-miR-221-5p after soybean 7S globulin treatment. Therefore, we conducted a preliminary study on the mechanisms of ssc-miR-221-5p in soybean 7S globulin-injured IPEC-J2 cells. Our research indicated that ssc-miR-221-5p may inhibit ROS production to alleviate soybean 7S globulin-induced apoptosis and inflammation in IPEC-J2 cells, thus protecting the cellular mechanical barrier, increasing cell proliferation, and improving cell viability. This study provides a theoretical basis for the prevention and control of diarrhea of weaned piglets.

De novo genome assembly of a high-protein soybean variety HJ117.

OBJECTIVES: Soybean is an important feed and oil crop in the world due to its high protein and oil content. China has a collection of more than 43,000 soybean germplasm resources, which provides a rich genetic diversity for soybean breeding. However, the rich genetic diversity poses great challenges to the genetic improvement of soybean. This study reports on the de novo genome assembly of HJ117, a soybean variety with high protein content of 52.99%. These data will prove to be valuable resources for further soybean quality improvement research, and will aid in the elucidation of regulatory mechanisms underlying soybean protein content. DATA DESCRIPTION: We generated a contiguous reference genome of 1041.94 Mb for HJ117 using a combination of Illumina short reads (23.38 Gb) and PacBio long reads (25.58 Gb), with high-quality sequence coverage of approximately 22.44× and 24.55×, respectively. HJ117 was developed through backcross breeding, using Jidou 12 as the recurrent parent and Chamoshidou as the donor parent. The assembly was further assisted by 114.5 Gb Hi-C data (109.9×), resulting in a contig N50 of 19.32 Mb and scaffold N50 of 51.43 Mb. Notably, Core Eukaryotic Genes Mapping Approach (CEGMA) assessment and Benchmarking Universal Single-Copy Orthologs (BUSCO) assessment results indicated that most core eukaryotic genes (97.18%) and genes in the BUSCO dataset (99.4%) were identified, and 96.44% of the genomic sequences were anchored onto twenty pseudochromosomes.

Genome-wide association study and high-quality gene mining related to soybean protein and fat.

BACKGROUND: Soybean is one of the most important oil crops in the world, and its protein and fat are the primary sources of edible oil and vegetable protein. The effective components in soybean protein and fat have positive effects on improving human immunity, anti-tumor, and regulating blood lipids and metabolism. Therefore, increasing the contents of protein and fat in soybeans is essential for improving the quality of soybeans. RESULTS: This study selected 292 soybean lines from different regions as experimental materials, based on SLAF-seq sequencing technology, and performed genome-wide association study (GWAS) on the phenotype data from 2019-2021 Planted at the experimental base of Jilin Agricultural University, such as the contents of protein and fat of soybeans. Through the GLM model and MLM model, four SNP sites (Gm09_39012959, Gm12_35492373, Gm16_9297124, and Gm20_24678362) that were significantly related to soybean fat content were associated for three consecutive years, and two SNP sites (Gm09_39012959 and Gm20_24678362) that were significantly related to soybean protein content were associated. By the annotation and enrichment of genes within the 100 Kb region of SNP loci flanking, two genes (Glyma.09G158100 and Glyma.09G158200) related to soybean protein synthesis and one gene (Glyma.12G180200) related to lipid metabolism were selected. By the preliminary verification of expression levels of genes with qPCR, it is found that during the periods of R6 and R7 of the accumulation of soybean protein and fat, Glyma.09G158100 and Glyma.09G158200 are positive regulatory genes that promote protein synthesis and accumulation, while Glyma.12G180200 is the negative regulatory gene that inhibits fat accumulation. CONCLUSIONS: These results lay the basis for further verifying the gene function and studying the molecular mechanisms regulating the accumulation of protein and fat in soybean seeds.

Overexpressing GmCGS2 Improves Total Amino Acid and Protein Content in Soybean Seed.

Soybean (Glycine max (L.) Merr.) is an important source of plant protein, the nutritional quality of which is considerably affected by the content of the sulfur-containing amino acid, methionine (Met). To improve the quality of soybean protein and increase the Met content in seeds, soybean cystathionine γ-synthase 2 (GmCGS2), the first unique enzyme in Met biosynthesis, was overexpressed in the soybean cultivar "Jack", producing three transgenic lines (OE3, OE4, and OE10). We detected a considerable increase in the content of free Met and other free amino acids in the developing seeds of the three transgenic lines at the 15th and 75th days after flowering (15D and 75D). In addition, transcriptome analysis showed that the expression of genes related to Met biosynthesis from the aspartate-family pathway and S-methyl Met cycle was promoted in developing green seeds of OE10. Ultimately, the accumulation of total amino acids and soluble proteins in transgenic mature seeds was promoted. Altogether, these results indicated that GmCGS2 plays an important role in Met biosynthesis, by providing a basis for improving the nutritional quality of soybean seeds.

A Soybean Sucrose Non-Fermenting Protein Kinase 1 Gene, GmSNF1, Positively Regulates Plant Response to Salt and Salt-Alkali Stress in Transgenic Plants.

Soybean is one of the most widely grown oilseed crops worldwide. Several unfavorable factors, including salt and salt-alkali stress caused by soil salinization, affect soybean yield and quality. Therefore, exploring the molecular basis of salt tolerance in plants and developing genetic resources for genetic breeding is important. Sucrose non-fermentable protein kinase 1 (SnRK1) belongs to a class of Ser/Thr protein kinases that are evolutionarily highly conserved direct homologs of yeast SNF1 and animal AMPKs and are involved in various abiotic stresses in plants. The GmPKS4 gene was experimentally shown to be involved with salinity tolerance. First, using the yeast two-hybrid technique and bimolecular fluorescence complementation (BiFC) technique, the GmSNF1 protein was shown to interact with the GmPKS4 protein. Second, the GmSNF1 gene responded positively to salt and salt-alkali stress according to qRT-PCR analysis, and the GmSNF1 protein was localized in the nucleus and cytoplasm using subcellular localization assay. The GmSNF1 gene was then heterologously expressed in yeast, and the GmSNF1 gene was tentatively identified as having salt and salt-alkali tolerance function. Finally, the salt-alkali tolerance function of the GmSNF1 gene was demonstrated by transgenic Arabidopsis thaliana, soybean hairy root complex plants overexpressing GmSNF1 and GmSNF1 gene-silenced soybean using VIGS. These results indicated that GmSNF1 might be useful in genetic engineering to improve plant salt and salt-alkali tolerance.

Genome-Wide Identification and Analysis of the Hsp40/J-Protein Family Reveals Its Role in Soybean (Glycine max) Growth and Development.

The J-protein family comprises molecular chaperones involved in plant growth, development, and stress responses. Little is known about this gene family in soybean. Hence, we characterized J-protein genes in soybean, with the most highly expressed and responsive during flower and seed development. We also revealed their phylogeny, structure, motif analysis, chromosome location, and expression. Based on their evolutionary links, we divided the 111 potential soybean J-proteins into 12 main clades (I-XII). Gene-structure estimation revealed that each clade had an exon-intron structure resembling or comparable to others. Most soybean J-protein genes lacked introns in Clades I, III, and XII. Moreover, transcriptome data obtained from a publicly accessible soybean database and RT-qPCR were used to examine the differential expression of DnaJ genes in various soybean tissues and organs. The expression level of DnaJ genes indicated that, among 14 tissues, at least one tissue expressed the 91 soybean genes. The findings suggest that J-protein genes could be involved in the soybean growth period and offer a baseline for further functional research into J-proteins' role in soybean. One important application is the identification of J-proteins that are highly expressed and responsive during flower and seed development in soybean. These genes likely play crucial roles in these processes, and their identification can contribute to breeding programs to improve soybean yield and quality.

GmWAK1, Novel Wall-Associated Protein Kinase, Positively Regulates Response of Soybean to Phytophthora sojae Infection.

Phytophthora root rot is a destructive soybean disease worldwide, which is caused by the oomycete pathogen Phytophthora sojae (P. sojae). Wall-associated protein kinase (WAK) genes, a family of the receptor-like protein kinase (RLK) genes, play important roles in the plant signaling pathways that regulate stress responses and pathogen resistance. In our study, we found a putative Glycine max wall-associated protein kinase, GmWAK1, which we identified by soybean GmLHP1 RNA-sequencing. The expression of GmWAK1 was significantly increased by P. sojae and salicylic acid (SA). Overexpression of GmWAK1 in soybean significantly improved resistance to P. sojae, and the levels of phenylalanine ammonia-lyase (PAL), SA, and SA-biosynthesis-related genes were markedly higher than in the wild-type (WT) soybean. The activities of enzymatic superoxide dismutase (SOD) and peroxidase (POD) antioxidants in GmWAK1-overexpressing (OE) plants were significantly higher than those in in WT plants treated with P. sojae; reactive oxygen species (ROS) and hydrogen peroxide (H2O2) accumulation was considerably lower in GmWAK1-OE after P. sojae infection. GmWAK1 interacted with annexin-like protein RJ, GmANNRJ4, which improved resistance to P. sojae and increased intracellular free-calcium accumulation. In GmANNRJ4-OE transgenic soybean, the calmodulin-dependent kinase gene GmMPK6 and several pathogenesis-related (PR) genes were constitutively activated. Collectively, these results indicated that GmWAK1 interacts with GmANNRJ4, and GmWAK1 plays a positive role in soybean resistance to P. sojae via a process that might be dependent on SA and involved in alleviating damage caused by oxidative stress.

Soybean seed protein storage vacuoles for expression of recombinant molecules.

Soybean is one of the most important protein sources for human consumption and livestock feed. Soy production also allows the biosynthesis of edible oils, biodiesel, and biofertilizers. With the advent of modern agricultural biotechnology, soybean plants have also converted into bioreactors of therapeutic proteins and industrial enzymes. Soybean's characteristics, such as protein storage vacuoles (PSVs) and other unique organelles, allow the plant to be exploited as an accumulator of heterologous proteins under high stability and scalability conditions, and that maintains its basic functions. This review reports the main aspects of heterologous protein accumulation in soybean PSVs.

Amino acids induce high seed-specific expression driven by a soybean (Glycine max) glycinin seed storage protein promoter.

KEY MESSAGE: We characterize GFP expression driven by a soybean glycinin promoter in transgenic soybean. We demonstrate specific amino acid-mediated induction of this promoter in developing soybean seeds in vitro. In plants, gene expression is primarily regulated by promoter regions which are located upstream of gene coding sequences. Promoters allow transcription in certain tissues and respond to environmental stimuli as well as other inductive phenomena. In soybean, seed storage proteins (SSPs) accumulate during seed development and account for most of the monetary and nutritional value of this crop. To better study the regulatory functions of a SSP promoter, we developed a cotyledon culture system where media and media addenda were evaluated for their effects on cotyledon development and promoter activity. Stably transformed soybean events containing a glycinin SSP promoter regulating the green fluorescent protein (GFP) were generated. Promoter activity, as visualized by GFP expression, was only observed in developing in planta seeds and in vitro-cultured isolated embryos and cotyledons from developing seeds when specific media addenda were included. Asparagine, proline, and especially glutamine induced glycinin promoter activity in cultured cotyledons from developing seeds. Other amino acids did not induce the glycinin promoter. Here, we report, for the first time, induction of a reintroduced glycinin SSP promoter by specific amino acids in cotyledon tissues during seed development.

RNAi and CRISPR-Cas silencing E3-RING ubiquitin ligase AIP2 enhances soybean seed protein content.

The majority of plant protein in the world's food supply is derived from soybean (Glycine max). Soybean is a key protein source for global animal feed and is incorporated into plant-based foods for people, including meat alternatives. Soybean protein content is genetically variable and is usually inversely related to seed oil content. ABI3-interacting protein 2 (AIP2) is an E3-RING ubiquitin ligase that targets the seed-specific transcription factor ABI3. Silencing both soybean AIP2 genes (AIP2a and AIP2b) by RNAi enhanced seed protein content by up to seven percentage points, with no significant decrease in seed oil content. The protein content enhancement did not alter the composition of the seed storage proteins. Inactivation of either AIP2a or AIP2b by a CRISPR-Cas9-mediated mutation increased seed protein content, and this effect was greater when both genes were inactivated. Transactivation assays in transfected soybean hypocotyl protoplasts indicated that ABI3 changes the expression of glycinin, conglycinin, 2S albumin, and oleosin genes, indicating that AIP2 depletion increased seed protein content by regulating activity of the ABI3 transcription factor protein. These results provide an example of a gene-editing prototype directed to improve global food security and protein availability in soybean that may also be applicable to other protein-source crops.

A polygalacturonase gene PG031 regulates seed coat permeability with a pleiotropic effect on seed weight in soybean.

KEY MESSAGE: A QTL gene PG031 regulates the seed coat permeability and seed weight. The critical SNP that can explain the variation of permeability in soybean population can be used for seed improvement. Seed coat permeability is a critical trait for soybean and is tightly associated with seed storage longevity, germination, soy-food processing, and other commercially important traits. However, the molecular mechanism of such an important trait in soybean is largely unclear. In the present study, we uncovered a polygalacturonase (PG) gene, PG031, which controls seed coat permeability in soybean. PG031 exhibited tissue expression specificity in flowers while it was strongly induced in the seed coat and radical upon imbibition. Subcellular localization localized PG031 to the cell wall, suggesting its role specific to the cell wall of the seed coat. Natural variation analysis reveals three haplotypes (PG031289H, PG031289Y, and PG031Hap3) and the single nucleotide polymorphism (SNP) variation for H289Y may explain the variation in permeability in cultivated soybean population. Overexpression of impermeable allele PG031289H significantly reduced the seed coat permeability and 100-seed weight in transgenic seeds through decreasing intracellular spaces of the osteosclereid layer and parenchyma of the seed coat to decline water accessing the seed. PG031 was also located within a quantitative trait locus (QTL) explaining ~ 15% of total phenotypic variation in permeability, nominating it the QTL gene controlling permeability. PG031289Y allele associated with high permeability and high seed weight is experiencing ongoing artificial selection. The results provide insight into the genetic mechanism of seed coat permeability and indicate its potential for the improvement of permeability-associated seed traits in soybean.

Genome-wide analysis of sucrose synthase family in soybean and their expression in response to abiotic stress and seed development.

The sucrose synthase (SS) is an important enzyme family which play a vital role in sugar metabolism to improve the fruit quality of the plants. In many plant species, the members of SS family have been investigated but the detailed information is not available in legumes particularly and Glycine max specifically. In the present study, we found thirteen SS members (GmSS1-GmSS13) in G. max genome. High conserved regions were present in the GmSS sequences that may due to the selection pressure during evolutionary events. The segmental duplication was the major factor to increase the number of GmSS family members. The identified thirteen GmSS genes were divided into Class I, Class II and Class III with variable numbers of genes in each class. The protein interaction of GmSS gave the co-expression of sucrose synthase with glucose-1-phosphate adenylyltransferase while SLAC and REL test found number of positive sites in the coding sequences of SS family members. All the GmSS family members except GmSS7 and few of class III members, were highly expressed in all the soybean tissues. The expression of the class I members decreased during seed development, whireas, the class II members expression increased during the seed developing, may involve in sugar metabolism during seed development. Solexa sequencing libraries of acidic condition (pH 4.2) stress samples showed that the expression of class I GmSS genes increased 1- to 2-folds in treated samples than control. The differential expression pattern was observed between the members of a paralogous. This study provides detailed genome-wide analysis of GmSS family in soybean that will provide new insights for future evolutionary and soybean breeding to improve the plant growth and development.

Growth and transpiration of soybean genotypes with HaHB4® transcription factor for drought tolerance.

The expression of HaHB4® transcription factor reduces soybean sensitivity to abiotic stresses, such as water deficit. Studies that quantify the tolerance of HaHB4® soybean to the soil water content in comparison with cultivars currently sown in Brazil are lacking. The objective of this study was to determine the level of drought tolerance of soybean genotypes expressing the HaHB4® transcription factor (TS18-6-610108 and TS18-6-610084) and commercial cultivars (TMG 7063 IPRO and BS IRGA 1642 IPRO) subjected to water deficit during the vegetative phase. We used the fraction of transpirable soil water (FTSW) approach. Parameters related to leaf transpiration, dry matter accumulation, water use efficiency (WUE), and transpiration coefficient (TC) were evaluated in the four soybean genotypes and two treatments (T1-100% replacement of transpired water and T2-without replacement of transpired water). The FTSW threshold for the decline in transpiration was evaluated to identify the onset of water stress in soybean. TS18-6-610108 and BS IRGA 1642 IPRO maintains potential transpiration at low FTSW values. The TS18-6-610108 genotype has 14% higher WUE than the sensitive cultivar under water deficit. Under well-irrigated conditions, the HaHB4® genotypes showed the highest TC values, which indicate well-functioning physiological processes.

A Multi-Year, Multi-Cultivar Approach to Differential Expression Analysis of High- and Low-Protein Soybean (Glycine max).

Soybean (Glycine max (L.) Merr.) is among the most valuable crops based on its nutritious seed protein and oil. Protein quality, evaluated as the ratio of glycinin (11S) to ß-conglycinin (7S), can play a role in food and feed quality. To help uncover the underlying differences between high and low protein soybean varieties, we performed differential expression analysis on high and low total protein soybean varieties and high and low 11S soybean varieties grown in four locations across Eastern and Western Canada over three years (2018-2020). Simultaneously, ten individual differential expression datasets for high vs. low total protein soybeans and ten individual differential expression datasets for high vs. low 11S soybeans were assessed, for a total of 20 datasets. The top 15 most upregulated and the 15 most downregulated genes were extracted from each differential expression dataset and cross-examination was conducted to create shortlists of the most consistently differentially expressed genes. Shortlisted genes were assessed for gene ontology to gain a global appreciation of the commonly differentially expressed genes. Genes with roles in the lipid metabolic pathway and carbohydrate metabolic pathway were differentially expressed in high total protein and high 11S soybeans in comparison to their low total protein and low 11S counterparts. Expression differences were consistent between East and West locations with the exception of one, Glyma.03G054100. These data are important for uncovering the genes and biological pathways responsible for the difference in seed protein between high and low total protein or 11S cultivars.

Genome-Wide Analysis of the Soybean Calmodulin-Binding Protein 60 Family and Identification of GmCBP60A-1 Responses to Drought and Salt Stresses.

Calmodulin-binding protein 60 (CBP60) members constitute a plant-specific protein family that plays an important role in plant growth and development. In the soybean genome, nineteen CBP60 members were identified and analyzed for their corresponding sequences and structures to explore their functions. Among GmCBP60A-1, which primarily locates in the cytomembrane, was significantly induced by drought and salt stresses. The overexpression of GmCBP60A-1 enhanced drought and salt tolerance in Arabidopsis, which showed better state in the germination of seeds and the root growth of seedlings. In the soybean hairy roots experiment, the overexpression of GmCBP60A-1 increased proline content, lowered water loss rate and malondialdehyde (MDA) content, all of which likely enhanced the drought and salt tolerance of soybean seedlings. Under stress conditions, drought and salt response-related genes showed significant differences in expression in hairy root soybean plants of GmCBP60A-1-overexpressing and hairy root soybean plants of RNAi. The present study identified GmCBP60A-1 as an important gene in response to salt and drought stresses based on the functional analysis of this gene and its potential underlying mechanisms in soybean stress-tolerance.

Two TGA Transcription Factor Members from Hyper-Susceptible Soybean Exhibiting Significant Basal Resistance to Soybean mosaic virus.

TGA transcription factors (TFs) exhibit basal resistance in Arabidopsis, but susceptibility to a pathogen attack in tomatoes; however, their roles in soybean (Glycine max) to Soybean mosaic virus (SMV) are unknown. In this study, 27 TGA genes were isolated from a SMV hyper-susceptible soybean NN1138-2, designated GmTGA1~GmTGA27, which were clustered into seven phylogenetic groups. The expression profiles of GmTGAs showed that the highly expressed genes were mainly in Groups I, II, and VII under non-induction conditions, while out of the 27 GmTGAs, 19 responded to SMV-induction. Interestingly, in further transient N. benthamiana-SMV pathosystem assay, all the 19 GmTGAs overexpressed did not promote SMV infection in inoculated leaves, but they exhibited basal resistance except one without function. Among the 18 functional ones, GmTGA8 and GmTGA19, with similar motif distribution, nuclear localization sequence and interaction proteins, showed a rapid response to SMV infection and performed better than the others in inhibiting SMV multiplication. This finding suggested that GmTGA TFs may support basal resistance to SMV even from a hyper-susceptible source. What the mechanism of the genes (GmTGA8, GmTGA19, etc.) with basal resistance to SMV is and what their potential for the future improvement of resistance to SMV in soybeans is, are to be explored.

Silencing Autophagy-Related Gene 2 (ATG2) Results in Accelerated Senescence and Enhanced Immunity in Soybean.

Autophagy plays a critical role in nutrient recycling and stress adaptations. However, the role of autophagy has not been extensively investigated in crop plants. In this study, soybean autophagy-related gene 2 (GmATG2) was silenced, using virus-induced silencing (VIGS) mediated by Bean pod mottle virus (BPMV). An accelerated senescence phenotype was exclusively observed for the GmATG2-silenced plants under dark conditions. In addition, significantly increased accumulation of both ROS and SA as well as a significantly induced expression of the pathogenesis-related gene 1 (PR1) were also observed on the leaves of the GmATG2-silenced plants, indicating an activated immune response. Consistent with this, GmATG2-silenced plants exhibited a significantly enhanced resistance to Pseudomonas syringae pv. glycinea (Psg) relative to empty vector control plants (BPMV-0). Notably, the activated immunity of the GmATG2-silenced plants was independent of the MAPK signaling pathway. The fact that the accumulation levels of ATG8 protein and poly-ubiquitinated proteins were significantly increased in the dark-treated GmATG2-silenced plants relative to the BPMV-0 plants indicated that the autophagic degradation is compromised in the GmATG2-silenced plants. Together, our results indicated that silencing GmATG2 compromises the autophagy pathway, and the autophagy pathway is conserved in different plant species.

Genome-Wide Identification, Characterization and Expression Analysis of Soybean CHYR Gene Family.

The CHYR (CHY ZINC-FINGER AND RING FINGER PROTEIN) proteins have been functionally characterized in iron regulation and stress response in Arabidopsis, rice and Populus. However, their roles in soybean have not yet been systematically investigated. Here, in this study, 16 GmCHYR genes with conserved Zinc_ribbon, CHY zinc finger and Ring finger domains were obtained and divided into three groups. Moreover, additional 2-3 hemerythrin domains could be found in the N terminus of Group III. Phylogenetic and homology analysis of CHYRs in green plants indicated that three groups might originate from different ancestors. Expectedly, GmCHYR genes shared similar conserved domains/motifs distribution within the same group. Gene expression analysis uncovered their special expression patterns in different soybean tissues/organs and under various abiotic stresses. Group I and II members were mainly involved in salt and alkaline stresses. The expression of Group III members was induced/repressed by dehydration, salt and alkaline stresses, indicating their diverse roles in response to abiotic stress. In conclusion, our work will benefit for further revealing the biological roles of GmCHYRs.

Ampelopsin Confers Endurance and Rehabilitation Mechanisms in Glycine max cv. Sowonkong under Multiple Abiotic Stresses.

The present investigation aims to perceive the effect of exogenous ampelopsin treatment on salinity and heavy metal damaged soybean seedlings (Glycine max L.) in terms of physiochemical and molecular responses. Screening of numerous ampelopsin concentrations (0, 0.1, 1, 5, 10 and 25 µM) on soybean seedling growth indicated that the 1 µM concentration displayed an increase in agronomic traits. The study also determined how ampelopsin application could recover salinity and heavy metal damaged plants. Soybean seedlings were irrigated with water, 1.5% NaCl or 3 mM chosen heavy metals for 12 days. Our results showed that the application of ampelopsin raised survival of the 45-day old salinity and heavy metal stressed soybean plants. The ampelopsin treated plants sustained high chlorophyll, protein, amino acid, fatty acid, salicylic acid, sugar, antioxidant activities and proline contents, and displayed low hydrogen peroxide, lipid metabolism, and abscisic acid contents under unfavorable status. A gene expression survey revealed that ampelopsin application led to the improved expression of GmNAC109, GmFDL19, GmFAD3, GmAPX, GmWRKY12, GmWRKY142, and GmSAP16 genes, and reduced the expression of the GmERF75 gene. This study suggests irrigation with ampelopsin can alleviate plant damage and improve plant yield under stress conditions, especially those including salinity and heavy metals.