IntroductionIsoflavones possess significant health benefits, including anti-inflammatory, anti-cancer, and antioxidant properties. Soybean (Glycine max (L.) Merr) is an effective natural source of isoflavones for humans. Thus, it is important to breed soybean varieties with enhanced isoflavone content and to elucidate the secondary metabolic pathways involved. Small auxin-up RNA (SAUR) genes constitute the largest family responsive to auxin, however, few studies have addressed their roles in soybean flavonoid metabolism.MethodsIn this study, flavonoid-targeted metabolites were measured in GmSAUR:GmSAUR transgenic soybeans. Chromatin immunoprecipitation sequencing (ChIP-seq) was performed to identify potential target genes regulated by GmSAUR. Additionally, RNA‑Seq was performed to identify downstream genes regulated by 35S:GmMYB176, including GmC4H, GmIF7MaT, GmCYP450 84A1‑like, and GmCYP450 84A1.ResultsThe transgenic soybeans exhibited higher contents of daidzin and genistin compared to controls. ChIP-seq revealed that GmSAUR binds to the promoter region of GmMYB176, thereby promoting the expression of downstream genes including cinnamate 4-hydroxylase (GmC4H), isoflavone 7-O-beta-glucoside 6′′-O-malonyltransferase (GmIF7MaT), cytochrome P450 84A1-like (GmCYP450 84A1-like), and cytochrome P450 84A1 (GmCYP450 84A1). This regulatory cascade ultimately led to increased accumulation of soybean isoflavones.DiscussionThese findings indicate that the GmSAUR gene facilitates soybean isoflavone biosynthesis by modulating the expression of GmMYB176, providing new insights into the genetic improvement of isoflavone content in soybean.

Colletotrichum gloeosporioides infects the plant host via an appressorium, which generates turgor pressure by metabolizing certain internal compounds. The mechanical force created by the turgor pressure is required for the production of infection pegs that penetrate host epidermal cells. Thus, appressorium turgor pressure must be sufficient for C. gloeosporioides infection. The regulated metabolism of intracellular metabolites or compounds plays a key role in the process underlying increases in turgor pressure. In this study, we analyzed appressorial metabolites at different developmental stages using a non-targeted metabolomics approach. We identified 39 differentially accumulated metabolites associated with turgor pressure and classified them into six major groups: amino acids, fatty acids, phospholipids, glycerolipids, carbohydrates, and organic acids. Four notable metabolic pathways related to turgor pressure were identified: degradation of carbohydrates, degradation of lipids, amino acid (arginine) synthesis, and phospholipid metabolism. Moreover, the phospholipid metabolism may be important for the development of appressoria and the required increase in turgor pressure. Three representative inhibitors of phospholipid metabolism (Neomycin, Doxorubicin, and alexidine dihydrochloride) were selected to study the relationship of phospholipid metabolism on the development of appressorium and changes in turgor pressure in C. gloeosporioides. Neomycin was shown to be a potent inhibitor of C. gloeosporioides and could effectively control poplar anthracnose. It was found that phospholipid metabolism is associated with the required increase in appressorium turgor. Our findings provide new insights into the mechanism underlying appressorium turgor pressure formation as well as potential targets for improving the control of C. gloeosporioides.

Alkaline stress is a major constraint on crop growth and development and negatively impacts soybean (Glycine max) production and yield. Despite the remarkable progress that has been made in investigating beneficial microbes that facilitate plant growth and development, the role of rhizobacteria in regulating alkaline tolerance in soybean remains poorly understood. Here, we isolated Klebsiella sp. strain B7 from the Suaeda glauca roots and found that it enhances the alkaline tolerance of soybean by secreting pyruvic acid. Metabolome and RT-qPCR analysis of soybean roots indicated that high levels of pyruvic acid secreted by B7 activated the expression of genes involved in pyruvic acid metabolism and increased L-malic acid accumulation in soybean roots, thereby effectively mitigating reactive oxygen species induced by alkaline stress. Overexpression of these pyruvic acid metabolism-associated genes greatly enhanced alkaline tolerance of soybean and ATP-citrate lyase activity, further confirming the positive role of pyruvic acid in L-malic acid biosynthesis and alkaline tolerance in soybean. Notably, the B7 application to alkaline soil enhanced the soybean yield. Moreover, B7 recruited more beneficial microbes and shaped the composition of the rhizosphere bacterial community of soybean plants. These findings highlight the vital function of rhizobacteria strain B7 in enhancing alkaline tolerance in soybean, thus providing further evidence for the crucial role of plant growth-promoting rhizobacteria in the abiotic stress response of soybean.

Pine wood nematode (PWN, Bursaphelenchus xylophilus) is fatal to the pine trees around the world. Its northward and westward expansion in China endangers Larix spp., yet its molecular response remains understudied. We conducted transcriptomic analysis (RNA-seq) on three economically important larch species (Larix principis-rupprechtii, L. olgensis, and L. kaempferi) infected by geographically distinct PWN isolates (northern Fushun and southern Changde strains) at 1 and 3 days post-inoculation. Comparative RNA-seq analysis of 36 samples revealed that genes such as oxidative stress, and secondary metabolite production were differentially expressed in Larix spp. upon infection by the PWNs. Furthermore, compared to the Changde strain, infection with the Fushun PWN strain can elicit a consistently stronger and more distinct transcriptional defense response across all tested larch species. These results provide insights into the molecular mechanisms of plant defense against PWNs, offering genetic target for resistance breeding and informing the development of targeted control measures against this pathogen.

Several studies have been conducted on plant responses to nutrient stressors; however, the mechanism underlying low-sulfur (LS) stress responses is still unclear. Here, we elucidated the function of COE2 in Arabidopsis response to sulfur deficiency using a series of phenotypic, physiological, biochemical, and molecular studies of the loss-of-function of COE2 (coe2 mutant). Under low sulfur conditions, WT seedlings had considerably longer roots than the coe2 seedlings. Although the chlorophyll fluorescence of coe2 and WT was lower under low sulfur, the reduction was more pronounced in the WT seedlings, indicating WT sensitivity to LS stress. Next, RNA-sequencing analysis was performed to investigate the roles of the COE2 in Arabidopsis response to sulfur deficiency at the molecular level. The coe2 and WT leaves responded to the induction of genes related to jasmonic acid, abscisic acid, and water deprivation, which are all crucial for leaf growth and defense. WT roots had more upregulated genes than the coe2 roots; thus, activation of these genes is tightly linked to WT and coe2 root responses to LS stress. We further evaluated the involvement of AtPSBO1 (a photosynthetic-inducible gene) in coe2 growth regulation under LS conditions. Compared with the coe2 seedlings, plants expressing 35S::PSBO1 exhibit increased sensitivity to sulfur deficiency in the leaves and roots, suggesting COE2 functions in chloroplast and root development under LS conditions. This study highlights the crucial roles of COE2 in root-shoot coordination in response to sulfur deficiency.

Colletotrichum gloeosporioides , which is an important plant pathogen infecting gramineous and woody plant species, is common in tropical, subtropical and temperate regions. Appressorium formation is a key step during the infection of plant hosts by C . gloeosporioides . Thus, inhibiting this step has become a primary goal of researchers attempting to develop improved methods for preventing and controlling anthracnose and related fungal diseases of plants. This review summarises the recent advances in functional genomics research related to appressorium formation in C . gloeosporioides , with a particular focus on mitogen-activated protein kinase signalling pathways, the cyclic AMP-dependent protein kinase A signalling pathway and metabolic pathways. The elucidation of pathogenicity-related mechanisms in C . gloeosporioides may result in new disease prevention and control measures involving novel fungicides and molecular breeding using candidate target genes.

Salt-alkali stress is one of the most widespread and devastating abiotic stress. Alternative splicing is a response pathway to such stress. However, the role of microexons in response to salt-alkali stress in soybean remains obscure. In this study, we identified microexons related to salt-alkali stress. We focused on analyzing the conserved sequence patterns of 27-30 bp microexons, and consistently observed conserved GT and AG sequences at the 5' and 3' ends of these microexons. Additionally, we found that the AP2 protein domain had the most abundant microexons. Interestingly, the majority of microexons in the AP2 transcription factor were 9 bp in length, encoding a conserved valine (V), tyrosine (Y), or leucine (L), suggesting their indispensable role. Furthermore, we cloned two transcripts of three AP2 genes with and without the salt-alkali stress-induced microexon and generated stable transgenic soybeans. Surprisingly, we discovered that the depletion of microexons in the AP2 gene enhances salt-alkali resistance. Collectively, this characterization of microexon suggests a new scenario explaining soybean salt-alkali stress resistance.

Plant cells exhibit an extraordinary regenerative potential, achieving cellular totipotency by dedifferentiating to form new tissues. While significant progress has been made in understanding cell fate mechanisms, the regulatory networks governing callus cell development remain insufficiently explored, particularly regarding cell classification, morphology, and regulatory processes. This study provides a detailed investigation into the developmental dynamics and transcriptomic profiles of callus cells in Arabidopsis at key stages: initiation, proliferation, and greening. Employing single-cell RNA sequencing and UMAP-based clustering, we annotated cell clusters based on highly enriched gene expressions. Developmental trajectories were further mapped through pseudotime analysis, revealing distinct transcription factor networks. Additionally, functional analysis of key regulatory genes was conducted using mutant and overexpression lines, affirming their roles in callus development. Gene Ontology analysis highlighted the involvement of environmental factors-low oxygen and salinity promoted callus formation, while light inhibited it, though essential for greening. These findings shed light on the complex regulatory landscape of plant tissue regeneration and guide future research avenues.

Poplar anthracnose caused by Colletotrichum gloeosporioides is one of the major forest diseases worldwide, causing incalculable losses in forestry production every year. In this study, an endophytic bacterium with significant antagonistic activity against C. gloeosporioides was isolated from Populus canadensis leaves, identified and named Pseudomonas atacamensis GZ-3. The specific biological functions of strain GZ-3 were analyzed using whole genome sequencing. The volatile organic compounds (VOCs) produced by strain GZ-3 had good antifungal activity against C. gloeosporioides. These VOCs could effectively inhibit mycelial growth and conidial germination of C. gloeosporioides. In addition, metabolomics analysis revealed that VOCs could help poplar resist infection of C. gloeosporioides by inducing the synthesis of flavonoids in poplar. 3-methyl-1-butanol and 2-heptanone were the main antifungal compounds in VOCs. Therefore, Pseudomonas atacamensis GZ-3 had good potential for biocontrol. This study provided a new strategy for the biological control of poplar anthracnose.

Plant specialized metabolites are commonly stored in glycosylated forms within plant cells, with their homeostasis regulated by glycosyltransferases and β-glucosidases (BGLUs, also known as β-glucoside hydrolases (E.C.3.2.1.21)). Soyasaponins, the predominant triterpenoid compounds (C30) in soybean seeds, contain two sugar moieties attached at the C3 and C22 positions. While glycosyltransferases involved in soyasaponin biosynthesis have been well characterized, the role of BGLUs in soyasaponin homeostasis remains unclear. In this study, we identified GmSSBG1 (Soyasaponin β-glucosidase1; Glyma.07G258700) as a candidate gene potentially involved in soyasaponin homeostasis through gene to gene co-expression analysis. Biochemical assays demonstrated that GmSSBG1 specifically hydrolyzes arabinose residues at the C22 position of A0- and B0-series soyasaponins. Loss-of-function mutations in GmSSBG1 led to a significant accumulation of A0- and B0-series soyasaponins in mutant seeds, which correlated with a pronounced decrease in resistance to the soybean pod borer (Leguminivora glycinivorella). Our findings provide critical insights into the regulatory mechanisms underlying soyasaponin homeostasis and lay a theoretical foundation for molecular breeding strategies aimed at developing soybean lines with enhanced resistance to soybean pod borer, even to other insect pests.

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.

Colletotrichum gloeosporioides is the main pathogen that causes poplar anthracnose. This hemibiotrophic fungus, which can severely decrease the economic benefits and ecological functions of poplar trees, infects the host by forming an appressorium. Hox7 is an important regulatory factor that functions downstream of the Pmk1 MAPK signaling pathway. In this study, we investigated the effect of deleting CgHox7 on C. gloeosporioides. The conidia of the ΔCgHox7 deletion mutant germinated on a GelBond membrane to form non-melanized hyphal structures, but were unable to form appressoria. The deletion of CgHox7 weakened the ability of hyphae to penetrate a cellophane membrane and resulted in decreased virulence on poplar leaves. Furthermore, deleting CgHox7 affected the oxidative stress response. In the initial stage of appressorium formation, the accumulation of reactive oxygen species differed between the ΔCgHox7 deletion mutant and the wild-type control. Moreover, CgHox7 expression was necessary for maintaining cell wall integrity. Considered together, these results indicate that CgHox7 is a transcription factor with crucial regulatory effects on appressorium formation and the pathogenicity of C. gloeosporioides.

Poplar is an economically and ecologically valuable tree species. Anthracnose, which severely affects poplar tree growth, is mainly caused by Colletotrichum gloeosporioides. In the infestation cycle of poplar anthracnose, the entry of C. gloeosporioides into the host tissue depends on the formation of an appressorium. The subsequent development of the appressorium determines the pathogenesis of poplar anthracnose and the degree of damage. Previous studies have found that the transcription factor CgSte12 affects appressorium formation and development by regulating the expression of a series of genes, including the sterol-synthesis-related gene CgCYP51, which influences appressorium formation and development. In this study, knockout and functional analyses of CgCYP51 revealed decreases in differentiation, darkening rate, and turgor pressure of appressoria in mutants. Additionally, compared with the wild-type appressorium, mutant appressoria secreted less mucus and exhibited abnormal penetration pore formation, ultimately leading to decreased pathogenicity. Moreover, CgCyp51 affected the sensitivity of C. gloeosporioides to sterol biosynthesis inhibitors. Considered together, the study findings indicate CgCYP51 is a key CgSte12-regulated gene that affects C. gloeosporioides appressorium formation and development. Furthermore, the study data provide new insights into the molecular basis of C. gloeosporioides appressorium formation and development.

Colletotrichum gloeosporioides is the causal agent of poplar anthracnose, which induces major economic losses and adversely affects the ecosystem services of poplar forests. The appressorium serves as a penetration structure for many pathogenic fungi, including C. gloeosporioides. The production of mucilage and the formation of penetration pegs are critically important for the appressorium-mediated penetration of host tissues. We previously found that CgPmk1 is a key protein involved in appressorium formation, penetration, and pathogenicity. Although CgSte12, which is a transcription factor that functions downstream of CgPmk1, regulates the formation of penetration pegs, its role in C. gloeosporioides appressorium development and pathogenicity has not been elucidated. Here, we developed C. gloeosporioides CgSTE12 mutants and characterized the molecular and cellular functions of CgSTE12. The results showed that mycelial growth and morphology were not affected in the CgSTE12 knockout mutants, which produced normal melanized appressoria. However, these mutants had less mucilage secreted around the appressoria, impaired appressorial cone formation, and the inability to form penetration pores and pegs, which ultimately led to a significant loss of pathogenicity. Our comparative transcriptome analysis revealed that CgSte12 controls the expression of genes involved in appressorium development and function, including genes encoding cutinases, NADPH oxidase, spermine biosynthesis-related proteins, ceramide biosynthesis-related proteins, fatty acid metabolism-related proteins, and glycerophospholipid metabolism-related proteins. Overall, our findings indicate that CgSte12 is a critical regulator of appressorium development and affects C. gloeosporioides pathogenicity by modulating the structural integrity of appressoria.

Soybean [Glycine max (L.) Merr.], an essential staple food and oil crop worldwide, boasts abundant vegetable proteins and fats beneficial for both human and animal consumption. However, the soybean pod borer (Leguminivora glycinivorella) (SPB) stands as the most destructive soybean insect pest in northeast China and other northeastern Asian regions, leading to significant annual losses in soybean yield and economic burden. Therefore, this study aims to investigate the introduction of a previously tested codon-optimized cry1c gene, cry1c*, into the soybean genome and assess its effect on the SPB infestation by generating and characterizing stable transgenic soybeans overexpressing cry1c*. The transgenic soybean lines that constitutively overexpressed cry1c* exhibited a significant reduction in the percentage of damaged seeds, reaching as low as 5% in plants under field conditions. Additionally, feeding transgenic leaves to the larvae of S. exigua, S. litura, and M. separta resulted in inhibited larval growth, decreased larval body weight, and lower survival rates compared to larvae fed on wild-type leaves. These findings showed that the transgenic lines maintained their resistance to SPB and other lepidopteran pests, especially the transgenic line KC1. Southern blotting and genome-wide resequencing analysis revealed that T-DNA integration occurred as a single copy between loci 50,868,122 and 50,868,123 of chromosome 10 in the transgenic line KC1. Therefore, the transgenic line KC1, overexpressing high levels of cry1c* in leaves and seeds, holds strong potential for commercial use in the integrated management of SPB and other lepidopteran pests.

Colletotrichum gloeosporioides is a model plant pathogenic fungus, and the appressoria are the main infection structures integral to the pathogenic process. Septin proteins play fundamental roles in facilitating shape alteration and organizing the F-actin cytoskeleton, thereby aiding the invasive growth of various fungi. Herein, we examined the roles of four septin-coding genes (CgSEP3, CgSEP4, CgSEP5, and CgSEP6) in C. gloeosporioides. Our findings reveal the diverse functions of septins in C. gloeosporioides, which encompass the regulation of vegetative growth, conidiation, cell wall integrity, and stress responses. Critically, septins are involved in the formation, invasion, and expansion of infection structures and they directly influence virulence on unwounded hosts. Interestingly, the deletion of CgSEP4 resulted in the formation of hooked and bent germ tubes and caused a significant decrease in appressorium turgor pressure, which has not been reported in other fungi. Our findings demonstrated that CgSEP3 and CgSEP6 were regulated by ROS signal transduction during the formation of infection structure. Moreover, the knockout of the key component, CgSEP5, significantly decreased growth rate compared to the wild type, completely blocking the penetration of infection structures and subsequently abolishing virulence on poplar leaves. By subcellular localization of GFP fusions, it was proved that CgSEP5 may regulate the formation of appressorial pegs in C. gloeosporioides through forming a ring-like structure inside the appressorium. Collectively, our research underscores the pivotal role of septins in fungal pathogenicity, by orchestrating the formation and development of infection structures. We speculate that CgSEP5 function as a promising anti-fungal target, and believe these findings provide a substantial reference for future investigations into the mechanisms underpinning the invasion of fungi appressoria on woody plants.

Poplar anthracnose, which is one of the most important tree diseases, is primarily caused by Colletotrichum gloeosporioides, which has been detected in poplar plantations in China and is responsible for serious economic losses. The characteristics of 84K poplar that have made it one of the typical woody model plants used for investigating stress resistance include its rapid growth, simple reproduction, and adaptability. In this study, we found that the resistance of 84K poplar to anthracnose varied considerably depending on how the samples were inoculated of the two seedlings in each tissue culture bottle, one (84K-Cg) was inoculated for 6 days, whereas the 84K-DCg samples were another seedling inoculated at the 6th day and incubated for another 6 days under the same conditions. It was showed that the average anthracnose spot diameter on 84K-Cg and 84K-DCg leaves was 1.23 ± 0.0577 cm and 0.67 ± 0.1154 cm, respectively. Based on the transcriptome sequencing analysis, it was indicated that the upregulated phenylpropanoid biosynthesis-related genes in 84K poplar infected with C. gloeosporioides, including genes encoding PAL, C4H, 4CL, HCT, CCR, COMT, F5H, and CAD, are also involved in other KEGG pathways (i.e., flavonoid biosynthesis and phenylalanine metabolism). The expression levels of these genes were lowest in 84K-Cg and highest in 84K-DCg. It was found that PAL-related genes may be crucial for the induced resistance of 84K poplar to anthracnose, which enriched in the phenylpropanoid biosynthesis. These results will provide the basis for future research conducted to verify the contribution of phenylpropanoid biosynthesis to induced resistance and explore plant immune resistance-related signals that may regulate plant defense capabilities, which may provide valuable insights relevant to the development of effective and environmentally friendly methods for controlling poplar anthracnose.

Colletotrichum gloeosporioides , which penetrates the plant hosts via special infection structures called appressoria, causes anthracnose in many plants worldwide. However, the biological mechanism underlying appressorium formation is not fully characterized in this pathogen. In the present study, the overall gene expression pattern of appressorium formation was studied by targeted gene deletion and expression analysis during the conidial germination of wildtype and Δ CgMsb2Sho1 double mutant strains of C . gloeosporioides on hydrophobic surfaces. Significant transcriptional changes were detected for nearly 41.9% and 45.4% of the genes in the wildtype and Δ CgMsb2Sho1 strains, respectively. Two genes encoding the cAMP‐dependent protein kinase regulatory subunit and adenylate cyclase had higher expression in the wild type than in the Δ CgMsb2Sho1 mutant; cAMP then activates the mitogen‐activated protein kinase (MAPK) signalling pathway. However, we revealed that genes involved in the MAPK signalling pathway may inhibit appressorium formation by encoding proteins that disrupt the downstream signal transduction process after recognition of the host by the cell membrane surface receptor proteins mucin (Msb2) and the sensor protein Sho, encoded by CgMsb2 and CgSho1 , respectively. Finally, the regulation of transcription factors on lipid metabolism and plant cell wall‐degrading enzymes was affected. The results provide a comprehensive overview of the changes in the expression of important genes during the early stage of appressorium formation, which may be useful for understanding the molecular mechanism of appressorium formation in C . gloeosporioides .

Soybean production is severely hampered by saline-alkaline stress caused by saline-alkalization. Plants have aldehydrogenase (ALDH) family members that convert reactive aldehydes to carboxylic acids to remove active aldehyde molecules. However, little is known about the increased saline-alkali tolerance caused by the ALDH function in soybean. Here, we introduced a previously identified ALDH coding gene AhALDH3H1 from Arachis hypogaea into the soybean genome to investigate its critical role in response to saline-alkali stress. Transgenic soybean with increased aldehyde dehydrogenase activity showed significant tolerance to saline-alkali stress. It reduced malondialdehyde (MDA) content compared to its receptor, suggesting that over-expression of AhALDH3H1 accelerated soybean tolerance to saline-alkali stress by increasing aldehyde dehydrogenase activity, which is responsible for scavenging toxic MDA. To further analyze the inner mechanisms that allow transgenic plants to tolerate saline-alkali stress, we sequenced the transcriptome and metabolome of P3 (wild type, WT) and transgenic lines which were separately treated with water and a saline-alkali solution. When subjected to saline-alkali stress, the integrated analysis of the transcriptome and metabolome suggested that several genes related to cell wall structure crucial for preserving cell wall extensibility and plasticity were largely responsible for restoring homeostasis within the transgenic cells compared to WT. Metabolites, including both necessary ingredients for cell wall genesis and harmful production produced during the saline-alkali stress response, could be transported efficiently with the help of the ABC transporter, reducing the negative effects of saline-alkali stress. These findings suggest that introducing AhALDH3H1 increases transgenic soybean tolerance to saline-alkali stress may through cell wall structure maintenance and metabolites transport.

Our findings revealed that the inoculation of Colletotrichum gloeosporioides has a greater effect on the fungal community than the bacterial community. In addition, coumarins, organic acids, and flavonoids may have recruitment effects on phyllosphere microorganisms, while indoles may have inhibitory effects on these organisms. These findings may provide the theoretical basis for the prevention and control of poplar anthracnose.