Isoflavonoid Biosynthesis Research Articles

Comprehensive Analysis of Secondary Metabolites of Four Medicinal Thyme Species Used in Folk Medicine and Their Antioxidant Activities In Vitro

Thyme is a colloquial term for number of aromatic species belonging to the genus Thymus L., known for their expressed biological activities and therefore used worldwide for seasoning and in folk medicine. In the present paper, the content of the total polyphenols (TP), total flavonoids (TF), and antioxidant capacity were assessed in the extracts of four traditionally used thyme species. Moreover, a comprehensive metabolomic study of thyme bioactive compounds was performed, and the obtained data were processed using multivariate statistical tests. The results clearly demonstrated the positive correlation between the content of the TP, TF, and antioxidant activity, and TF was more significant than TP. The findings revealed that four selected thyme species contained 528 secondary metabolites, including 289 flavonoids and 146 phenolic acids. Thymus marschallianus had a higher concentration of active ingredients, which improve its antioxidant capacity. Differentially accumulated metabolites were formed by complex pathways such as flavonoid, flavone, flavonol, isoflavonoid, and anthocyanin biosynthesis. Correlation analysis showed that 59 metabolites (including 28 flavonoids, 18 phenolic acids, and 7 terpenoid compounds) were significantly correlated with obtained values of the antioxidant capacity. The results suggested that selected thyme species exhibit a great diversity in antioxidant-related components, whereas flavonoids may be responsible for the high antioxidant capacity of all studied thyme species. The present study greatly expands our understanding of the complex phytochemical profiles and related applications of selected medicinal plants.

ASSVd infection inhibits the vegetative growth of apple trees by affecting leaf metabolism.

Apple scar skin viroid (ASSVd) can infect apple trees and cause scar skin symptoms. However, the associated physiological mechanisms are unclear in young saplings. In this study, ASSVd-infected and control 'Odysso' and 'Tonami' apple saplings were examined to clarify the effects of ASSVd on apple tree growth and physiological characteristics as well as the leaf metabolome. The results indicated that leaf ASSVd contents increased significantly after grafting and remained high in the second year. Leaf size, tree height, stem diameter, branch length, and leaf photosynthetic efficiency decreased significantly in viroid-infected saplings. In response to the ASSVd infection, the chlorophyll a and b contents decreased significantly in 'Odysso', but were unchanged in 'Tonami'. Moreover, the N, P, K, Fe, Mn, and Ca contents decreased significantly in the leaves of viroid-infected 'Odysso' or 'Tonami'. Similarly, the CAT and POD contents decreased significantly in the viroid-infected saplings, but the SOD content increased in the viroid-infected 'Tonami' saplings. A total of 15 and 40 differentially abundant metabolites were respectively identified in the metabolome analyses of 'Odysso' and 'Tonami' leaves. Specifically, in the viroid-infected 'Odysso' and 'Tonami' samples, the L-2-aminobutyric acid, 6″-O-malonyldaidzin, and D-xylose contents increased, while the coumarin content decreased. These metabolites are related to the biosynthesis of isoflavonoids and phenylpropanoids as well as the metabolism of carbohydrates and amino acids. These results imply that ASSVd affects apple sapling growth by affecting physiological characteristics and metabolism of apple leaves. The study data may be useful for future investigations on the physiological mechanisms underlying apple tree responses to ASSVd.

New insights into substrate preference and aryl ring migration mechanism for isoflavonoid biosynthesis

Isoflavonoids are an essential source of bioactive compounds. Due to their enormous potential for human health, the demand for isoflavonoids is rising. Isoflavonoid synthase (IFS), a unique P450 monooxygenase, catalyzes the rate-limiting step in the biosynthesis of isoflavonoids. Here, IFS enzymes from five different plants are characterized for isoflavonoid biosynthesis in yeast. Although IFS generally prefers liquiritigenin as a substrate, this is the first time an IFS from Pueraria candollei has been characterised with a preference for naringenin. Co-expression of 2-hydroxyisoflavonoid dehydratase (HID) with IFS significantly improves isoflavonoids biosynthesis. However, a preference of IFS towards particular HID enzymes types was observed in this study. Additionally, the protein conformational basis of aryl ring migration mechanism has been explored for Onobrychis viciifolia IFS, P. candollei IFS, and CYP82D26 and compared with other P450 enzymes that accept the same substrates but do not produce isoflavonoids. Protein structure and docking analysis based on key amino acids, heme orientation, and IFS promiscuity has led us to suggest a new mechanism for aryl ring migration. Characterisation of IFS for efficiency, substrate preference, and understanding of aryl ring migration mechanism will help advance heterologous biosynthesis and protein engineering efforts.

Selection and Validation of Suitable Reference Genes for Gene Expression Studies in Callerya speciosa (Champ. ex Benth.) Schot under Different Experimental Conditions

The accuracy of reverse transcription quantitative real-time PCR (RT-qPCR) strongly depends on the stability of reference gene. It is essential to select a suitable reference gene in order to obtain reliable RT-qPCR results when gene expression profiles were evaluated. Callerya speciosa, a traditional Chinese medicine, has a long cultivation history in south China. However, suitable reference genes in C. speciosa have not yet been investigated for accurate gene expression quantification under different experimental conditions. In this study, eight candidate reference genes, including GAPDH (glyceraldehyde-3-phosphate dehydrogenase), 60S (60S ribosomal protein L34), ACTIN (actin), TUA2 (tubulin alpha chain), TUB1 (tubulin beta-1 chain), TIF5 (eukaryotic translation initiation factor 5A), UBQ (polyubiquitin), and EF2 (elongation factor 2), were selected from the transcriptome databases, and their expression stability under six different experimental conditions (developmental stages, tissues, MeJA treatment, GA3 treatment, CPPU treatment, and PP333 treatment) was evaluated using ΔCT, geNorm, NormFinder, BestKeeper, and RefFinder programs. The results showed that GAPDH was the optimal reference gene for all different experimental conditions, whereas ACTIN showed the best stability under most of the hormone treatments. TUA2 and EF2 were the two least suitable ones as reference genes in C. speciosa. The expression pattern of CsMYB36, a gene associated with the regulation of isoflavonoid biosynthesis in C. speciosa, verified the accuracy of our experimental results. These results provided a theoretical basis for subsequent research on the regulation of functional gene expression in C. speciosa and other Callerya species in the future.

Phylogenetic Analysis and Protein Modelling of Isoflavonoid Synthase Highlights Key Catalytic Sites towards Realising New Bioengineering Endeavours

Isoflavonoid synthase (IFS) is a critical enzyme for the biosynthesis of over 2400 isoflavonoids. Isoflavonoids are an important class of plant secondary metabolites that have a range of pharmaceutical and nutraceutical properties. With growing interest in isoflavonoids from both research and industrial perspectives, efforts are being forwarded to enhance isoflavonoid production in-planta and ex-planta; therefore, in-silico analysis and characterisation of available IFS protein sequences are needed. The present study is the first-ever attempt toward phylogenetic analysis and protein modelling of available IFS protein sequences. Phylogenetic analysis has shown that IFS amino acid sequences have 86.4% pairwise identity and 26.5% identical sites, and the sequences were grouped into six different clades. The presence of a β-hairpin and extra loop at catalytic sites of Trifolium pratense, Beta vulgaris and Medicago truncatula, respectively, compared with Glycyrrhiza echinata are critical structural differences that may affect catalytic function. Protein docking highlighted the preference of selected IFS for liquiritigenin compared with naringenin and has listed T. pratense as the most efficient candidate for heterologous biosynthesis of isoflavonoids. The in-silico characterisation of IFS represented in this study is vital in realising the new bioengineering endeavours and will help in the characterisation and selection of IFS candidate enzymes for heterologous biosynthesis of isoflavonoids.

A Comparison of the Flavonoid Biosynthesis Mechanisms of Dendrobium Species by Analyzing the Transcriptome and Metabolome.

Dendrobium huoshanense, Dendrobium officinale, and Dendrobium moniliforme, as precious Chinese medicinal materials, have a variety of medicinal properties. Flavonoids are important medicinal components of Dendrobium, but their accumulation rules and biosynthesis mechanisms remain unclear. To explore the similarities and differences of flavonoid accumulation and biosynthesis in these three Dendrobium species, we performed flavonoid content determination, widely-targeted metabolomics and transcriptome sequencing on 1–4 years old Dendrobium species. The results showed that in different growth years, D. huoshanense stems had the highest flavonoid content in the second year of growth, while D. officinale and D. moniliforme stems had the highest flavonoid content in the third year of growth. A total of 644 differentially accumulated metabolites (DAMs) and 10,426 differentially expressed genes (DEGs) were identified by metabolomic and transcriptomic analysis. It was found that DAMs and DEGs were not only enriched in the general pathway of “flavonoid biosynthesis”, but also in multiple sub-pathways such as “Flavone biosynthesis”, and “Flavonol biosynthesis” and “Isoflavonoid biosynthesis”. According to a combined transcriptome and metabolome analysis, the expression levels of the F3′H gene (LOC110096779) and two F3′5′H genes (LOC110101765 and LOC110103762) may be the main genes responsible for the differences in flavonoid accumulation. As a result of this study, we have not only determined the optimal harvesting period for three Dendrobium plants, but also identified the key genes involved in flavonoid biosynthesis and provided a basis for further study of the molecular mechanism of flavonoid synthesis.

Integrated analyses of transcriptome and metabolome provides new insights into the primary and secondary metabolism in response to nitrogen deficiency and soil compaction stress in peanut roots.

Peanut (Arachis hypogaea L.) is an important oil crop globally because of its high edible and economic value. However, its yield and quality are often restricted by certain soil factors, especially nitrogen (N) deficiency, and soil compaction. To explore the molecular mechanisms and metabolic basis behind the peanut response to N deficiency and soil compaction stresses, transcriptome and metabolome analyses of peanut root were carried out. The results showed that N deficiency and soil compaction stresses clearly impaired the growth and development of peanut's aboveground and underground parts, as well as its root nodulation. A total of 18645 differentially expressed genes (DEGs) and 875 known differentially accumulated metabolites (DAMs) were identified in peanut root under differing soil compaction and N conditions. The transcriptome analysis revealed that DEGs related to N deficiency were mainly enriched in “amino acid metabolism”, “starch and sucrose metabolism”, and “TCA cycle” pathways, while DEGs related to soil compaction were mainly enriched in “oxidoreductase activity”, “lipids metabolism”, and “isoflavonoid biosynthesis” pathways. The metabolome analysis also showed significant differences in the accumulation of metabolisms in these pathways under different stress conditions. Then the involvement of genes and metabolites in pathways of “amino acid metabolism”, “TCA cycle”, “lipids metabolism”, and “isoflavonoid biosynthesis” under different soil compaction and N deficiency stresses were well discussed. This integrated transcriptome and metabolome analysis study enhances our mechanistic knowledge of how peanut plants respond to N deficiency and soil compaction stresses. Moreover, it provides new leads to further investigate candidate functional genes and metabolic pathways for use in improving the adaptability of peanut to abiotic stress and accelerating its breeding process of new stress-resistant varieties.

Mepiquat chloride inhibits soybean growth but improves drought resistance.

Soybeans are an important economic crop. As the most widely used growth regulator globally, the molecular mechanism of mepiquat chloride (DPC) in soybean remains unknown. In this study, RNA sequencing technology combined with ultra-performance liquid chromatography and tandem mass spectrometry were used to analyze the changes in the leaf transcriptome and metabolomics of soybean leaves at the seedling stage under DPC stress. The results showed that differentially expressed genes related to photosynthesis and cell wall synthesis were significantly downregulated at the transcriptional level. In addition, the syntheses of gibberellin, zeatin, brassinolide, and other plant hormones were inhibited in the signal transduction pathway of plant hormones, thereby inhibiting plant growth. In contrast, at the metabolic level, the expression levels of flavonoid differential metabolites were significantly increased, and the proportions of flavonoids in the two varieties were 61.5 and 66%, respectively. The combined analysis of transcriptome and metabolomics showed that the differential expressed genes and metabolites were mainly enriched in the isoflavonoid biosynthesis and flavonoid biosynthesis pathways. Principally, DPC inhibited plant growth but improved drought resistance. Our study is the first to report the molecular mechanism of DPC regulation in soybean, providing useful insights into the rational application of DPC in soybean.

Comprehensive transcriptomic and metabolomic profiling reveals the differences between alfalfa sprouts germinated with or without light exposure.

Alfalfa sprouts are among the most nutritionally rich foods, and light exposure is a critical factor in determining their biomass and quality. However, detailed metabolic and molecular differences between yellow and green alfalfa sprouts remain unclear. In this study, comprehensive metabolomic and transcriptomic analyses were integrated to evaluate the nutrient composition of alfalfa sprouts during germination with or without light exposure. Differentially expressed genes and differentially accumulated metabolites in green and yellow alfalfa sprouts were significantly enriched in secondary metabolic pathways, such as the isoflavonoid biosynthesis pathway. Green alfalfa sprouts contained a wide variety of lipids, flavonoids, phenolic acids, and terpenoids, among which the top three upregulated were calycosin, methyl gallate, and epicatechin 3-gallate, whereas yellow alfalfa sprouts contained relatively more isoquercitrin. These results provide new insights into the nutritional value and composition of alfalfa sprouts under different germination regimes.

A Review of Synthesis Methods of Chalcones, Flavonoids, and Coumarins

Chalcones are the principal precursors for the biosynthesis of flavonoids and isoflavonoids. A three-carbon α, β-unsaturated carbonyl system constitutes chalcones. Chalcones are the condensation products of an aromatic aldehyde with acetophenones in attendance of catalyst. A lot of methods and schemes have been reported for the synthesis of these compounds. Amongst all, Aldol condensation and Claisen-Schmidt condensation reactions are the most cited synthetic protocols in literature, nevertheless, Suzuki reaction, Witting reaction, and Photo-Fries rearrangement have also been utilized as synthetic protocols towards the chalcone framework. Several catalysts have been developed in the synthesis of the chalcone framework among which SOCl 2 natural phosphate, lithium nitrate, amino grafted zeolites, zinc oxide, water, K 2 CO 3 , PEG400, silica sulfuric acid, ZrCl 4, and ionic liquid are the most cited ones. The development of better synthetic techniques for the synthesis of α, β- unsaturated carbonyl compounds is still remaining high demand. Keywords: Chalcones, Aldol condensation, Claisen-Schmidt condensation, Suzuki reaction, Witting reaction, Photo-Fries rearrangement DOI: 10.7176/CMR/14-3-01 Publication date: August 31 st 2022

Transcriptome and Metabonomics Combined Analysis Revealed the Defense Mechanism Involved in Hydrogen-Rich Water-Regulated Cold Stress Response of Tetrastigma hemsleyanum.

The poor resistance to cold stress conditions has become the bottleneck problem in Tetrastigma hemsleyanum (T. hemsleyanum) planting industry. Exogenous hydrogen (H2) plays an important role in improving stress resistance in plants. However, the key factors and regulatory network of plants in response to hydrogen-rich water (HRW) treatment under environmental stress are not clear. Here, we conducted integrative analyses of metabolome and transcriptome profiles to reveal the defense mechanism involved in the HRW-regulated cold stress response of T. hemsleyanum. The application of 75% HRW could alleviate stress damage by decreasing stomatal apparatus density and significantly increasing photosynthetic efficiency and mitigating physiological indexes of resistance, such as Pn, Cond, MDA, SOD, etc., which were changed by cold stress conditions. A total of 7,883 DEGs and 439 DEMs were identified. DEGs were the most relevant to phenylpropanoid, isoflavonoid, monoterpenoid, and flavonoid biosynthesis pathways. Using gene co-expression analysis (WGCNA), we identified one gene module that showed a strong correlation between total antioxidant capacity and transpiration rate. Trend analysis indicated that the phenylpropanoid biosynthesis pathway played a major role in the transcription and metabolism process of HRW treatment under cold stress. Based on the integrated analysis of genes and metabolites, the results showed cold stress upregulated the expression of PAL, CHS, COMT, CCR, AtBG1, etc., resulting in the accumulation of coniferyl alcohol and eriodictyol contents in T. hemsleyanum under cold stress, but the 75% HRW treatment could attenuate the enhancement. The study not only identified the main strategy of HRW protection against cold stress but also provided candidate genes for flavonoid biosynthesis, so as to better improve cold tolerance through molecular breeding techniques.

Morphological and Biochemical Factors Associated with Constitutive Defense to Thrips in Alfalfa

Plants have evolved a series of inducible or constitutive defense mechanisms in response to herbivore attack. Constitutive plant defenses are morphological and biochemical traits of the plants themselves, regardless of the presence of herbivores. We bred an alfalfa variety (Caoyuan No.4) with high thrips resistance, but the mechanisms underlying Caoyuan No.4 resistance to thrips are not well understood. To explore the constitutive defense of Caoyuan No.4, the morphological and biochemical traits associated with constitutive defense to thrips in alfalfa were analyzed using a thrips-susceptible alfalfa accession (Caoyuan No.2) as a control. The results showed that Caoyuan No.4 had thicker palisade tissue and parenchyma tissue, wider collenchyma, phloem, cambium and lignin layer, and smaller epidermal cells and stomatal aperture compared to Caoyuan No.2. Moreover, Caoyuan No.4 showed more non-glandular trichomes in both leaves and stems, but less glandular trichomes and more wax in stems. In addition, the results of the widely targeted metabolomics analysis showed that metabolites related to flavonoid, isoflavonoid, flavone and flavonol biosynthesis, as well as cysteine and methionine metabolism, differed between CaoyuanNo.2 and Caoyuan No.4. These findings shed new light on the constitutive insect defense of plants associated with physical or biochemical traits and may provide convenient markers for breeding thrips-resistant alfalfa cultivars.

Transcriptomic and Non-Targeted Metabolomic Analyses Reveal the Flavonoid Biosynthesis Pathway in Auricularia cornea.

Flavonoids, which are abundant in plants, are recognized for their antioxidant and anticancer roles in clinical applications. However, little is known about the molecular basis of flavonoid biosynthesis in fungi. In this study, we found that inclusion of leachate of Korshinsk peashrub (Caragana korshinskii) in the fermentation medium increased the total flavonoid content of the edible fungus Auricularia cornea by 23.6% relative to that grown in a control medium. Combined transcriptomic and non-targeted metabolomic analysis of the flavonoid biosynthesis pathway in A. cornea illustrated that there are important metabolites in the phenylpropanoid, coumarin and isoflavonoid biosynthesis pathways. In addition, we found that certain homologous genes encode phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO) and chalcone isomerase (CHI) in these biosynthesis pathways. These results, in this study, provide a new line for studying the regulation of flavonoid production in edible fungi.

Metabonomics analysis of flavonoids in seeds and sprouts of two Chinese soybean cultivars

A popular food in China, soybean seeds and sprouts contained many biologically active substances which are beneficial to the human body, such as flavonoids. Northeast of China is the main producing area of soybean. The experimental materials came from the main soybean producing areas in Northeast China, this study compared flavonoids of two China cultivars of soybeans, Heinong52(HN52) and Heinong71(HN71). Here, we also considered the effects of germination on the chemical profile of flavonoids. Using a LC–ESI–MS/MS system, 114 differential flavonoid metabolites were identified. A total of 18 metabolites were significantly different between the two soybean varieties before germination, of which 14 were up-regulated and 4 were down-regulated. After germination, 33 significantly different metabolites were found in the two soybean sprouts, of which 19 were up-regulated and 14 were down-regulated. These experimental results revealed significant up-regulation of metabolites in soybean sprouts compared with soybean seeds, thus suggesting that soybean germination may increase content of flavonoid metabolites. There are six main pathways for the synthesis of flavonoids: isoflavonoid biosynthesis, flavonoid biosynthesis, flavone and flavonol biosynthesis, biosynthesis of secondary metabolites, and biosynthesis of phenylpropanoids. Soybean seeds lack flavone and flavanol biosynthesis and develop the capacity for this biosynthetic pathway after germination as sprouts. Isoflavonoid biosynthesis is the most abundantly utilized pathway.

Ionomics, transcriptomics and untargeted metabolomics analyses provide new insights into the Cd response and accumulation mechanisms of mulberry

Recently, the phytoremediation and safe utilization of cadmium (Cd) polluted soil by mulberry planting and sericulture have attracted researchers' attention. However, the understandings of related molecular mechanisms of mulberry response to Cd are still limited. Meanwhile, there are few reports on the diversity of the Cd tolerance and accumulation ability of different mulberry cultivars. Here, we obtained two mulberry cultivars with significant differences in Cd accumulation: high Cd accumulation cultivar FC and low Cd accumulation cultivar G12. Ionomics analysis showed that the distribution pattern of Na is the most similar to that of Cd in both G12 and FC. In transcriptomics analysis, the results of GO and KEGG enrichment analyses showed that cell wall organization-related genes were significantly affected by Cd stress, while cell division-related genes were significantly down-regulated. The expression levels of several Cd-chelation and transporter genes (AGT2, MT2, HIPP26, MTP9 and DTX43/44) were higher in all groups of G12 than those of FC, while the expression level of PCR2 and ABCC2 were higher in all groups of FC than those of G12. In untargeted metabolomics analysis, compared with FC, the metabolites of G12 were less affected by Cd stress in phenylpropanoid, flavonoid and isoflavonoid biosynthesis pathways. Notably, the content of rutin in FC was significantly lower than that of G12. These results provide theoretical support and candidate cultivars for mulberry application in Cd polluted soil, and candidate genes for mulberry genetic modification and molecular breeding in the future.

Bioactive constituents of Iris hybrida (Iridaceae): Processing effect.

Iris genus plants are a valuable source of bioactive compounds, which are an important component for pharmaceutical development. The present article shows the potential for mineral nutrition with application of magnesium sulfate, iron chelates and potassium oxide affecting the phenolic compound contents in Iris hybrida 'Tsikavynka', I. hybrida 'Tambo' and I. hybridа 'Widecombe Fire'. The effect of mineral processing was specific to plant organs and varied in the component composition. The Iris rhizomes had an increased total phenolic compound content after treatment (up to 10% of the total isoflavonoid content, up to 8% of phenolic acids, up to 5% of γ-pyrones and up to 13% of flavonoids), determined using UV-vis spectroscopy. A positive effect of nutrition on the biosynthesis and content of individual isoflavonoids (tectoridin, nigricin d-glucoside, genistin, iristectorigenin B, nigricin, irigenin and irisolidone) and xanthone mangiferin in Iris rhizomes by HPLC was established. In addition, an increase in the chlorogenic acid amount in Iris leaves was noted. The results demonstrate the sensitivity of Iris phenylpropanoid metabolism to mineral nutrition and can be used to predict medical plant cultivation with increased content of bioactive constituents.

Metabonomics Analysis of Stem Extracts from Dalbergia sissoo.

Dalbergia sissoo is a woody plant with economic and medicinal value. As the pharmacological qualities and properties of the wood from this plant primarily depend on its extractives, in this study, the metabolomic analysis of extractives from its stems was carried out using UPLC-MS/MS. A total of 735 metabolites were detected from two groups of samples, heartwood and sapwood, with the largest number of terpenoids in type and the largest number of flavonoids in quantity. The PCA and cluster analysis showed significant differences in the metabolite composition between the two groups. The differential metabolites were mainly organic oxygen compounds, flavonoids, and isoflavones. Among the 105 differential metabolites, 26 metabolites were significantly higher in relative content in sapwood than in heartwood, while the other 79 metabolites were significantly higher in relative content in heartwood than in sapwood. KEGG metabolic pathway enrichment analysis showed that these differential metabolites were mainly enriched in three metabolic pathways: Flavonoid biosynthesis, isoflavonoid biosynthesis, and flavonoid and flavonol biosynthesis. This study provides a reference for metabolomics studies in Dalbergia and other woody plants.

N-Acetyl-d-glucosamine Inhibition of Hexokinase Results in Downregulation of the Phenylpropanoid Metabolic Pathway and Decreased Resistance to Brown Rot in Peach Fruit.

To explore the role of hexokinase (HXK) on disease resistance in peach fruit, peaches were treated with N-acetyl-d-glucosamine (NAG), a known HXK inhibitor, and then inoculated with Monilinia fructicola. We demonstrate that NAG significantly inhibits HXK activity, which in turn results in significantly reduced resistance to M. fructicola infection. In the HXK-inhibited fruit, the sucrose content was higher and the glucose and fructose contents were lower than in the control fruit. By transcriptome analysis, we found 347 differentially expressed genes (DEGs) between NAG-treated and control peaches, most of which were involved in the mitogen-activated protein kinase signaling pathway in plants, plant-pathogen interaction, plant hormone signal transduction, and the phenylpropanoid biosynthesis pathway. In particular, the DEGs related to phenylpropanoid metabolism, such as peroxidase, flavonoid, and isoflavonoid biosynthesis were significantly downregulated. Nontargeted metabolomic analysis revealed 44 differential metabolites, 9 of which were increased and 35 of which were decreased in the NAG-treated fruit. The decreased metabolites were secondary metabolites, including polyphenols, flavonoids, terpenoids, and glycosides. The relationship between HXK and phenylpropanoid metabolism was further investigated, and we found that in HXK-inhibited fruits the activities of phenylalanine ammonia-lyase, 4-coumarate-CoA ligase, and cinnamate 4-hydroxylase were significantly decreased over the control fruit, as well as the total phenol and total flavone contents were also significantly decreased. These results demonstrate that the inhibition of HXK activity decreases the disease resistance of peach fruits by affecting sugar metabolism and the phenylpropanoid pathway.

Medicarpin confers powdery mildew resistance in Medicago truncatula and activates the salicylic acid signalling pathway

Powdery mildew (PM) caused by the obligate biotrophic fungal pathogen Erysiphe pisi is an economically important disease of legumes. Legumes are rich in isoflavonoids, a class of secondary metabolites whose role in PM resistance is ambiguous. Here we show that the pterocarpan medicarpin accumulates at fungal infection sites, as analysed by fluorescein‐tagged medicarpin, and provides penetration and post‐penetration resistance against E. pisi in Medicago truncatula in part through the activation of the salicylic acid (SA) signalling pathway. Comparative gene expression and metabolite analyses revealed an early induction of isoflavonoid biosynthesis and accumulation of the defence phytohormones SA and jasmonic acid (JA) in the highly resistant M. truncatula genotype A17 but not in moderately susceptible R108 in response to PM infection. Pretreatment of R108 leaves with medicarpin increased SA levels, SA‐associated gene expression, and accumulation of hydrogen peroxide at PM infection sites, and reduced fungal penetration and colony formation. Strong parallels in the levels of medicarpin and SA, but not JA, were observed on medicarpin/SA treatment pre‐ or post‐PM infection. Collectively, our results suggest that medicarpin and SA may act in concert to restrict E. pisi growth, providing new insights into the metabolic and signalling pathways required for PM resistance in legumes.

Transcriptome-wide N6-methyladenosine (m6A) methylation profiling of fresh-cut potato browning inhibition by nitrogen

Although fresh-cut potato is extremely popular, it is highly prone to browning, severely affecting its quality and shelf life. Nitrogen treatment can inhibit fresh-cut potato browning. Potato browning from the perspective of transcriptome-wide N6-methyladenosine (m6A) methylation has not yet been reported. This study performed transcriptome-wide m6A-sequencing of fresh-cut potato from nitrogen-treated group (1 h N2) and control group (1 h CK and 0 h CK) to reveal the m6A methylation modification features, and determine the methylation sites of the differentially methylated genes (DMGs). Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that the DMGs in 1 h CK vs 0 h CK and 1 h N2 vs 1 h CK were mainly enriched in the isoflavonoid and betalain biosynthesis pathways, respectively. The m6A modifications in potato were mainly enriched around the 3′-untranslated region and stop codons of the coding sequence. The DMG expression was further verified by quantitative real-time polymerase chain reaction (qPCR), while the methylation sites were determined using SELECT qPCR. This study is the first to reveal the transcriptome-wide m6A profile of potato, which may provide new insight into inhibiting fresh-cut potato browning using nitrogen treatment from the perspective of m6A methylation.