Search Results (88)

RNA interference (RNAi) is an effective mechanism for reducing gene expression, therefore enabling the exploration of gene function. Its application in hairy root transient transformation facilitates rapid preliminary evaluation of gene function, specifically tackling the difficulties linked to steady transformation in soybean. The mechanisms of RNAi have been thoroughly investigated in model plants. Nonetheless, it remains ambiguous whether silencing proceeds at the transcriptional or post-transcriptional level in hairy roots. We analyzed RNA levels and phenotype by RNAi targeting four genes (GmIAA14, GmCPC, GmPHT1, and GmTTG1) in soybean roots. A reduction in mRNA levels is generally associated with an altered lateral root phenotype. However, the analysis of chromatin-bound/nascent RNAs during the identical RNAi events revealed no substantial decrease in their abundance. Moreover, the functional impairment of GmDCL2 or GmDCL3 did not influence RNA interference, indicating either redundancy or the presence of alternate routes for short RNA-mediated RNA interference in soybean hairy roots. Collectively, our findings underscore possible issues associated with the use of the hairy root transformation method for gene transcription studies. The lack of investigation into RNAi effects at the nascent RNA level, even in model plants, requires a re-evaluation of the current knowledge of RNAi mechanisms. Full article

Phosphoglycerate kinase (PGK) is a vital glycolytic enzyme that provides energy and carbon skeletons to support fatty acid synthesis. However, the PGK gene family has not been characterized in soybean (Glycine max), and its role in soybean oil accumulation remains unclear. Here, we identified six GmPGK genes in soybean, all of which encode proteins containing conserved PGK domains. Phylogenetic analysis clustered soybean PGK proteins into three groups. Analysis of GmPGK promoters revealed relatively abundant cis-elements related to plant growth, development, and phytohormone response. Expression profiling showed that GmPGK5 transcript abundance increases progressively with oil accumulation during seed development, and is significantly higher in the high-oil variety NN1138-2. Overexpression of GmPGK5 significantly increased total fatty acid content in soybean hairy roots. A single nucleotide polymorphism (SNP) located at Chr15:49447855 within the GmPGK5 promoter was significantly associated with both seed oil content and seed weight in natural soybean accessions. Based on this SNP, a derived cleaved amplified polymorphic sequence (dCAPS) marker was developed to facilitate soybean molecular breeding. Our findings suggest that GmPGK5 may positively regulate fatty acid accumulation in soybean. The identified natural variation and dCAPS marker provide potential valuable tools for marker-assisted selection to improve soybean oil content and seed weight. Full article

Soil salinity is rapidly spreading across agricultural regions and has become one of the most critical constraints on soybean growth, yield, and sustainable production. Despite the central role of transcription factors (TFs) in coordinating plant responses to abiotic stresses, the molecular mechanisms by which RWP-RK domain-containing TFs regulate salt-tolerant responses in soybean remain poorly understood. Our previous genome-wide characterization identified 28 RWP-RK TFs in soybean exhibiting abiotic stress-responsive expression, yet their biological functions under salt stress have not been experimentally validated. Here, we investigated a 981-bp GmRWP-RK1 encoding region and demonstrated its regulatory role in enhancing salt tolerance by activating antioxidant defence, Na⁺/K⁺ homeostasis, and transcriptional control of salt-responsive genes using a cross-species overexpression approach. The two Arabidopsis lines (OE1 & OE4) overexpressing GmRWP-RK1 demonstrated significantly improved salt tolerance, as evidenced by ~18% greater survival and enhanced germination compared to non-transgenic plants under salinity stress. This phenotype was supported by stronger antioxidant protection, as indicated by elevated proline levels, reduced MDA accumulation, and increased SOD and POD activities. At the molecular level, the transgenic lines also showed up-regulated expression of key stress-responsive genes (AtACS10, AtSUMO1, AtGBF1), confirming the regulatory influence of GmRWP-RK1 on salt-adaptation pathways. Consistent with the Arabidopsis results, GmRWP-RK1 overexpression in soybean hairy roots also led to improved salt-stress tolerance by accumulating significantly reduced ROS contents (27.38% lower H₂O₂ and 33.98% lower O₂⁻), and maintained a balanced Na⁺/K⁺ ratio compared to that of non-transgenic hairy roots under salinity. Furthermore, GmRWP-RK1-overexpressing transgenic soybean hairy roots showed increased expression of stress-responsive genes, especially GmATG-5, GmOLP-1, and GmOLP-2. Overall, our results support a possible role of GmRWP-RK1 in soybean salt tolerance and provide a foundation for future functional and breeding-oriented studies. Full article

Heavy metal-associated isoprenylated plant protein (HIPP) family genes are known to be involved in plant development and stress responses. Even though the HIPPs have been identified and characterized in some plants, the roles of these genes in plant abiotic stress tolerance remain unclear in G. soja (Glycine soja), especially in response to alkaline stress. In the present study, a total of 79 potential HIPP family genes were obtained in G. soja using the Hidden Markov Model. Bioinformatics analysis was used to explore their physicochemical properties, gene structure, phylogenetic relationships, cis-acting elements, chromosomal location and collinearity. Expression profiling showed that 18 HIPP family genes were displayed significantly different transcript levels under alkaline stress, among which GsHIPP79 was selected for functional characterization. The results showed that GsHIPP79 exhibited enhanced alkaline stress tolerance in transgenic Arabidopsis plants, as evidenced by it exhibiting higher chlorophyll contents, strengthening the antioxidant defense system, and regulating the expression of stress-responsive marker genes. Moreover, overexpression of GsHIPP79 in transgenic soybean hairy roots conferred enhanced alkaline stress tolerance. In conclusion, this study provided valuable information on HIPP family genes in G. soja and identified the positive roles of GsHIPP79 in response to alkaline stress tolerance. Full article

Soybean is a critical oil and protein crop for both food and forage production; however, its growth and development are severely impacted by drought stress. Nevertheless, the molecular regulatory mechanisms underlying drought tolerance in soybean remain poorly understood. In this study, two soybean varieties, Jindou 21 (JD21, drought-tolerant) and Suinong 26 (SN26, drought-sensitive), were used as experimental materials and subjected to 15% PEG6000 to simulate drought stress. Roots and leaves were sampled at 0 h, 6 h, and 12 h after treatment to determine physiological indicators and conduct RNA-seq analysis. The results showed that JD21 exhibited a lower malondialdehyde (MDA) content but higher soluble sugar and proline contents than SN26. A total of 2603 and 3128 osmotic-stress-responsive genes were identified in the roots and leaves of SN26 and JD21, respectively. Additionally, 256 genes in the roots and 215 genes in the leaves showed consistent differential expression between the two varieties across the three treatment time points. KEGG enrichment analysis revealed that the differentially expressed genes were significantly enriched in pathways related to glutathione metabolism, arginine and proline metabolism, glycolysis/gluconeogenesis, and starch and sucrose metabolism. Within these pathways, the functions of GmGST, GmAMD1, GmADH1, GmENO, GmsacA, and GmSUS3 were validated through transgenic hairy root assays, demonstrating that these genes play positive regulatory roles in osmotic stress response. This study provides valuable data for elucidating plant PEG-induced osmotic-stress-response mechanisms and offers theoretical support for drought-resistant soybean breeding. Full article

The SQS_PSY family (Squalene/phytoene synthase family), with squalene synthase (SQS) and phytoene synthase (PSY) as core members, comprises enzymes that catalyze the head-to-head condensation of isoprenoid precursors. These enzymes play pivotal roles in mediating plant responses to both biotic and abiotic stresses; nevertheless, their specific functions in soybean defense against Phytophthora sojae infection remain elusive. In the present study, a comprehensive bioinformatics approach was utilized to identify 12 SQS_PSY family members in the soybean genome, followed by subsequent analyses of chromosomal distribution, phylogenetic relationships, gene structures, conserved motifs, and cis-acting regulatory elements within promoter regions. Notably, multiple cis-elements responsive to biotic and abiotic stresses were detected in the promoter regions of SQS_PSY genes in soybean, implying their potential involvement in stress-responsive pathways. To elucidate their roles in defense, resistant and susceptible soybean cultivars were inoculated with P. sojae, and RNA-seq was conducted on sampled tissues. Integrated with quantitative real-time PCR (qRT-PCR) validation, our findings demonstrated that GmSQS1 exhibited differential expression patterns between resistant and susceptible cultivars at multiple time points post-inoculation. Furthermore, the functional role of GmSQS1 in enhancing soybean resistance to P. sojae was confirmed using a transgenic hairy root system. Collectively, this study preliminarily validated the functions of the SQS_PSY genes in soybean and offers novel insights into their potential application for improving resistance against Phytophthora root rot. Full article

Using the cotyledonary node method, four traits related to callus induction rate were identified in 185 soybean germplasm resources. Cultivation of callus tissue is crucial for soybean (Glycine max (L.) Merr.) genetic transformation and functional genomics studies. Identifying genes associated with the induction rate of soybean callus tissue is therefore essential for biotechnological breeding and for understanding the molecular genetic mechanisms of soybean regeneration. The efficiency of genetic transformation impacts the breeding rate of soybeans, with its success rate dependent on the soybean regeneration system. Subsequently, whole genome association analysis (GWAS) and multidimensional functional validation were conducted. GWAS identified 66 significantly associated SNP loci corresponding to the four traits. Expression analysis in extreme phenotypes highlighted four candidate genes: Glyma.12G164100 (GmARF1), Glyma.12G164700 (GmPPR), Glyma.02G006200 (GmERF1), and Glyma.19G128800 (GmAECC1), which positively regulate callus formation. Overexpression and gene-editing assays in hairy roots confirmed that these genes significantly enhanced callus formation rate and density, with GmARF1 exerting the most prominent effect. Hormone profiling revealed elevated levels of gibberellin (GA), auxin (IAA), cytokinin (CTK), and other phytohormones in transgenic lines, consistent with enhanced responsiveness to exogenous GA. Overall, the results suggest that these four candidate genes may promote soybean regeneration, with GmARF1 showing the most pronounced effect. These results provide valuable genetic resources for improving soybean regeneration efficiency and accelerating genetic transformation-based breeding. Full article

Soil salinity severely threatens soybean productivity worldwide. While transcriptional responses to salt stress are well-documented, the role of post-transcriptional regulation, particularly alternative splicing (AS), remains underexplored. This study combines physiological phenotyping, transcriptome-wide analysis, and molecular genetics to uncover the mechanisms behind the differences in salt tolerance between the salt-sensitive variety Huachun 6 (HC6) and the resistant variety Fiskeby III. Under salt stress, Fiskeby III exhibited superior survival rates and maintained ion homeostasis, as evidenced by a lower Na⁺/K⁺ ratio, compared with HC6. Transcriptomic and splicing analysis revealed extensive salt-induced alternative splicing reprogramming. Genes undergoing differential AS were enriched in pathways related to stress response, ion transport, and RNA splicing. Based on the overlap with both differentially expressed genes (DEG) and alternative splicing (DAS) genes under salt stress, a key splicing factor, GmSR34b, was identified as a central regulator of AS under salt stress. Under NaCl stress, the expression of GmSR34b in leaves peaked at 1 h and a salt stress-specific splicing variant was rapidly induced. A comparative analysis showed that the Fiskeby III cultivar prioritized maintenance of the full-length transcript during prolonged stress, whereas the HC6 cultivar accumulated higher levels of the splicing variant. This indicates differences in the regulation of alternative splicing between these two cultivars. Functional validation confirmed that overexpression of GmSR34b in soybean hairy roots inhibited salt tolerance. This study provides novel insights into the molecular mechanisms of salt tolerance in soybean, suggesting potential strategies for breeding resilient crops through the manipulation of splicing regulators. Full article

Soybean (Glycine max) is a globally important grain and oil crop, but its yield and quality are severely limited by soybean cyst nematode (SCN, Heterodera glycines Ichinohe), a devastating soil-borne pathogen. Here, we evaluated SCN race 3 resistance in 306 soybean germplasms and combined a genome-wide association study (GWAS) with transcriptome analysis to identify key resistance-related genes. GWAS using 30× resequencing data (632,540 SNPs) revealed 77 significant quantitative trait loci (QTLs) associated with SCN resistance, while transcriptome comparison between the extreme resistant accession Dongnong L10 and susceptible Heinong 37 identified 4185 upregulated and 3195 downregulated genes. Integrating these results, we characterized the GmRF2-like gene as a candidate resistance gene. Subcellular localization showed GmRF2-like encodes a nuclear-localized protein. Functional validation via soybean hairy root transformation demonstrated that overexpression of GmRF2-like significantly inhibits SCN race 3 infection. Collectively, our findings confirm that GmRF2-like plays a positive role in soybean resistance to SCN race 3, providing critical insights for dissecting the molecular mechanism of SCN resistance and facilitating the development of resistant soybean varieties. Full article

Soybean cyst nematode (SCN, Heterodera glycines) is one of the major pathogens of soybean worldwide. We utilized the CHIP-Seq (chromatin immunoprecipitation sequencing) and RNA-Seq (RNA sequencing) data from the transgenic GmMYB29 strain (Glycine Max roots). We then performed enrichment analysis using KEGG and GO to identify potential candidate genes within the promoter-binding region. A targeted regulatory relationship between the GmMYB29 and GmPP2C-37like genes was further identified using the dual-luciferase Assay (Luciferase, LUC) and yeast one-hybrid Assay (Y1H). Hairy roots with target gene overexpression and gene-edited hairy roots were generated, and their resistance to soybean cyst nematode (SCN) was evaluated. Meanwhile, the presence of reciprocal genes with GmPP2C-37like was determined by the yeast two-hybrid library screening method. The targeting relationship between GmMYB29 and GmPP2C-37like genes was further validated through the Y1H assay and LUC assay. Based on phenotypic assessments of SCN, transgenic soybean roots overexpressing GmPP2C-37like exhibited significantly enhanced resistance to SCN 3 compared to wild-type. Further analysis revealed that GmPP2C-37like collaborates with other regulatory factors to modulate soybean resistance against SCN. Yeast two-hybrid library (Y2H) screening identified 18 interacting proteins. These findings not only illuminate the functional role of GmPP2C-37like but also provide a foundation for dissecting its molecular network. Moreover, the results offer promising candidate genes for enhancing SCN resistance and optimizing soybean resilience through targeted genetic strategies. Full article

Heavy-metal-associated (HMA) isoprenylated plant proteins (HIPPs) play crucial roles in plant responses to biotic/abiotic stresses and heavy-metal homeostasis. However, the involvement of HIPP genes in the response of soybean (Glycine max) to aluminum (Al) toxicity remains unexplored. This study aimed to comprehensively characterize the GmHIPP gene family and investigate its role in Al toxicity. A total of 76 GmHIPP genes were identified in the soybean genome. Phylogenetic and synteny analyses revealed that HIPP evolution was highly conserved among soybean, Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa). Cis-element analysis indicated that GmHIPP genes might be involved in phytohormone response, abiotic and biotic stresses, and plant growth. RNA-seq analysis further revealed that the expression of 20 GmHIPPs was up-regulated, and three GmHIPPs were down-regulated under Al stress in roots. Among them, six genes (GmHIPP9/13/29/43/58/73) were highly induced by Al, with GmHIPP29 exhibiting particularly high expression in root tips. Subcellular localization demonstrated that GmHIPP29 is a plasma membrane-localized protein. GmHIPP29-overexpression significantly increased Al accumulation in the cell sap of the transgenic soybean hairy root tips, leading to increased Al sensitivity. Collectively, these results demonstrate that GmHIPP29 acts as a negative regulator of Al tolerance by promoting Al accumulation in soybean roots. Full article

The soybean cyst nematode (Heterodera glycines, SCN) is the leading pathogen causing economic losses in soybean production worldwide. Using resistant cultivars is the most sustainable control method, yet the molecular basis of this resistance remains unclear. Heinong 531 (HN531), a high-yield soybean variety rich in seed oil, shows broad resistance to multiple SCN races. In this research, we studied HN531’s resistance to SCN races 3 and 5 through phenotypic assessment and comparative transcriptomics. Although initial infection rates were similar between resistant HN531 and the susceptible Dongsheng 1 (DS1), HN531 limited later nematode development inside roots, with fewer progressing to the J2 stage and maturing females. RNA-seq at 5 days post-infection revealed 1459 differentially expressed genes (DEGs) in HN531, mainly involved in secondary metabolite pathways, especially phenylpropanoid biosynthesis. We pinpointed a β-glucosidase gene (Glyma.12G053800, BGLU) upregulated after SCN infection and naturally more expressed in HN531 roots than DS1. Functional tests using Agrobacterium rhizogenes-mediated hairy root transformation showed that overexpressing Glyma.12G053800 in the susceptible DS1 significantly decreased SCN development and adult female counts by around 65%, without affecting initial infection. These findings suggest Glyma.12G053800 contributes to SCN resistance via phenylpropanoid-driven secondary metabolism, offering new insights into nematode resistance pathways and a valuable genetic resource for breeding broad-spectrum resistant soybean varieties. Full article

Soil alkalinity severely restricts the cultivation of Lupinus angustifolius, a valuable legume. Wild soybean (Glycine soja) is a leguminous plant with extremely strong alkaline resistance (pH 8.5). Transferring the alkali-tolerant genes from wild soybeans into lupinus can effectively enhance the alkali tolerance. In this study, we combined transcriptome profiling and genetic transformation to elucidate the molecular basis of alkaline stress response in lupinus. RNA-seq analysis of root tips under acid (HCl, pH 4.0) and alkali (NaHCO₃, pH 8.5) stress revealed 104,353 annotated unigenes, with differential expression patterns highlighting enrichment in cellular component, binding, and catalytic activity categories. KEGG pathway analysis indicated that early responses involved ribosome-related pathways, while later stages activated plant hormone signaling and MAPK pathways. Notably, no homeodomain-leucine zipper (HD-Zip) family genes were identified in the lupinus genome. Therefore, we transferred GsHZ4, an alkali-resistant HD-Zip transcription factor from wild soybean into lupinus hairy roots via Agrobacterium rhizogenes-mediated transformation. Overexpression of GsHZ4 significantly enhanced antioxidant enzyme activities (CAT, POD, and SOD) and reduced malondialdehyde content under NaHCO₃ stress. Furthermore, the promoter of GsHZ4 expression was strongly induced by indole-3-acetic acid (IAA). Key alkali-responsive genes (LaKIN, LaMYB34, LaDnaJ1, LaDnaJ20, LaNAC22, and LaNAC35) were upregulated in transgenic lines, suggesting that GsHZ4 integrates into the endogenous stress-regulation network. Our findings demonstrate that heterologous expression of GsHZ4 can enhance alkaline tolerance of lupinus, providing a novel strategy for breeding stress-resistant varieties and expanding lupinus cultivation in saline–alkali soils. Full article

Fusarium oxysporum-induced root rot severely threatens global soybean production, yet limited understanding of resistance mechanisms constrains breeding progress. This study conducted comparative transcriptomic analysis between highly resistant (Xiaoheiqi) and susceptible (L83-4752) soybean accessions following pathogen inoculation across four time points (8–17 days post-infection). RNA-seq analysis identified 1496 differentially expressed genes following pathogen challenge. KEGG pathway enrichment analysis revealed significant enrichment in MAPK signaling pathway (12 genes) and plant–pathogen interaction pathway (13 genes). Eight genes co-occurred in both pathways, with GmCML (Glyma.10G178400) exhibiting the most dramatic differential expression among these candidates. This gene encodes a 151-amino acid calmodulin-like protein showing 185-fold higher expression in resistant plants at 17 days post-inoculation, confirmed by qRT-PCR validation. Functional validation through transgenic hairy root overexpression demonstrated that GmCML significantly enhanced disease resistance by coordinately activating antioxidant defense systems. Overexpression of GmCML in transgenic soybean enhanced resistance to F. oxysporum by modulating the activity of antioxidant enzymes (superoxide dismutase, SOD; peroxidase, POD; catalase, CAT) and the accumulation of osmoregulatory substances (proline and soluble sugars). Population genetic analysis of 295 diverse soybean accessions revealed three GmCML haplotypes based on promoter region polymorphisms. Two favorable variants (Hap2 and Hap3) conferred significantly lower disease indices and exhibited evidence of positive selection during domestication, indicating evolutionary importance in disease resistance. This research provides the first comprehensive characterization of GmCML’s role in soybean–Fusarium interactions, establishing this calmodulin-like protein as a regulatory hub linking calcium signaling to coordinated defense responses. The identified natural variants and functional mechanisms offer validated targets for both marker-assisted breeding and genetic engineering approaches to enhance soybean disease resistance. Full article

Fatty acid transporters (FAXs) play an important role in fatty acid synthesis by facilitating transport fatty acids from the plastid to the endoplasmic reticulum. This process is essential for providing precursor substances necessary for triglycerides (TAGs). Although FAX genes have been identified in variety of plant species, the identification and molecular functions of the GmFAX gene members in soybean are still unclear. In this study, soybean FAX genes were identified through the utilization of the Phytozome (v13) and NCBI online websites. Subsequently, phylogenetic trees, expression patterns, gene structures, and qRT-PCR were analyzed. A total of eight GmFAX members were identified at the whole genome level, and further phylogenetic analysis revealed that these members can be categorized into four subfamilies. In addition, all members of GmFAX contain a highly conserved domain Tmemb_14. Through qRT-PCR analysis, it was found that the expression level of the GmFAX8 gene is relatively high in leaves and stems. Further investigation revealed that the total fatty acid content in hairy roots overexpressing the GmFAX8 gene was significantly greater than that observed in the control strain. The results presented above suggest that the GmFAX8 gene may play an important role in the accumulation of oil within soybeans. Full article