Modification Of Flavonoids Research Articles

Advances in Flavonoid Research: Sources, Biological Activities, and Developmental Prospectives.

At present, the occurrence of a large number of infectious and non-communicable diseases poses a serious threat to human health as well as to drug development for the treatment of these diseases. One of the most significant challenges is finding new drug candidates that are therapeutically effective and have few or no side effects. In this respect, the active compounds in medicinal plants, especially flavonoids, are potentially useful compounds with a wide range of pharmacological activities. They are naturally present in nature and valuable in the treatment of many infectious and non-communicable diseases. Flavonoids are divided into fourteen categories and are mainly derived from plant extraction, chemical synthesis and structural modification, and biosynthesis. The structural modification of flavonoids is an important way to discover new drugs, but biosynthesis is currently considered the most promising research direction with the potential to revolutionize the new production pipeline in the synthesis of flavonoids. However, relevant problems such as metabolic pathway analyses and cell synthesis protocols for flavonoids need to be addressed on an urgent basis. In the present review, new research techniques for assessing the biological activities of flavonoids and the mechanisms of their biological activities are elucidated and their modes of interaction with other drugs are described. Moreover, novel drug delivery systems, such as nanoparticles, bioparticles, colloidals, etc., are gradually becoming new means of addressing the issues of poor hydrophilicity, lipophilicity, poor chemical stability, and low bioavailability of flavonoids. The present review summarizes the latest research progress on flavonoids, existing problems with their therapeutic efficacy, and how these issues can be solved with the research on flavonoids.

Investigation on production and reaction conditions of sucrose synthase based glucosylation cascade towards flavonoid modification

Enzyme-based glycosylation is of great interest in the context of natural products decoration. Yet, its industrial application is hindered by optimisation difficulties and hard-to-standardise productivities. In this study, five sugar nucleotide-dependent glucosyltransferases from different origins (bacterial, plant and fungal) were coupled with soy sucrose synthase (GmSuSy) to create a set of diverse cascade biocatalysts for flavonoid glucosylation, which evaluation brought new insights into the field. Investigations into co-expression conditions and reaction settings enabled to define optimal induction temperature (25 °C) and uridine diphosphate (UDP) concentration (0.5 mM) for all tested pairs of enzymes. Moreover, the influence of pH and substrate concentration on the monoglucosylated product distribution was detected and analysed. The utilisation of crude protein extracts as a cost-effective source of catalysts unveiled their glycosidase activity against flavonoid glucosides, resulting in decreased productivity, which, to our knowledge, has not previously been discussed in such a context. Additionally, examination of the commercially available EziG immobilisation resins showed that selection of suitable carrier for solid catalyst production can be problematic and not only enzyme’s but also reagent’s properties have to be considered. Flavonoids, due to their complexation and hydrophobic properties, can adsorb on different types of surfaces, including divalent metal ions required for IMAC based immobilisation, necessitating detailed examination of the resins while the catalysis design.

Constructing the metabolic network of wheat kernels based on structure-guided chemical modification and multi-omics data

Metabolic network construction plays a pivotal role in unraveling the regulatory mechanism of biological activities, although it often proves to be challenging and labor-intensive, particularly with non-model organisms. In this study, we develop a computational approach that employs reaction models based on the structure-guided chemical modification and related compounds to construct a metabolic network in wheat. This construction results in a comprehensive structure-guided network, including 625 identified metabolites and additional 333 putative reactions compared with the Kyoto Encyclopedia of Genes and Genomes database. Using a combination of gene annotation, reaction classification, structure similarity, and correlations from transcriptome and metabolome analysis, a total of 229 potential genes related to these reactions are identified within this network. To validate the network, the functionality of a hydroxycinnamoyltransferase (TraesCS3D01G314900) for the synthesis of polyphenols and a rhamnosyltransferase (TraesCS2D01G078700) for the modification of flavonoids are verified through in vitro enzymatic studies and wheat mutant tests, respectively. Our research thus supports the utility of structure-guided chemical modification as an effective tool in identifying causal candidate genes for constructing metabolic networks and further in metabolomic genetic studies.

Identification and characterisation of MdUGT78T2 as a galactosyltransferase with dual activity on flavonol and anthocyanidin substrates in red-skinned apple fruit (Malus domestica L.).

Anthocyanidin and flavonol glycosides have been linked to the health-promoting effects associated with apple consumption. However, very few enzymes involved in flavonoid glycosylation have been characterised to date. Here, we present the identification and phylogenetic analysis of 234 putative glycosyltransferases involved in flavonoid biosynthesis, and detail the biochemical and structural characterisation of MdUGT78T2 as a strict galactosyltransferase involved in the formation of quercetin-3-O-galactoside and cyanidin-3-O-galactoside, the major glycoconjugates of flavonoids in apple. The enzyme is also active on other flavonoids but with a lower catalytic efficiency. Our data, complemented with gene expression analysis suggest that MdUGT78T2 synthesises the glycoconjugates at both the early and late stages of fruit development. This newly discovered type of catalytic activity can potentially be exploited for in vitro modification of flavonoids to increase their stability in food products and to modify apple fruits and other commercial crops through breeding approaches to enhance their health benefits.

Improvement of the emulsifying properties of mixed emulsifiers by optimizing ultrasonic-assisted processing

Optimizing ultrasound (ULD)-assisted flavonoid modification is an important component of enhancing its application potential. In this work, diverse flavonoids, such as quercetin (Que), apigenin (Api), and morin (Mor), were used to modify protein in myofibrillar protein (MP)/cellulose nanocrystal (CN) complexes using ULD-assisted method. Compared with the MP/CNs group, the triiodide contents of MP-Que/CNs, MP-Api/CNs, and MP-Mor/CNs increased by 1175.84%, 479.05%, and 2281.50% respectively. The findings revealed that the actual intensity of ULD was drastically reduced by the molecular weight decrease of these flavonoids. For olive oil emulsions prepared with mixed emulsifiers, the low interfacial diffusion rates (0.03 mN·m·s−1/2) and weak emulsifying activity (8.33 m2/g) of the MP/CN complexes were significantly improved by the flavonoids after ULD-assisted treatment. Notably, the emulsions prepared using MP-Api/CNs contained smaller oil droplets and exhibited better emulsifying properties, compared to emulsions prepared with MP-Mor/CNs or MP-Que/CNs. This study is essential for ULD-assisted treatment since the processing impact may be increased by choosing the most suitable flavonoid.

Differences in phytohormone and flavonoid metabolism explain the sex differences in responses of Salix rehderiana to drought and nitrogen deposition.

Due to global warming and the increase in nitrogen oxide emissions, plants experience drought and nitrogen (N) deposition. However, little is known about the acclimation to drought and N deposition of Salix species, which are dioecious woody plants. Here, an investigation into foliar N deposition combined with drought was conducted by assessing integrated phenotypes, phytohormones, transcriptomics, and metabolomics of male and female Salix rehderiana. The results indicated that there was greater transcriptional regulation in males than in females. Foliar N deposition induced an increase in foliar abscisic acid (ABA) levels in males, resulting in the inhibition of stomatal conductance, photosynthesis, carbon (C) and N accumulation, and growth, whereas more N was assimilated in females. Growth as well as C and N accumulation in drought-stressed S. rehderiana females increased after N deposition. Interestingly, drought decreased flavonoid biosynthesis whereas N deposition increased it in females. Both drought and N deposition increased flavonoid methylation in males and glycosylation in females. However, in drought-exposed S. rehderiana, N deposition increased the biosynthesis and glycosylation of flavonoids in females but decreased glycosylation in males. Therefore, foliar N deposition affects the growth and drought tolerance of S. rehderiana by altering the foliar ABA levels and the biosynthesis and modification of flavonoids. This work provides a basis for understanding how S. rehderiana may acclimate to N deposition and drought in the future.

Engineering of flavonoid 3'-O-methyltransferase for improved biosynthesis of chrysoeriol in Escherichia coli.

O-Methylation catalyzed by O-methyltransferases (OMTs) is an important modification of flavonoids for improving the transport efficiency across membranes and metabolic stability in mammalian cells. Chrysoeriol, also known as 3'-O-methylated luteolin, is a methylated flavonoid compound with health-promoting activities. The generation of chrysoeriol from luteolin can be catalyzed by a rice-derived 3'-OMT named ROMT-9, which has a high regiospecificity and activity toward flavonoids in vitro. Herein, we explored the potential of ROMT-9 for in vivo biosynthesis of chrysoeriol in Escherichia coli and adopted semi-rational enzyme engineering guided by homology modeling and molecular docking to improve the bio-production. Two positive variants including L34Q and W284A were obtained which promoted chrysoeriol formation to more than 85 mg/L from 200 mg/L of luteolin in 24 h compared with a titer of 55 mg/L for the strain expressing the native enzyme. Further biochemical analysis confirmed that such improvement in production stemmed from a higher enzyme expression level for the L34Q variant and higher efficiency in substrate binding and catalysis for the W284A variant. This study provides some insights into the engineering of other flavonoid OMTs and will facilitate high-level biosynthesis of methylated flavonoids in engineered microorganisms. KEY POINTS: • Biosynthesis of chrysoeriol from luteolin in E. coli using ROMT-9 • Engineering of ROMT-9 for better bio-production • ROMT-9 variants promote production via better expression or better catalysis.

Use of Micronization and Complex Coacervation to Preserve Antioxidant Properties of Flavonoids.

The plant flavonoids taxifolin and rutin are among the best known and best studied antioxidants. In addition to their antioxidant properties, other pharmacobiological properties have been established for these substances. At the same time, taxifolin and rutin are chemically labile. They are prone to oxidative degradation and have poor water solubility. Under conditions of their real consumption, all this can lead to a significant reduction or complete loss of bioactivity of these flavonoids. Flavonoid modification and encapsulation techniques can be used to overcome these barrier factors. The use of micronization process for taxifolin and rutin allows changing the lipophilicity values of antioxidants. For micronized taxifolin, the log P value is 1.3 (1.12 for the control forms), and for rutin, it was 0.15 (-0.64 for the control forms). The antioxidant activity of micronized flavonoids has increased about 1.16 times compared to control forms. The present study evaluates the possibility of using encapsulation of premyconized flavonoids by complex coacervation, in order to preserve their antioxidant properties. The results of an in vitro digestion study show that the encapsulated forms of antioxidants retain their bioactivity and bioavailability better than their original forms. The bioavailability indices for the encapsulated forms of flavonoids are more than 1.6 times higher than for their original forms. The digested fractions of the encapsulated properties reveal better antioxidant properties than their original forms in in vitro tests evaluating the antioxidant properties on cultures of the protozoan Paramecium caudatum and human neuroblastoma SH-SY5Y cells. Encapsulated rutin indicates the highest activity, 0.64 relative to PMA. Thus, the studies represent the feasibility of using encapsulation to protect flavonoids during digestion and ensure the preservation of their antioxidant properties.

Nucleophilic modification of flavonoids for enhanced solubility and photostability towards uniform colouration, bio-activation and ultraviolet-proof finishing of silk fabric

This study targets on the molecular-decoration of flavonoids (FLs) with a reactive ultraviolet absorber (UV-ABS) to enhance their low-solubility and photo-sensitivity, further serving for uniform and ultraviolet (UV)-proof treatment of silk. Quercetin (QUE) and rutin (RUT) showing structural discrepancy in disaccharide moiety are compared. The chemical structures of modified flavonoids (MOD-FLs), adsorption behaviour/mechanism of MOD-FLs on silk, and the colour features and functionalities of treated silk were explored. Results reveal that the absorbance of FLs in water significantly increases after modification indicating the enhanced solubility, which is reconfirmed by the water/n-butanol phase separation experiment. Two intense adsorption bands of MOD-FLs in UVA/UVB range imply their potential use as UV-proof finisher for textile. The exhaustion of MOD-FLs on silk gradually descends as pH increases. MOD-FLs display a closer trend to Psuedo 2nd-order kinetic model than FLs indicating the dominance of chemical adsorption due to the ionic nature of UV-ABS part. MOD-QUE show superior building-up property than MOD-RUT on silk, which is beneficial to achieving diverse intensity of colour and functionality on silk. Fewer aggregates of MOD-FLs are observed on silk surface than FLs. After modification, the colour fading of QUE and RUT treated silk after 8 h light exposure significantly reduce by 71.96% and 42.91%, respectively. MOD-FLs treated silk disinfect over 80 % of E. coli and S. aureus. However, a significant antioxidant decrease of FLs occurs after modification. MOD-FLs (5% owf) imparts silk with high UPF values (50+). In general, the strategy for enhancing the water solubility and photostability of FLs is developed to better serve as bio-based modifiers for textile which is also transferable to the modification of other natural molecules.

Creating diverse glycosides of 2′-hydroxyflavone through microbial glycosylation

Four new 2′-hydroxyflavone glycosides, namely hydroxyflavone-2′-O-β-D-glucuronide (1), hydroxyflavone-2′-O-α-L-rhamnoside (2), hydroxyflavone-2′-O-β-D-glucoside (3), and hydroxyflavone-2′-O-4″-O-methyl-β-D-glucoside (4), were biosynthesized through microbial glycosylation using Streptomyces coeruleorubidus NRRL B-2569, Streptomyces toxytricini NRRL 15443, Escherichia coli BL21(DE3)/pWZ8, and Beauveria bassiana ATCC 7159, respectively. Compounds 1–4 were structurally characterized through extensive analysis of 1D and 2D NMR spectroscopic data. The water solubility of glycosylated products 1–4 were enhanced by 7 to 15 times compared to the substrate 2′-hydroxyflavone. Moreover, antioxidant assays revealed that compounds 1 and 2 exhibited stronger 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity than the substrate, decreasing the logIC50 by 68.7% and 80.7%, respectively. Therefore, this research provides several effective biocatalysts that can be used for structural modification of flavonoids for enhanced water solubility and biological activities.

Synthesis, biological and computational studies of flavonoid acetamide derivatives.

This study reports the synthesis and characterization of a novel class of flavonoid acetamide derivatives (FA) of quercetin, apigenin, fisetin, kaempferol, and luteolin. Flavonoids display numerous biological properties but are limited by aqueous insolubility, enzymatic degradation, instability, and low bioavailability. FAs were synthesized, with 80–82% yields, through the sequential modification of the flavonoid hydroxyl groups into the acetamide moieties. Bioavailability, antioxidant, and ADMET are structure–activity-dependent properties that vary across different classes of flavonoids and dictate the prevalent biological applications of the flavonoids. Thus, the FAs were evaluated for their bioavailability, antioxidant, and ADMET toxicity properties versus the unmodified flavonoids (UFs). In vitro bioavailability analysis shows that the UFs have bio-availabilities in the range of 10.78–19.29% against that of the FAs in the range of 20.70–34.87%. The antioxidant capacity was measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH·) assay with recorded IC50 values of 2.19–13.03 μM for the UFs. Conversely, the FAs had high DPPH IC50 values ranging from 33.83 to 67.10 μM and corresponding to lower antioxidant activity. The FAs showed favorable ADMET properties. The modification of flavonoids into FAs significantly improves the bioavailability and the ADMET toxicity properties, albeit with decreased antioxidant activity. This work highlights the effect of the global modification of the flavonoids with the acetamide groups on the bioavailability, antioxidant, and ADMET toxicity properties which are critical determinants in the biological applications of the flavonoids.

Non-targeted metabolomic analysis of the variations in the metabolites of two genotypes of Glycyrrhiza uralensis Fisch. under drought stress

Drought stress is one of the important abiotic stresses that restrict the growth of plants. Plants acclimatize themselves to the stress by reprogramming their metabolic pathways. Therefore, understanding the metabolic mechanisms of plants is of great importance to meet the challenge of water shortage. Licorice (Glycyrrhiza uralensis Fisch.) is a commonly used herb that is widely distributed around the world. Nevertheless, the variation of integrated metabolites in licorice under drought stress are still unclear. In this study, the non-targeted metabolomic technology was applied for the first time to comparatively investigate the metabolites changes under different degree of drought stress (slight, moderate, and intense) of two morphologically distinct genotypes of licorice from Gansu (G1) and Xinjiang (G2) provinces. Under the drought conditions, the length and dry weight of the roots of licorice were affected. Different degrees of drought caused antioxidant enzyme activities in both of the two genotypes to increase first and then decrease, and the increase in the G1 genotype was greater than that of the G2 genotype. The amino acid metabolism, flavonoid metabolism and other metabolic pathways were disrupted by drought stress. Additionally, a higher amount of amino acids such as tryptophan and tyrosine was observed in the root of the G2 genotype, and the increase of phospholipid content in the G2 genotype depended on the deepening of drought stress. Slight or moderate drought stress was beneficial to the accumulation of flavonoids. The glycosylation modification of flavonoids and accumulation patterns of jasmonic acid and abscisic acid had differences between genotypes. Collectively, this study provided new insights into the potential biochemical pathways of licorice or other legumes in response to drought and a fundamental framework to improvement the medicinal value of licorice.

Identification and Structure-Activity Relationships of Dietary Flavonoids as Human Macrophage Migration Inhibitory Factor (MIF) Inhibitors.

Dietary flavonoids are known to have anti-inflammatory and anticancer effects, but their influences on human macrophage migration inhibitory factor (MIF), a vital proinflammatory cytokine recognized as a therapeutic target for infectious diseases and cancers, have been rarely reported. Here, we identified 24 dietary flavonoids that could inhibit the tautomerase activity of MIF, five of which exerted IC50 values lower than the positive control ISO-1 in the micromolar range: morin (IC50 = 11.01 ± 0.45 μM) and amentoflavone (IC50 = 13.32 ± 0.64 μM) exhibited the most potent efficacy followed by apigenin (IC50 = 42.74 ± 4.20 μM), naringin (IC50 = 51.38 ± 2.12 μM), and fisetin (IC50 = 51.99 ± 0.63 μM). X-ray crystallography, molecular docking, and cellular experiments were utilized to illustrate the molecular binding details and structure-activity relationships. Scaffold modifications of flavonoids significantly influenced the potency. What stands out for morin is the unique 2'-OH substitution. In addition, amentoflavone situated at the MIF trimer pore may impact MIF-CD74 signaling. The results also showed that flavonoids could suppress cell chemotaxis and nitric oxide production in RAW264.7 cells. Our results elucidate the molecular mechanism of flavonoids acting on MIF and shed light on developing lead compounds against MIF-involved diseases.

Flavonoid-Modifying Capabilities of the Human Gut Microbiome-An In Silico Study.

Flavonoids are a major group of dietary plant polyphenols and have a positive health impact, but their modification and degradation in the human gut is still widely unknown. Due to the rise of metagenome data of the human gut microbiome and the assembly of hundreds of thousands of bacterial metagenome-assembled genomes (MAGs), large-scale screening for potential flavonoid-modifying enzymes of human gut bacteria is now feasible. With sequences of characterized flavonoid-transforming enzymes as queries, the Unified Human Gastrointestinal Protein catalog was analyzed and genes encoding putative flavonoid-modifying enzymes were quantified. The results revealed that flavonoid-modifying enzymes are often encoded in gut bacteria hitherto not considered to modify flavonoids. The enzymes for the physiologically important daidzein-to-equol conversion, well studied in Slackia isoflavoniconvertens, were encoded only to a minor extent in Slackia MAGs, but were more abundant in Adlercreutzia equolifaciens and an uncharacterized Eggerthellaceae species. In addition, enzymes with a sequence identity of about 35% were encoded in highly abundant MAGs of uncultivated Collinsella species, which suggests a hitherto uncharacterized daidzein-to-equol potential in these bacteria. Of all potential flavonoid modification steps, O-deglycosylation (including derhamnosylation) was by far the most abundant in this analysis. In contrast, enzymes putatively involved in C-deglycosylation were detected less often in human gut bacteria and mainly found in Agathobacter faecis (formerly Roseburia faecis). Homologs to phloretin hydrolase, flavanonol/flavanone-cleaving reductase and flavone reductase were of intermediate abundance (several hundred MAGs) and mainly prevalent in Flavonifractor plautii. This first comprehensive insight into the black box of flavonoid modification in the human gut highlights many hitherto overlooked and uncultured bacterial genera and species as potential key organisms in flavonoid modification. This could lead to a significant contribution to future biochemical-microbiological investigations on gut bacterial flavonoid transformation. In addition, our results are important for individual nutritional recommendations and for biotechnological applications that rely on novel enzymes catalyzing potentially useful flavonoid modification reactions.

Citric acid and enzyme-assisted modification of flavonoids from celery (Apium graveolens) extract and their anti-inflammatory activity in HMC-1.2 cells.

Apium graveolens (celery) of the family Apiaceae contains bioactive compounds including luteolin and apigenin. The purpose of this study was to increase the extraction yield of apigenin and luteolin in celery extract using green technology and to evaluate their biological activities. The results showed that CA and β-glucosidase-assisted celery extraction transformed apiin in the celery to apigenin with an increase in luteolin concentration. The CA and β-glucosidase-treated celery extract (CAGE) improved the anti-inflammatory properties of celery extract by inhibiting the expression and production of inflammatory cytokines (IL-6, IL-8, IL-31, and TNF-α) in IL-33-stimulated mast cells (HMC-1.2 cells). Their mechanism of action was tied to the inhibition of ERK, JNK, IKKα, IκBα, and NF-κB activation by CAGE in the stimulated cells. In conclusion, CA and enzyme treatment can be considered as a useful biotechnology tool for the improvement of bioactive compounds in celery and hence improve on their bioactivity. PRACTICAL APPLICATIONS: Apium graveolens commonly called celery is an edible agricultural product cultivated throughout the world and known as a "superfood." Celery contains bioactive compounds including apigenin and luteolin that contribute to their described biological activities. However, extracting celery using normal extraction procedures such as hot water and ethanol methods yields only a small amount of apigenin and luteolin. In the present study, we introduced an eco-friendly method using citric and β-glucosidase to obtain apigenin and luteolin-rich celery extract with improved anti-inflammatory activities. The present work will spark studies on the conversion of less biologically active compounds in functional food materials to more active compounds using CA and β-glucosidase, and the development of functional food with specifically enriched bioactive substances at the industrial levels.

Synthetic Biology towards Improved Flavonoid Pharmacokinetics.

Flavonoids are a structurally diverse class of natural products that have been found to have a range of beneficial activities in humans. However, the clinical utilisation of these molecules has been limited due to their low solubility, chemical stability, bioavailability and extensive intestinal metabolism in vivo. Recently, the view has been formed that site-specific modification of flavonoids by methylation and/or glycosylation, processes that occur in plants endogenously, can be used to improve and adapt their biophysical and pharmacokinetic properties. The traditional source of flavonoids and their modified forms is from plants and is limited due to the low amounts present in biomass, intrinsic to the nature of secondary metabolite biosynthesis. Access to greater amounts of flavonoids, and understanding of the impact of modifications, requires a rethink in terms of production, more specifically towards the adoption of plant biosynthetic pathways into ex planta synthesis approaches. Advances in synthetic biology and metabolic engineering, aided by protein engineering and machine learning methods, offer attractive and exciting avenues for ex planta flavonoid synthesis. This review seeks to explore the applications of synthetic biology towards the ex planta biosynthesis of flavonoids, and how the natural plant methylation and glycosylation pathways can be harnessed to produce modified flavonoids with more favourable biophysical and pharmacokinetic properties for clinical use. It is envisaged that the development of viable alternative production systems for the synthesis of flavonoids and their methylated and glycosylated forms will help facilitate their greater clinical application.

Integrative Analysis of Transcriptome and Metabolome Reveals Salt Stress Orchestrating the Accumulation of Specialized Metabolites in Lycium barbarum L. Fruit

Salt stress seriously affects yield and quality of crops. The fruit of Lycium barbarum (LBF) is extensively used as functional food due to its rich nutrient components. It remains unclear how salt stress influences the quality of LBF. In this study, we identified 71 differentially accumulated metabolites (DAMs) and 1396 differentially expressed genes (DEGs) among ripe LBF with and without 300 mM of NaCl treatment. Pearson correlation analysis indicated that the metabolomic changes caused by salt stress were strongly related to oxidoreductases; hydrolases; and modifying enzymes, in particular, acyltransferases, methyltransferases and glycosyltransferases. Further analysis revealed that salt stress facilitated flavonoid glycosylation and carotenoid esterification by boosting the expression of structural genes in the biosynthetic pathways. These results suggested that salt stress prompts the modification of flavonoids and carotenoids to alleviate ROS damage, which in turn improves the quality of LBF. Our results lay a solid foundation for uncovering the underlying molecular mechanism of salt stress orchestrating LBF quality, and the candidate genes identified will be a valuable gene resource for genetic improvement of L. barbarum.

Transcriptome profiling reveals candidate flavonoid-related genes during formation of dragon's blood from Dracaena cochinchinensis (Lour.) S.C.Chen under conditions of wounding stress

Ethnopharmacological relevanceDragon's Blood (Resina Draconis) is a red resin that has been used in traditional medicine to promote blood circulation, regenerate muscles, reduce swelling and pain, stop bleeding, etc., and its main chemical constituents are flavonoids. Dracaena cochinchinensis (Lour.) S.C.Chen is the only plant defined by the Pharmacopoeia of the People's Republic of China as a source of dragon's blood. Aim of the studyWe aimed to reveal genes involved in the biosynthesis and accumulation of flavonoids of D. cochinchinensis which is under wounding stress by performing a de novo transcriptome analysis. Materials and methodsD. cochinchinensis samples were collected for transcriptome sequencing and bioinformatics analysis at 0 days (0 d), 3 days (3 d), 6 days (6 d), and 10 days (10 d) after induction wounding stress, and tissues were microscopically observed after wounding stress. ResultsA total of 63,244 unigenes were obtained through bioinformatics analysis, and genes associated with the biosynthesis of flavonoids were identified. Through the analysis of DEGs after wounding stress in D. cochinchinensis, based on gene expression consistent with flavonoid accumulation levels, 20 genes in connection with the flavonoid synthesis pathway and 56 genes that may be responsible for flavonoid modification and transport, and also revealed TFs (MYB, bHLH) that may be responsible for flavonoid biosynthesis. Analysis of DEGs between the four periods revealed that after wounding stress, the greatest number of significant DEGs were enriched during the first 3 days, while fewer DEGs were enriched after day 3, which corresponding to only about 1/10 (353/3883) the number of DEGs during the first 3 days. In addition, putative unigenes involved in lignin biosynthesis, such as CSE, HCT, CCR, F5H, and CAD, were significantly down-regulation after D. cochinchinensis wounding stress, but the putative unigenes responsible for flavonoid biosynthesis, such as CHS, CHI, DFR, F3'5'H, F3H, ANR, FLS, and ANS were significantly up-regulation. ConclusionWe performed de novo transcriptome analysis of D.cochinchinensis under wounding stress, candidate genes and TFs involved in the biosynthesis and accumulation of flavonoids were identified, which is the first report on the transcript variants in flavonoid form accumulation in D. cochinchinensis under wounding stress. According to the results of DEGs analysis, wounding stress attenuated lignin biosynthesis meanwhile promoted flavonoid biosynthesis. In addition, we also compared the transcriptomics of the two different original plants (D.cochinchinensis and D.cambodiana) that form dragon's blood in order to provide further understanding of the formation of dragon's blood.

Cloning and Functional Characterization of Chalcone Isomerase Genes Involved in Anthocyanin Biosynthesis in <i>Clivia miniata</i>

Chalcone isomerase (CHI), catalyzing isomerization of chalcones, is a crucial enzyme in flavonoid biosynthesis. Three <i>CHI</i> genes were isolated from <i>Clivia miniata</i> and designated as <i>CmCHI1, CmCHI2</i> and <i>CmCHI3</i>, respectively. Multiple sequence alignments and phylogenetic analysis showed that CmCHI1 and CmCHI2 were members of type I CHI proteins, whereas CmCHI3 belonged to type IV CHI proteins. Subcellular localization analysis found that all three CmCHIs had diffused distribution in the cytoplasm similar to green fluorescent protein (GFP). Anthocyanin biosynthesis and gene expression analysis demonstrated that <i>CmCHIs</i> were highly expressed in anthocyanin accumulated tissues. To further functionally characterize the role of CmCHIs, an <i>in vitro</i> enzymatic activity assay was carried out using the purified recombinant proteins. Results showed that CmCHI1 and CmCHI2 could completely convert the substrate naringenin chalcone (NC) into the product naringenin (NA), whereas CmCHI3 seemed nonfunctional as no increment of NA was detected. Further genetic transformation of <i>Arabidopsis tt5-1</i> mutant validated that CmCHI1 and CmCHI2 rather than CmCHI3 could complement the <i>chi</i> deficient phenotypes. In summary, <i>CmCHI1</i> and <i>CmCHI2</i> are the real active <i>CHI</i> genes in <i>Clivia miniata</i>. The results not only broaden our knowledge on flavonoid biosynthesis in <i>C. miniata</i> but also lay a new foundation for further flavonoid modification in <i>C. miniata</i>.

Metabolomics analysis and metabolite-agronomic trait associations using kernels of wheat (Triticum aestivum) recombinant inbred lines.

SummaryPlants produce numerous metabolites that are important for their development and growth. However, the genetic architecture of the wheat metabolome has not been well studied. Here, utilizing a high‐density genetic map, we conducted a comprehensive metabolome study via widely targeted LC‐MS/MS to analyze the wheat kernel metabolism. We further combined agronomic traits and dissected the genetic relationship between metabolites and agronomic traits. In total, 1260 metabolic features were detected. Using linkage analysis, 1005 metabolic quantitative trait loci (mQTLs) were found distributed unevenly across the genome. Twenty‐four candidate genes were found to modulate the levels of different metabolites, of which two were functionally annotated by in vitro analysis to be involved in the synthesis and modification of flavonoids. Combining the correlation analysis of metabolite‐agronomic traits with the co‐localization of methylation quantitative trait locus (mQTL) and phenotypic QTL (pQTL), genetic relationships between the metabolites and agronomic traits were uncovered. For example, a candidate was identified using correlation and co‐localization analysis that may manage auxin accumulation, thereby affecting number of grains per spike (NGPS). Furthermore, metabolomics data were used to predict the performance of wheat agronomic traits, with metabolites being found that provide strong predictive power for NGPS and plant height. This study used metabolomics and association analysis to better understand the genetic basis of the wheat metabolism which will ultimately assist in wheat breeding.