Search Results (18)

Seed physical dormancy, also known as hard-seededness, is a characteristic commonly found in higher plants, which functions to prevent water and oxygen from passing through the impermeable seed coat. Background: Notably, seed dormancy has emerged as a critical factor in the domestication of leguminous plants. Alfalfa (Medicago sativa L.) is a globally cultivated high-quality legume forage crop, while the seeds from different varieties maintain varying degrees of hard-seededness. However, the molecular mechanisms underlying physical dormancy in alfalfa seeds remain poorly understood. In particular, the regulatory mechanisms at the transcriptomic level remain unclear, which has hindered the breeding process of varieties with low hard-seededness. Methods: In this study, we performed global transcriptome analysis to discover the genes specifically expressed in the alfalfa seed coat and provide insights into alfalfa seeds’ physical dormancy domestication traits. RNA sequencing was performed on various alfalfa tissues, including roots, stems, leaves, flowers, and seed coats. Results: This analysis led to the identification of 4740 seed coat-specific expressed genes, including key genes such as KNOX4 (a class II KNOTTED-like homeobox gene), qHs1 (encoding endo-1,4-β-glucanase), GmHs1-1 (encoding a calcineurin-like metallophosphoesterase), and KCS12 (β-ketoacyl-CoA synthase). In addition, several seed coat-specific transcription factor families were identified, including ERF, B3, and NAC, among others. Furthermore, a comparison of gene expression profiles between seeds with and without physical dormancy revealed 60 upregulated and 197 downregulated genes associated with physical dormancy. Crucially, functional enrichment analysis demonstrated that these genes are predominantly associated with lipid metabolism pathways, particularly those involved in the formation of “monolayer-surrounding lipid storage bodies.” Conclusions: This key finding suggests that the establishment of physical dormancy is closely linked to the biosynthesis and deposition of specialized lipid-based layers in the seed coat, which likely constitute the primary barrier to water penetration. Our study thus provides fundamental insights and a valuable genetic resource for future functional studies aimed at deciphering and manipulating physical dormancy in alfalfa. Full article

This review presents soybean responses to drought, heat, and salinity within a signal–transcript–chromatin framework. In this framework, calcium/reactive oxygen species and abscisic acid cues converge on abscisic acid-responsive element binding factor (ABF/AREB), dehydration-responsive element binding protein (DREB), NAC, and heat shock factor (HSF) families. These processes are modulated by locus-specific chromatin and non-coding RNA layers. Base-resolved methylomes reveal a high level of CG methylation in the gene body, strong CHG methylation in heterochromatin, and dynamic CHH ‘islands’ at the borders of transposable elements. CHH methylation increases over that of transposable elements during seed development, and GmDMEa editing is associated with seed size. Chromatin studies in soybean and model species implicate the reconfiguration of salt-responsive histone H3 lysine 27 trimethylation (H3K27me3) in G. max and heat-linked H2A.Z dynamics at thermoresponsive promoters characterized in Arabidopsis and other plants, suggesting that a conserved chromatin layer likely operates in soybean. miR169–NF-YA, miR398–Cu/Zn Superoxide Dismutases(CSD)/copper chaperone of CSD(CCS), miR393–transporter inhibitor response1/auxin signaling F-box (TIR1/AFB), and miR396–growth regulating factors (GRF) operate across leaves, roots, and nodules. Overexpression of lncRNA77580 enhances drought tolerance, but with context-dependent trade-offs under salinity. Single-nucleus and spatial atlases anchor these circuits in cell types and microenvironments relevant to stress and symbiosis. We present translational routes, sentinel epimarkers (bisulfite amplicons, CUT&Tag), haplotype-by-epigenotype prediction, and precise cis-regulatory editing to accelerate marker development, genomic prediction and the breeding of resilient soybean varieties with stable yields. Full article

Soybean is a major source of plant-based protein and vegetable oil, but its productivity is severely limited by soil salinity. Transcription factors including NAC family play pivotal roles in regulating stress-responsive pathways. Here, we identified and characterized a salt-induced NAC transcription factor, GmNAC03, in soybean. Overexpression of GmNAC03 significantly improved salt tolerance at both the germination and seedling stages. Physiological analyses revealed that antioxidant enzyme activities, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), were elevated in GmNAC03 transgenic lines, accompanied by reduced malondialdehyde (MDA) accumulation, indicating enhanced oxidative stress resistance. To further explore its regulatory mechanisms, RNA-seq analysis was performed, which showed that GmNAC03 overexpression affected pathways related to amino acid metabolism, particularly glutamine and aspartate family amino acid biosynthesis, as well as phenylpropanoid biosynthesis. Differentially expressed genes were enriched in alanine, aspartate, and glutamate metabolism, suggesting a role for GmNAC03 in metabolic reprogramming under salt stress. Together, these findings demonstrate that GmNAC03 functions as a positive regulator of salt tolerance in soybean by modulating antioxidant defense and amino acid metabolic pathways. This work provides new insights into the molecular basis of NAC-mediated stress adaptation and offers a potential target for breeding soybean varieties with enhanced salinity resistance. Full article

In response to abiotic stress, plants utilize hub protein-mediated signaling networks, with members of the SIMILAR TO RCD ONE (SRO) protein family playing a pivotal role in regulating stress resistance pathways. This study investigates the functional role of the soybean GmRCD1 protein and its interaction mechanisms to elucidate its molecular regulatory network in stress resistance responses. By employing yeast two-hybrid technology to screen a soybean cDNA library under high-salt stress conditions, 17 potential interacting proteins were identified, which include NAC transcription factors (e.g., GmNAC058), ubiquitin–proteasome proteins, and ribosomal proteins. Subsequent validation using GST pull-down and bimolecular fluorescence complementation assays confirmed the direct interaction between GmRCD1 and GmNAC058, which is mediated by the RST domain of GmRCD1 and the C-terminal disordered region (amino acids 288–323) of GmNAC058. Subcellular localization studies revealed that both proteins are nuclear-localized, aligning with their roles in transcriptional regulation. Furthermore, PAR binding assays demonstrated that both GmRCD1 and AtRCD1 can bind to PAR polymers; however, PARP activity analysis revealed that neither protein exhibits catalytic activity, indicating their participation in stress responses via non-enzymatic mechanisms. This study represents the first to elucidate the interaction network and structural basis between soybean GmRCD1 and GmNAC058, providing crucial theoretical support for understanding the multifunctional roles of plant hub proteins in stress resistance regulation and for molecular breeding in soybean. Full article

Soybean, a globally significant and versatile crop, serves as a vital source of both oil and protein. However, environmental factors such as soil salinization pose substantial challenges to its cultivation, adversely affecting both yield and quality. Enhancing the salt tolerance of soybeans can mitigate yield losses and promote the development of the soybean industry. Members of the plant-specific transcription factor family NAC play crucial roles in plant adaptation to abiotic stress conditions. In this study, we screened the soybean GmNAC family genes potentially involved in the salt stress response and identified 18 GmNAC genes that may function during the early stages of salt stress. Among these, the GmNAC035 gene exhibited a rapid increase in expression within one hour of salt treatment, with its expression being induced by abscisic acid (ABA) and methyl jasmonate (MeJA), suggesting its significant role in the soybean salt stress response. We further elucidated the role of GmNAC035 in soybean salt tolerance. GmNAC035, a nuclear-localized transcriptional activator, enhances salt tolerance when overexpressed in Arabidopsis, reducing oxidative damage and boosting the expression of stress-responsive genes. It achieves this by regulating key stress response pathways, including the SOS pathway, calcium signaling, and ABA signaling. These findings highlight the potential of GmNAC035 as a genetic engineering target to improve crop salt tolerance. Full article

Flooding stress poses a significant challenge to soybean cultivation, impacting plant growth, development, and ultimately yield. In this study, we investigated the responses of two distinct soybean cultivars: flooding-tolerant Nanxiadou 38 (ND38) and flooding-sensitive Nanxiadou 45 (ND45). To achieve this, healthy seedlings were cultivated with the water surface consistently maintained at 5 cm above the soil surface. Our objective was to elucidate the physiological and molecular adaptations of the two cultivars. Under flooding stress, seedlings of both cultivars exhibited significant dwarfing and a notable decrease in root length. While there were no significant differences in the dry weight of aboveground shoots, the dry weight of underground shoots in ND38 was strikingly decreased following flooding. Additionally, total chlorophyll content decreased significantly following flooding stress, indicating impaired photosynthetic performance of the cultivars. Moreover, malondialdehyde (MDA) levels increased significantly after flooding, particularly in the ND45 cultivar, suggesting heightened oxidative stress. Expression analysis of methylation and demethylation genes indicated that MET1 and DME play crucial roles in response to flooding stress in soybeans. Meanwhile, analysis of the hemoglobin family (GLBs), aquaporin family (AQPs), glycolytic pathway-related genes, and NAC transcription factor-related genes identified GLB1-1 and GLB1-2, GLB2-2, PIP2-6, PIP2-7, TIP2-2, TIP4-1, TIP5-1, Gm02G222400 (fructose-bisphosphate aldolase), Gm19G017200 (glucose-6-phosphate isomerase), and Gm04G213900 (alcohol dehydrogenase 1) as key contributors to flooding tolerance in both soybean cultivars. These findings provide crucial insights into the physiological and molecular mechanisms underlying flooding tolerance in soybeans, which could guide future molecular breeding strategies for the development of flooding-tolerant soybean cultivars. Full article

Phytoalexins are plant defense metabolites that are biosynthesized transiently in response to pathogens. Despite that their biosynthesis is highly restricted in plant tissues, the transcription factors that negatively regulate phytoalexin biosynthesis remain largely unknown. Glyceollins are isoflavonoid-derived phytoalexins that have critical roles in protecting soybean crops from the oomycete pathogen Phytophthora sojae. To identify regulators of glyceollin biosynthesis, we used a transcriptomics approach to search for transcription factors that are co-expressed with glyceollin biosynthesis in soybean and stilbene synthase phytoalexin genes in grapevine. We identified and functionally characterized the WRKY family protein GmWRKY72, which is one of four WRKY72-type transcription factors of soybean. Overexpressing and RNA interference silencing of GmWRKY72 in the soybean hairy root system decreased and increased expression of glyceollin biosynthetic genes and metabolites, respectively, in response to wall glucan elicitor from P. sojae. A translational fusion with green fluorescent protein demonstrated that GFP-GmWRKY72 localizes mainly to the nucleus of soybean cells. The GmWRKY72 protein directly interacts with several glyceollin biosynthetic gene promoters and the glyceollin transcription factor proteins GmNAC42-1 and GmMYB29A1 in yeast hybrid systems. The results show that GmWRKY72 is a negative regulator of glyceollin biosynthesis that may repress biosynthetic gene expression by interacting with transcription factor proteins and the DNA of glyceollin biosynthetic genes. Full article

NAC transcription factors are commonly involved in the plant response to drought stress. A transcriptome analysis of root samples of the soybean variety ‘Jiyu47’ under drought stress revealed the evidently up-regulated expression of GmNAC19, consistent with the expression pattern revealed by quantitative real-time PCR analysis. The overexpression of GmNAC19 enhanced drought tolerance in Saccharomyces cerevisiae INVSc1. The seed germination percentage and root growth of transgenic Arabidopsis thaliana were improved in comparison with those of the wild type, while the transgenic soybean composite line showed improved chlorophyll content. The altered contents of physiological and biochemical indices (i.e., soluble protein, soluble sugar, proline, and malondialdehyde) related to drought stress and the activities of three antioxidant enzymes (i.e., superoxide dismutase, peroxidase, and catalase) revealed enhanced drought tolerance in both transgenic Arabidopsis and soybean. The expressions of three genes (i.e., P5CS, OAT, and P5CR) involved in proline synthesis were decreased in the transgenic soybean hairy roots, while the expression of ProDH involved in the breakdown of proline was increased. This study revealed the molecular mechanisms underlying drought tolerance enhanced by GmNAC19 via regulation of the contents of soluble protein and soluble sugar and the activities of antioxidant enzymes, providing a candidate gene for the molecular breeding of drought-tolerant crop plants. Full article

Glyceollins, isoflavonoid-derived antimicrobial metabolites, are the major phytoalexins in soybean (Glycine max). They play essential roles in providing resistance to the soil-borne pathogen Phytophthora sojae and have unconventional anticancer and neuroprotective activities that render them desirable for pharmaceutical development. Our previous studies revealed that the transcription factors GmMYB29A2 and GmNAC42-1 have essential roles in activating glyceollin biosynthesis, yet each cannot activate the transcription of all biosynthesis genes in the absence of a pathogen elicitor treatment. Here, we report that co-overexpressing both transcription factors is also insufficient to activate glyceollin biosynthesis. To understand this insufficiency, we compared the transcriptome profiles of hairy roots overexpressing each transcription factor with glyceollin-synthesizing roots treated with wall glucan elicitor (WGE) from P. sojae. GmMYB29A2 upregulated most of the WGE-regulated genes that encode enzymatic steps spanning from primary metabolism to the last step of glyceollin biosynthesis. By contrast, GmNAC42-1 upregulated glyceollin biosynthesis genes only when overexpressed in the presence of WGE treatment. This is consistent with our recent discovery that, in the absence of WGE, GmNAC42-1 is bound by GmJAZ1 proteins that inhibit its transactivation activity. WGE, and not GmMYB29A2 or GmNAC42-1, upregulated the heat shock family gene GmHSF6-1, the homolog of Arabidopsis HSFB2a that directly activated the transcription of several glyceollin biosynthesis genes. Our results provide important insights into what biosynthesis genes will need to be upregulated to activate the entire glyceollin biosynthetic pathway. Full article

Soybean transcription factor GmNAC plays important roles in plant resistance to environmental stresses. In this study, GmNAC3 was cloned in the drought tolerant soybean variety “Jiyu47”, with the molecular properties of GmNAC3 characterized to establish its candidacy as a NAC transcription factor. The yeast self-activation experiments revealed the transcriptional activation activity of GmNAC3, which was localized in the nucleus by the subcellular localization analysis. The highest expression of GmNAC3 was detected in roots in the podding stage of soybean, and in roots of soybean seedlings treated with 20% PEG6000 for 12 h, which was 16 times higher compared with the control. In the transgenic soybean hairy roots obtained by the Agrobacterium-mediated method treated with 20% PEG6000 for 12 h, the activities of superoxide dismutase, peroxidase, and catalase and the content of proline were increased, the malondialdehyde content was decreased, and the expressions of stress resistance-related genes (i.e., APX2, LEA14, 6PGDH, and P5CS) were up-regulated. These expression patterns were confirmed by transgenic Arabidopsis thaliana with the overexpression of GmNAC3. This study provided strong scientific evidence to support further investigation of the regulatory function of GmNAC3 in plant drought resistance and the molecular mechanisms regulating the plant response to environmental stresses. Full article

NAC transcription factors (TFs) could regulate drought stresses in plants; however, the function of NAC TFs in soybeans remains unclear. To unravel NAC TF function, we established that GmNAC12, a NAC TF from soybean (Glycine max), was involved in the manipulation of stress tolerance. The expression of GmNAC12 was significantly upregulated more than 10-fold under drought stress and more than threefold under abscisic acid (ABA) and ethylene (ETH) treatment. In order to determine the function of GmNAC12 under drought stress conditions, we generated GmNAC12 overexpression and knockout lines. The present findings showed that under drought stress, the survival rate of GmNAC12 overexpression lines increased by more than 57% compared with wild-type plants, while the survival rate of GmNAC12 knockout lines decreased by at least 46%. Furthermore, a subcellular localisation analysis showed that the GmNAC12 protein is concentrated in the nucleus of the tobacco cell. In addition, we used a yeast two-hybrid assay to identify 185 proteins that interact with GmNAC12. Gene ontology (GO) and KEGG analysis showed that GmNAC12 interaction proteins are related to chitin, chlorophyll, ubiquitin–protein transferase, and peroxidase activity. Hence, we have inferred that GmNAC12, as a key gene, could positively regulate soybean tolerance to drought stress. Full article

Senescence is modulated by ORESARA1 (ORE1), a NAC transcription factor that interacts with hormones to fully induce senescence. The At-ore1 gene acts as a suppressor of leaf senescence; however, its exact role in this respect has not been clearly defined. In this study, the function of At-ore1 during leaf senescence was analyzed in soybeans. The precocious leaf senescence of the ore1-1 line was associated with greater chlorophyll loss, leaf necrosis, and redox imbalance in the early vegetative stage during the hyper-accumulation of endogenous abscisic acid (ABA) by enhancing the expression of GmNECD3-related ABA synthesis. At-ore1 induced ABA regulation of the H₂O₂-GmARF2-GmNAC081 signaling circuit, which relays the At-ore1-induced cell death signal mediation to the caspase-1-like vacuolar processing enzyme (VPE) expression, triggering programmed cell death. In contrast, it was found that At-ore1 functions in IAA to delay leaf-senescence-mediated suppression of the expression of ABA, ROS, and senescence-associated gene 39 (GmSAG39). The IAA-induced GmNAC065 expression controls soybean leaves’ longevity, as discovered by screening At-ore1 expression in ore1-6 for a more stay-green leaf phenotype by helping to increase seed yields. These results uncover a mechanism that modulates ore1 plants’ amplitude expression involved in the ABA/IAA balance in the activation of GmNAC081- or GmNAC065-dependent H₂O₂ levels, which are crucial in the senescence or delayed leaf senescence of soybeans. Full article

The membrane-bound NAC transcription (NTL) factors have been demonstrated to participate in the regulation of plant development and the responses to multiple environmental stresses. This study is aimed to functionally characterize soybean NTL transcription factors in response to Al-toxicity, which is largely uncharacterized. The qRT-PCR assays in the present study found that thirteen out of fifteen GmNTL genes in the soybean genome were up-regulated by Al toxicity. However, among the Al-up-regulated GmNTLs selected from six duplicate gene pairs, only overexpressing GmNTL1, GmNTL4, and GmNTL10 could confer Arabidopsis Al resistance. Further comprehensive functional characterization of GmNTL4 showed that the expression of this gene in response to Al stress depended on root tissues, as well as the Al concentration and period of Al treatment. Overexpression of GmNTL4 conferred Al tolerance of transgenic Arabidopsis in long-term (48 and 72 h) Al treatments. Moreover, RNA-seq assay identified 517 DEGs regulated by GmNTL4 in Arabidopsis responsive to Al stress, which included MATEs, ALMTs, PMEs, and XTHs. These results suggest that the function of GmNTLs in Al responses is divergent, and GmNTL4 might confer Al resistance partially by regulating the expression of genes involved in organic acid efflux and cell wall modification. Full article

Abiotic stress factors, such as drought and salinity, are known to negatively affect plant growth and development. To cope with these adverse conditions, plants have utilized certain defense mechanisms involved in various aspects, including morphological, biochemical and molecular alterations. Particularly, a great deal of evidence for the biological importance of the plant-specific NAM, ATAF1/2, CUC2 (NAC) transcription factors (TFs) in plant adaptation to abiotic stress conditions has been reported. A previous in planta study conducted by our research group demonstrated that soybean (Glycine max) GmNAC085 mediated drought resistance in transgenic Arabidopsis plants. In this study, further characterization of GmNAC085 function in association with salt stress was performed. The findings revealed that under this condition, transgenic soybean plants overexpressing GmNAC085 displayed better germination rates than wild-type plants. In addition, biochemical and transcriptional analyses showed that the transgenic plants acquired a better defense system against salinity-induced oxidative stress, with higher activities of antioxidant enzymes responsible for scavenging hydrogen peroxide or superoxide radicals. Higher transcript levels of several key stress-responsive genes involved in the proline biosynthetic pathway, sodium ion transporter and accumulation of dehydrins were also observed, indicating better osmoprotection and more efficient ion regulation capacity in the transgenic lines. Taken together, these findings and our previous report indicate that GmNAC085 may play a role as a positive regulator in plant adaptation to drought and salinity conditions. Full article

Leaf senescence is a genetically regulated developmental process that can be triggered by a variety of internal and external signals, including hormones and environmental stimuli. Among the senescence-associated genes controlling leaf senescence, the transcriptional factors (TFs) comprise a functional class that is highly active at the onset and during the progression of leaf senescence. The plant-specific NAC (NAM, ATAF, and CUC) TFs are essential for controlling leaf senescence. Several members of Arabidopsis AtNAC-SAGs are well characterized as players in elucidated regulatory networks. However, only a few soybean members of this class display well-known functions; knowledge about their regulatory circuits is still rudimentary. Here, we describe the expression profile of soybean GmNAC-SAGs upregulated by natural senescence and their functional correlation with putative AtNAC-SAGs orthologs. The mechanisms and the regulatory gene networks underlying GmNAC081- and GmNAC030-positive regulation in leaf senescence are discussed. Furthermore, new insights into the role of GmNAC065 as a negative senescence regulator are presented, demonstrating extraordinary functional conservation with the Arabidopsis counterpart. Finally, we describe a regulatory circuit which integrates a stress-induced cell death program with developmental leaf senescence via the NRP-NAC-VPE signaling module. Full article