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Abstract Lichen-forming fungi (LFF) are prolific producers of functionally and structurally diverse secondary metabolites, most of which are taxonomically exclusive and play lineage-specific roles. To date, widely distributed, evolutionarily conserved biosynthetic pathways in LFF are not known. However, this idea stems from polyketide derivatives, since most biochemical research on lichens has concentrated on polyketide synthases (PKSs). Here, we present the first systematic identification and comparison of terpene biosynthetic genes of LFF using all the available Lecanoromycete reference genomes and 22 de novo sequenced ones (111 in total, representing 60 genera and 23 families). We implemented genome mining and gene networking approaches to identify and group the biosynthetic gene clusters (BGCs) into networks of similar BGCs. Our large-scale analysis led to the identification of 724 terpene BGCs with varying degrees of pairwise similarity. Most BGCs in the dataset were unique with no similarity to a previously known fungal or bacterial BGC or among each other. Remarkably, we found two BGCs that were widely distributed in LFF. Interestingly, both conserved BGCs contain the same core gene, i.e., putatively a squalene/phytoene synthase (SQS), involved in sterol biosynthesis. This indicates that early gene duplications, followed by gene losses/gains and gene rearrangement are the major evolutionary factors shaping the composition of these widely distributed SQS BGCs across LFF. We provide an in-depth overview of these BGCs, including the transmembrane, conserved, variable and LFF-specific regions. Our study revealed that lichenized fungi do have a highly conserved BGC, providing the first evidence that a biosynthetic gene may constitute essential genes in lichens.

Abstract Coevolutionary antagonism generates relentless selection that can favour genetic exchange, including transfer of antibiotic synthesis and resistance genes among bacteria, and sexual recombination of disease resistance alleles in eukaryotes. We report an unusual link between biological conflict and DNA transfer in bdelloid rotifers, microscopic animals whose genomes show elevated levels of horizontal gene transfer from non-metazoan taxa. When rotifers were challenged with a fungal pathogen, horizontally acquired genes were over twice as likely to be upregulated as other genes — a stronger enrichment than observed for abiotic stressors. Among hundreds of upregulated genes, the most markedly overrepresented were clusters resembling bacterial polyketide and nonribosomal peptide synthetases that produce antibiotics. Upregulation of these clusters in a pathogen-resistant rotifer species was nearly ten times stronger than in a susceptible species. By acquiring, domesticating, and expressing non-metazoan biosynthetic pathways, bdelloids may have evolved to resist natural enemies using antimicrobial mechanisms absent from other animals.

Multi-modular enzyme complexes known as non-ribosomal peptide synthetases (NRPSs) and polyketide synthetases (PKSs) have been widely reported in bacteria that produce secondary bioactive metabolites such as non-ribosomal peptides (NRPs) and polyketides (PKs), respectively. These NRPS/PKS pathways contribute to synthesizing several antibiotics, such as vancomycin, rifamycin, and bleomycin, which are vital in human medicine. The present study aimed to isolate gut-associated bacteria from mud crab Scylla serrata, and detect NRPS and PKS gene clusters associated with it. This study included 36 bacterial isolates from five mud crab gut samples. Biosynthetic gene clusters (NRPS and PKS), were detected by PCR using degenerative primers specific to these genes. Three isolates (FKP2–4, FKP4–1, and FKP2–16) were positive for NRPS and two for PKS (FKP2–4 and FKP4–1) genes. The isolates were subjected to 16S rRNA gene amplification and sequenced. In silico analysis of the sequences using the Basic Local Alignment Search Tool (BLAST) identified the isolates FKP2–4, FKP4–1, and FKP2–16 as Acinetobacter variabilis, Vagococcus fluvialis, and Staphylococcus arlettae, respectively, after comparing with the existing sequences available in the National Center for Biotechnology Information (NCBI) database. Compared to the control, it was observed that these isolates exhibited intriguing antagonistic activities against Escherichia coli and Staphylococcus aureus. However, these isolates failed to show significant activity against Candida albicans. Exopolysaccharide production by the isolated organisms was tested using Zobell marine agar (ZMA) with 5% sucrose, but none of the colonies were mucoid or slimy.

Lichen-forming fungi (LFF) are prolific producers of functionally and structurally diverse secondary metabolites, most of which are taxonomically exclusive and play lineage-specific roles. To date, widely distributed, evolutionarily conserved biosynthetic pathways in LFF are not known. However, this idea stems from polyketide derivatives, since most biochemical research on lichens has concentrated on polyketide synthases (PKSs). Here, we present the first systematic identification and comparison of terpene biosynthetic genes of LFF using all the available Lecanoromycete reference genomes and 22 de novo sequenced ones (111 in total, representing 60 genera and 23 families). We implemented genome mining and gene networking approaches to identify and group the biosynthetic gene clusters (BGCs) into networks of similar BGCs. Our large-scale analysis led to the identification of 724 terpene BGCs with varying degrees of pairwise similarity. Most BGCs in the dataset were unique with no similarity to a previously known fungal or bacterial BGC or among each other. Remarkably, we found two BGCs that were widely distributed in LFF. Interestingly, both conserved BGCs contain the same core gene, i.e., putatively a squalene/phytoene synthase (SQS), involved in sterol biosynthesis. This indicates that early gene duplications, followed by gene losses/gains and gene rearrangement are the major evolutionary factors shaping the composition of these widely distributed SQS BGCs across LFF. We provide an in-depth overview of these BGCs, including the transmembrane, conserved, variable and LFF-specific regions. Our study revealed that lichenized fungi do have a highly conserved BGC, providing the first evidence that a biosynthetic gene may constitute essential genes in lichens., (© 2024. The Author(s).)

Aging is the main risk factor of cognitive neurodegenerative diseases such as Alzheimer's disease, with epigenome alterations as a contributing factor. Here, we compared transcriptomic/epigenomic changes in the hippocampus, modified by aging and by tauopathy, an AD-related feature. We show that the cholesterol biosynthesis pathway is severely impaired in hippocampal neurons of tauopathic but not of aged mice pointing to vulnerability of these neurons in the disease. At the epigenomic level, histone hyperacetylation was observed at neuronal enhancers associated with glutamatergic regulations only in the tauopathy. Lastly, a treatment of tau mice with the CSP-TTK21 epi-drug that restored expression of key cholesterol biosynthesis genes counteracted hyperacetylation at neuronal enhancers and restored object memory. As acetyl-CoA is the primary substrate of both pathways, these data suggest that the rate of the cholesterol biosynthesis in hippocampal neurons may trigger epigenetic-driven changes, that may compromise the functions of hippocampal neurons in pathological conditions.

Summary: The dynamics of N6-methyladenosine (m6A) mRNA modification are tightly controlled by the m6A methyltransferase complex and demethylases. Here, we find that auxin treatment alters m6A modification on auxin-responsive genes. Mechanically, TRANSMEMBRANE KINASE 4 (TMK4), a component of the auxin signaling pathway, interacts with and phosphorylates FKBP12-INTERACTING PROTEIN 37 (FIP37), a core component of the m6A methyltransferase complex, in an auxin-dependent manner. Phosphorylation of FIP37 enhances its interaction with RNA, thereby increasing m6A modification on its target genes, such as NITRILASE 1 (NIT1), a gene involved in indole-3-acetic acid (IAA) biosynthesis. 1-Naphthalacetic acid (NAA) treatment accelerates the mRNA decay of NIT1, in a TMK4- and FIP37-dependent manner, which leads to inhibition of auxin biosynthesis. Our findings identify a regulatory mechanism by which auxin modulates m6A modification through the phosphorylation of FIP37, ultimately affecting mRNA stability and auxin biosynthesis in plants.

Carnation (Dianthus caryophyllus L.) is a typical ethylene-sensitive cut flower. Variations in carnation vase life and sensitivity to ethylene have been reported, but no detailed analysis has been performed to date. In order to investigate the ethylene sensitivity of different cut carnation varieties and study the effect of ethylene on postharvest physiological changes of different carnation varieties, 14 varieties were used to explore ethylene sensitivity, and six varieties were used to analyze the release pattern of endogenous ethylene and the expression pattern of related genes. The results showed that among the 14 carnation varieties, 'Master' had the strongest ethylene sensitivity and 'Snow White' had the weakest ethylene sensitivity. Ethylene release changes of 'Master' are the terminal ascending type, and 'Cloud Shium', 'Little Pink', 'Seashell', 'Freedom' and 'Snow White' are the similar ethylene leap type. Ethylene biosynthesis genes DcACS1 and DcACO1 of 'Master' were up-regulated the most, and DcACO1 of 'Snow White' was the least up-regulated. The transient silencing and overexpression of DcACS1 and DcACO1 were performed and it was found that transient silencing can significantly delay aging, and overexpression significantly accelerates aging. This study laid the foundation for further research on the molecular mechanism of ethylene regulation of postharvest senescence of cut flowers of carnation, and also indicated the direction for further breeding and artificial screening of new storage tolerant carnation species by gene editing technology.

High-temperature stress (HTS) affects the growth and production of vegetable crops, including eggplant (Solanum melongena L.). Jasmonic acid (JA) plays key roles in regulating resistance to biotic and abiotic stresses in plants. Nonetheless, reports on the role of JA in heat tolerance in eggplant are rare. Herein, the effects of JA on heat tolerance in eggplant and the functions of the JA biosynthetic genes SmLOX4 and SmLOX5 were analyzed. The results showed that the JA content increased under high-temperature treatment (HTT) and exogenous methyl jasmonate (MeJA) treatment reduced the damage caused by HTT to eggplant. The expression of SmLOX4 and SmLOX5 was induced by HTT and significantly positively correlated with JA biosynthesis. SmLOX4 and SmLOX5 were localized in chloroplasts. The silencing of SmLOX4 and SmLOX5 by virus-induced gene silencing suppressed the heat tolerance of eggplant, whereas the overexpression of SmLOX4 and SmLOX5 enhanced the heat tolerance of Arabidopsis thaliana. JA content and the expression of JA signaling-related genes decreased in the SmLOX4- and SmLOX5-silenced plants but increased in the OE-SmLOX4 and OE-SmLOX5 transgenic plants. These results revealed that SmLOX4 and SmLOX5 improved eggplant heat tolerance by mediating JA biosynthesis and JA signaling pathways., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Poplar Cytospora canker, caused by Cytospora chrysosperma , is one of the most destructive and widespread poplar diseases worldwide, especially in northern China. However, our current understanding of its pathogenic mechanisms remains limited. Here, we showed that trehalose biosynthetic genes, such as trehalose-6-phosphate synthase 1 (Tps1), trehalose-6-phosphate phosphatase (Tps2), and the regulatory subunit (Tps3), play important roles in the development and virulence of C. chrysosperma . The targeted deletion mutants showed reduced trehalose synthesis and were defective in hyphal growth and conidiation. Deletion of any of the three genes attenuated virulence in poplar twigs and stronger poplar defense responses were triggered once inoculated by the mutants. Additionally, the mutants exhibited increased sensitivity to H 2 O 2 and cell wall stressors. Taken together, the finding suggests that trehalose biosynthetic genes contribute to fungal development, stress responses, and full virulence in C. chrysosperma .

Red peel color, a desirable trait in pears, is determined by anthocyanin accumulation. While REVEILLE (RVEs) transcription factors regulate anthocyanin biosynthesis in some plant species, their role in pear peel has not been well-studied. This study investigates the function of RVEs in anthocyanin biosynthesis in 'Zaosu' (low anthocyanin content) and its red bud mutant, 'Red Zaosu' (high anthocyanin content) fruit peel. Consistent with higher anthocyanin content, 'Red Zaosu' pears exhibited increased PbRVE6 expression compared to 'Zaosu', while PbRVE3a and PbRVE3b levels remained unchanged. Additionally, PbRVE6 was localized to the nucleus. Overexpression of PbRVE6 in 'Zaosu' pear pericarp significantly increased anthocyanin content and upregulated key anthocyanin pathway genes PbANS and PbUFGT. Conversely, VIGS silencing of PbRVE6 in 'Red Palacer' pears led to decreased expression of PbANS and PbUFGT and a slight reduction in anthocyanin content. Yeast one-hybrid and dual-luciferase assays confirmed that PbRVE6 can bind and activate the promoters of PbANS and PbUFGT. These findings demonstrate that PbRVE6 promotes anthocyanin accumulation in pear peel by directly regulating PbANS and PbUFGT expression. This study provides a valuable foundation for understanding the regulatory network of anthocyanins in pear peels, and offers potential genetic resources for the production of new pear germplasm., (© 2024 Scandinavian Plant Physiology Society.)

Flavonoids in Musaceae are involved in pigmentation and stress responses, including cold resistance, and are a component of the healthy human diet. Identification and analysis of the sequence and copy number of flavonoid biosynthetic genes are valuable for understanding the nature and diversity of flavonoid evolution in Musaceae species. In this study, we identified 71-80 flavonoid biosynthetic genes in chromosome-scale genome sequence assemblies of Musaceae, including those of Ensete glaucum , Musella lasiocarpa , Musa beccarii , M. acuminata , M. balbisiana and M. schizocarpa, checking annotations with BLAST and determining the presence of conserved domains. The number of genes increased through segmental duplication and tandem duplication. Orthologues of both structural and regulatory genes in the flavonoid biosynthetic pathway are highly conserved across Musaceae. The flavonoid 3',5'-hydroxylase gene F3'5'H was amplified in Musaceae and ginger compared with grasses (rice, Brachypodium, Avena longiglumis, and sorghum). One group of genes from this gene family amplified near the centromere of chromosome 2 in the x  = 11 Musaceae species. Flavonoid biosynthetic genes displayed few consistent responses in the yellow and red bracts of Musella lasiocarpa when subjected to low temperatures. The expression levels of MlDFR2/3 (dihydroflavonol reductase) increased while MlLAR (leucoanthocyanidin reductase) was reduced by half. Overall, the results establish the range of diversity in both sequence and copy number of flavonoid biosynthetic genes during evolution of Musaceae. The combination of allelic variants of genes, changes in their copy numbers, and variation in transcription factors with the modulation of expression under cold treatments and between genotypes with contrasting bract-colours suggests the variation may be exploited in plant breeding programmes, particularly for improvement of stress-resistance in the banana crop., Competing Interests: The authors declare no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Annals of Botany Company.)

The Fusarium head blight (FHB) pathogen Fusarium graminearum produces the trichothecene mycotoxin deoxynivalenol and reduces wheat yield and grain quality. Spring wheat ( Triticum aestivum ) genotype CB037 was transformed with constitutive expression (CE) constructs containing sorghum ( Sorghum bicolor ) genes encoding monolignol biosynthetic enzymes caffeoyl coenzyme A (CoA) 3- O -methyltransferase ( SbCCoAOMT ), 4-coumarate-CoA ligase ( Sb4CL ), or coumaroyl shikimate 3-hydroxylase ( SbC3'H ) or monolignol pathway transcriptional activator SbMyb60 . Spring wheats were screened for type I (resistance to initial infection, using spray inoculations) and type II (resistance to spread within the spike, using single-floret inoculations) resistances in the field (spray) and greenhouse (spray and single floret). Following field inoculations, disease index, percentage of Fusarium -damaged kernels (FDK), and deoxynivalenol measurements of CE plants were similar to or greater than those of CB037. For greenhouse inoculations, the area under the disease progress curve (AUDPC) and FDK were determined. Following screens, focus was placed on two each of SbC3'H and SbCCoAOMT CE lines because of trends toward a decreased AUDPC and FDK observed following single-floret inoculations. These four lines were as susceptible as CB037 following spray inoculations. However, single-floret inoculations showed that these CE lines had a significantly reduced AUDPC ( P < 0.01) and FDK ( P ≤ 0.02) compared with CB037, indicating improved type II resistance. None of these CE lines had increased acid detergent lignin compared with CB037, indicating that lignin concentration may not be a major factor in FHB resistance. The SbC3'H and SbCCoAOMT CE lines are valuable for investigating phenylpropanoid-based resistance to FHB., Competing Interests: The author(s) declare no conflict of interest.

Abstract Rhizoctonia solani is a polyphagous necrotrophic fungal pathogen that causes sheath blight disease in rice. It deploys effector molecules as well as carbohydrate‐active enzymes and enhances the production of reactive oxygen species for killing host tissues. Understanding R. solani ability to sustain growth under an oxidative‐stress‐enriched environment is important for developing disease control strategies. Here, we demonstrate that R. solani upregulates methionine biosynthetic genes, including Rs_MET13 during infection in rice, and double‐stranded RNA‐mediated silencing of these genes impairs the pathogen's ability to cause disease. Exogenous treatment with methionine restores the disease‐causing ability of Rs_MET13‐silenced R. solani and facilitates its growth on 10 mM H2O2‐containing minimal‐media. Notably, the Rs_MsrA gene that encodes methionine sulfoxide reductase A, an antioxidant enzyme involved in the repair of oxidative damage of methionine, is upregulated upon H2O2 treatment and also during infection in rice. Rs_MsrA‐silenced R. solani is unable to cause disease, suggesting that it is important for the repair of oxidative damage in methionine during host colonization. We propose that spray‐induced gene silencing of Rs_MsrA and designing of antagonistic molecules that block MsrA activity can be exploited as a drug target for effective control of sheath blight disease in rice.

Chemical study of the nematicidal biocontrol fungus Pochonia chlamydosporia PC-170 led to discovery of six resorcylic acid lactones (RALs), including three nematicidal glycosylated RALs, monocillin VI glycoside (1), colletogloeolactone A (2) and monocillin II glycoside (3), and three antibacterial non-glycosylated RALs, monocillin VI (4), monocillin IV (5) and monocillin II (6). The planar structure of the new compound monocillin VI glycoside (1) was elucidated using HRESIMS and NMR data, and its monosaccharide configuration was further determined through sugar hydrolysis experiment and GC–MS analysis method. Furthermore, their two biosynthetic-related PKS genes, pchE and pchI, were identified through the gene knockout experiment. The glycosylated RALs 1–3 exhibited nematicidal activity against Meloidogyne incognita, with LC50 values of 94, 152 and 64 μg/mL, respectively, and thus had great potential in the development of new nematicidal natural products to control M. incognita in the future.

Floral scent is an essential and genetically complex trait in herbaceous peonies (Paeonia lactiflora Pall.); however, specific genes related to metabolic and regulatory networks remain scantily studied. Our study integrated metabolite profiling and RNA-sequencing to screen floral scent biosynthetic genes. Hence, the major molecules identified by headspace collection combined with cultivar-specific GC-MS analysis were geraniol, β-caryophyllene, 2-phenylethanol (2-PE), citronellol, and 1,8-cineole. Genes related to terpenoids and 2-PE biosynthesis were identified after the assembly and annotation of the P. lactiflora transcriptomes. Eight angiosperm-specific terpene synthases (TPSs) from the TPS-a and TPS-b clades, as well as enzymes linked to 2-PE synthesis such as aromatic amino acid decarboxylase (AADC), phenylacetaldehyde reductase (PAR), and geranial reductase (GER) were identified. The biochemical analysis of the enzymes encoded by PlPAR1 and PlGER1 generated 2-PE from phenylacetaldehyde (PAld). The pairwise alignment of AADC1 reveals a splice variant lacking a 124 bp fragment, thus highlighting the possible role of alternative splicing in modulating floral scent composition. This study offers insights into the molecular-level biosynthesis of terpenoids and 2-PE in Peonia taxa, and provides the basis for the functional characterization, breeding, and bioengineering of prospective candidate genes for the production of floral volatiles in the Paeonia genus.

The biosynthesis of cellulose, lignin, and hemicelluloses in plant secondary cell walls (SCWs) is regulated by a hierarchical transcriptional regulatory network. This network features orthologous transcription factors shared between poplar and Arabidopsis, highlighting a foundational similarity in their genetic regulation. However, knowledge on the discrepant behavior of the transcriptional-level molecular regulatory mechanisms between poplar and Arabidopsis remains limited. In this study, we investigated the function of PagMYB128 during wood formation and found it had broader impacts on SCW formation compared to its Arabidopsis ortholog, AtMYB103. Transgenic poplar trees overexpressing PagMYB128 exhibited significantly enhanced xylem development, with fiber cells and vessels displaying thicker walls, and an increase in the levels of cellulose, lignin, and hemicelluloses in the wood. In contrast, plants with dominant repression of PagMYB128 demonstrated the opposite phenotypes. RNA sequencing and reverse transcription - quantitative polymerase chain reaction showed that PagMYB128 could activate SCW biosynthetic gene expression, and chromatin immunoprecipitation along with yeast one-hybrid, and effector-reporter assays showed this regulation was direct. Further analysis revealed that PagSND1 (SECONDARY WALL-ASSOCIATED NAC-DOMAIN PROTEIN1) directly regulates PagMYB128 but not cell wall metabolic genes, highlighting the pivotal role of PagMYB128 in the SND1-driven regulatory network for wood development, thereby creating a feedforward loop in SCW biosynthesis., (© 2024 Institute of Botany, Chinese Academy of Sciences.)

Human Milk Oligosaccharides (HMOs) are abundant carbohydrates fundamental to infant health and development. Although these oligosaccharides were discovered more than half a century ago, their biosynthesis in the mammary gland remains largely uncharacterized. Here, we use a systems biology framework that integrates glycan and RNA expression data to construct an HMO biosynthetic network and predict glycosyltransferases involved. To accomplish this, we construct models describing the most likely pathways for the synthesis of the oligosaccharides accounting for >95% of the HMO content in human milk. Through our models, we propose candidate genes for elongation, branching, fucosylation, and sialylation of HMOs. Our model aggregation approach recovers 2 of 2 previously known gene-enzyme relations and 2 of 3 empirically confirmed gene-enzyme relations. The top genes we propose for the remaining 5 linkage reactions are consistent with previously published literature. These results provide the molecular basis of HMO biosynthesis necessary to guide progress in HMO research and application with the goal of understanding and improving infant health and development.

Abstract Background Streptomyces are well known for their potential to produce various pharmaceutically active compounds, the commercial development of which is often limited by the low productivity and purity of the desired compounds expressed by natural producers. Well-characterized promoters are crucial for driving the expression of target genes and improving the production of metabolites of interest. Results A strong constitutive promoter, stnYp, was identified in Streptomyces flocculus CGMCC4.1223 and was characterized by its effective activation of silent biosynthetic genes and high efficiency of heterologous gene expression. The promoter stnYp showed the highest activity in model strains of four Streptomyces species compared with the three frequently used constitutive promoters ermEp*, kasOp*, and SP44. The promoter stnYp could efficiently activate the indigoidine biosynthetic gene cluster in S. albus J1074, which is thought to be silent under routine laboratory conditions. Moreover, stnYp was found suitable for heterologous gene expression in different Streptomyces hosts. Compared with the promoters ermEp*, kasOp*, and SP44, stnYp conferred the highest production level of diverse metabolites in various heterologous hosts, including the agricultural-bactericide aureonuclemycin and the antitumor compound YM-216391, with an approximately 1.4 − 11.6-fold enhancement of the yields. Furthermore, the purity of tylosin A was greatly improved by overexpressing rate-limiting genes through stnYp in the industrial strain. Further, the yield of tylosin A was significantly elevated to 10.30 ± 0.12 g/L, approximately 1.7-fold higher than that of the original strain. Conclusions The promoter stnYp is a reliable, well-defined promoter with strong activity and broad suitability. The findings of this study can expand promoter diversity, facilitate genetic manipulation, and promote metabolic engineering in multiple Streptomyces species.

BackgroundDatura stramonium (Jimsonweed) is a medicinally and pharmaceutically important plant in the nightshade family (Solanaceae) known for its production of various toxic, hallucinogenic, and therapeutic tropane alkaloids. Recently, we published a tissue-culture based transformation protocol for D. stramonium that enables more thorough functional genomics studies of this plant. However, the tissue culture process can lead to undesirable phenotypic and genomic consequences independent of the transgene used. Here, we have assembled and annotated a draft genome of D. stramonium with a focus on tropane alkaloid biosynthetic genes. We then use mRNA sequencing and genome resequencing of transformants to characterize changes following tissue culture.ResultsOur draft assembly conforms to the expected 2 gigabasepair haploid genome size of this plant and achieved a BUSCO score of 94.7% complete, single-copy genes. The repetitive content of the genome is 61%, with Gypsy-type retrotransposons accounting for half of this. Our gene annotation estimates the number of protein-coding genes at 52,149 and shows evidence of duplications in two key alkaloid biosynthetic genes, tropinone reductase I and hyoscyamine 6 β-hydroxylase. Following tissue culture, we detected only 186 differentially expressed genes, but were unable to correlate these changes in expression with either polymorphisms from resequencing or positional effects of transposons.ConclusionsWe have assembled, annotated, and characterized the first draft genome for this important model plant species. Using this resource, we show duplications of genes leading to the synthesis of the medicinally important alkaloid, scopolamine. Our results also demonstrate that following tissue culture, mutation rates of transformed plants are quite high (1.16 × 10- 3 mutations per site), but do not have a drastic impact on gene expression.

Soybean, a vital protein-rich crop, offers bioactivity that can mitigate various chronic human diseases. Nonetheless, soybean breeding poses a challenge due to the negative correlation between enhanced protein levels and overall productivity. Our previous studies demonstrated that applying gaseous phytohormone, ethylene, to soybean leaves significantly boosts the accumulation of free amino acids, particularly asparagine (Asn). Current studies also revealed that ethylene application to soybeans significantly enhanced both essential and non-essential amino acid contents in leaves and stems. Asn plays a crucial role in ammonia detoxification and reducing fatigue. However, the molecular evidence supporting this phenomenon remains elusive. This study explores the molecular mechanisms behind enhanced Asn accumulation in ethylene-treated soybean leaves. Transcriptional analysis revealed that ethylene treatments to soybean leaves enhance the transcriptional levels of key genes involved in Asn biosynthesis, such as aspartate aminotransferase (AspAT) and Asn synthetase (ASN), which aligns with our previous observations of elevated Asn levels. These findings shed light on the role of ethylene in upregulating Asn biosynthetic genes, subsequently enhancing Asn concentrations. This molecular insight into amino acid metabolism regulation provides valuable knowledge for the metabolic farming of crops, especially in elevating nutraceutical ingredients with non-genetic modification (GM) approach for improved protein content.