RESUMEN
KEY MESSAGE: Combined transcriptomic and metabolic analyses reveal that fruit of Rubus chingii Hu launches biosynthesis of phenolic acids and flavonols at beginning of fruit set and then coordinately accumulated or converted to their derivatives. Rubus chingii Hu (Chinese raspberry) is an important dual functional food with nutraceutical and pharmaceutical values. Comprehensively understanding the mechanisms of fruit development and bioactive components synthesis and regulation could accelerate genetic analysis and molecular breeding for the unique species. Combined transcriptomic and metabolic analyses of R. chingii fruits from different developmental stages, including big green, green-to-yellow, yellow-to-orange, and red stages, were conducted. A total of 89,188 unigenes were generated and 57,545 unigenes (64.52%) were annotated. Differential expression genes (DEGs) and differentially accumulated metabolites (DAMs) were mainly involved in the biosynthesis of secondary metabolites. The fruit launched the biosynthesis of phenolic acids and flavonols at the very beginning of fruit set and then coordinately accumulated or converted to their derivatives. This was tightly regulated by expressions of the related genes and MYB and bHLH transcription factors. The core genes products participated in the biosynthesis of ellagic acid (EA) and kaempferol-3-O-rutinoside (K-3-R), such as DAHPS, DQD/SDH, PAL, 4CL, CHS, CHI, F3H, F3'H, FLS, and UGT78D2, and their corresponding metabolites were elaborately characterized. Our research reveals the molecular and chemical mechanisms of the fruit development of R. chingii. The results provide a solid foundation for the genetic analysis, functional genes isolation, fruit quality improvement and modifiable breeding of R. chingii.
Asunto(s)
Ácido Elágico , Frutas/crecimiento & desarrollo , Frutas/metabolismo , Regulación de la Expresión Génica de las Plantas , Rubus/crecimiento & desarrollo , Ácido Elágico/metabolismo , Flavonoles/biosíntesis , Flavonoles/genética , Frutas/genética , Perfilación de la Expresión Génica , Hidroxibenzoatos/metabolismo , Quempferoles/genética , Quempferoles/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Control de Calidad , Rubus/genética , Rubus/metabolismo , Terpenos/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Abundant gene clusters of natural products are observed in the endophytic fungus Phomopsis liquidambaris; however, most of them are silent. Herein, a plug-and-play DNA assembly tool has been applied for flavonoid synthesis in P. liquidambaris. A shuttle plasmid was constructed based on S. cerevisiae, E. coli, and P. liquidambaris with screening markers URA, Amp, and hygR, respectively. Each fragment or cassette was successively assembled by overlap extension PCR with at least 40-50 bp homologous arms in S. cerevisiae for generating a new vector. Seven native promoters were screened by the DNA assembly based on the fluorescence intensity of the mCherry reporter gene in P. liquidambaris, and two of them were new promoters. The key enzyme chalcone synthase was the limiting step of the pathway. The naringenin and kaempferol pathways were refactored and activated with the titers of naringenin and kaempferol of 121.53 mg/L and 75.38 mg/L in P. liquidambaris using fed-batch fermentation, respectively. This study will be efficient and helpful for the biosynthesis of secondary metabolites.
Asunto(s)
Ascomicetos , Endófitos , Flavanonas/biosíntesis , Quempferoles/biosíntesis , Ascomicetos/genética , Ascomicetos/metabolismo , Endófitos/genética , Endófitos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Flavanonas/genética , Quempferoles/genética , Plásmidos/genética , Plásmidos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Apocynum venetum L. is a traditional Chinese medicinal herb with great potential to treat angiocardiopathy. Its major medicinal constituents are flavonoids. However, the natural habitats of A. venetum are typically affected by salt stress, which can modify both biomass and accumulation of medicinal compounds. In this study, the effects of salt stress on growth and development of A. venetum, accumulation of flavonoids and expression patterns of genes involved in flavonoid biosynthesis were evaluated. In general, the growth and development of seedlings (seedling height, root length, leaf length, leaf width and seed germination) were inhibited by salt stress. Unlike typical halophytes, there was no optimal NaCl concentration range that promoted growth and development, but seedlings had an elevated DW/FW ratio under salt stress (induced by irrigation with 50, 100, 200 or 400 mm NaCl). Furthermore, quercetin and kaempferol were significantly accumulated in A. venetum seedlings under salt stress, resulting in a balanced content and reduced FW. Moreover, the expression of AvCHS, AvCHI and AvF3GT was inhibited by salt stress; however, AvF3'H, AvF3H and AvFLS, which are involved in the flavonol synthesis pathway, were up-regulated under salt stress, consistent with a decrease in total flavonoids and an increase of flavonols (quercetin and kaempferol). In summary, cultivation of A. venetum in saline soils appeared to be feasible and improved the medicinal quality of A. venetum (quercetin and kaempferol accumulation under salt stress), thus this species can effectively utilize saline soil resources.
Asunto(s)
Apocynum , Quempferoles , Quercetina , Estrés Salino , Plantones , Cloruro de Sodio , Apocynum/efectos de los fármacos , Apocynum/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Crecimiento y Desarrollo/efectos de los fármacos , Quempferoles/genética , Quempferoles/metabolismo , Quercetina/genética , Quercetina/metabolismo , Estrés Salino/fisiología , Plantones/efectos de los fármacos , Plantones/crecimiento & desarrollo , Cloruro de Sodio/farmacologíaRESUMEN
Widely distributed in tea plants, the flavonoid flavonol and its glycosylated derivatives have important roles in determining tea quality. However, the biosynthesis and accumulation of these compounds has not been fully studied, especially in response to nitrogen (N) supply. In the present study, 'Longjing 43' potted tea seedlings were subjected to N deficiency (0g/pot), normal N (4g/pot) or excess N (16g/pot). Quantitative analyses using Ultra Performance Liquid Chromatography-Triple Quadrupole Mass Spectrometry (UPLC-QqQ-MS/MS) revealed that most flavonol glycosides (e.g., Quercetin-3-glucoside, Kaempferol-3-rgalactoside and Kaempferol-3-glucosyl-rhamnsoyl-glucoside) accumulated to the highest levels when treated with normal N. Results from metabolomics using Gas Chromatography-Mass Spectrometer (GC-MS) suggested that the levels of carbohydrate substrates of flavonol glycosides (e.g., sucrose, sucrose-6-phosphate, D-fructose 1,6-bisphosphate and glucose-1-phosphate) were positively correlated with flavonol glycoside content in response to N availability. Furthermore, Quantitative Real-time PCR analysis of 28 genes confirmed that genes related to flavonoid (e.g., flavonol synthase 1, flavonol 3-O-galactosyltransferase) and carbohydrate (e.g., sucrose phosphate synthase, sucrose synthase and glucokinase) metabolism have important roles in regulating the biosynthesis and accumulation of flavonol glycosides. Collectively, our results suggest that normal N levels promote the biosynthesis of flavonol glycosides through gene regulation and the accumulation of substrate carbohydrates, while abnormal N availability has inhibitory effects, especially excess N.
Asunto(s)
Camellia sinensis/metabolismo , Glicósidos/biosíntesis , Quempferoles/biosíntesis , Nitrógeno , Hojas de la Planta/metabolismo , Quercetina/análogos & derivados , Plantones/metabolismo , Camellia sinensis/genética , Glicósidos/genética , Quempferoles/genética , Nitrógeno/metabolismo , Nitrógeno/farmacología , Hojas de la Planta/genética , Quercetina/biosíntesis , Quercetina/genética , Plantones/genéticaRESUMEN
Flavonols are a flavonoid subfamily widely distributed in plants, including several ones of great importance in human and animal diet (apple, tomato, broccoli, onion, beans, tea). These polyphenolic nutraceuticals exert potent antimicrobial (membrane potential disruptors), antioxidant (free-radical scavengers), pharmacokinetic (CYP450 modulators), anti-inflammatory (lipoxygenase inhibitors), antiangiogenic (VEGF inhibitors) and antitumor (cyclin inhibitors) activities. Biotechnological production of these nutraceuticals, for example via heterologous biosynthesis in industrial actinomycetes, is favored since in plants these polyphenols appear as inactive glycosylated derivatives, in low concentrations or as part of complex mixtures with other polyphenolic compounds. In this work, we describe the de novo biosynthesis of three important flavonols, myricetin, kaempferol and quercetin, in the industrially relevant actinomycetes Streptomyces coelicolor and S. albus. De novo biosynthesis of kaempferol, myricetin and quercetin in actinomycetes has not been described before.
Asunto(s)
Suplementos Dietéticos , Flavonoides , Quempferoles , Microorganismos Modificados Genéticamente , Quercetina , Streptomyces coelicolor , Flavonoides/biosíntesis , Flavonoides/genética , Quempferoles/biosíntesis , Quempferoles/genética , Microorganismos Modificados Genéticamente/genética , Microorganismos Modificados Genéticamente/metabolismo , Quercetina/biosíntesis , Quercetina/genética , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismoRESUMEN
Anthocyanins and flavonols are secondary metabolites that can function in plant defence against herbivores. In Arabidopsis thaliana, anthocyanin and flavonol biosynthesis are regulated by MYB transcription factors. Overexpression of MYB75 (oxMYB75) in Arabidopsis results in increasing anthocyanin and flavonol levels which enhances plant resistance to generalist caterpillars. However, how these metabolites affect specialist herbivores has remained unknown. Performance of a specialist aphid (Brevicoryne brassicae) was unaffected after feeding on oxMYB75 plants, whereas a specialist caterpillar (Pieris brassicae) gained significantly higher body mass when feeding on this plant. An increase in anthocyanin and total flavonol glycoside levels correlated negatively with the body mass of caterpillars fed on oxMYB75 plants. However, a significant reduction of kaempferol-3,7-dirhamnoside (KRR) corresponded to an increased susceptibility of oxMYB75 plants to caterpillar feeding. Pieris brassicae caterpillars also grew less on an artificial diet containing KRR or on oxMYB75 plants that were exogenously treated with KRR, supporting KRR's function in direct defence against this specialist caterpillar. The results show that enhancing the activity of the anthocyanin pathway in oxMYB75 plants results in re-channelling of quercetin/kaempferol metabolites which has a negative effect on the accumulation of KRR, a novel defensive metabolite against a specialist caterpillar.
Asunto(s)
Antocianinas/genética , Antibiosis , Proteínas de Arabidopsis/genética , Flavonoles/genética , Regulación de la Expresión Génica de las Plantas , Factores de Transcripción/genética , Animales , Antocianinas/metabolismo , Áfidos/crecimiento & desarrollo , Áfidos/fisiología , Proteínas de Arabidopsis/metabolismo , Mariposas Diurnas/crecimiento & desarrollo , Mariposas Diurnas/fisiología , Flavonoles/metabolismo , Cadena Alimentaria , Herbivoria , Quempferoles/genética , Quempferoles/metabolismo , Larva/fisiología , Ninfa/fisiología , Factores de Transcripción/metabolismoRESUMEN
Ovarian cancer is 1 of the most significant malignancies in the Western world, and the antiangiogenesis strategy has been postulated for prevention and treatment of ovarian cancers. Kaempferol is a natural flavonoid present in many fruits and vegetables. The antiangiogenesis potential of kaempferol and its underlying mechanisms were investigated in two ovarian cancer cell lines, OVCAR-3 and A2780/CP70. Kaempferol mildly inhibits cell viability but significantly reduces VEGF gene expression at mRNA and protein levels in both ovarian cancer cell lines. In chorioallantoic membranes of chicken embryos, kaempferol significantly inhibits OVCAR-3-induced angiogenesis and tumor growth. HIF-1alpha, a regulator of VEGF, is downregulated by kaempferol treatment in both ovarian cancer cell lines. Kaempferol also represses AKT phosphorylation dose dependently at 5 to 20 muM concentrations. ESRRA is a HIF-independent VEGF regulator, and it is also downregulated by kaempferol in a dose-dependent manner. Overall, this study demonstrated that kaempferol is low in cytotoxicity but inhibits angiogenesis and VEGF expression in human ovarian cancer cells through both HIF-dependent (Akt/HIF) and HIF-independent (ESRRA) pathways and deserves further studies for possible application in angio prevention and treatment of ovarian cancers.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Proteínas de Choque Térmico/metabolismo , Factor 1 Inducible por Hipoxia/metabolismo , Quempferoles/farmacología , Neoplasias Ováricas/metabolismo , Factores de Transcripción/metabolismo , Factores de Crecimiento Endotelial Vascular/metabolismo , Análisis de Varianza , Inhibidores de la Angiogénesis/administración & dosificación , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Embrión de Pollo , Membrana Corioalantoides/irrigación sanguínea , Relación Dosis-Respuesta a Droga , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Choque Térmico/genética , Humanos , Factor 1 Inducible por Hipoxia/genética , Quempferoles/administración & dosificación , Quempferoles/genética , Quempferoles/metabolismo , Neoplasias Ováricas/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Factores de Crecimiento Endotelial Vascular/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Receptor Relacionado con Estrógeno ERRalfaRESUMEN
Kaempferol is a dietary flavonoid that is thought to function as a selective estrogen receptor modulator. In this study, we established that kaempferol also functions as an inverse agonist for estrogen-related receptors alpha and gamma (ERRalpha and ERRgamma). We demonstrated that kaempferol binds to ERRalpha and ERRgamma and blocks their interaction with coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha). Kaempferol also suppressed the expressions of ERR-target genes pyruvate dehydrogenase kinase 2 and 4 (PDK2 and PDK4). This evidence suggests that kaempferol may exert some of its biological effect through both estrogen receptors and estrogen-related receptors.