Phytophthora root rot, caused by Phytophthora sojae, is a devastating soilborne disease of soybean (Glycine max). However, the epigenetic regulation of soybean responses to P. sojae remains incompletely understood. Here, using genetic, molecular and biochemical approaches, we characterized the functions of LIKE HETEROCHROMATIN PROTEIN 1 (GmLHP1) and its downstream regulatory network. We demonstrated that GmLHP1, as a reader of H3K27me3, negatively regulates soybean resistance to P. sojae. GmLHP1 binds to H3K27me3 peptides in vitro and colocalizes with H3K27me3 marks genome-wide in vivo. The integrated chromatin immunoprecipitation sequencing and RNA sequencing analysis identified the ethylene biosynthesis pathway gene 1-AMINO-CYCLOPROPANE-1-CARBOXYLATE SYNTHASE 18 (GmACS18) as being enriched for H3K27me3 and bound by GmLHP1, leading to its transcriptional downregulation. Notably, GmLHP1 associates with the GmACS18 promoter by directly binding to AATTAA motifs and recognizing H3K27me3 marks. Moreover, GmACS18 enhances defense against P. sojae by accumulating the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Further analysis unveiled that recognition of H3K27me3 by GmLHP1 is essential for regulating soybean resistance to P. sojae through repressing GmACS18 transcription and decreasing ACC accumulation. Our findings reveal a novel epigenetic regulatory mechanism in which the H3K27me3 reader GmLHP1 blocks soybean resistance to P. sojae by repressing ethylene precursor ACC accumulation.

Phytophthora root rot, a devastating disease caused by Phytophthora sojae, poses a significant threat to worldwide soybean (Glycine max) production. Therefore, enhancing crop resistance to this pathogen is a major breeding objective. However, the signalling mechanisms underlying the response of soybean plants to P. sojae infection, and the networks and targets of key transcription factors TFs, are not yet fully understood. Here, we reveal the mechanisms and function of GmERF109, which differs in expression between soybean cultivars resistant and susceptible to P. sojae race 1 and encodes an AP2/ERF transcription factor. Molecular evaluation and disease resistance analysis show that GmERF109 is a nucleus-localized transcription factor that positively regulates soybean resistance to P. sojae. We also demonstrate that GmERF109 targets and activates the expression of GmG4DT-like, a gene whose role in the biosynthesis of the phytoalexin glyceollin was confirmed through overexpression and RNA interference (RNAi) analyses. GmG4DT-like also enhances P. sojae resistance. GmG4DT-like and GmERF109 greatly increased the content of the glyceollin I isomer. Overall, our results suggest that GmERF109 enhances glyceollin accumulation by positively regulating the expression of its target gene GmG4DT-like, thereby improving soybean resistance to P. sojae. These findings provide novel insights into soybean resistance to Phytophthora root rot and will be useful in efforts to create resistant soybean cultivars.

The transcription factor GmPEIN3 enhances resistance to Phytophthora sojae via ethylene-mediated antioxidant regulation and improves favorable agronomic traits in soybean. Phytophthora sojae is a devastating pathogen of soybean, and developing cultivars that combine high yield with strong disease resistance is a primary breeding objective. However, genes that coordinately regulate these two crucial traits have rarely been identified. In a previous study, using a combination of subtractive suppression hybridization (SSH) and cDNA microarray analysis on the highly resistant soybean variety ‘Suinong 10’, we identified a promising candidate, GmPEIN3 (Glycine max P. sojae-responsive EIN3), an EIN-type transcription factor. In this work, we confirmed that the expression of GmPEIN3 was potently induced by P. sojae infection in resistant soybean germplasm. Overexpression of GmPEIN3 in soybean resulted in enhanced resistance to P. sojae, while RNA-interference (RNAi) silencing led to increased susceptibility. Transcriptome analysis revealed that GmPEIN3 modulates plant hormone signal transduction and phenylpropanoid biosynthesis pathways. Consistently, ethylene (ET) levels, along with the activities and transcript levels of key antioxidant enzymes (SOD, POD, CAT, and GPX), were markedly higher in GmPEIN3-overexpressing (GmPEIN3-OE) transgenic soybean than in wild type (WT). Mechanistically, molecular assays demonstrated that GmPEIN3 directly binds to and transactivates the promoters of the ET biosynthesis gene GmACO3 and antioxidant-related genes GmSODB and GmPOD21, thereby promoting ET production and enhancing antioxidant defense capacity. Moreover, GmPEIN3-OE lines exhibited significant increases in effective pod number, seeds per plant, and 100-seed weight, whereas these traits were markedly reduced in RNAi lines. Taken together, these results suggest that GmPEIN3 plays a positive dual role in soybean, enhancing P. sojae resistance through the modulation of ethylene signaling and antioxidant defense systems, and simultaneously improving yield-related agronomic traits.

Protein phosphorylation and ubiquitination play central roles in signal transduction. SnRK1 is a key kinase that mediates plant responses to environmental cues. From a wild soybean cDNA library, we identified GsSRF2, which encodes a RING-domain E3 ligase that interacts with GsSnRK1. GsSRF2 is predominantly expressed in roots and responds dynamically to salt stress. BiFC, pull-down, and co-IP assays this interaction in the cytoplasm and at the plasma membrane. Biochemical analyses revealed that GsSnRK1 phosphorylates GsSRF2 at Thr514. GnSnRK1-mediated phosphorylation enhanced GsSRF2 ubiquitination and promoted its proteasomal degradation. Coexpression assays in soybean hairy roots demonstrated that the GsSnRK1-GsSRF2 module significantly improved salt tolerance, whereas the GsSnRK1-GsSRF2(T514A) and kinase-dead GsSnRK1(K49M)-GsSRF2 modules reduced salt resistance in composite plants. Together, these results indicate that GsSnRK1 positively regulates soybean salt tolerance by modulating GsSRF2 ligase activity and protein stability, providing valuable genetic resources for crop improvement.

Summary statement We designed a bioinformatics pipeline combining PSPredictor and Metapredict to systematically identify phase separation proteins in soybean, yielding a total of 5515 candidates. Experimental validation confirmed that four nuclear‐localized proteins are capable of forming dynamic liquid‐like condensates through liquid–liquid phase separation both in vivo and in vitro. In addition, saline‐alkali tolerance assays revealed that GmBAF60b, GmFCA and GmPUX5 significantly enhance stress tolerance by lowering Na + /K + ratios and boosting antioxidant enzyme activities. These findings provide key genetic resources and mechanistic insights into LLPS‐mediated stress adaptation in crops.

Nitrogen (N) limitation significantly constrains crop growth, yield and quality. Developing crop varieties with high N deficiency tolerance represents a critical strategy for reducing N fertilizer application and promoting sustainable agriculture. Semi-wild soybean offers valuable genetic resources for the improvement of soybean varieties. Nevertheless, the mechanisms underlying N deficiency tolerance remain poorly understood. In this study, we employed a comprehensive analytical approach-including Pearson's correlation analysis, principal component analysis, subordinate function analysis, and cluster analysis-to evaluate the N starvation tolerance of 50 semi-wild soybean varieties. Shoot fresh weight, root-shoot ratio, SPAD2 value and leaf nitrate content were identified as key indicators for assessing N starvation tolerance. The variety V03 was identified as the most N starvation-tolerant. Comparative physiological analyses revealed that V03 enhances tolerance to N deficiency by optimizing root architecture and sustaining the activity of nitrogen metabolism enzymes-such as nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (GOGAT)-in root and leaf tissues. Transcriptomic analysis indicated that V03 exhibits a broader transcriptional response (with more N Starvation-induced DEGs) and functional reprogramming in root tissues, showing stronger enrichment in stress-responsive processes, regulatory functions, and plasma membrane-related terms as well as environmental information processing pathways. Furthermore, V03 displayed more pronounced changes in the expression of genes related to N transport, N assimilation and transcription factor (TF) compared to the N starvation-sensitive variety V46. This study provides a robust and comprehensive methodology for evaluating N deficiency tolerance in semi-wild soybean. Our findings offer new insights into the physiological adaptions and molecular regulatory network governing N uptake and metabolism, which may support future breeding efforts aimed at enhancing NUE in leguminous crops.

Saline–alkali stress is becoming a major global issue due to environmental degradation, hindering plant growth and reducing both seed yield and quality of many crops, including soybean. Improving stress tolerance through genetic resources is crucial for sustainable production. In this study, overexpression of the Arabidopsis SRRM1L gene increased soybean (Glycine max) tolerance to saline–alkali stress by reducing reactive oxygen species and malondialdehyde accumulation and promoting antioxidant enzyme activities, whereas knockdown of GmSRRM1L1/2 genes reduced stress resistance and overexpression of GmSRRM1L1/2 increased it. These findings highlight that the AtSRRM1L and GmSRRM1L proteins are valuable genetic resources for breeding stress-resistant cultivars without yield penalty.

Soybean (Glycine max) is a major economic and food crop whose yield is severely affected by frogeye leaf spot (FLS), caused by Cercospora sojina. Current knowledge of resistance genes remains insufficient for effective molecular breeding. In this study, a recombinant inbred line (RIL) population derived from a cross between the resistant parent, Henong 60 (H60), and the susceptible parent, Dongnong L13 (DN L13), was evaluated under field conditions in Acheng (AC) and Xiangyang (XY). Plants were artificially inoculated with physiological race 7 of C. sojina, and disease severity at the R3 growth stage was recorded. Genotyping using the SoySNP660K chip yielded 54,836 high-quality single-nucleotide polymorphism (SNP) markers. A genome-wide association study (GWAS) was performed using the 3VmrMLM model by integrating dual-environment phenotypic data, and four quantitative trait nucleotides (QTNs) significantly associated with resistance to FLS were identified on chromosomes 8 (1), 17 (1), and 20 (2). By the analysis of genomic annotation, functional enrichment, metabolic pathway analyses, haplotype-phenotype association and quantitative real-time PCR (qRT-PCR), Glyma.20G155700 and Glyma.17G070500 are intended to be candidate genes related to soybean resistance to race 7 of FLS. The findings of this study provide insights into the genetic mechanisms underlying resistance to FLS in soybean. The identified molecular markers and candidate genes may provide useful resources for marker-assisted breeding and the development of disease-resistant germplasm.

Soybean frogeye leaf spot (FLS) is a global fungal disease that adversely affects both the yield and quality of soybean. The available resistant loci and genes for FLS are limited, thereby constraining the molecular breeding of soybean for FLS resistance. In this study, a recombinant inbred lines (RIL) population (RIL3613), derived from the cross of Dongnong L13 and Heihe 36, was utilized to analyze the genetics of resistance and identify resistant loci. By combining the relative lesion area (RLA) data from each individual of RIL3613 collected across two environments with a high-density bin genetic map, a total of 19 quantitative trait loci (QTL) were identified, distributed across 9 chromosomes, with phenotypic variation explained (PVE) ranging from 1.12% to 18.93%. Notably, 8 QTL were consistently located in both environments, prompting a search for candidate genes within these stable QTL. Through parental sequence variation analysis, expression level determination, haplotype analysis, and gene function annotation, one gene (Glyma.15G245300) within qFLSm-15-1, which encodes cytochrome P450, was selected as a candidate gene. This study identified several novel loci and genes that may enhance molecular breeding efforts aimed at improving resistance to FLS in soybean.

Wild soybean (Glycine soja) is a leguminous species known for its ability to thrive in challenging and barren environments. It has been reported that the nitrate transporters (NRTs) play critical roles for plants to survive in the nutrient-poor soils. However, the molecular mechanisms of GsNRTs in governing nitrogen (N) uptake remain largely elusive. In the present study, we identified a NRT2.4-like protein (GsNRT2.4a) as an interactor of GsSnRK1.1 kinase. Our biophysical and physiological analyses indicate that GsNRT2.4a functions as an active NRT, and GsSnRK1.1 kinase phosphorylates the Ser518 residue at the carboxyl region of GsNRT2.4a. Under N starvation conditions, the double mutant nrt2.1/nrt2.2 (2nrtm) and the quadruple mutant nrt2.1/nrt2.2/kin10/kin11 (2kinm/2nrtm) exhibited compromised growth of Arabidopsis. However, introduction of GsNRT2.4a or GsSnRK1.1/GsNRT2.4a genes into the mutants rescued their defective growth to different extent. Furthermore, we determined that GsSnRK1.1 plays a pivotal role in modulating GsNRT2.4a activity in planta by phosphorylating GsNRT2.4a at the Ser518 site, thereby collaboratively modulating plant growth under N starvation. Our findings suggest that GsNRT2.4a is essential for optimising nitrate uptake in plants, and it also elucidates a novel regulatory mechanism of GsSnRK1.1-GsNRT2.4a module for potential enhancement of nitrogen use efficiency (NUE) in plants.

The flowering time of soybean [Glycine max (L.) Merr.] is extremely sensitive to photoperiod, which importantly influences its yield potential and restricts the geographical range of soybean cultivars to specific latitudes. Molecular breeding to modulate flowering time and reduce sensitivity to day length is an effective approach to enhance the adaptability and productivity of soybean. Here, we characterized reproductive meristem 16a (GmREM16a), a member of the AP2/B3-like transcription factor family. The GmREM16a protein contains 2 B3 domains, and the expression of its encoding gene is responsive to photoperiod and circadian rhythm. The overexpression of GmREM16a in soybean accelerated flowering by regulating the expression of flowering-related genes. The GmREM16a protein was able to directly bind to the promoters of GmSOC1, GmFT2a, and GmFT5a and upregulate their expression. Yeast two-hybrid screening revealed that GmCSN5 interacts with GmREM16a. GmCSN5 is the fifth subunit of the COP9 signalosome (constitutively photomorphogenic signalosome, CSN) that regulates the activity of CULLIN-RING E3 ubiquitin ligases and regulates protein degradation. Protein degradation assays in vivo and in vitro showed that GmCSN5 promotes the degradation of GmREM16a protein via the ubiquitin-proteasome pathway. Taken together, these findings indicate that the transcription factor GmREM16a promotes flowering by regulating the expression of flowering-related genes. Additionally, GmCSN5 interacts with GmREM16a to regulate its stability in soybean. The GmREM16a-GmSCN5 module may represent a pathway involved in the regulation of flowering time in soybean and is a useful genetic resource for improving the adaptability of soybean through molecular breeding approaches.

Drought stress represents a prevalent environmental challenge that significantly impedes plant growth. The Chinese hog-peanut (Amphicarpaea edgeworthii Benth.), an amphicarpic legume, can produce both aerial seeds (ASs) and subterranean seeds (SSs). However, it is largely unknown whether there are differences between the seedlings from ASs and SSs in response to drought stress. In this study, the 30-day old AS and SS seedlings of A. edgeworthii are subjected to drought stress by withholding watering for five or ten days. Then, we identify the morphological and physio-biochemical characteristics of seedlings from both ASs and SSs under drought stress. Following ten days of drought treatment, the contents of proline (PRO) and malondialdehyde (MDA), the root shoot ratio, and the rate of water loss were significantly increased, whereas the chlorophyll content and the relative water content were significantly decreased in both AS and SS seedlings. Moreover, compared to AS seedlings, SS seedlings accumulated more hydrogen peroxide (H2O2) while exhibiting significantly lower peroxidase (POD) and superoxide dismutase (SOD) activities after exposure to ten days of drought stress. These findings indicate that SS seedlings are more susceptible to drought stress. To identify drought-associated genes and reveal the mechanisms underlying drought adaptability in AS and SS seedlings, we performed an RNA-seq-based transcriptomic analysis in AS and SS seedlings exposed to drought stress. We identified 1317 and 2029 differentially expressed genes (DEGs) in AS seedlings five and ten days post-drought treatment, respectively, and 1793 DEGs in SS seedlings ten days post-drought treatment compared to the normal treatment (CK). These DEGs were commonly enriched in response-related GO terms. Furthermore, hundreds of transcription factor (TF) genes were identified among the DEGs in AS and SS seedlings after drought treatment. Notably, the ERF, bHLH, NAC, and C2H2 families were predominant in AS seedlings five days following drought treatment, while the bHLH, ERF, MYB-related, and WRKY families were prevalent in both AS and SS seedlings ten days following drought treatment. These findings suggest that the identified TFs may play crucial roles in the response of AS and SS seedlings of A. edgeworthii to drought stress.

Salineu2013alkali stress is becoming a major global issue due to environmental degradation, hindering plant growth and reducing both seed yield and quality of many crops, including soybean. Improving stress tolerance through genetic resources is crucial for sustainable production. In this study, overexpression of the Arabidopsis SRRM1L gene increased soybean (Glycine max) tolerance to salineu2013alkali stress by reducing reactive oxygen species and malondialdehyde accumulation and promoting antioxidant enzyme activities, whereas knockdown of GmSRRM1L1/2 genes reduced stress resistance and overexpression of GmSRRM1L1/2 increased it. These findings highlight that the AtSRRM1L and GmSRRM1L proteins are valuable genetic resources for breeding stress-resistant cultivars without yield penalty.

Transcriptomic analysis revealed that GmERF1 expression is induced by Phytophthora sojae and positively regulates soybean resistance to P. sojae via salicylic acid signal transduction pathway. Soybean (Glycine max) root rot caused by Phytophthora sojae is a major disease constraining the global soybean industry. Therefore, improving crop resistance to this pathogen remains a key objective in breeding efforts. However, the mechanisms by which soybeans respond to P. sojae infection, as well as the specific regulatory networks of key transcription factors (TFs), remain to be elucidated. Here, we report that Ethylene Response Factor 1 (GmERF1), encoding an AP2/ERF transcription factor, exhibits significant differences in expression between resistant and susceptible soybean cultivars. Molecular evaluation and disease resistance analysis show that GmERF1 could improve soybean resistance to P. sojae. Further transcriptomic analysis and quantitative analysis of salicylic acid (SA) signal transduction genes indicate that GmERF1 could positively regulate the expression of Non-expressor of Pathogenesis-Related genes 1 (GmNPR1), TGACG sequence-specific binding factor (GmTGA) and Pathogenesis-Related gene 1 (GmPR1). Taken together, these results suggest that GmERF1 positively regulates soybean resistance to P. sojae by enhancing SA signaling, providing novel insights into soybean resistance to Phytophthora root rot.

Phytophthora root and stem rot is a destructive soybean disease worldwide, and thus improving soybean resistance to P. sojae is a major breeding target. However, the complex regulatory networks governing host defense remain unclear. Our previous study showed that GmWRKY40 positively regulates resistance of soybean to P. sojae. Here, to explore its molecular mechanism, we found that GmWRKY40 is induced by P. sojae in resistant cultivars and that the protein localizes in nucleus. RNA-seq and metabolomic analyses revealed that GmWRKY40 modulates the jasmonate (JA) signaling pathway. We then found that GmWRKY40 directly suppresses the key JA repressor GmJAZ1 by binding to the promoter. This leads to higher endogenous JA levels, and the overall state of enhanced resistance is also characterized by elevated SOD and POD antioxidant enzyme activity. Furthermore, we demonstrated that GmWRKY40 interacts with GmWRKY36, a transcription factor identified as a negative regulator of P. sojae infection in this research. Taken together, our study delineates a novel regulatory module where GmWRKY40 enhances resistance to P. sojae through a dual mechanism: activating the JA pathway by repressing its suppressor GmJAZ1, and engaging in a potentially antagonistic interaction with the negative regulator GmWRKY36, ultimately enhancing soybean resistance to P. sojae.

Soybean frogeye leaf spot (FLS) disease has been reported globally and is caused by the fungus Cercospora sojina, which affects the growth, seed yield, and quality of soybean. Among the 15 physiological microspecies of C. sojina soybean in China, Race 7 is one of the main pathogenic microspecies. A few genes are involved in resistance to FLS, and they cannot meet the need to design molecular breeding methods for disease resistance. In this study, a soybean recombinant inbred line (RIL3613) population and a germplasm resource (GP) population were planted at two sites, Acheng (AC) and Xiangyang (XY). Phenotypic data on the percentage of leaf area diseased (PLAD) in soybean leaves were obtained via image recognition technology after the inoculation of seven physiological species and full onset at the R3 stage. Quantitative trait loci (QTLs) and quantitative trait nucleotides (QTNs) were mapped via linkage analysis and genome-wide association studies (GWASs), respectively. The resistance genes of FLS were subsequently predicted in the linkage disequilibrium region of the collocated QTN. We identified 114 QTLs and 18 QTNs in the RIL3613 and GP populations, respectively. A total of 14 QTN loci were colocalized in the two populations, six of which presented high phenotypic contributions. Through haplotype–phenotype association analysis and expression quantification, three genes (Glyma.06G300100, Glyma.06G300600, and Glyma.13G172300) located near molecular markers AX-90524088 and AX-90437152 (QTNs) are associated with FLS Chinese Race 7, identifying them as potential candidate resistance genes. These results provide a theoretical basis for the genetic mining of soybean antigray spot No. 7 physiological species. These findings also provide a theoretical basis for understanding the genetic mechanism underlying FLS resistance in soybeans.

Alternative splicing (AS) plays important roles in modulating environmental stress responses in plants. However, little is known about the functions of bicarbonate-induced AS in cultivated soybean (Glycine max L. Merr.). In this study, we combined PacBio isoform sequencing (Iso-seq) and Illumina RNA sequencing (RNA-seq) to elucidate the bicarbonate-induced AS events in soybean root and leaf tissues. Compared to RNA-seq, Iso-seq identified more novel genes and transcripts, as well as more AS events, indicating that Iso-seq is more efficient in AS detection. Combining these two technologies, we found that intron retention (IR) is the most frequent AS event type. We identified a total of 913 and 1974 bicarbonate stress-responsive differentially alternative spliced genes (DAGs) in soybean leaves and roots respectively, from our RNA-seq results. Additionally, we determined a transcription factor (GmNTL9) and a splicing factor (GmRSZ22), and validated their roles in bicarbonate stress response by AS. Overall, our study opens an avenue for evaluating plant AS regulatory networks, and the obtained global landscape of alternative splicing provides valuable insights into the AS-mediated bicarbonate-responsive mechanisms in plant species.

The properties and applications of soybean protein isolates (SPIs) have been extensively investigated. In this study, we determined the optimal conditions for the preparation of the DND358 soybean protein isolate (DND358-SPI), assessed its physicochemical and functional properties, and investigated its potential applications in the food industry. According to the results, the highest extraction rate of DND358-SPI was observed when the pH was 9.5, the temperature was 55 °C, the duration was 80 min, and the material-to-liquid ratio was 1:20 (w/v). With regard to the functional properties, the water-holding capacity (WHC) and oil-binding capacity (OBC) of DND358-SPI were higher than those of other varieties, reaching 4.73% and 11.04%, respectively. In addition, the hardness, adhesiveness, chewiness, and resilience of DND358-SPI were higher than those of other varieties, reaching 159.27 g, 186.07 g, 6.78 mj, and 1.88, respectively. These findings indicate that DND358-SPI can reduce cholesterol levels and may be used to produce cholesterol-lowering food products.

Soybean beta-conglycinin is a major allergen that adversely affects the nutritional properties of soybean. Soybean deficient in beta-conglycinin is associated with low allergenicity and high nutritional value. Long intergenic noncoding RNAs (lincRNAs) regulate gene expression and are considered important regulators of essential biological processes. Despite increasing knowledge of the functions of lincRNAs, relatively little is known about the effects of lincRNAs on the accumulation of soybean beta-conglycinin. The current study presents the identification of a lincRNA lincCG1 that was mapped to the intergenic noncoding region of the beta-conglycinin alpha-subunit locus. The full-length lincCG1 sequence was cloned and found to regulate the expression of soybean seed storage protein (SSP) genes via both cis- and trans-acting regulatory mechanisms. Loss-of-function lincCG1 mutations generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system led to the deficiency of the allergenic alpha '-, alpha-, and beta-subunits of soybean beta-conglycinin as well as higher content of proteins, sulfur-containing amino acids, and free arginine. The dominant null allele LincCG1, and consequently, the beta-conglycinin-deficient phenotype associated with the lincCG1-gene-edited line was stably inherited by the progenies in a Mendelian fashion. The dominant null allele LincCG1 may therefore be exploited for engineering/developing novel hypoallergenic soybean varieties. Furthermore, Cas9-free and beta-conglycinin-deficient homozygous mutant lines were obtained in the T1 generation. This study is the first to employ the CRISPR/Cas9 technology for editing a lincRNA gene associated with the soybean allergenic protein beta-conglycinin. Moreover, this study reveals that lincCG1 plays a crucial role in regulating the expression of the beta-conglycinin subunit gene cluster, besides highlighting the efficiency of employing the CRISPR/Cas9 system for modulating lincRNAs, and thereby regulating soybean seed components.

Wild soybean (Glycine soja), a relative of cultivated soybean, shows high adaptability to adverse environmental conditions. We identified and characterized a wild soybean transcription factor gene, GsWRKY40, that promotes plant salt stress. GsWRKY40 was highly expressed in wild soybean roots and was up-regulated by salt treatment. GsWRKY40 was localized in nucleus and demonstrated DNA-binding activities but without transcriptional activation. Mutation and overexpression of GsWRKY40 altered salt tolerance of Arabidopsis plants. To understand the molecular mechanism of GsWRKY40 in regulating plant salt resistance, we screened a cDNA library and identified a GsWRKY40 interacting protein GsbHLH92 by using yeast two-hybrid approach. The physical interaction of GsWRKY40 and GsbHLH92 was confirmed by co-immunoprecipitation (co-IP), GST pull-down, and bimolecular fluorescence complementation (BiFC) techniques. Intriguingly, co-overexpression of GsWRKY40 and GsbHLH92 resulted in higher salt tolerance and lower ROS levels than overexpression of GsWRKY40 or GsbHLH92 in composite soybean plants, suggesting that GsWRKY40 and GsbHLH92 may synergistically regulate plant salt resistance through inhibiting ROS production. qRT-PCR data indicated that the expression level of GmSPOD1 gene encoding peroxidase was cooperatively regulated by GsWRKY40 and GsbHLH92, which was confirmed by using a dual luciferase report system and yeast one-hybrid experiment. Our study reveals a pathway that GsWRKY40 and GsbHLH92 collaboratively up-regulate plant salt resistance through impeding GmSPOD1 expression and reducing ROS levels, providing a novel perspective on the regulatory mechanisms underlying plant tolerance to abiotic stresses