Chalcone Synthase Research Articles

GmSTOP1-3 regulates flavonoid synthesis to reduce ROS accumulation and enhance aluminum tolerance in soybean

Aluminum (Al) toxicity is a significant limiting factor for crop production in acid soils. The functions and regulatory mechanisms of transcription factor STOP1 (Sensitive to Proton Rhizotoxicity 1) family genes in Al-tolerance have been widely studied in many plant species, except for soybean. Here, expression of GmSTOP1-3 was significantly enhanced by Al stress in soybean roots. Overexpression of GmSTOP1-3 resulted in enhanced root elongation and decreased Al content, which was accompanied by increased antioxidant capacity under Al treatment. Furthermore, RNA-seq identified 498 downstream genes of GmSTOP1-3, including genes involved in flavonoid biosynthesis. Among them, the expression of chalcone synthase (GmCHS) and isoflavone synthase (GmIFS) were highly enhanced by GmSTOP1-3 overexpression. Further quantitative flavonoid metabolome analysis showed that overexpression of GmSTOP1-3 significantly increased the content of naringenin chalcone, naringenin, and genistein in soybean roots under Al treatment, which positively correlated with the expression level of the genes relative to flavonoid biosynthesis. Notably, genistein had a significant positive correlation with the expression levels of GmIFS. Combination of Dual Luciferase Complementation (LUC) and Electrophoretic Mobility Shift Assays (EMSA) revealed that GmSTOP1-3 directly bound to the promoters of GmCHS/GmIFS and activated both genes’ transcription. Taken together, these results suggest that GmSTOP1-3 enhances soybean Al tolerance partially through regulating the flavonoid synthesis.

Overexpression Analysis of PtrLBD41 Suggests Its Involvement in Salt Tolerance and Flavonoid Pathway in Populus trichocarpa.

Soil salinization is a significant environmental stress factor, threatening global agricultural yield and ecological security. Plants must effectively cope with the adverse effects of salt stress on survival and successful reproduction. Lateral Organ Boundaries (LOB) Domain (LBD) genes, a gene family encoding plant-specific transcription factors (TFs), play important roles in plant growth and development. Here, we identified and functionally characterized the LBD family TF PtrLBD41 from Populus trichocarpa, which can be induced by various abiotic stresses, including salt, dehydration, low temperature, and Abscisic Acid (ABA). Meanwhile, transgenic plants overexpressing PtrLBD41 showed a better phenotype and higher tolerance than the wild-type (WT) plants under salt stress treatment. Transcriptome analysis found that the differentially expressed genes (DEGs) between the WT and overexpression (OE) line were enriched in the flavonoid biosynthetic process, in which chalcone synthases (CHS), naringenin 3-dioxygenase (F3H), and chalcone isomerase (CHI) were significantly up-regulated under salt stress conditions through qRT-PCR analysis. Therefore, we demonstrate that PtrLBD41 plays an important role in the tolerance to salt stress in P. trichocarpa.

Ultraviolet-B Radiation Stimulates Flavonoid Biosynthesis and Antioxidant Systems in Buckwheat Sprouts.

Abiotic stress not only elevates the synthesis of secondary metabolites in plant sprouts but also boosts their antioxidant capacity. In this study, the mechanisms of flavonoid biosynthesis and antioxidant systems in buckwheat sprouts exposed to ultraviolet-B (UV-B) radiation were investigated. The findings revealed that UV-B treatment significantly increased flavonoid content in buckwheat sprouts, with 3-day-old sprouts exhibiting a flavonoid content 1.73 times greater than that of the control treatment. UV-B radiation significantly increased the activities of key enzymes involved in flavonoid biosynthesis (phenylalanine ammonia-lyase, 4-coumarate-CoA ligase, cinnamate 4-hydroxylase, and chalcone synthase) and the relative expression levels of the corresponding genes. Although UV-B radiation caused damage to the cell membranes of buckwheat sprouts, promoting increases in hydrogen peroxide and malondialdehyde content and inhibiting the growth of sprouts, importantly, UV-B radiation also significantly increased the activities of catalase, peroxidase, and superoxide dismutase as well as the relative expression levels of the corresponding genes, thus enhancing the antioxidant system of buckwheat sprouts. This enhancement was corroborated by a notable increase in ABTS, DPPH, and FRAP radical scavenging activities in 3-day-old sprouts subjected to UV-B radiation. Additionally, UV-B radiation significantly increased chlorophyll a and chlorophyll b contents in sprouts. These results suggest that UV-B radiation is advantageous for cultivating buckwheat sprouts with increased flavonoid content and enhanced antioxidant capacity.

Manipulating flavonoid biosynthesis in Trigonella persica through controlled spectral lighting

Trigonella persica Boiss., is renowned for its rich phytochemical profile, particularly the presence of the flavonol quercetin. This study explored the effects of various light treatments, including blue, red, blue-red (1:1) radiation (BRR), and pink fluorescent light (PFL), on the biochemical and molecular mechanisms governing quercetin and flavonoid biosynthesis in T. persica at different growth stages. Our results showed that light treatments significantly influenced the activity of key enzymes phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) during germination and vegetative growth, with blue light inducing higher PAL and TAL activities compared to control conditions. High-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) analyses revealed that 48-h-old sprouts grown under red light exhibited the highest levels of flavonoid components and phenolic acids, with catechin as the predominant flavonoid. Notably, BRR treatment led to elevated concentrations of the bioavailable quercetin-3-rhamnoside in 48-h-old sprouts, while 15-day-old plants grown under PFL conditions showed a significant accumulation of the sulfated quercetin-3-sulfate. Real-time PCR analysis demonstrated that BRR upregulated the expression of flavonoid biosynthesis genes PAL, chalcone synthase (CHS), and chalcone isomerase (CHI) in sprouts, whereas PFL treatment induced higher expression of these genes, as well as cinnamate 4-hydroxylase (C4H), in aerial parts. These findings suggest that targeted light treatments, particularly blue and red LED light, can enhance the accumulation of bioavailable quercetin-3-rhamnoside during T. persica germination and sprouts exhibit higher levels of flavonoids and phenolic acids than aerial parts during different vegetative growth stages.

Chromosome-Scale Genome of the Fern Cibotium barometz Unveils a Genetic Resource of Medicinal Value

Ferns represent the second-largest group of vascular plants, yet their genomic resources lag far behind. Here, we present a chromosome-scale genome assembly of Cibotium barometz (L.) J. Sm., a medicinally important fern species. The 3.49 Gb genome, assembled into 66 chromosomes with 99.41% sequence anchorage, revealed an exceptionally high proportion (83.93%) of repetitive elements, dominated by recently expanded LTR retrotransposons. We identified 30,616 protein-coding genes, providing insights into fern-specific gene families. Genomic analyses uncover the evolutionary dynamics of 513 key biosynthetic genes, particularly those involved in terpenoid and flavonoid production. Expression profiling across tissues revealed tissue-specific regulation of these pathways, with notable upregulation of chalcone synthase genes in roots. Our structural analysis of 1-deoxy-d-xylulose-5-phosphate synthase, a key enzyme in terpenoid biosynthesis, demonstrated high conservation across land plants while highlighting fern-specific adaptations. The identification of multiple isoforms for key enzymes points to potential gene-duplication events or the evolution of fern-specific variants. This genome provides a foundation for understanding fern biology, evolution, and the molecular basis of their medicinal properties. It also offers valuable resources for conservation efforts and pharmacological research, paving the way for sustainable utilization of this valuable medicinal plant and advancing our understanding of plant diversity and natural product biosynthesis.

Combined application of earthworms and plant growth promoting rhizobacteria improve metal uptake, photosynthetic efficiency and modulate secondary metabolites levels under chromium metal toxicity in Brassica juncea L

Chromium (Cr) toxicity impairs essential morphological and metabolic activities in plants. The present investigation was carried out to evaluate the beneficial role of plant growth promoting rhizobacterial strains namely Pseudomonas aeruginosa (M1), Burkholderia gladioli (M2) and earthworms (Eisenia fetida) in alleviating Cr toxicity in 10 days old Brassica juncea L. The findings delineated that addition of earthworms and PGPR restored growth, boosted Cr uptake and showed upregulation of metal transporter genes (SULTR 1-4). Supplementation of rhizospheric amendments reinstated Cr induced impairment in photosynthetic attributes. Gaseous exchange attributes, the efficiency of PS II, the content of total phenols, anthocyanin and flavonoids was enhanced with application of earthworms along with PGPR. Confocal imaging of primary photosynthetic pigment (chlorophyll), accessory photosynthetic pigment (carotenoids) and total phenols showed maximum fluorescence with combined inoculation of earthworms and both microbial strains (M1M2). The gene expression analysis revealed that Phyotene synthase (PSY), Photosystem II core protein psb A, psb B were down regulated in Cr stressed seedlings which upon supplementation with earthworms and PGPR were upregulated. Further, Phenylalanine ammonialyase (PAL), chalcone synthase (CHS) were upregulated with addition of earthworms and PGPR. Increased nitric oxide content, enhanced activity and upregulation of nitrate reductase (NR) gene was observed with addition of PGPR and earthworms

ChBBX6 and ChBBX18 are positive regulators of anthocyanins biosynthesis and carotenoids degradation in Cerasus humilis

B-box zinc-finger transcription factor (BBX) plays important regulatory roles in plant secondary metabolism. Here, we identified 21 BBXs that could be further categorized into five subfamilies from Cerasus humilis. Two segmentally duplicated Subfamily IV members, ChBBX6 and ChBBX18, were found to share high homology with reported anthocyanin-related BBXs and express highly in fruits with high anthocyanins but low carotenoids contents. Their transient overexpression in apple and C. humilis fruits both led to significantly increased anthocyanins accumulation and significantly upregulated expression of anthocyanins-related genes. However, their overexpression resulted in decreased carotenoids accumulation and greatly upregulated the expression of carotenoids-related genes especially degradation-related genes. Additionally, their overexpression both greatly improved the ABA content in C. humilis fruits. Through yeast one-hybrid and dual-luciferase reporter assays, we found that both ChBBX6 and ChBBX18 could bind to and activate the promoters of chalcone synthase (ChCHS), flavanone 3-hydroxylase (ChF3H), and 9-cis-epoxycarotenoid dioxygenase 5 (ChNCED5). Our study demonstrates that ChBBX6 and ChBBX18 are positive regulators of anthocyanins biosynthesis and carotenoids degradation and can provide basis for understanding the roles of BBX genes in C. humilis.

Proteomic analysis identified proteins that are differentially expressed in the flavonoid and carotenoid biosynthetic pathways of Camellia Nitidissima flowers

BackgroundCamellia nitidissima Chi is a popular ornamental plant because of its golden flowers, which contain flavonoids and carotenoids. To understand the regulatory mechanism of golden color formation, the metabolites of C. nitidissima petals at five different developmental stages were detected, a proteome map of petals was first constructed via tandem mass tag (TMT) analysis, and the accuracy of the sequencing data was validated via parallel reaction monitoring (PRM).ResultsNineteen color components were detected, and most of these components were carotenoids that gradually accumulated, while some metabolites were flavonoids that were gradually depleted. A total of 97,647 spectra were obtained, and 6,789 quantifiable proteins were identified. Then, 1,319 differentially expressed proteins (DEPs) were found, 55 of which belong to the flavonoid and carotenoid pathways, as revealed by pairwise comparisons of protein expression levels across the five developmental stages. Notably, most DEPs involved in the synthesis of flavonoids, such as phenylalanine ammonium lyase and 4-coumarate-CoA ligase, were downregulated during petal development, whereas DEPs involved in carotenoid synthesis, such as phytoene synthase, 1-deoxy-D-xylulose-5-phosphate synthase, and β-cyclase, tended to be upregulated. Furthermore, protein‒protein interaction (PPI) network analysis revealed that these 55 DEPs formed two distinct PPI networks closely tied to the flavonoid and carotenoid synthesis pathways. Phytoene synthase and chalcone synthase exhibited extensive interactions with numerous other proteins and displayed high connectivity within the PPI networks, suggesting their pivotal biological functions in flavonoid and carotenoid biosynthesis.ConclusionProteomic data on the flavonoid and carotenoid biosynthesis pathways were obtained, and the regulatory roles of the DEPs were analyzed, which provided a theoretical basis for further understanding the golden color formation mechanism of C. nitidissima.

Trivalent chromium stress trigger accumulation of secondary metabolites in rice plants: Integration of biochemical and transcriptomic analysis

Trivalent chromium [Cr (III)] is a frequently detected environmental contaminant that negatively affects plant metabolism, growth, and yield. Plant secondary metabolites are non-nutrient compounds involved in diverse physiological functions in response to abiotic stresses. This study performed biochemical and transcriptomic analyses to clarify the protective role and mechanism of secondary metabolites in rice seedlings under Cr(III) stress. The content of measured secondary metabolites i.e., total soluble phenolics, flavonoids, lignin, and anthocyanin in rice tissues was significantly enhanced under Cr(III) exposure. Additionally, the activities of several enzymes including chalcone synthase, phenylalanine ammonialyase, peroxidase, and anthocyanidin synthase, were positively activated. Transcriptomic analysis revealed a number of differentially expressed genes (DEGs) in Cr(III)-treated rice seedlings and their expression patterns are tissue-specific. Additionally, the DEGs involved in secondary metabolism pathways were evident after KEGG enrichment analysis. Cr(III) exposure resulted in distinct genetic regulation strategies in rice tissues, with different DEGs activating various sub-pathways in the secondary metabolism pathways to cope with Cr(III) stress. Furthermore, the integrated correlation analysis of the secondary metabolites and transcriptome data identified key genes involved in different steps of the sub-pathways of secondary metabolism pathway in rice plants under Cr(III) stress. This study indicates that the accumulation of secondary metabolites in rice plants is a survival and detoxification strategy to reduce the adverse effects of toxic compounds. Future research should explore how key genes in secondary metabolism pathways aid in Cr(III) detoxification and enhance crop stress tolerance.

Combined Transcriptome and Metabolome Analyses Provide New Insights into the Changes in the Flesh Color of Anthocyanins in Strawberry (Fragaria × ananassa (Weston) Duchesne ex Rozier).

Strawberries are bright in color, sweet and sour in taste, and rich in nutrients and flavonoid compounds such as anthocyanins and proanthocyanidins. The synthesis and accumulation of anthocyanins are the decisive factors that make strawberries appear bright red. From the perspective of plant breeding, a change in flesh color is an important goal. In this study, two strawberry plants with different flesh colors were selected, and transcriptome and metabolome analyses were performed during the color change period (S1) and ripening period (S2). RNA-seq revealed a total of 13,341 differentially expressed genes (DEGs) between and within materials, which were clustered into 5 clusters. A total of 695 metabolites were detected via metabolome analysis, and 243 differentially regulated metabolites (DRMs) were identified. The anthocyanin biosynthesis, starch and sucrose metabolism and glycolysis/gluconeogenesis pathways were determined to be important regulatory pathways for changes in strawberry flesh color through a joint analysis of RNA-seq data and the metabolome. The leucoanthocyanidin reductase (LAR) and chalcone synthase (CHS) gene is a key gene related to anthocyanins, cinnamic acid, and phenylalanine. In addition, through joint RNA-seq and metabolome analyses combined with weighted gene co-expression network analysis (WGCNA), we identified 9 candidate genes related to strawberry flesh color. Our research findings have laid the groundwork for a more comprehensive understanding of the molecular mechanisms governing the color transformation in strawberry flesh. Additionally, we have identified novel genetic resources that can be instrumental in advancing research related to strawberry color change.

R2R3-MYB repressor, BrMYB32, regulates anthocyanin biosynthesis in Chinese cabbage.

Anthocyanin-enriched Chinese cabbage has health-enhancing antioxidant properties. Although various regulators of anthocyanin biosynthesis have been identified, the role of individual repressors in this process remains underexplored. This study identifies and characterizes the R2R3-MYB BrMYB32 in Chinese cabbage (Brassica rapa), which acts as a repressor in anthocyanin biosynthesis. BrMYB32 expression is significantly upregulated under anthocyanin inductive conditions, such as sucrose and high light treatment. Transgenic tobacco plants overexpressing BrMYB32 show decreased anthocyanin levels and downregulation of anthocyanin biosynthesis genes in flowers, highlighting BrMYB32's repressive role. Located in the nucleus, BrMYB32 interacts with the TRANSPARENT TESTA 8 (BrTT8), a basic helix-loop-helix protein, but no interaction was detected with the R2R3-MYB protein PRODUCTION OF ANTHOCYANIN PIGMENT 1 (BrPAP1). Functional assays in Chinese cabbage cotyledons and tobacco leaves demonstrate that BrMYB32 represses the transcript level of anthocyanin biosynthesis genes, thereby inhibiting pigment accumulation. Promoter activation assays further reveal that BrMYB32 inhibits the transactivation of CHALCONE SYNTHASE and DIHYDROFLAVONOL REDUCTASE through the C1 and C2 motifs. Notably, BrMYB32 expression is induced by BrPAP1, either alone or in co-expression with BrTT8, and subsequently regulates the expression of these activators. It verifies that BrMYB32 not only interferes with the formation of an active MYB-bHLH-WD40 complex but also downregulates the transcript levels of anthocyanin biosynthesis genes, thereby fine-tuning anthocyanin biosynthesis. Our findings suggest a model in which anthocyanin biosynthesis in Chinese cabbage is precisely regulated by the interplay between activators and repressors.

Integrated Analysis of Metabolome and Transcriptome Reveals the Effect of Burdock Fructooligosaccharide on the Quality of Chinese Cabbage (Brassica rapa L. ssp. Pekinensis).

Burdock fructooligosaccharide (BFO) is fructose with a low polymerization degree, which could improve the immunity to pathogens, quality, and stress resistance of vegetables. Still, there are no studies on applying BFO in Chinese cabbage. In this study, the effects of exogenous BFO sprayed with different concentrations (0, 5, 10, 20, 30 g·L-1) on the growth and soluble sugar content of Chinese cabbage seedlings were determined. The result showed that 10 g·L-1 was the appropriate spraying concentration. Based on metabolome analysis, a total of 220 differentially accumulated metabolites (DAMs) were found, among which flavonoid metabolites, glucosinolate metabolites, and soluble sugar-related metabolites were the key metabolites involved in improving the quality of Chinese cabbage caused by BFO. Further combination analysis with transcriptome, trans-cinnamate 4-monooxygenase (CYP73A5), and chalcone synthase 1 (CHS1) were more closely associated with the DAMs of flavonoid biosynthesis. Sulfotransferases 18 (SOT18), Branched-chain amino acid amino transferases 6 (BCAT6), and cytochrome P450 monooxygenase (CYP83A1) were the key genes in glucosinolate biosynthesis. Hexokinase (HxK1), beta-glucosidase 8 (BGL08), invertase 3 (INV3), beta-glucosidase 3B (BGL3B), and sucrose phosphate synthase 1 (SPS1) were significantly upregulated, potentially playing crucial roles in the soluble sugar metabolism. In conclusion, these results provided an understanding of the effects of BFO on the expression of genes and the accumulation of metabolites related to quality formation in Chinese cabbage.

Elucidating the metabolic roles of isoflavone synthase-mediated protein-protein interactions in yeast.

Transient plant enzyme complexes formed via protein-protein interactions (PPIs) play crucial regulatory roles in secondary metabolism. Complexes assembled on cytochrome P450s (CYPs) are challenging to characterize metabolically due to difficulties in decoupling the PPIs' metabolic impacts from the CYPs' catalytic activities. Here, we developed a yeast-based synthetic biology approach to elucidate the metabolic roles of PPIs between a soybean-derived CYP, isoflavone synthase (GmIFS2), and other enzymes in isoflavonoid metabolism. By reconstructing multiple complex variants with an inactive GmIFS2 in yeast, we found that GmIFS2-mediated PPIs can regulate metabolic flux between two competing pathways producing deoxyisoflavonoids and isoflavonoids. Specifically, GmIFS2 can recruit chalcone synthase (GmCHS7) and chalcone reductase (GmCHR5) to enhance deoxyisoflavonoid production or GmCHS7 and chalcone isomerase (GmCHI1B1) to enhance isoflavonoid production. Additionally, we identified and characterized two novel isoflavone O-methyltransferases interacting with GmIFS2. This study highlights the potential of yeast synthetic biology for characterizing CYP-mediated complexes.

Unveiling the Molecular Mechanisms of Browning in Camellia hainanica Callus through Transcriptomic and Metabolomic Analysis.

Camellia hainanica is one of the camellia plants distributed in tropical regions, and its regeneration system and genetic transformation are affected by callus browning. However, the underlying mechanism of Camellia hainanica callus browning formation remains largely unknown. To investigate the metabolic basis and molecular mechanism of the callus browning of Camellia hainanica, histological staining, high-throughput metabolomics, and transcriptomic assays were performed on calli with different browning degrees (T1, T2, and T3). The results of histological staining revealed that the brown callus cells had obvious lignification and accumulation of polyphenols. Widely targeted metabolomics revealed 1190 differentially accumulated metabolites (DAMs), with 53 DAMs annotated as phenylpropanoids and flavonoids. Comparative transcriptomics revealed differentially expressed genes (DEGs) of the T2 vs. T1 associated with the biosynthesis and regulation of flavonoids and transcription factors in Camellia hainanica. Among them, forty-four enzyme genes associated with flavonoid biosynthesis were identified, including phenylalaninase (PAL), 4-coumaroyl CoA ligase (4CL), naringenin via flavanone 3-hydroxylase (F3H), flavonol synthase (FLS), Chalcone synthase (CHS), Chalcone isomerase (CHI), hydroxycinnamoyl-CoA shikimate transferase (HCT), Dihydroflavonol reductase (DFR), anthocyanin reductase (LAR), anthocyanin synthetase (ANS), and anthocyanin reductase (ANR). Related transcription factors R2R3-MYB, basic helix-loop-helix (bHLH), and WRKY genes also presented different expression patterns in T2 vs. T1. These results indicate that the browning of calli in Camellia hainanica is regulated at both the transcriptional and metabolic levels. The oxidation of flavonoids and the regulation of related structural genes and transcription factors are crucial decisive factors. This study preliminarily revealed the molecular mechanism of the browning of the callus of Camellia hainanensis, and the results can provide a reference for the anti-browning culture of Camellia hainanica callus.

CRISPR/Cas9 mediated CHS2 mutation provides a new insight into resveratrol biosynthesis by causing a metabolic pathway shift from flavonoids to stilbenoids in Vitis davidii cells

Abstract Resveratrol is an important phytoalexin that adapts to and responds to stressful conditions, plays various roles in health and medical therapies. However, it is only found in a limited number of plant species in low concentrations, which hinders its development and utilization. Chalcone synthase (CHS) and stilbene synthase (STS) catalyze the same substrates to produce flavonoids and resveratrol, respectively. However, it remains unclear how CHS and STS compete in metabolite synthesis. In this study, two CHS2 mutant cell lines (MT1 and MT2) were generated using CRISPR/Cas9 genome editing. These CHS2 mutant cell lines exhibited abundant mutations in CHS2, leading to the premature termination of protein translation and subsequent CHS2 knockout. Amplicon sequencing confirmed comprehensive CHS2 knockout in MT1, whereas the wild-type sequence remained predominant in the MT2 cell line. Transcriptome and RT-qPCR results showed a significant downregulation of genes involved in flavonoid biosynthesis, including CHS2, CHS3, F3H, F3’H, DFR, FLS, LDOX, among others, resulting in decreased flavonoid accumulation, such as anthocyanins, proanthocyanidins, quercetin, and kaempferol. Conversely, STS genes involved in stilbenoid biosynthesis were upregulated competing with the flavonoid pathway. Consequently, there was a marked increase in stilbenoids, including resveratrol, piceatannol, piceid and pterostilbene, with a 4.1-fold increase in resveratrol and a 5.3-fold increase in piceid (a derivative of resveratrol) observed in CHS2 mutant cell lines. This research demonstrates that CHS2 mutation induces a shift from flavonoid biosynthesis towards stilbenoid biosynthesis, offering new insights into metabolite biosynthesis and regulation, as well as an alternative solution for natural resveratrol production, and a novel breeding approach for eliminating non-target agronomic traits using CRISPR-Cas9.

In silico profiling, docking analysis, and protein interactions of secondary metabolites in Musa spp. Against the SGE1 protein of Fusarium oxysporum f. sp. cubense

Banana Fusarium Wilt (BFW), caused by Fusarium oxysporum f. sp. cubense (Foc), threatens banana crops globally, with the pathogen's virulence partially regulated by the Sge1 transcription factor, which enhances disease severity. Certain Musa species display resistance to Foc, suggesting inherent genetic traits that confer immunity against Sge1Foc. This study utilized bioinformatics tools to investigate the mechanisms underlying this resistance in Musa accuminata subsp. aalaccensis. Through in silico analyses, we explored interactions between Musa spp. and Foc, focusing on the Sge1 protein. Tools such as Anti-SMASH, AutoDockVina 4.0, STRING, and Phoenix facilitated the profiling of secondary metabolites in Musa spp. and the identification of biosynthetic gene clusters involved in defense. Our results indicate that secondary metabolites, including saccharides, terpenes, and polyketides, are crucial to the plant's immune response. Molecular docking studies of selected Musa metabolites, such as 3-Phenylphenol, Catechin, and Epicatechin, revealed 3-Phenylphenol as having the highest binding affinity to the Sge1Foc protein (-6.7 kcal/mol).Further analysis of gene clusters associated with secondary metabolite biosynthesis in Musa spp. identified key domains like Chalcone synthase, Phenylalanine ammonia-lyase, Aminotran 1–2, and CoA-ligase, which are integral to phenylpropanoid production—a critical pathway for secondary metabolites. The study highlights that the phenylpropanoid pathway and secondary metabolite biosynthesis are vital for Musa spp. resistance to Foc. Flavonoids and lignin may inhibit Sge1 protein formation, potentially disrupting Foc's cellular processes. These findings emphasize the role of phenylpropanoid pathways and secondary metabolites in combating BFW and suggest that targeting these pathways could offer innovative strategies for enhancing resistance and controlling BFW in banana crops. This research lays the groundwork for developing sustainable methods to protect banana cultivation and ensure food security.

Comparative transcriptomic and metabolomic analysis reveals mechanisms of selenium-regulated anthocyanin synthesis in waxy maize (Zea mays L.).

Anthocyanins in maize (Zea mays L.) kernels determine the plant's color and can enhance its resistance. Selenium (Se) significantly impacts plant growth, development, and secondary metabolic regulation. However, the molecular mechanisms by which Se regulates anthocyanin synthesis in waxy corn remain unclear. This study employed integrated transcriptomic and metabolomic analyses to investigate the mechanisms through which selenium influences anthocyanin synthesis in yellow and purple waxy corn. The results showed that maize varieties with higher anthocyanin content had higher selenium enrichment capacity in their kernels. Under selenium stress, HN2025 exhibited 1,904 more differentially expressed genes (DEGs) and 140 more differential metabolites compared to HN5. The expression levels of anthocyanin synthesis-related genes and transcription factors such as phenylalanine ammonia-lyase, flavonoid 3-hydroxylase (F3H), dihydroflavonol reductase (DFR), chalcone synthase (CHS), cinnamate-4-hydroxylase (C4H), anthocyanin 5,3-O-glucosyltransferases, and anthocyanidin reductase, MYB, and bHLH were strongly induced in HN2025. Metabolomic analysis revealed significant enrichment in anthocyanin biosynthesis, flavonoid and flavonol biosynthesis, glutathione metabolism, phenylalanine biosynthesis, and phenylalanine metabolism under selenium treatment. Three up-regulated PAL genes and one C4H gene were significantly enriched with DAMs in phenylalanine metabolism, phenylpropanoid biosynthesis, flavonoid biosynthesis, and anthocyanin biosynthesis, resulting in significant differences between HN5 and HN2025 in selenium-induced anthocyanin metabolism-related pathways. These findings provide a theoretical basis for understanding the effects of selenium on the molecular regulatory mechanisms of anthocyanin biosynthesis in maize kernels.

Duplication and sub-functionalization of flavonoid biosynthesis genes plays important role in Leguminosae root nodule symbiosis evolution.

Gene innovation plays an essential role in trait evolution. Rhizobial symbioses, the most important N2-fixing agent in agricultural systems that exists mainly in Leguminosae, is one of the most attractive evolution events. However, the gene innovations underlying Leguminosae root nodule symbiosis (RNS) remain largely unknown. Here, we investigated the gene gain event in Leguminosae RNS evolution through comprehensive phylogenomic analyses. We revealed that Leguminosae-gain genes were acquired by gene duplication and underwent a strong purifying selection. Kyoto Encyclopedia of Genes and Genomes analyses showed that the innovated genes were enriched in flavonoid biosynthesis pathways, particular downstream of chalcone synthase (CHS). Among them, Leguminosae-gain type Ⅱ chalcone isomerase (CHI) could be further divided into CHI1A and CHI1B clades, which resulted from the products of tandem duplication. Furthermore, the duplicated CHI genes exhibited exon-intron structural divergences evolved through exon/intron gain/loss and insertion/deletion. Knocking down CHI1B significantly reduced nodulation in Glycine max (soybean) and Medicago truncatula; whereas, knocking down its duplication gene CHI1A had no effect on nodulation. Therefore, Leguminosae-gain type Ⅱ CHI participated in RNS and the duplicated CHI1A and CHI1B genes exhibited RNS functional divergence. This study provides functional insights into Leguminosae-gain genetic innovation and sub-functionalization after gene duplication that contribute to the evolution and adaptation of RNS in Leguminosae.

An in-depth study of anthocyanin synthesis in the exocarp of virescens and nigrescens oil palm: metabolomic and transcriptomic analysis.

Oil palm (Elaeis guineensis Jacq.) is a very important tropical woody oil plant with high commercial and ornamental value. The exocarp of the oil palm fruit is rich in anthocyanosides and proanthocyanidins, which not only give it a bright colour, but also mark the maturity of the fruit. The study of the dynamic change pattern of anthocyanoside content and important anthocyanoside metabolism-related regulatory genes during oil palm ripening is conducive to the improvement of the ornamental value of oil palm and the determination of the optimal harvesting period of the fruits. We analyzed the virescens oil palm (AS) and nigrescens oil palm (AT) at 95 days (AS1, AT1), 125 days (AS2, AT2) and 185 days (AS3, AT3) after pollination were used as experimental materials for determining the changes in the total amount of anthocyanins as well as their metabolomics and transcriptomics studies by using the LC-MS/MS technique and RNA-Seq technique. The results showed that the total anthocyanin content decreased significantly from AS1 (119µg/g) to AS3 (23µg/g), and from AT1 (1302µg/g) to AT3 (170µg/g), indicating a clear decreasing trend during fruit development. Among them, the higher flavonoids in AS and AT included anthocyanins such as peonidin-3-O-rutinoside (H35), pelargonidin-3-O-rutinoside (H21), and cyanidin-3-O-glucoside (H7), as well as condensed tannins such as procyanidin B2 (H47), procyanidin C1 (H49), and procyanidin B3 (H48). Notably, nine genes involved in the anthocyanin biosynthetic pathway exhibited up-regulated expression during the pre-development stage of oil palm fruits, particularly during the AS1 and AT1 periods. These genes include: Chalcone synthase (CHS; LOC105036364); Flavanone 3-hydroxylase (F3H; LOC105054663); Dihydroflavonol 4-reductase (DFR; LOC105040724, LOC105048473); Anthocyanidin synthase (ANS; LOC105035842), UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT; LOC105039612); Flavonoid 3',5'-hydroxylase (F3'5'H; LOC105036086, LOC105044124, LOC105045493). In contrast, five genes demonstrated up-regulated expression as the fruits developed, specifically during the AS3 and AT3 periods. These genes include: Chalcone synthase (CHS; LOC105036921, LOC105035716); Chalcone isomerase (CHI; LOC105045978); UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT; LOC105046326); Flavonoid 3'-hydroxylase (F3'H; LOC105036086). Most of differentially expressed genes exhibited up-regulation during the early stages of fruit development, which may contribute to the elevated anthocyanin content observed in oil palm fruits of both types during the pre-developmental period. Furthermore, the expression levels of most genes were found to be higher in the AT fruit type compared to the AS fruit type, suggesting that the differential expression of these genes may be a key factor underlying the differences in anthocyanoside production in the exocarp of oil palm fruits from these two fruit types. The findings of this study provide a theoretical foundation for the identification and characterization of genes involved in anthocyanin synthesis in oil palm fruits, as well as the development of novel variations using molecular biology approaches.

Naringenin chalcone carbon double-bond reductases mediate dihydrochalcone biosynthesis in apple leaves.

Dihydrochalcones (DHCs) are flavonoids produced as a side branch of the phenylpropanoid pathway. DHCs are found at high concentrations in apples (Malus spp.) but not in pears (Pyrus spp.) or other members of the Rosaceae. Biosynthesis of DHCs in apple has been hypothesized to occur via reduction of p-coumaroyl CoA by a Malus × domestica hydroxycinnamoyl CoA double-bond reductase (MdHCDBR) followed by the action chalcone synthase to produce phloretin or via direct reduction of naringenin chalcone to phloretin via an unknown enzyme. In this study, we report that genetic downregulation of MdHCDBR does not reduce DHC concentrations in apple leaves. We used comparative transcriptome analysis to identify candidate naringenin chalcone reductases (NCRs), designated MdNCR1a-c, expressed in apple leaves but not fruit. These MdNCR1 genes form an expanded gene cluster found exclusively in apple. Transient expression of MdNCR1 genes in Nicotiana benthamiana leaves indicated they produced DHCs at high concentrations in planta. Recombinant MdNCR1 utilized naringenin chalcone to produce phloretin at high efficiency. Downregulation of NCR genes in transgenic apple reduced foliar DHC levels by 85-95%. Reducing DHC production redirected flux to the production of flavonol glycosides. In situ localization indicated that NCR proteins were likely found in the vacuolar membrane. Active site analysis of AlphaFold models indicated that MdNCR1a-c share identical substrate binding pockets, but the pockets differ substantially in related weakly active/inactive NCR proteins. Identifying the missing enzyme required for DHC production provides opportunities to manipulate DHC content in apple and other fruits and has other applications, e.g., in biofermentation and biopharming.