Integrated Metabolomic and Transcriptomic Analysis Reveals Differential Mechanism of Flavonoid Biosynthesis in Two Cultivars of Angelica sinensis

Angelica sinensis is a traditional Chinese medicinal plant that has been primarily used as a blood tonic. It largely relies on its bioactive metabolites, which include ferulic acid, volatile oils, polysaccharides and flavonoids. In order to improve the yield and quality of A. sinensis, the two cultivars Mingui 1 (M1), with a purple stem, and Mingui 2 (M2), with a green stem, have been selected in the field. Although a higher root yield and ferulic acid content in M1 than M2 has been observed, the differences of flavonoid biosynthesis and stem-color formation are still limited. In this study, the contents of flavonoids and anthocyanins were determined by spectrophotometer, the differences of flavonoids and transcripts in M1 and M2 were conducted by metabolomic and transcriptomic analysis, and the expression level of candidate genes was validated by qRT-PCR. The results showed that the contents of flavonoids and anthocyanins were 1.5- and 2.6-fold greater in M1 than M2, respectively. A total of 26 differentially accumulated flavonoids (DAFs) with 19 up-regulated (UR) and seven down-regulated (DR) were obtained from the 131 identified flavonoids (e.g., flavonols, flavonoid, isoflavones, and anthocyanins) in M1 vs. M2. A total 2210 differentially expressed genes (DEGs) were obtained from the 34,528 full-length isoforms in M1 vs. M2, and 29 DEGs with 24 UR and 5 DR were identified to be involved in flavonoid biosynthesis, with 25 genes (e.g., CHS1, CHI3, F3H, DFR, ANS, CYPs and UGTs) mapped on the flavonoid biosynthetic pathway and four genes (e.g., RL1, RL6, MYB90 and MYB114) belonging to transcription factors. The differential accumulation level of flavonoids is coherent with the expression level of candidate genes. Finally, the network of DAFs regulated by DEGs was proposed. These findings will provide references for flavonoid production and cultivars selection of A. sinensis.

The Flavonoid Biosynthesis Network in Plants

Flavonoids are an important class of secondary metabolites widely found in plants, contributing to plant growth and development and having prominent applications in food and medicine. The biosynthesis of flavonoids has long been the focus of intense research in plant biology. Flavonoids are derived from the phenylpropanoid metabolic pathway, and have a basic structure that comprises a C15 benzene ring structure of C6-C3-C6. Over recent decades, a considerable number of studies have been directed at elucidating the mechanisms involved in flavonoid biosynthesis in plants. In this review, we systematically summarize the flavonoid biosynthetic pathway. We further assemble an exhaustive map of flavonoid biosynthesis in plants comprising eight branches (stilbene, aurone, flavone, isoflavone, flavonol, phlobaphene, proanthocyanidin, and anthocyanin biosynthesis) and four important intermediate metabolites (chalcone, flavanone, dihydroflavonol, and leucoanthocyanidin). This review affords a comprehensive overview of the current knowledge regarding flavonoid biosynthesis, and provides the theoretical basis for further elucidating the pathways involved in the biosynthesis of flavonoids, which will aid in better understanding their functions and potential uses.

Chalcone Scaffolds, Bioprecursors of Flavonoids: Chemistry, Bioactivities, and Pharmacokinetics

Chalcones are secondary metabolites belonging to the flavonoid (C6-C3-C6 system) family that are ubiquitous in edible and medicinal plants, and they are bioprecursors of plant flavonoids. Chalcones and their natural derivatives are important intermediates of the flavonoid biosynthetic pathway. Plants containing chalcones have been used in traditional medicines since antiquity. Chalcones are basically α,β-unsaturated ketones that exert great diversity in pharmacological activities such as antioxidant, anticancer, antimicrobial, antiviral, antitubercular, antiplasmodial, antileishmanial, immunosuppressive, anti-inflammatory, and so on. This review provides an insight into the chemistry, biosynthesis, and occurrence of chalcones from natural sources, particularly dietary and medicinal plants. Furthermore, the pharmacological, pharmacokinetics, and toxicological aspects of naturally occurring chalcone derivatives are also discussed herein. In view of having tremendous pharmacological potential, chalcone scaffolds/chalcone derivatives and bioflavonoids after subtle chemical modification could serve as a reliable platform for natural products-based drug discovery toward promising drug lead molecules/drug candidates.

Flavonoid Biosynthetic Pathway: Genetics and Biochemistry

Plants are sessile organisms which are capable of producing a large array of metabolites, required for their adaption and survival. Flavonoids are low molecular weight metabolites with C6–C3–C6 carbon backbones and are categorised into different classes on the basis of structural organization and polymerization. The biosynthesis and distribution of flavonoids depends on the development stage of the plant as well as on diverse environmental conditions. They play a significant role as pigments, phytoalexins, attractants of pollinators and promotes auxin transport. In plants, antioxidant and antimicrobial activities are attributed to interaction of flavonoids with various enzymes, transcription factor and signalling pathways. This review aims to provide the current understanding of structure, their types, biosynthesis and regulation of flavonoid pathway that provide the insights to the key regulating factors and their interactions which makes them the most promising and interesting targets for plant breeding programs to enhance the value-added products in plants. In this review the deep knowledge of flavonoid regulation by micro-RNAs has been provided that attracts the biotechnologists to develop new molecular approaches so as to engineer various plant metabolic pathways to enhance the health-promoting metabolites in plants for human consumption.

Molecular Dissection of the Gene OsGA2ox8 Conferring Osmotic Stress Tolerance in Rice

Gibberellin 2-oxidase (GA2ox) plays an important role in the GA catabolic pathway and the molecular function of the OsGA2ox genes in plant abiotic stress tolerance remains largely unknown. In this study, we functionally characterized the rice gibberellin 2-oxidase 8 (OsGA2ox8) gene. The OsGA2ox8 protein was localized in the nucleus, cell membrane, and cytoplasm, and was induced in response to various abiotic stresses and phytohormones. The overexpression of OsGA2ox8 significantly enhanced the osmotic stress tolerance of transgenic rice plants by increasing the number of osmotic regulators and antioxidants. OsGA2ox8 was differentially expressed in the shoots and roots to cope with osmotic stress. The plants overexpressing OsGA2ox8 showed reduced lengths of shoots and roots at the seedling stage, but no difference in plant height at the heading stage was observed, which may be due to the interaction of OsGA2ox8 and OsGA20ox1, implying a complex feedback regulation between GA biosynthesis and metabolism in rice. Importantly, OsGA2ox8 was able to indirectly regulate several genes associated with the anthocyanin and flavonoid biosynthetic pathway and the jasmonic acid (JA) and abscisic acid (ABA) biosynthetic pathway, and overexpression of OsGA2ox8 activated JA signal transduction by inhibiting the expression of jasmonate ZIM domain-containing proteins. These results provide a basis for a future understanding of the networks and respective phenotypic effects associated with OsGA2ox8.

Expression Characterization of Flavonoid Biosynthetic Pathway Genes and Transcription Factors in Peanut Under Water Deficit Conditions

Drought is one of the hostile environmental stresses that limit the yield production of crop plants by modulating their growth and development. Peanut (Arachis hypogaea) has a wide range of adaptations to arid and semi-arid climates, but its yield is prone to loss due to drought. Other than beneficial fatty acids and micronutrients, peanut harbors various bioactive compounds including flavonoids that hold a prominent position as antioxidants in plants and protect them from oxidative stress. In this study, understanding of the biosynthesis of flavonoids in peanut under water deficit conditions was developed through expression analysis and correlational analysis and determining the accumulation pattern of phenols, flavonols, and anthocyanins. Six peanut varieties (BARD479, BARI2011, BARI2000, GOLDEN, PG1102, and PG1265) having variable responses against drought stress have been selected. Higher water retention and flavonoid accumulation have been observed in BARI2011 but downregulation has been observed in the expression of genes and transcription factors (TFs) which indicated the maintenance of normal homeostasis. ANOVA revealed that the expression of flavonoid genes and TFs is highly dependent upon the genotype of peanut in a spatiotemporal manner. Correlation analysis between expression of flavonoid biosynthetic genes and TFs indicated the role of AhMYB111 and AhMYB7 as an inhibitor for AhF3H and AhFLS, respectively, and AhMYB7, AhTTG1, and AhCSU2 as a positive regulator for the expression of Ah4CL, AhCHS, and AhF3H, respectively. However, AhbHLH and AhGL3 revealed nil-to-little relation with the expression of flavonoid biosynthetic pathway genes. Correlational analysis between the expression of TFs related to the biosynthesis of flavonoids and the accumulation of phenolics, flavonols, and anthocyanins indicated coregulation of flavonoid synthesis by TFs under water deficit conditions in peanut. This study would provide insight into the role of flavonoid biosynthetic pathway in drought response in peanut and would aid to develop drought-tolerant varieties of peanut.

Effect of chilling acclimation on germination and seedlings response to cold in different seed coat colored wheat (Triticum aestivum L.)

Background Flavonoids can protect plants against extreme temperatures and ROS due to their antioxidant activities. We found that deep-purple seed coat color was controlled by two gene interaction (12:3:1) from the cross between yellow and deep-purple seed coat colored inbreds. F2:3 seeds were grouped in 3 by seed coat color and germinated under chilling (4 °C) and non-acclimated conditions (18 °C) for a week, followed by normal conditions (18 °C) for three weeks and a subsequent chilling stress (4 °C) induction. We analyzed mean daily germination in each group. Additionally, to study the acclimation in relationship to the different seed coat colors on the germination ability and seedling performances under the cold temperatures, we measured the chlorophyll content, ROS scavenging activity, and expression levels of genes involved in ROS scavenging, flavonoid biosynthetic pathway, and cold response in seedlings. Results The results of seed color segregation between yellow and deep purple suggested a two-gene model. In the germination study, normal environmental conditions induced the germination of yellow-seed, while under chilling conditions, the germination ratio of deep purple-seed was higher than that of yellow-colored seeds. We also found that the darker seed coat colors were highly responsive to cold acclimation based on the ROS scavenging enzymes activity and gene expression of ROS scavenging enzymes, flavonoid biosynthetic pathway and cold responsive genes. Conclusions We suggest that deep purple colored seed might be in a state of innate pre-acquired stress response state under normal conditions to counteract stresses in a more effective way. Whereas, after the acclimation, another stress should enhance the cold genes expression response, which might result in a more efficient chilling stress response in deep purple seed seedlings. Low temperature has a large impact on the yield of crops. Thus, understanding the benefit of seed coat color response to chilling stress and the identification of limiting factors are useful for developing breeding strategies in order to improve the yield of wheat under chilling stress.

Integrated metabolome and transcriptome revealed the flavonoid biosynthetic pathway in developing Vernonia amygdalina leaves

Background Vernonia amygdalina as a tropical horticultural crop has been widely used for medicinal herb, feed, and vegetable. Recently, increasing studies revealed that this species possesses multiple pharmacological properties. Notably, V. amygdalina leaves possess an abundance of flavonoids, but the specific profiles of flavonoids and the mechanisms of fl avonoid bi osynthesis in developing leaves are largely unknown. Methods The total flavonoids of V. amygdalina leaves were detected using ultraviolet spectrophotometer. The temporal flavonoid profiles of V. amygdalina leaves were analyzed by LC-MS. The transcriptome analysis of V. amygdalina leaves was performed by Illumina sequencing. Functional annotation and differential expression analysis of V. amygdalina genes were performed by Blast2GO v2.3.5 and RSEM v1.2.31, respectively. qRT-PCR analysis was used to verify the gene expressions in developing V. amygdalina leaves. Results By LC-MS analysis, three substrates (p-coumaric acid, trans-cinnamic acid, and phenylalanine) for flavonoid biosynthesis were identified in V. amygdalina leaves. Additionally, 42 flavonoids were identified from V. amygdalina leaves, including six dihydroflavones, 14 flavones, eight isoflavones, nine flavonols, two xanthones, one chalcone, one cyanidin, and one dihydroflavonol. Glycosylation and methylation were common at the hydroxy group of C3, C7, and C4’ positions. Moreover, dynamic patterns of different flavonoids showed diversity. By Illumina sequencing, the obtained over 200 million valid reads were assembled into 60,422 genes. Blast analysis indicated that 31,872 genes were annotated at least in one of public databases. Greatly increasing molecular resources makes up for the lack of gene information in V. amygdalina. By digital expression profiling and qRT-PCR, we specifically characterized some key enzymes, such as Va-PAL1, Va-PAL4, Va-C4H1, Va-4CL3, Va-ACC1, Va-CHS1, Va-CHI, Va-FNSII, and Va-IFS3, involved in flavonoid biosynthesis. Importantly, integrated metabolome and transcriptome data of V. amygdalina leaves, we systematically constructed a flavonoid biosynthetic pathway with regards to material supplying, flavonoid scaffold biosynthesis, and flavonoid modifications. Our findings contribute significantly to understand the underlying mechanisms of flavonoid biosynthesis in V. amygdalina leaves, and also provide valuable information for potential metabolic engineering.

Functional Annotation of a Full-Length Transcriptome and Identification of Genes Associated with Flower Development in Rhododendronsimsii (Ericaceae)

Rhododendronsimsii is one of the top ten famous flowers in China. Due to its historical value and high aesthetic, it is widely popular among Chinese people. Various colors are important breeding objectives in Rhododendron L. The understanding of the molecular mechanism of flower color formation can provide a theoretical basis for the improvement of flower color in Rhododendron L. To generate the R. simsii transcriptome, PacBio sequencing technology has been used. A total of 833,137 full-length non-chimeric reads were obtained and 726,846 high-quality full-length transcripts were found. Moreover, 40,556 total open reading frames were obtained; of which 36,018 were complete. In gene annotation analyses, 39,411, 18,565, 16,102 and 17,450 transcriptions were allocated to GO, Nr, KEGG and COG databases, correspondingly. To identify long non-coding RNAs (lncRNAs), we utilized four computational methods associated with Protein families (Pfam), Cooperative Data Classification (CPC), Coding Assessing Potential Tool (CPAT) and Coding Non Coding Index (CNCI) databases and observed 6170, 2265, 4084 and 1240 lncRNAs, respectively. Based on the results, most genes were enriched in the flavonoid biosynthetic pathway. The eight key genes on the anthocyanin biosynthetic pathway were further selected and analyzed by qRT-PCR. The F3′H and ANS showed an upward trend in the developmental stages of R. simsii. The highest expression of F3′5′H and FLS in the petal color formation of R. simsii was observed. This research provided a huge number of full-length transcripts, which will help to proceed genetic analyses of R. simsii. native, which is a semi-deciduous shrub.

Comparative transcriptome and flavonoids components analysis reveal the structural genes responsible for the yellow seed coat color of Brassica rapa L.

Background Seed coat color is an important horticultural trait in Brassica crops, which is divided into two categories: brown/black and yellow. Seeds with yellow seed coat color have higher oil quality, higher protein content and lower fiber content. Yellow seed coat color is therefore considered a desirable trait in hybrid breeding of Brassica rapa, Brassica juncea and Brassica napus. Methods Comprehensive analysis of the abundance transcripts for seed coat color at three development stages by RNA-sequencing (RNA-seq) and corresponding flavonoids compounds by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were carried out in B. rapa. Results We identified 41,286 unigenes with 4,989 differentially expressed genes between brown seeds (B147) and yellow seeds (B80) at the same development stage. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified 19 unigenes associated with the phenylpropanoid, flavonoid, flavone and flavonol biosynthetic pathways as involved in seed coat color formation. Interestingly, expression levels of early biosynthetic genes (BrCHS, BrCHI, BrF3H, BrF3’H and BrFLS) in the flavonoid biosynthetic pathway were down-regulated while late biosynthetic genes (BrDFR, BrLDOX and BrBAN) were hardly or not expressed in seeds of B80. At the same time, BrTT8 and BrMYB5 were down-regulated in B80. Results of LC-MS also showed that epicatechin was not detected in seeds of B80. We validated the accuracy of our RNA-seq data by RT-qPCR of nine critical genes. Epicatechin was not detected in seeds of B80 by LC-MS/MS. Conclusions The expression levels of flavonoid biosynthetic pathway genes and the relative content of flavonoid biosynthetic pathway metabolites clearly explained yellow seed color formation in B. rapa. This study provides a foundation for further research on the molecular mechanism of seed coat color formation.