Search Results (43)

Legume–rhizobium symbiosis represents a crucial biological nitrogen fixation system. The AP2/ERF transcription factor ERN1 plays a vital role in nodulation of model legumes; however, its function in peanut (Arachis hypogaea), a typical crack-entry infection legume, remains unclear. To explore this, we performed transcriptome sequencing of peanut roots at 3 days post-inoculation (dpi) with rhizobium. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that differentially expressed genes (DEGs) were mainly enriched in DNA-binding transcription factor activity, plant–pathogen interaction, and plant hormone signal transduction pathways. The most strongly up-regulated gene was AhERN1, which was highly expressed in peanut roots and nodules. Subcellular localization indicated that AhERN1 was a nuclear-localized protein, and yeast transcriptional activation assays confirmed that AhERN1 functions as a transcriptional activator relying on its C-terminal domain. Furthermore, hairy root overexpression of AhERN1 significantly increased the number of peanut nodules. Collectively, these results reveal that AhERN1 acts as a positive regulator to promote rhizobium-induced nodule development in peanut, providing new insights into the regulatory mechanism of nodulation in dalbergoid legumes. Full article

Phenolic acids are the major bioactive compounds in Perilla frutescens (L.) Britt; however, the regulatory roles of NAC transcription factors (TFs) in their biosynthesis remain unclear. Here, we performed a genome-wide identification and characterization of the NAC family in P. frutescens and explored their involvement in phenolic acid production. A total of 108 PfNAC genes were identified and classified into 17 subfamilies. Expression and promoter analyses suggested potential roles in secondary metabolism. PfNAC29 is located in the plasma membrane and necleus, while PfNAC40 and PfNAC80 are located in the nucleus.Yeast one-hybrid and dual-luciferase assays demonstrated that these TFs bind to the CATGTG motif in the PfC4H promoter and activate its transcription. Overexpression in transgenic hairy roots significantly increased rosmarinic acid, caffeic acid, and ferulic acid accumulation, accompanied by upregulation of key biosynthetic genes. These results indicate that PfNAC29, PfNAC40, and PfNAC80 act as positive regulators of phenolic acid biosynthesis and provide promising targets for metabolic engineering in medicinal plants. Full article

Background: There is an abundance of the neuroactive β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP) in grass pea (Lathyrus sativus), pea (Pisum sativum), and several Chinese traditional herbs such as Panax notoginseng. It is well known for its dose- and context-dependent effects on its toxicological characteristics (inducing neurodegenerative neurolathyrism upon excessive consumption) or for its pharmacological effects (including neuroprotection and wound healing). Therefore, reducing β-ODAP levels improves the safety profile of β-ODAP-containing species for utilization, whereas increasing them facilitates their isolation and purification. LsBAHD3 acyltransferase, named after the first letter of BEAT benzylalcohol O-acetyltransferase (BEAT), anthocyanin O-hydroxycinnamoyltransferase (AHCT), anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT), and deacetylvindoline 4-Oacetyltransferase (DAT), was proven to be β-ODAP synthetase. Methods: In this report, the interaction of miR172f with LsBAHD3 was investigated through bioinformatic analysis and transient co-expression assays in Nicotiana benthamiana. Functions of miR172f in β-ODAP biosynthesis were also investigated through knockdown in the hairy roots of L. sativus and via transcriptomic analysis. Results: The results suggest that the knockdown of miR172f in hairy roots of L. sativus increased β-ODAP content via targets to LsBAHD3. In this process, protein ubiquitination, cysteine and methionine metabolism, enzyme regulator activity, and so on were associated with β-ODAP biosynthesis. Conclusions: These results identify miR172f as a novel regulator of β-ODAP biosynthesis through targeting of LsBAHD3, offering new insight into the gene expression of β-ODAP synthetase and the genetic network governing β-ODAP biosynthesis in L. sativus. 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

Arbuscular mycorrhizal fungi (AMF) play a pivotal role in plant adaptation to arid ecosystems, yet their widespread agricultural use is constrained by the scarcity of high-quality, locally adapted inoculum. This study established a reliable monoxenic culture system for mass-producing an indigenous AMF isolate from the date palm (Phoenix dactylifera L.) rhizosphere in the Figuig oasis, southeastern Morocco. The isolate was identified as Rhizophagus irregularis based on spore morphology and Large Subunit ribosomal DNA (LSU rDNA) phylogeny. Two propagule types, surface-sterilized spores and mycorrhizal root fragments of Plantago lanceolata L., were compared for initiation of in vitro cultures on Ri T-DNA-transformed carrot (Daucus carota L.) hairy roots. By week 16, cultures initiated from mycorrhizal root fragments produced 1414 ± 65 spores per plate and showed significantly higher performance than spore-derived cultures in terms of propagule viability, root colonization, and hairy root growth. Propagule viability reached 84% and 68%, root colonization frequencies were 95% and 72%, and hairy root lengths averaged 81 and 63 cm in root fragment- and spore-derived cultures, respectively (p < 0.01). In a subsequent whole-plant assay using P. lanceolata, in vitro-produced spores induced markedly higher mycorrhizal colonization frequency (91.0 ± 1.6% compared with 74.8 ± 1.9%) and intensity (70.0 ± 1.6% compared with 55.0 ± 1.6%) than spores obtained from conventional trap cultures (p < 0.001). These results demonstrate that monoxenic root-organ culture using root fragments is a robust, reproducible method for generating abundant, contaminant-free, and functionally superior inoculum of native R. irregularis. This advance provides a solid platform for developing tailored bio-inoculants to enhance crop resilience and sustainability in arid and semi-arid agroecosystems. Full article

Seed coat color in peanut (Arachis hypogaea L.) is a critical agronomic trait that affects both nutritional quality and market appeal. In this study, we identified two bHLH transcription factor genes, AhWSC1a and AhWSC1b, homologues of Arabidopsis TRANSPARENT TESTA 8, as indispensable gatekeepers of basal flavonoid pigmentation. QTL-seq analysis of a recombinant inbred line population derived from a black-testa parent (S3) and a white-testa parent (S2) revealed that recessive loss-of-function mutations in both AhWSC1a/1b abolish proanthocyanidin biosynthesis, resulting in a white testa. Integrated metabolomic and transcriptomic profiling confirmed the absence of proanthocyanidins and a strong repression of late anthocyanin-pathway genes (DFR, LDOX) in the mutants. Molecular assays further demonstrated that AhWSC1 physically interacts with the R2R3-MYB regulator AhTc1 to form a functional MBW complex that activates AhDFR and AhLDOX transcription. In this research, we also found that the black testa phenotype may arise from elevated AhTc1 expression associated with a structural variant (SV); however, in the SV background, the introduction of ahwsc1a/1b mutant leads to a significant suppression of AhTc1 expression. Notably, because AhWSC1 is transcriptionally silent in hairy-root systems, overexpression of AhTc1 alone failed to induce these late-stage anthocyanin biosynthesis genes, highlighting AhWSC1 as an indispensable, rate-limiting hub of anthocyanin biosynthesis pathway regulation. Collectively, our findings establish AhWSC1a and AhWSC1b as master regulators of peanut testa pigmentation, elucidate the molecular basis of classical white testa inheritance, and provide genetic targets for precision-breeding of nutritionally enhanced cultivars. 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

Powdery mildew poses a significant threat to watermelon production. The development of disease-resistant varieties through gene editing represents a major focus in current breeding research. In this study, we identified an MLO family gene in watermelon, denoted by ClMLO5b, which is phylogenetically closely related to cucumber CsaMLO8 and melon CmMLO5. Homology modeling revealed high conservation of the three-dimensional protein structures among these orthologs. Expression analysis demonstrated that ClMLO5b is significantly up-regulated upon powdery mildew infection, and the protein localizes to the plasma membrane. To validate its function, we first employed an Agrobacterium rhizogenes-mediated hairy root transformation system to rapidly verify the editing efficiency of two CRISPR/Cas9 targets designed for ClMLO5b. Subsequently, stable transgenic watermelon plants were generated via Agrobacterium tumefaciens-mediated transformation, and a mutant line with homozygous substitutions at target site 2 was obtained. Disease resistance assays showed that, compared to wild-type plants, the Clmlo5b exhibited strongly inhibited mycelial growth, significantly reduced disease severity, and a substantial decrease in spore production after inoculation with powdery mildew. Our findings confirm that ClMLO5b is a key susceptibility gene in watermelon and provide both a promising genetic target and valuable breeding material for developing powdery mildew-resistant watermelon 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

Glycerol-3-phosphate acyltransferases (GPATs) catalyze the initial and rate-limiting step of glycerolipid biosynthesis, yet their contribution to salt tolerance in the soybean (Glycine max (L.) Merr.) plants remains largely uncharacterized. In this study, a total of 27 GmGPAT genes were identified, and their evolutionary relationships, chromosomal distribution, conserved motifs, and cis-regulatory elements were comprehensively analyzed. Through transcriptomic and qPCR analyses, many GmGPATs were found to be predominantly expressed in roots, with GmGPAT1, a plastid-targeted isoform, displaying the most rapid and pronounced transcriptional activation under salt stress. GFP-fusion experiments in transient expression assays confirmed plastid localization of GmGPAT1. Heterologous expression in Escherichia coli together with enzyme kinetics analyses validated its enzymatic function as a GPAT family member. The soybean hairy-root lines overexpressing GmGPAT1 exhibited enhanced root elongation, increased biomass, and improved photosynthetic efficiency under 120 mM NaCl stress, while CRISPR/Cas9 knockout mutants showed pronounced growth inhibition. Physiological assays demonstrated that GmGPAT1 overexpression mitigated oxidative damage by limiting reactive oxygen species (ROS) accumulation and lipid peroxidation, increasing antioxidant enzyme activities (CAT, SOD, POD), and elevating the ratios of AsA/DHA and GSH/GSSG. These changes contributed to redox homeostasis and improved Na⁺ extrusion capacity. A genome-wide association study (GWAS) involving 288 soybean accessions identified a single nucleotide polymorphism in the GmGPAT1 promoter that was significantly correlated with salt tolerance, and the beneficial Hap1 allele emerged as a promising molecular marker for breeding. Together, these analyses emphasize the status of GmGPAT1 as a major regulator of salt stress adaptation through the coordinated modulation of lipid metabolism and redox balance, extend the functional annotation of the soybean GPAT family, and highlight new genetic resources that can be leveraged to enhance tolerance to salt stress in soybean cultivars. Full article

As the ancestor and close relative of soybeans, wild soybeans exhibit strong salt tolerance and are ideal materials for discovering salt-tolerant genes. Expansins are a type of cell wall-loosening protein that plays an active role in regulating plant salt tolerance. We previously obtained the wild soybean expansin gene GsEXPB1, which is specifically transcribed in roots and actively responds to salt stress. Overexpression of this gene significantly promotes the growth of soybean hairy roots under salt stress. To further elucidate the function of the gene in regulating plant tolerance to salt stress, this study obtained soybean hairy roots that overexpress the GsEXPB1 gene and silence its homologous gene GmEXPB4 through RNAi. Under salt stress, the overexpression of the GsEXPB1 gene significantly promoted the growth of soybean hairy roots, while the hairy roots that were silenced for the GmEXPB4 gene exhibited an opposite phenotype. Physiological assay results indicate that GsEXPB1 enhances the tolerance of soybean hairy roots to salt stress by regulating the antioxidant system and Na⁺/K⁺ content. In soybean lines overexpressing GsEXPB1, the germination rate of seeds and root growth indicators under salt stress were significantly improved compared to those of wild-type plants. Meanwhile, GsEXPB1 enhances the tolerance of transgenic lines to salt stress by actively regulating the antioxidant system, osmotic adjustment system, chlorophyll content, cell wall components, and Na⁺/K⁺ levels, significantly promoting growth and increasing the number of flowers and grain weight. This study reveals the physiological mechanism by which GsEXPB1 enhances soybean salt tolerance, providing a theoretical basis and relevant references for the application of this gene in the breeding of new soybean salt-tolerant varieties. Full article

Auxin is the most important phytohormone, regulating root growth and development in plants. ARFs function as central regulators in auxin signaling in model plants, and the functions of ARF activators have been widely investigated, while the functions of ARF repressors remain elusive. In this study, we investigated the family of GmARFs in soybean through a genome-wide expression pattern and functional study of roots highly expressing ARFs. In total, we identified 59 GmARF members in the soybean genome. GmARFs harbor canonical B3 DNA-binding (B3), ARF (Aux rep), and PB1 functional protein domains. We identified two potential repressor genes, GmARF9b (Glyma.03G070500) and GmARF2a (Glyma.05G200800), which are specifically or highly expressed in the roots. Histochemical staining suggested that both genes are primarily expressed in the stele, root tips, and lateral root primordia. Subcellular localization analysis showed they were mainly localized in the nucleus. Overexpression of GmARF9b and GmARF2a significantly inhibited root growth using a transgenic hairy root assay. Root section analysis further suggested that GmARF9b and GmARF2a negatively regulated cortical cell layers and the longitudinal cell length of roots, thereby modulating root growth. Overall, this study has preliminarily identified two GmARFs that inhibit root growth in soybean. This discovery has deepened our understanding of the functions of ARFs in root development and has provided guidance for breeding crop roots with improved nutrient use efficiency. Full article

Agastache rugosa is also known as Korean mint, and it has numerous health benefits due to its rich source of phenolic compounds. The main objective of this study was to produce a ZmLC-overexpressing transgenic hairy root line via Agrobacterium rhizogenes-mediated transformation. The overexpressing transgenic lines were screened using qRT-PCR after exposure to light conditions. The best hairy root line was selected, and the expression levels of phenylpropanoid biosynthetic pathway genes and phenylpropanoid compound accumulation were analysed using qRT-PCR and HPLC, respectively. In addition, antioxidant activities (RPA, ABTS, and DPPH), total phenolic content, and total flavonoid content were analysed. The ZmLC-overexpressing transgenic line upregulated all the phenylpropanoid pathway genes, which led to the higher accumulation of phenylpropanoid compounds in the transgenic line than in the control line. In addition, the total phenolic and flavonoid content was significantly higher in the transgenic line. The antioxidant activity assay showed that the transgenic hairy root line had significantly higher activity than that of the control lines. Thus, ZmLC positively enhances the phenylpropanoid biosynthetic pathway and antioxidant activities in A. rugosa. The results show that ZmLC can be used to enhance phenylpropanoid compounds and antioxidant activities in transgenic A. rugosa hairy root lines via the genetic engineering approach. Full article

Hypericum perforatum transformed shoot lines (TSL) regenerated from corresponding hairy roots and non-transformed shoots (NTS) were comparatively evaluated for their phenolic compound contents and in vitro inhibitory capacity against target enzymes (monoamine oxidase-A, cholinesterases, tyrosinase, α-amylase, α-glucosidase, lipase, and cholesterol esterase). Molecular docking was conducted to assess the contribution of dominant phenolic compounds to the enzyme-inhibitory properties of TSL samples. The TSL extracts represent a rich source of chlorogenic acid, epicatechin and procyanidins, quercetin aglycone and glycosides, anthocyanins, naphthodianthrones, acyl-phloroglucinols, and xanthones. Concerning in vitro bioactivity assays, TSL displayed significantly higher acetylcholinesterase, tyrosinase, α-amylase, pancreatic lipase, and cholesterol esterase inhibitory properties compared to NTS, implying their neuroprotective, antidiabetic, and antiobesity potential. The docking data revealed that pseudohypericin, hyperforin, cadensin G, epicatechin, and chlorogenic acid are superior inhibitors of selected enzymes, exhibiting the lowest binding energy of ligand–receptor complexes. Present data indicate that H. perforatum transformed shoots might be recognized as an excellent biotechnological system for producing phenolic compounds with multiple health benefits. Full article

MicroRNAs (miRNAs) are a group of endogenous small non-coding RNAs in plants. They play critical functions in various biological processes during plant growth and development. Salvia miltiorrhiza is a well-known traditional Chinese medicinal plant with significant medicinal, economic, and academic values. In order to elucidate the role of miRNAs in S. miltiorrhiza, six small RNA libraries from mature roots, young roots, stems, mature leaves, young leaves and flowers of S. miltiorrhiza and one degradome library from mixed tissues were constructed. A total of 184 miRNA precursors, generating 137 known and 49 novel miRNAs, were genome-widely identified. The identified miRNAs were predicted to play diversified regulatory roles in plants through regulating 891 genes. qRT-PCR and 5′ RLM-RACE assays validated the negative regulatory role of smi-miR159a in SmMYB62, SmMYB78, and SmMYB80. To elucidate the function of smi-miR159a in bioactive compound biosynthesis, smi-miR159a transgenic hairy roots were generated and analyzed. The results showed that overexpression of smi-miR159a caused a significant decrease in rosmarinic acid and salvianolic acid B contents. qRT-PCR analysis showed that the targets of smi-miR159a, including SmMYB62, SmMYB78, and SmMYB80, were significantly down-regulated, accompanied by the down-regulation of SmPAL1, SmC4H1, Sm4CL1, SmTAT1, SmTAT3, SmHPPR1, SmRAS, and SmCYP98A14 genes involved in phenolic acid biosynthesis. It suggests that smi-miR159a is a significant negative regulator of phenolic acid biosynthesis in S. miltiorrhiza. Full article