Flavonoid Metabolism Research Articles

NMR and LC–MS-based metabolomics to investigate the efficacy of a commercial bio stimulant for the treatment of wheat (Triticum aestivum)

IntroductionBio stimulants are substances and/or microorganisms that are used to improve plant growth and crop yields by modulating physiological processes and metabolism of plants. While research has primarily focused on the broad effects of bio stimulants in crops, understanding their cellular and molecular influences in plants, using metabolomic analysis, could elucidate their effectiveness and offer possibilities for fine-tuning their application. One such bio stimulant containing galacturonic acid as elicitor is used in agriculture to improve wheat vigor and strengthen resistance to lodging.ObjectiveHowever, whether a metabolic response is evolved by plants treated with this bio stimulant and the manner in which the latter might regulate plant metabolism have not been studied.MethodTherefore, the present study used 1H-NMR and LC–MS to assess changes in primary and secondary metabolites in the roots, stems, and leaves of wheat (Triticum aestivum) treated with the bio stimulant. Orthogonal partial least squares discriminant analysis effectively distinguished between treated and control samples, confirming a metabolic response to treatment in the roots, stems, and leaves of wheat.ResultsFold-change analysis indicated that treatment with the bio stimulation solution appeared to increase the levels of hydroxycinnamic acid amides, lignin, and flavonoid metabolism in different plant parts, potentially promoting root growth, implantation, and developmental cell wall maturation and lignification.ConclusionThese results demonstrate how non-targeted metabolomic approaches can be utilized to investigate and monitor the effects of new agroecological solutions based on systemic responses.

Function of MYB8 in larch under PEG simulated drought stress

Larch, a prominent afforestation, and timber species in northeastern China, faces growth limitations due to drought. To further investigate the mechanism of larch’s drought resistance, we conducted full-length sequencing on embryonic callus subjected to PEG-simulated drought stress. The sequencing results revealed that the differentially expressed genes (DEGs) primarily played roles in cellular activities and cell components, with molecular functions such as binding, catalytic activity, and transport activity. Furthermore, the DEGs showed significant enrichment in pathways related to protein processing, starch and sucrose metabolism, benzose-glucuronic acid interconversion, phenylpropyl biology, flavonoid biosynthesis, as well as nitrogen metabolism and alanine, aspartic acid, and glutamic acid metabolism. Consequently, the transcription factor T_transcript_77027, which is involved in multiple pathways, was selected as a candidate gene for subsequent drought stress resistance tests. Under PEG-simulated drought stress, the LoMYB8 gene was induced and showed significantly upregulated expression compared to the control. Physiological indices demonstrated an improved drought resistance in the transgenic plants. After 48 h of PEG stress, the transcriptome sequencing results of the transiently transformed LoMYB8 plants and control plants exhibited that genes were significantly enriched in biological process, cellular component and molecular function. Function analyses indicated for the enrichment of multiple KEGG pathways, including energy synthesis, metabolic pathways, antioxidant pathways, and other relevant processes. The pathways annotated by the differential metabolites mainly encompassed signal transduction, carbohydrate metabolism, amino acid metabolism, and flavonoid metabolism.

Molecular Mechanisms of Chlorophyll Deficiency in Ilex × attenuata 'Sunny Foster' Mutant.

Ilex × attenuata 'Sunny Foster' represents a yellow leaf mutant originating from I. × attenuata 'Foster#2', a popular ornamental woody cultivar. However, the molecular mechanisms underlying this leaf color mutation remain unclear. Using a comprehensive approach encompassing cytological, physiological, and transcriptomic methodologies, notable distinctions were discerned between the mutant specimen and its wild type. The mutant phenotype displayed aberrant chloroplast morphology, diminished chlorophyll content, heightened carotenoid/chlorophyll ratios, and a decelerated rate of plant development. Transcriptome analysis identified differentially expressed genes (DEGs) related to chlorophyll metabolism, carotenoid biosynthesis and photosynthesis. The up-regulation of CHLD and CHLI subunits leads to decreased magnesium chelatase activity, while the up-regulation of COX10 increases heme biosynthesis-both impair chlorophyll synthesis. Conversely, the down-regulation of HEMD hindered chlorophyll synthesis, and the up-regulation of SGR enhanced chlorophyll degradation, resulting in reduced chlorophyll content. Additionally, genes linked to carotenoid biosynthesis, flavonoid metabolism, and photosynthesis were significantly down-regulated. We also identified 311 putative differentially expressed transcription factors, including bHLHs and GLKs. These findings shed light on the molecular mechanisms underlying leaf color mutation in I. × attenuata 'Sunny Foster' and provide a substantial gene reservoir for enhancing leaf color through breeding techniques.

Physiology and transcriptome analysis of Artemisia argyi adaptation and accumulation to soil cadmium

The soil pollution caused by cadmium (Cd) poses a significant threat to the environment. Therefore, identifying plants that can effectively remediate Cd-contaminated soils is urgently needed. In this study, physiological, cytological, and transcriptome analyses were performed to comprehensively understand the changes in Artemisia argyi under Cd stress. Physiological and cytological analyses indicated that A. argyi maintained normal growth with intact cell structure under Cd stress levels up to 10 mg/kg. Cytological analysis showed that Cd precipitation in leaf cells occurred in the cytoplasm and intercellular spaces. As the levels of Cd stress increased, proline accumulation in leaves increased, whereas soluble protein and soluble sugar initially increased, followed by a subsequent decline. The translocation factor was above 1 under 0.6 mg/kg Cd stress but decreased when it exceeded this concentration. Transcriptome analyses revealed several crucial Cd-influenced pathways, including amino acid, terpenoid, flavonoid, and sugar metabolisms. This study not only proved that A. argyi could enrich Cd in soil but also revealed the response of A. argyi to Cd and its resistance mechanisms, which provided insight into the cleaner production of A. argyi and the remediation of Cd-contaminated soil.

Genome-Wide Analysis of Transcription Factor R2R3-MYB Gene Family and Gene Expression Profiles during Anthocyanin Synthesis in Common Walnut (Juglans regia L.).

The R2R3-MYB gene family, encoding plant transcriptional regulators, participates in many metabolic pathways of plant physiology and development, including flavonoid metabolism and anthocyanin synthesis. This study proceeded as follows: the JrR2R3-MYB gene family was analyzed genome-wide, and the family members were identified and characterized using the high-quality walnut reference genome "Chandler 2.0". All 204 JrR2R3-MYBs were established and categorized into 30 subgroups via phylogenetic analysis. JrR2R3-MYBs were unevenly distributed over 16 chromosomes. Most JrR2R3-MYBs had similar structures and conservative motifs. The cis-acting elements exhibit multiple functions of JrR2R3-MYBs such as light response, metabolite response, and stress response. We found that the expansion of JrR2R3-MYBs was mainly caused by WGD or segmental duplication events. Ka/Ks analysis indicated that these genes were in a state of negative purifying selection. Transcriptome results suggested that JrR2R3-MYBs were widely entangled in the process of walnut organ development and differentially expressed in different colored varieties of walnuts. Subsequently, we identified 17 differentially expressed JrR2R3-MYBs, 9 of which may regulate anthocyanin biosynthesis based on the results of a phylogenetic analysis. These genes were present in greater expression levels in 'Zijing' leaves than in 'Lvling' leaves, as revealed by the results of qRT-PCR experiments. These results contributed to the elucidation of the functions of JrR2R3-MYBs in walnut coloration. Collectively, this work provides a foundation for exploring the functional characteristics of the JrR2R3-MYBs in walnuts and improving the nutritional value and appearance quality of walnuts.

Rapid production of abundant transgenic pomegranate (Punica granatum) hairy roots

Pomegranates (Punica granatum) are known for their high levels of health-beneficial compounds that belong to the hydrolyzable tannin (HT) and flavonoid families. However, a significant gap in our understanding exists of the biosynthetic and regulatory genes related to the accumulation of these compounds. To this end, the induction and transformation of hairy roots presents an exciting opportunity to decipher genetically the functions of candidate genes involved in the HT and flavonoid metabolism. This study aims at improving methods for rapid and abundant hairy root production from pomegranate explants by increasing seed germination rates and expediting the attainment of requisite biomass for multifaceted analyses. We found that treating seeds with sandpaper and concentrated sulfuric acid significantly enhanced their germination rates. Interestingly, no correlation between seed-coat texture and seed germination rate was observed among the 20 pomegranate accessions evaluated in this study. Notably, transferring hairy roots from agar plates to soil promoted rapid root biomass growth compared to maintaining them on agar plates. Root biomass as well as levels of gallic acid (a precursor for HT biosynthesis) and punicalagins (the major HTs in roots) across 15 pomegranate accessions were also analyzed. Three of these accessions exhibited higher seed germination percentages along with augmented biomass and elevated punicalagin levels. These pomegranate accessions emerge as promising germplasm for future genetic transformation and functional genomics studies.

UDP-glucosyltransferase 71C4 controls the flux of phenylpropanoid metabolism to shape cotton seed development

Seeds play a crucial role in plant reproduction, making it essential to identify genes that affect seed development. In this study, we focused on UDP-glucosyltransferase 71C4 (UGT71C4) in cotton, a member of the glycosyltransferase family that shapes seed width and length, thereby influencing seed index and seed cotton yield. Overexpression of UGT71C4 results in seed enlargement owing to its glycosyltransferase activity on flavonoids, which redirects metabolic flux from lignin to flavonoid metabolism. This shift promotes cell proliferation in the ovule via accumulation of flavonoid glycosides, significantly enhancing seed cotton yield and increasing the seed index from 10.66 g to 11.91 g. By contrast, knockout of UGT71C4 leads to smaller seeds through activation of the lignin metabolism pathway and redirection of metabolic flux back to lignin synthesis. This redirection leads to increased ectopic lignin deposition in the ovule, inhibiting ovule growth and development, and alters yield components, increasing the lint percentage from 41.42% to 43.40% and reducing the seed index from 10.66 g to 8.60 g. Our research sheds new light on seed size development and reveals potential pathways for enhancing seed yield.

Transcriptome and methylome analyses unveil the effects of low and high temperatures on the postharvest senescence of plum fruit

Temperature strongly affects plum fruit senescence, but the epigenetic mechanism is unclear. Analysis of data on firmness, weigh loss rate, and decay rate showed that high temperature (HT) accelerates postharvest senescence, while low temperature (LT) delays it. Transcriptome analysis suggests that genes related to cell wall metabolism are associated with fruit softening. DNA methylation analysis shows that HT promotes fruit senescence accompanied by an increase in DNA methylation level, while LT delays senescence accompanied by a decrease in DNA methylation level. Further analysis suggests that PsDRM2 and PsDML1 may be responsible for regulating changes in methylation level. Association analysis showed that HT accelerates senescence through DNA methylation-mediated regulation of flavonoid metabolism, whereas LT delays senescence through DNA methylation-mediated regulation of lipid metabolism. These findings highlight the importance of temperature and DNA methylation in controlling postharvest senescence and provide valuable insights for developing effective strategies to control fruit senescence.

Evolutionary analysis of MADS-box genes in buckwheat species and functional study of FdMADS28 in flavonoid metabolism

The MADS-box gene family is a transcription factor family that is widely expressed in plants. It controls secondary metabolic processes in plants and encourages the development of tissues like roots and flowers. However, the phylogenetic analysis and evolutionary model of MADS-box genes in Fagopyrum species has not been reported yet. This study identified the MADS-box genes of three buckwheat species at the whole genome level, and conducted systematic evolution and physicochemical analysis. The results showed that these genes can be divided into four subfamilies, with fragment duplication being the main way for the gene family expansion. During the domestication process from golden buckwheat to tartary buckwheat and the common buckwheat, the Ka/Ks ratio indicated that most members of the family experienced strong purification selection pressure, and with individual gene pairs experiencing positive selection. In addition, we combined the expression profile data of the MADS genes, mGWAS data, and WGCNA data to mine genes FdMADS28/48/50 that may be related to flavonoid metabolism. The results also showed that overexpression of FdMADS28 could increase rutin content by decreasing Kaempferol pathway content in hairy roots, and increase the resistance and growth of hairy roots to PEG and NaCl. This study systematically analyzed the evolutionary relationship of MADS-box genes in the buckwheat species, and elaborated on the expression patterns of MADS genes in different tissues under biotic and abiotic stresses, laying an important theoretical foundation for further elucidating their role in flavonoid metabolism.

Haplotype-resolved chromosomal-level genome assembly reveals regulatory variations in mulberry fruit anthocyanin content.

Understanding the intricate regulatory mechanisms underlying the anthocyanin content (AC) in fruits and vegetables is crucial for advanced biotechnological customization. In this study, we generated high-quality haplotype-resolved genome assemblies for two mulberry cultivars: the high-AC 'Zhongsang5801' (ZS5801) and the low-AC 'Zhenzhubai' (ZZB). Additionally, we conducted a comprehensive analysis of genes associated with AC production. Through genome-wide association studies (GWAS) on 112 mulberry fruits, we identified MaVHAG3, which encodes a vacuolar-type H+-ATPase G3 subunit, as a key gene linked to purple pigmentation. To gain deeper insights into the genetic and molecular processes underlying high AC, we compared the genomes of ZS5801 and ZZB, along with fruit transcriptome data across five developmental stages, and quantified the accumulation of metabolic substances. Compared to ZZB, ZS5801 exhibited significantly more differentially expressed genes (DEGs) related to anthocyanin metabolism and higher levels of anthocyanins and flavonoids. Comparative analyses revealed expansions and contractions in the flavonol synthase (FLS) and dihydroflavonol 4-reductase (DFR) genes, resulting in altered carbon flow. Co-expression analysis demonstrated that ZS5801 displayed more significant alterations in genes involved in late-stage AC regulation compared to ZZB, particularly during the phase stage. In summary, our findings provide valuable insights into the regulation of mulberry fruit AC, offering genetic resources to enhance cultivars with higher AC traits.

Comparative physiological, biochemical, metabolomic, and transcriptomic analyses reveal the formation mechanism of heartwood for Acacia melanoxylon

Acacia melanoxylon is well known as a valuable commercial tree species owing to its high-quality heartwood (HW) products. However, the metabolism and regulatory mechanism of heartwood during wood development remain largely unclear. In this study, both microscopic observation and content determination proved that total amount of starches decreased and phenolics and flavonoids increased gradually from sapwood (SW) to HW. We also obtained the metabolite profiles of 10 metabolites related to phenolics and flavonoids during HW formation by metabolomics. Additionally, we collected a comprehensive overview of genes associated with the biosynthesis of sugars, terpenoids, phenolics, and flavonoids using RNA-seq. A total of ninety-one genes related to HW formation were identified. The transcripts related to plant hormones, programmed cell death (PCD), and dehydration were increased in transition zone (TZ) than in SW. The results of RT-PCR showed that the relative expression level of genes and transcription factors was also high in the TZ, regardless of the horizontal or vertical direction of the trunk. Therefore, the HW formation took place in the TZ for A. melanoxylon from molecular level, and potentially connected to plant hormones, PCD, and cell dehydration. Besides, the increased expression of sugar and terpenoid biosynthesis-related genes in TZ further confirmed the close connection between terpenoid biosynthesis and carbohydrate metabolites of A. melanoxylon. Furthermore, the integrated analysis of metabolism data and RNA-seq data showed the key transcription factors (TFs) regulating flavonoids and phenolics accumulation in HW, including negative correlation TFs (WRKY, MYB) and positive correlation TFs (AP2, bZIP, CBF, PB1, and TCP). And, the genes and metabolites from phenylpropanoid and flavonoid metabolism and biosynthesis were up-regulated and largely accumulated in TZ and HW, respectively. The findings of this research provide a basis for comprehending the buildup of metabolites and the molecular regulatory processes of HW formation in A. melanoxylon.

Genome-wide characterization of post-transcriptional processes related to wood formation in Dalbergia odorifera

BackgroundAlternative polyadenylation (APA), alternative splicing (AS), and long non-coding RNAs (lncRNAs) play regulatory roles in post-transcriptional processes in plants. However, little is known about their involvement in xylem development in Dalbergia odorifera, a valuable rosewood species with medicinal and commercial significance. We addressed this by conducting Isoform Sequencing (Iso-Seq) using PacBio’s SMRT technology and combined it with RNA-seq analysis (RNA sequencing on Illumina platform) after collecting xylem samples from the transition zone and the sapwood of D. odorifera.ResultsWe identified 14,938 full-length transcripts, including 9,830 novel isoforms, which has updated the D. odorifera genome annotation. Our analysis has revealed that 4,164 genes undergo APA, whereas 3,084 genes encounter AS. We have also annotated 118 lncRNAs. Furthermore, RNA-seq analysis identified 170 differential alternative splicing (DAS) events, 344 genes with differential APA site usage (DE-APA), and 6 differentially expressed lncRNAs in the transition zone when compared to the sapwood. AS, APA, and lncRNAs are differentially regulated during xylem development. Differentially expressed APA genes were enriched for terpenoid and flavonoid metabolism, indicating their role in the heartwood formation. Additionally, DE-APA genes were associated with cell wall biosynthesis and terpenoid metabolism, implying an APA’s role in wood formation. A DAS gene (involved in chalcone accumulation) with a significantly greater inclusion of the last exon in the transition zone than in the sapwood was identified. We also found that differentially expressed lncRNAs targeted the genes related to terpene synthesis.ConclusionsThis study enhances our understanding of the molecular regulatory mechanisms underlying wood formation in D. odorifera, and provides valuable genetic resources and insights for its molecular-assisted breeding.

Integrated transcriptomic and metabolomic data reveal the cold stress responses molecular mechanisms of two coconut varieties.

Among tropical fruit trees, coconut holds significant edible and economic importance. The natural growth of coconuts faces a challenge in the form of low temperatures, which is a crucial factor among adverse environmental stresses impacting their geographical distribution. Hence, it is essential to enhance our comprehension of the molecular mechanisms through which cold stress influences various coconut varieties. We employed analyses of leaf growth morphology and physiological traits to examine how coconuts respond to low temperatures over 2-hour, 8-hour, 2-day, and 7-day intervals. Additionally, we performed transcriptome and metabolome analyses to identify the molecular and physiological shifts in two coconut varieties displaying distinct sensitivities to the cold stress. As the length of cold stress extended, there was a prominent escalation within the soluble protein (SP), proline (Pro) concentrations, the activity of peroxidase (POD) and superoxide dismutase (SOD) in the leaves. Contrariwise, the activity of glutathione peroxidase (GSH) underwent a substantial reduction during this period. The widespread analysis of metabolome and transcriptome disclosed a nexus of genes and metabolites intricately cold stress were chiefly involved in pathways centered around amino acid, flavonoid, carbohydrate and lipid metabolism. We perceived several stress-responsive metabolites, such as flavonoids, carbohydrates, lipids, and amino acids, which unveiled considerably, lower in the genotype subtle to cold stress. Furthermore, we uncovered pivotal genes in the amino acid biosynthesis, antioxidant system and flavonoid biosynthesis pathway that presented down-regulation in coconut varieties sensitive to cold stress. This study broadly enriches our contemporary perception of the molecular machinery that contributes to altering levels of cold stress tolerance amid coconut genotypes. It also unlocks several unique prospects for exploration in the areas of breeding or engineering, aiming to identifying tolerant and/or sensitive coconut varieties encompassing multi-omics layers in response to cold stress conditions.

Widely targeted metabolomics reveals differences in metabolites of Paeonia lactiflora cultivars.

Paeonia lactiflora contains diverse active constituents and exhibits various pharmacological activities. However, only partial identification of biologically active substances from P. lactiflora has been achieved using low-throughput techniques. Here, the roots of P. lactiflora, namely, Fenyunu (CK), Dafugui (DFG), and Red Charm (HSML), were studied. The primary and secondary metabolites were investigated using ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESIMS/MS). The chemical compounds and categories were detected using broadly targeted UPLC-MS/MS. Principal component analysis (PCA), orthogonal partial least-squares discriminant analysis (OPLS-DA), and hierarchical clustering analysis (HCA) were carried out for metabolites of different varieties of P. lactiflora. A total of 1237 compounds were detected and classified into 11 categories. HCA, PCA, and OPLS-DA of these metabolites indicated that each variety of P. lactiflora was clearly separated from the other groups. Differential accumulated metabolite analysis revealed that the three P. lactiflora varieties contained 116 differentially activated metabolites (DAMs) involved in flavonoid, flavone, and flavonol metabolism. KEGG pathway analysis revealed that, in 65 pathways, 336 differentially abundant metabolites (DMs) were enriched in the CK and DFG groups; moreover, the type and content of terpenoids were greater in the CK group than in the DFG group. The CK and HSML groups contained 457 DMs enriched in 61 pathways; the type and amount of flavonoids, terpenoids, and tannins were greater in the CK group than in the HSML group. The DFG and HSML groups contained 497 DMs enriched in 65 pathways; terpenoids and alkaloids were more abundant in the HSML variety than in the DFG variety. A total of 1237 compounds were detected, and the results revealed significant differences among the three P. lactiflora varieties. Among the three P. lactiflora varieties, phenolic acids and flavonoids composed the largest and most diverse category of metabolites, and their contents varied greatly. Therefore, CK is suitable for medicinal plant varieties, and DFG and HSML are suitable for ornamental plant varieties. Twelve proanthocyanidin metabolites likely determined the differences in color among the three varieties.

Genome-Wide Identification of Glutathione S-Transferase Genes in Eggplant (Solanum melongena L.) Reveals Their Potential Role in Anthocyanin Accumulation on the Fruit Peel.

Anthocyanins are ubiquitous pigments derived from the phenylpropanoid compound conferring red, purple and blue pigmentations to various organs of horticultural crops. The metabolism of flavonoids in the cytoplasm leads to the biosynthesis of anthocyanin, which is then conveyed to the vacuoles for storage by plant glutathione S-transferases (GST). Although GST is important for transporting anthocyanin in plants, its identification and characterization in eggplant (Solanum melongena L.) remains obscure. In this study, a total of 40 GST genes were obtained in the eggplant genome and classified into seven distinct chief groups based on the evolutionary relationship with Arabidopsis thaliana GST genes. The seven subgroups of eggplant GST genes (SmGST) comprise: dehydroascorbate reductase (DHAR), elongation factor 1Bγ (EF1Bγ), Zeta (Z), Theta(T), Phi(F), Tau(U) and tetra-chlorohydroquinone dehalogenase TCHQD. The 40 GST genes were unevenly distributed throughout the 10 eggplant chromosomes and were predominantly located in the cytoplasm. Structural gene analysis showed similarity in exons and introns within a GST subgroup. Six pairs of both tandem and segmental duplications have been identified, making them the primary factors contributing to the evolution of the SmGST. Light-related cis-regulatory elements were dominant, followed by stress-related and hormone-responsive elements. The syntenic analysis of orthologous genes indicated that eggplant, Arabidopsis and tomato (Solanum lycopersicum L.) counterpart genes seemed to be derived from a common ancestry. RNA-seq data analyses showed high expression of 13 SmGST genes with SmGSTF1 being glaringly upregulated on the peel of purple eggplant but showed no or low expression on eggplant varieties with green or white peel. Subsequently, SmGSTF1 had a strong positive correlation with anthocyanin content and with anthocyanin structural genes like SmUFGT (r = 0.9), SmANS (r = 0.85), SmF3H (r = 0.82) and SmCHI2 (r = 0.7). The suppression of SmGSTF1 through virus-induced gene silencing (VIGs) resulted in a decrease in anthocyanin on the infiltrated fruit surface. In a nutshell, results from this study established that SmGSTF1 has the potential of anthocyanin accumulation in eggplant peel and offers viable candidate genes for the improvement of purple eggplant. The comprehensive studies of the SmGST family genes provide the foundation for deciphering molecular investigations into the functional analysis of SmGST genes in eggplant.

Gut microbiome and metabolome profiling in Framingham heart study reveals cholesterol-metabolizing bacteria

Accumulating evidence suggests that cardiovascular disease (CVD) is associated with an altered gut microbiome. Our understanding of the underlying mechanisms has been hindered by lack of matched multi-omic data with diagnostic biomarkers. To comprehensively profile gut microbiome contributions to CVD, we generated stool metagenomics and metabolomics from 1,429 Framingham Heart Study participants. We identified blood lipids and cardiovascular health measurements associated with microbiome and metabolome composition. Integrated analysis revealed microbial pathways implicated in CVD, including flavonoid, γ-butyrobetaine, and cholesterol metabolism. Species from the Oscillibacter genus were associated with decreased fecal and plasma cholesterol levels. Using functional prediction and invitro characterization of multiple representative human gut Oscillibacter isolates, we uncovered conserved cholesterol-metabolizing capabilities, including glycosylation and dehydrogenation. These findings suggest that cholesterol metabolism is a broad property of phylogenetically diverse Oscillibacter spp., with potential benefits for lipid homeostasis and cardiovascular health.

UPLC-ESI-MS/MS analysis revealed the dynamic changes and conversion mechanism of non-volatile metabolites during green tea fixation

Fixation is crucial for green tea quality; previous studies were conducted on finished teas and focused on the volatile metabolites of green tea. However, non-volatile metabolites’ (NVMs) evolution and formation mechanisms during fixation remain unclear. Here, fixated tea samples were collected at seven different time points during the green tea fixation stage, the dynamic changes and formation mechanisms of NVMs investigated via ultra-performance liquid chromatography-tandem mass spectrometry and enzymatic activity analyses. The results showed that fixation contained enzymatic activity thermal and non-enzymatic thermal stages. Moreover, 1196 NVMs (10 classes) were detected, and significant NVM variations were observed during the "enzymatic activity thermal" fixation stages, with increasing trends in lipids and amino acids, except alkaloids. Furthermore, 160 common differential NVMs were identified. KEGG analysis revealed that unsaturated fatty acid, linoleic acid phenylpropanoid, and flavonoid metabolism pathways were essential during fixation and influenced green tea aroma, taste, and color formation. These results provide a theoretical basis to regulate green tea fixation technology and enrich the chemical theory of green tea processing.

Tea-Derived Polyphenols Enhance Drought Resistance of Tea Plants (Camellia sinensis) by Alleviating Jasmonate-Isoleucine Pathway and Flavonoid Metabolism Flow.

Extreme drought weather has occurred frequently in recent years, resulting in serious yield loss in tea plantations. The study of drought in tea plantations is becoming more and more intensive, but there are fewer studies on drought-resistant measures applied in actual production. Therefore, in this study, we investigated the effect of exogenous tea polyphenols on the drought resistance of tea plant by pouring 100 mg·L-1 of exogenous tea polyphenols into the root under drought. The exogenous tea polyphenols were able to promote the closure of stomata and reduce water loss from leaves under drought stress. Drought-induced malondialdehyde (MDA) accumulation in tea leaves and roots was also significantly reduced by exogenous tea polyphenols. Combined transcriptomic and metabolomic analyses showed that exogenous tea polyphenols regulated the abnormal responses of photosynthetic and energy metabolism in leaves under drought conditions and alleviated sphingolipid metabolism, arginine metabolism, and glutathione metabolism in the root system, which enhanced the drought resistance of tea seedlings. Exogenous tea polyphenols induced jasmonic acid-isoleucine (JA-ILE) accumulation in the root system, and the jasmonic acid-isoleucine synthetase gene (TEA028623), jasmonic acid ZIM structural domain proteins (JAMs) synthesis genes (novel.22237, TEA001821), and the transcription factor MYC2 (TEA014288, TEA005840) were significantly up-regulated. Meanwhile, the flavonoid metabolic flow was significantly altered in the root; for example, the content of EGCG, ECG, and EGC was significantly increased. Thus, exogenous tea polyphenols enhance the drought resistance of tea plants through multiple pathways.

Enrichment of Flavonoids in Short-Germinated Black Soybeans (Glycine max L.) Induced by Slight Acid Treatment.

Exogenous abiotic stimulant treatments are a straightforward and effective method for enhancing secondary metabolites in plants. In this study, the response surface optimization method was used to optimize the conditions for enriching flavonoids in short-germinated black soybeans under a slight acid treatment, and the mechanism of flavonoid accumulation during black soybean germination was explored. The results show that the use of a 126.2 mM citric acid-sodium citrate buffer (pH 5.10) as a slight acid treatment resulted in the highest flavonoid content when the black soybeans were germinated for 24 h. Under these conditions, the isoflavonoid (glycitin, daidzein, and genistein) increased significantly, and the flavonoid content reached 2.32 mg/g FW. The microacidified germination treatment significantly increased the activities and relative gene expression levels of key enzymes involved in flavonoid metabolism (4-coumarate-CoA ligase and cinnamic acid 4-hydroxylase, etc.). However, the slight acid treatment inhibited the growth of the black soybeans and caused damage to their cells. This was evidenced by significantly higher levels of malondialdehyde, superoxide anion, and hydrogen peroxide compared to the control group. Furthermore, the antioxidant system in the short-germinated soybeans was activated by the slight acid treatment, leading to a significant increase in the activities and relative gene expression levels of catalase and peroxidase. The results above show that a slight acid treatment was beneficial in inducing the accumulation of flavonoids during the growth of black soybean sprouts. This lays a technical foundation for producing black soybean products that are rich in flavonoids.

Peroxisomal 4-coumaroyl-CoA ligases participate in shikonin production in Lithospermum erythrorhizon.

4-Coumaroyl-CoA ligase (4CL) is a key enzyme in the phenylpropanoid pathway, which is involved in the biosynthesis of various specialized metabolites such as flavonoids, coumarins, lignans, and lignin. Plants have several 4CLs showing divergence in sequence: Class I 4CLs involved in lignin metabolism, Class II 4CLs associated with flavonoid metabolism, and atypical 4CLs and 4CL-like proteins of unknown function. Shikonin, a Boraginaceae-specific specialized metabolite in red gromwell (Lithospermum erythrorhizon), is biosynthesized from p-hydroxybenzoic acid, and the involvement of 4CL in its biosynthesis has long been debated. In this study, we demonstrated the requirement of 4CL for shikonin biosynthesis using a 4CL-specific inhibitor. In silico analysis of the L. erythrorhizon genome revealed the presence of at least 8 4CL genes, among which the expression of 3 (Le4CL3, Le4CL4, and Le4CL5) showed a positive association with shikonin production. Phylogenetic analysis indicated that Le4CL5 belongs to Class I 4CLs, while Le4CL3 and Le4CL4 belong to clades that are distant from Class I and Class II. Interestingly, both Le4CL3 and Le4CL4 have peroxisome targeting signal 1 in their C-terminal region, and subcellular localization analysis revealed that both localize to the peroxisome. We targeted each of the 3 Le4CL genes by CRISPR/Cas9-mediated mutagenesis and observed remarkably lower shikonin production in Le4CL3-ge and Le4CL4-ge genome-edited lines compared with the vector control. We, therefore, conclude that peroxisomal Le4CL3 and Le4CL4 are responsible for shikonin production and propose a model for metabolite-specific 4CL distribution in L. erythrorhizon.