RESUMEN
Gene expression and phytohormone contents were measured in response to elevating ascorbate in the absence of other confounding stimuli such as high light and abiotic stresses. Young Arabidopsis plants were treated with 25 mM solutions of l-galactose pathway intermediates l-galactose (l-gal) or l-galactono-1,4-lactone (l-galL), as well as L-ascorbic acid (AsA), with 25 mM glucose used as control. Feeding increased rosette AsA 2- to 4-fold but there was little change in AsA biosynthetic gene transcripts. Of the ascorbate recycling genes, only Dehydroascorbate reductase 1 expression was increased. Some known regulatory genes displayed increased expression and included ANAC019, ANAC072, ATHB12, ZAT10 and ZAT12. Investigation of the ANAC019/ANAC072/ATHB12 gene regulatory network revealed a high proportion of ABA regulated genes. Measurement of a subset of jasmonate, ABA, auxin (IAA) and salicylic acid compounds revealed consistent increases in ABA (up to 4.2-fold) and phaseic acid (PA; up to 5-fold), and less consistently certain jasmonates, IAA, but no change in salicylic acid levels. Increased ABA is likely due to increased transcripts for the ABA biosynthetic gene NCED3. There were also smaller increases in transcripts for transcription factors ATHB7, ERD1, and ABF3. These results provide insights into how increasing AsA content can mediate increased abiotic stress tolerance.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/fisiología , Ácido Ascórbico/metabolismo , Glutatión Transferasa/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Estrés Fisiológico/fisiología , Ácido Abscísico/metabolismo , Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/metabolismo , Ascorbato Oxidasa/genética , Ascorbato Oxidasa/metabolismo , Ácido Ascórbico/genética , Ciclopentanos/metabolismo , Galactosa/farmacología , Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes , Glutatión Transferasa/metabolismo , Ácidos Hexurónicos/metabolismo , Ácidos Indolacéticos/metabolismo , Oxilipinas/metabolismo , Reguladores del Crecimiento de las Plantas/genética , Sesquiterpenos/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Emerging evidence from human intervention trials indicates health benefits of consuming blackcurrant fruit, including improvements to cognitive performance, modulation of blood flow, regulation of blood glucose and inhibition of enzymes underpinning normal cognitive function. Of particular relevance is our previous demonstration of monoamine oxidase (MAO)-A and B inhibition after the consumption of a New Zealand "Blackadder" blackcurrant juice in humans. The current study uses a double-blind, placebo-controlled, randomised cross- over design to assess the pharmacodynamics of the effects on platelet MAO-B inhibition and associated substrates, plasma prolactin levels and blood glucose levels after consumption of a single serve of "Blackadder" blackcurrant juice standardised to 500â mg polyphenols. Eight healthy male (20--35 years) participants completed the trial. Measurements were obtained at baseline 15, 30, 45, 60, 100, 120, 150, 180, 240 mins and 24â h post dose. A fast, absolute and reversible inhibition of blood platelet MAO-B (P < 0.001) and a significant but delayed reduction in plasma prolactin (P < 0.001) were observed following the consumption of "Blackadder" blackcurrant juice when compared to a placebo control. No interpretable changes in substrates of MAO or associated metabolites were seen. These data provide a clear time course of the reversible inhibition of MAO-B after the single consumption of a of New Zealand "Blackadder" blackcurrant juice standardised at 500â mg of polyphenols and, therefore, provide a therapeutic window on which to base future nutritional interventions.
Asunto(s)
Inhibidores de la Monoaminooxidasa/administración & dosificación , Ácido gammalinolénico/farmacocinética , Adulto , Glucemia/efectos de los fármacos , Plaquetas , Estudios Cruzados , Método Doble Ciego , Humanos , Masculino , Polifenoles/farmacocinética , Prolactina/sangre , Adulto JovenRESUMEN
The polyphenol profile of apple (Malus × domestica) is dominated by the dihydrochalcone glycoside phloridzin, but its physiological role is yet to be elucidated. Biosynthesis of phloridzin occurs as a side branch of the main phenylpropanoid pathway, with the final step mediated by the phloretin-specific glycosyltransferase UGT88F1. Unexpectedly, given that UGTs are sometimes viewed as 'decorating enzymes', UGT88F1 knockdown lines were severely dwarfed, with greatly reduced internode lengths, narrow lanceolate leaves, and changes in leaf and fruit cellular morphology. These changes suggested that auxin transport had been altered in the knockdown lines, which was confirmed in assays showing that auxin flux from the shoot apex was increased in the transgenic lines. Metabolite analysis revealed no accumulation of the phloretin aglycone, as well as decreases in many non-target phenylpropanoid compounds. This decreased accumulation of metabolites appeared to be mediated by the repression of the phenylpropanoid pathway via a reduction in key transcript levels (e.g. phenylalanine ammonia lyase, PAL) and enzyme activities (PAL and chalcone synthase). Application of exogenous phloridzin to the UGT88F1 knockdown lines in tissue culture enhanced axial leaf growth and partially restored some aspects of 'normal' apple leaf growth. Together, our results strongly implicate dihydrochalcones as critical compounds in modulating phenylpropanoid pathway flux and establishing auxin patterning early in apple development.
Asunto(s)
Glicosiltransferasas/genética , Malus/metabolismo , Floretina/metabolismo , Proteínas de Plantas/genética , Aciltransferasas/genética , Aciltransferasas/metabolismo , Chalconas/metabolismo , Regulación de la Expresión Génica de las Plantas , Técnicas de Silenciamiento del Gen , Glicosiltransferasas/metabolismo , Ácidos Indolacéticos/metabolismo , Malus/efectos de los fármacos , Malus/genética , Fenilanina Amoníaco-Liasa/metabolismo , Florizina/farmacología , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Proteínas de Plantas/metabolismo , Plantas Modificadas GenéticamenteRESUMEN
Apple (Malus × domestica) accumulates bioactive ursane-, oleanane-, and lupane-type triterpenes in its fruit cuticle, but their biosynthetic pathway is still poorly understood. We used a homology-based approach to identify and functionally characterize two new oxidosqualene cyclases (MdOSC4 and MdOSC5) and one cytochrome P450 (CYP716A175). The gene expression patterns of these enzymes and of previously described oxidosqualene cyclases were further studied in 20 apple cultivars with contrasting triterpene profiles. MdOSC4 encodes a multifunctional oxidosqualene cyclase producing an oleanane-type triterpene, putatively identified as germanicol, as well as ß-amyrin and lupeol, in the proportion 82 : 14 : 4. MdOSC5 cyclizes 2,3-oxidosqualene into lupeol and ß-amyrin at a ratio of 95 : 5. CYP716A175 catalyses the C-28 oxidation of α-amyrin, ß-amyrin, lupeol and germanicol, producing ursolic acid, oleanolic acid, betulinic acid, and putatively morolic acid. The gene expression of MdOSC1 was linked to the concentrations of ursolic and oleanolic acid, whereas the expression of MdOSC5 was correlated with the concentrations of betulinic acid and its caffeate derivatives. Two new multifuntional triterpene synthases as well as a multifunctional triterpene C-28 oxidase were identified in Malus × domestica. This study also suggests that MdOSC1 and MdOSC5 are key genes in apple fruit triterpene biosynthesis.
Asunto(s)
Vías Biosintéticas , Sistema Enzimático del Citocromo P-450/metabolismo , Frutas/enzimología , Transferasas Intramoleculares/metabolismo , Malus/enzimología , Triterpenos/metabolismo , Secuencia de Aminoácidos , Vías Biosintéticas/genética , Clonación Molecular , Frutas/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Malus/genética , Filogenia , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente , Análisis de Componente Principal , Alineación de Secuencia , Análisis de Secuencia de Proteína , Escualeno/análogos & derivados , Escualeno/química , Escualeno/metabolismo , Nicotiana/genética , Triterpenos/químicaRESUMEN
Glycosides are an important potential source of aroma and flavour compounds for release as volatiles in flowers and fruit. The production of glycosides is catalysed by UDP-glycosyltransferases (UGTs) that mediate the transfer of an activated nucleotide sugar to acceptor aglycones. A screen of UGTs expressed in kiwifruit (Actinidia deliciosa) identified the gene AdGT4 which was highly expressed in floral tissues and whose expression increased during fruit ripening. Recombinant AdGT4 enzyme glycosylated a range of terpenes and primary alcohols found as glycosides in ripe kiwifruit. Two of the enzyme's preferred alcohol aglycones, hexanol and (Z)-hex-3-enol, contribute strongly to the 'grassy-green' aroma notes of ripe kiwifruit and other fruit including tomato and olive. Transient over-expression of AdGT4 in tobacco leaves showed that enzyme was able to glycosylate geraniol and octan-3-ol in planta whilst transient expression of an RNAi construct in Actinidia eriantha fruit reduced accumulation of a range of terpene glycosides. Stable over-expression of AdGT4 in transgenic petunia resulted in increased sequestration of hexanol and other alcohols in the flowers. Transgenic tomato fruit stably over-expressing AdGT4 showed changes in both the sequestration and release of a range of alcohols including 3-methylbutanol, hexanol and geraniol. Sequestration occurred at all stages of fruit ripening. Ripe fruit sequestering high levels of glycosides were identified as having a less intense, earthier aroma in a sensory trial. These results demonstrate the importance of UGTs in sequestering key volatile compounds in planta and suggest a future approach to enhancing aromas and flavours in flowers and during fruit ripening.
Asunto(s)
Actinidia/enzimología , Alcoholes/metabolismo , Glicosiltransferasas/metabolismo , Odorantes , Terpenos/metabolismo , Actinidia/metabolismo , Cromatografía Liquida , Cinética , Espectrometría de Masas , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Especificidad por SustratoRESUMEN
Poisonings due to consumption of honeys containing plant toxins have been reported widely. One cause is the neurotoxin tutin, an oxygenated sesquiterpene picrotoxane, traced back to honeybees (Apis mellifera) collecting honeydew produced by passionvine hoppers (Scolypopa australis) feeding on sap of the poisonous shrub tutu (Coriaria spp.). However, a pharmacokinetic study suggested that unidentified conjugates of tutin were also present in such honeys. We now report the discovery, using ion trap LC-MS, of two tutin glycosides and their purification and structure determination as 2-(ß-d-glucopyranosyl)tutin (4) and 2-[6'-(α-d-glucopyranosyl)-ß-d-glucopyranosyl]tutin (5). These compounds were used to develop a quantitative triple quadrupole LC-MS method for honey analysis, which showed the presence of tutin (3.6 ± 0.1 µg/g honey), hyenanchin (19.3 ± 0.5), tutin glycoside (4) (4.9 ± 0.4), and tutin diglycoside (5) (4.9 ± 0.1) in one toxic honey. The ratios of 4 and 5 to tutin varied widely in other tutin-containing honeys. The glycosidation of tutin may represent detoxification by one or both of the insects involved in the food chain from plant to honey.
Asunto(s)
Glicósidos/análisis , Miel/análisis , Picrotoxina/análogos & derivados , Sesquiterpenos/farmacología , Contaminación de Alimentos/análisis , Glicósidos/química , Glicósidos/envenenamiento , Estructura Molecular , Neurotoxinas/sangre , Neurotoxinas/farmacocinética , Resonancia Magnética Nuclear Biomolecular , Picrotoxina/análisis , Picrotoxina/química , Picrotoxina/farmacología , Sesquiterpenos/análisis , Sesquiterpenos/químicaRESUMEN
Previous clinical studies indicate that monoamine oxidase-B (MAO-B) inhibition by blackcurrants must be predominantly attributed to bioactives other than anthocyanins. In this natural products discovery study, MAO-A/B inhibitory phytochemicals were isolated from blackcurrants, and a double-blind crossover study investigated the efficacy of freeze-dried whole-fruit blackcurrant powder in inhibiting MAO-B compared with blackcurrant juice in healthy adults. Platelet MAO-B inhibition was comparable between powder (89% ± 6) and juice (91% ± 4), and it was positively correlated with MAO-modulated plasma catecholamines, subjective alertness, and reduced mental fatigue, assessed using the Bond-Lader questionnaire. Sarmentosin, a nitrile glycoside, and its hydroxycinnamoyl esters were identified as novel MAO-A/B inhibitors from blackcurrant in vitro, and sarmentosin was demonstrated to inhibit platelet MAO-B activity in vivo. These findings confirm sarmentosin as the primary bioactive for MAO-A/B inhibition in blackcurrants, as well as its bioavailability and stability during freeze-drying, and suggest that consuming blackcurrant powder and juice may positively affect mood in healthy adults.
Asunto(s)
Plaquetas , Estudios Cruzados , Inhibidores de la Monoaminooxidasa , Monoaminooxidasa , Extractos Vegetales , Ribes , Humanos , Ribes/química , Adulto , Masculino , Monoaminooxidasa/metabolismo , Inhibidores de la Monoaminooxidasa/farmacología , Inhibidores de la Monoaminooxidasa/química , Extractos Vegetales/química , Extractos Vegetales/farmacología , Femenino , Plaquetas/efectos de los fármacos , Plaquetas/enzimología , Plaquetas/metabolismo , Adulto Joven , Método Doble Ciego , Frutas/química , Persona de Mediana EdadRESUMEN
Heteroxylans in the plant cell wall have been proposed to have a role analogous to that of xyloglucans or heteromannans, forming growth-restraining networks by interlocking cellulose microfibrils. A xylan endotransglycosylase has been identified that can transglycosylate heteroxylan polysaccharides in the presence of xylan-derived oligosaccharides. High activity was detected in ripe fruit of papaya (Carica papaya), but activity was also found in a range of other fruits, imbibed seeds and rapidly growing seedlings of cereals. Xylan endotransglycosylase from ripe papaya fruit used a range of heteroxylans, such as wheat arabinoxylan, birchwood glucuronoxylan and various heteroxylans from dicotyledonous primary cell walls purified from tomato and papaya fruit, as donor molecules. As acceptor molecules, the enzyme preferentially used xylopentaitol over xylohexaitol or shorter-length acceptors. Xylan endotransglycosylase was active over a broad pH range and could perform transglycosylation reactions up to 55 °C. Xylan endotransglycosylase activity was purified from ripe papaya fruit by ultrafiltration and cation exchange chromatography. Highest endotransglycosylase activity was identified in fractions that also contained high xylan hydrolase activity and correlated with the presence of the endoxylanase CpaEXY1. Recombinant CpaEXY1 protein transiently over-expressed in Nicotiana benthamiana leaves showed both endoxylanase and xylan endotransglycosylase activities in vitro, suggesting that CpaEXY1 is a single enzyme with dual activity in planta. Purified native CpaEXY1 showed two- to fourfold higher endoxylanase than endotransglycosylase activity, suggesting that CpaEXY1 may act primarily as a hydrolase. We propose that xylan endotransglycosylase activity (like xyloglucan and mannan endotransglycosylase activities) could be involved in remodelling or re-arrangement of heteroxylans of the cellulose-non-cellulosic cell wall framework.
Asunto(s)
Pared Celular/enzimología , Glicosiltransferasas/metabolismo , Proteínas de Plantas/metabolismo , Plantas/enzimología , Polisacáridos/metabolismo , Secuencia de Aminoácidos , Carica/enzimología , Carica/metabolismo , Pared Celular/metabolismo , Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/metabolismo , Frutas/enzimología , Frutas/metabolismo , Glicosilación , Concentración de Iones de Hidrógeno , Hidrolasas/metabolismo , Cinética , Solanum lycopersicum/enzimología , Solanum lycopersicum/metabolismo , Datos de Secuencia Molecular , Hojas de la Planta/genética , Plantas/metabolismo , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Temperatura , Nicotiana/genética , Xilanos/metabolismoRESUMEN
Cysteine proteases (CPs) accumulate to high concentration in many fruit, where they are believed to play a role in fungal and insect defense. The fruit of Actinidia species (kiwifruit) exhibit a range of CP activities (e.g. the Actinidia chinensis variety YellowA shows less than 2% of the activity of Actinidia deliciosa variety Hayward). A major quantitative trait locus for CP activity was mapped to linkage group 16 in a segregating population of A. chinensis. This quantitative trait locus colocated with the gene encoding actinidin, the major acidic CP in ripe Hayward fruit encoded by the ACT1A-1 allele. Sequence analysis indicated that the ACT1A locus in the segregating A. chinensis population contained one functional allele (A-2) and three nonfunctional alleles (a-3, a-4, and a-5) each containing a unique frameshift mutation. YellowA kiwifruit contained two further alleles: a-6, which was nonfunctional because of a large insertion, and a-7, which produced an inactive enzyme. Site-directed mutagenesis of the act1a-7 protein revealed a residue that restored CP activity. Expression of the functional ACT1A-1 cDNA in transgenic plants complemented the natural YellowA mutations and partially restored CP activity in fruit. Two consequences of the increase in CP activity were enhanced degradation of gelatin-based jellies in vitro and an increase in the processing of a class IV chitinase in planta. These results provide new insight into key residues required for CP activity and the in vivo protein targets of actinidin.
Asunto(s)
Actinidia/genética , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Actinidia/metabolismo , Alelos , Quitinasas/metabolismo , Mapeo Cromosómico , ADN Complementario , Mutación del Sistema de Lectura , Gelatina/metabolismo , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Sitios de Carácter Cuantitativo , Análisis de Secuencia de ADNRESUMEN
Inflammatory bowel disease (IBD) is characterized by intestinal inflammation and is believed to involve complex interactions between genetic, immunological, and environmental factors. We measured changes in the proteome associated with bacterially induced intestinal inflammation in the interleukin 10 gene-deficient (Il10(-/-)) mouse model of IBD, established effects of the dietary polyunsaturated fatty acids (PUFAs) n-3 eicosapentaenoic acid (EPA) and n-6 arachidonic acid (AA) on protein expression (using oleic acid as a control fatty acid), and compared these changes with previously observed transcriptome changes in the same model. Ingenuity pathways analysis of proteomics data showed bacterially induced inflammation was associated with reduced expression of proteins from pathways of metabolism and digestion/absorption/excretion of nutrients/ions, and increased expression of cellular stress and immune response proteins. Both PUFA treatments showed anti-inflammatory activity; EPA appeared to act via the PPARα pathway, whereas AA appeared to increase energy metabolism and cytoskeletal organization and reduce cellular stress responses, possibly enabling a more robust response to inflammation. While there was agreement between proteomic and transcriptomic data with respect to pathways, there was limited concordance between individual gene and protein data, reflecting the importance of having both gene and protein data to better understand complex diseases such as IBD.
Asunto(s)
Colon/efectos de los fármacos , Colon/metabolismo , Grasas Insaturadas en la Dieta/farmacología , Interleucina-10/deficiencia , Proteoma/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Animales , Ácido Araquidónico/metabolismo , Análisis por Conglomerados , Colon/química , Grasas Insaturadas en la Dieta/metabolismo , Ácido Eicosapentaenoico/metabolismo , Perfilación de la Expresión Génica , Inflamación , Interleucina-10/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ácido Oléico/metabolismo , Proteínas , ProteómicaRESUMEN
Inflammatory bowel disease (IBD) is a collective term for conditions characterised by chronic inflammation of the gastrointestinal tract involving an inappropriate immune response to commensal micro-organisms in a genetically susceptible host. Previously, aqueous and ethyl acetate extracts of gold kiwifruit (Actinidia chinensis) or green kiwifruit (A. deliciosa) have demonstrated anti-inflammatory activity using in vitro models of IBD. The present study examined whether these kiwifruit extracts (KFE) had immune-modulating effects in vivo against inflammatory processes that are known to be increased in patients with IBD. KFE were used as a dietary intervention in IL-10-gene-deficient (Il10(-/-)) mice (an in vivo model of IBD) and the C57BL/6J background strain in a 3 × 2 factorial design. While all Il10(-/-) mice developed significant colonic inflammation compared with C57BL/6J mice, this was not affected by the inclusion of KFE in the diet. These findings are in direct contrast to our previous study where KFE reduced inflammatory signalling in primary cells isolated from Il10(-/-) and C57BL/6J mice. Whole-genome gene and protein expression level profiling indicated that KFE influenced immune signalling pathways and metabolic processes within the colonic tissue; however, the effects were subtle. In particular, expression levels across gene sets related to adaptive immune pathways were significantly reduced using three of the four KFE in C57BL/6J mice. The present study highlights the importance of investigating food components identified by cell-based assays with appropriate in vivo models before making dietary recommendations, as a food that looks promising in vitro may not be effective in vivo.
Asunto(s)
Actinidia/química , Colon/efectos de los fármacos , Frutas/química , Interleucina-10/genética , Interleucina-10/metabolismo , Extractos Vegetales/farmacología , Animales , Colon/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Extractos Vegetales/química , Proteínas/clasificación , Proteínas/genética , Proteínas/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Some apple (Malus × domestica Borkh.) varieties have attractive striping patterns, a quality attribute that is important for determining apple fruit market acceptance. Most apple cultivars (e.g. 'Royal Gala') produce fruit with a defined fruit pigment pattern, but in the case of 'Honeycrisp' apple, trees can produce fruits of two different kinds: striped and blushed. The causes of this phenomenon are unknown. RESULTS: Here we show that striped areas of 'Honeycrisp' and 'Royal Gala' are due to sectorial increases in anthocyanin concentration. Transcript levels of the major biosynthetic genes and MYB10, a transcription factor that upregulates apple anthocyanin production, correlated with increased anthocyanin concentration in stripes. However, nucleotide changes in the promoter and coding sequence of MYB10 do not correlate with skin pattern in 'Honeycrisp' and other cultivars differing in peel pigmentation patterns. A survey of methylation levels throughout the coding region of MYB10 and a 2.5 Kb region 5' of the ATG translation start site indicated that an area 900 bp long, starting 1400 bp upstream of the translation start site, is highly methylated. Cytosine methylation was present in all three contexts, with higher methylation levels observed for CHH and CHG (where H is A, C or T) than for CG. Comparisons of methylation levels of the MYB10 promoter in 'Honeycrisp' red and green stripes indicated that they correlate with peel phenotypes, with an enrichment of methylation observed in green stripes. CONCLUSIONS: Differences in anthocyanin levels between red and green stripes can be explained by differential transcript accumulation of MYB10. Different levels of MYB10 transcript in red versus green stripes are inversely associated with methylation levels in the promoter region. Although observed methylation differences are modest, trends are consistent across years and differences are statistically significant. Methylation may be associated with the presence of a TRIM retrotransposon within the promoter region, but the presence of the TRIM element alone cannot explain the phenotypic variability observed in 'Honeycrisp'. We suggest that methylation in the MYB10 promoter is more variable in 'Honeycrisp' than in 'Royal Gala', leading to more variable color patterns in the peel of this cultivar.
Asunto(s)
Antocianinas/biosíntesis , Frutas/fisiología , Malus/genética , Proteínas de Plantas/metabolismo , Factores de Transcripción/metabolismo , Alelos , Antocianinas/análisis , Citosina/metabolismo , Metilación de ADN , Frutas/metabolismo , Galactósidos/análisis , Regulación de la Expresión Génica de las Plantas , Malus/metabolismo , Malus/fisiología , Fenotipo , Pigmentación , Proteínas de Plantas/genética , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Transcripción Genética , Regulación hacia ArribaRESUMEN
Polyphenols within fruits and vegetables may contribute to health benefits due to their consumption, with the anthocyanin sub-set also adding colour. The Lemonade™ apple variety has green skin and white flesh, with low anthocyanin content, while some apple varieties have high anthocyanin content in both the skin and flesh. Effects of red compared with white-fleshed apples were studied in healthy human subjects in a randomized, placebo-controlled, cross-over intervention trial. Twenty-five healthy subjects consumed dried daily portions of the red-fleshed or placebo (white-fleshed) apple for two weeks, followed by one-week washout and further two-week crossover period. During the study, volunteers provided faecal samples for microbiota composition analysis and blood samples for peripheral blood mononuclear cell (PBMC) gene expression analysis. Subtle differences were observed in the faecal microbiota of subjects that were fed the different apples, with significant (p < 0.05) reductions in relative abundances of Streptococcus, Ruminococcus, Blautia, and Roseburia, and increased relative abundances of Sutterella, Butyricicoccus, and Lactobacillus in subjects after consuming the red apple. Changes in PBMC gene expression showed 18 mRNA transcripts were differentially expressed between the two groups, of which 16 were immunoglobulin related genes. Pathway analysis showed that these genes had roles in pathways such as immunoglobulin production, B cell-mediated immunity, complement activation, and phagocytosis. In conclusion, this study shows that anthocyanin-rich apples may influence immune function compared to control apples, with changes potentially associated with differences in the faecal microbiota.
Asunto(s)
Heces/microbiología , Frutas/química , Regulación de la Expresión Génica/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Malus/química , Polifenoles/farmacología , Adulto , Estudios Cruzados , Femenino , Regulación de la Expresión Génica/inmunología , Humanos , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Polifenoles/química , Adulto JovenRESUMEN
Whole kiwifruit ('Hayward' and 'Zesy002') were examined for their bioaminergic potential after being subjected to in vitro gastrointestinal digestion and colonic fermentation. Controls included the prebiotic inulin and water, a carbohydrate-free vehicle. The dopamine precursor l-dihydroxyphenylalanine (L-DOPA) and the serotonin precursor 5-hydroxytryptophan were increased in the kiwifruit gastrointestinal digesta ('Hayward' > 'Zesy002') in comparison to the water digesta. Fermentation of the digesta with human fecal bacteria for 18 h modulated the concentrations of bioamine metabolites. The most notable were the significant increases in L-DOPA ('Zesy002' > 'Hayward') and γ-aminobutyric acid (GABA) ('Hayward' > 'Zesy002'). Kiwifruit increased Bifidobacterium spp. and Veillonellaceae (correlating with L-DOPA increase), and Lachnospira spp. (correlating with GABA). The digesta and fermenta were incubated with Caco-2 cells for 3 h followed by gene expression analysis. Effects were seen on genes related to serotonin synthesis/re-uptake/conversion to melatonin, gut tight junction, inflammation and circadian rhythm with different digesta and fermenta from the four treatments. These indicate potential effects of the substrates and the microbially generated organic acid and bioamine metabolites on intestinal functions that have physiological relevance. Further studies are required to confirm the potential bioaminergic effects of gut microbiota-kiwifruit interactions.
RESUMEN
The groups of plant flavonoid metabolites termed anthocyanins and proanthocyanins (PA) are responsible for pigmentation in seeds, flowers and fruits. Anthocyanins and PAs are produced by a pathway of enzymes which are transcriptionally regulated by transcription factors (TFs) that form the MYB-bHLH-WD40 (MBW) complex. In this study, transcriptomic analysis of purple-pigmented kiwifruit skin and flesh tissues identified MYBC1, from subgroup 5 of the R2R3 MYB family, and WRKY44 (highly similar to Arabidopsis TTG2) as candidate activators of the anthocyanin pathway. Transient over-expression of MYBC1 and WRKY44 induced anthocyanin accumulation in tobacco leaves. Dual luciferase promoter activation assays revealed that both MYBC1 and WRKY44 were able to strongly activate the promoters of the kiwifruit F3'H and F3'5'H genes. These enzymes are branch points of the pathway which specifies the type of anthocyanin accumulated. Stable over-expression of MYBC1 and WRKY44 in kiwifruit calli activated the expression of F3'5'H and PA-related biosynthetic genes as well as increasing levels of PAs. These results suggest that while previously characterised anthocyanin activator MYBs regulate the overall anthocyanin biosynthesis pathway, the PA-related TFs, MYBC1 and WRKY44, more specifically regulate key branch points. This adds a layer of regulatory control that potentially balances anthocyanin and PA levels.
Asunto(s)
Actinidia/metabolismo , Antocianinas/biosíntesis , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/fisiología , Factores de Transcripción/fisiología , Actinidia/clasificación , Actinidia/genética , Secuencias de Aminoácidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Flavonoides/biosíntesis , Frutas/metabolismo , Filogenia , Pigmentos Biológicos/biosíntesis , Hojas de la Planta/metabolismo , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes/metabolismo , Especificidad de la Especie , Nicotiana/metabolismo , TranscriptomaRESUMEN
* High-temperature, low-light (HTLL) treatment of 35S:PAP1 Arabidopsis thaliana over-expressing the PAP1 (Production of Anthocyanin Pigment 1) gene results in reversible reduction of red colouration, suggesting the action of additional anthocyanin regulators. High-performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LCMS) and Affimetrix-based microarrays were used to measure changes in anthocyanin, flavonoids, and gene expression in response to HTLL. * HTLL treatment of control and 35S:PAP1 A. thaliana resulted in a reversible reduction in the concentrations of major anthocyanins despite ongoing over-expression of the PAP1 MYB transcription factor. Twenty-one anthocyanins including eight cis-coumaryl esters were identified by LCMS. The concentrations of nine anthocyanins were reduced and those of three were increased, consistent with a sequential process of anthocyanin degradation. Analysis of gene expression showed down-regulation of flavonol and anthocyanin biosynthesis and of transport-related genes within 24 h of HTLL treatment. No catabolic genes up-regulated by HTLL were found. * Reductions in the concentrations of anthocyanins and down-regulation of the genes of anthocyanin biosynthesis were achieved by environmental manipulation, despite ongoing over-expression of PAP1. Quantitative PCR showed reduced expression of three genes (TT8, TTG1 and EGL3) of the PAP1 transcriptional complex, and increased expression of the potential transcriptional repressors AtMYB3, AtMYB6 and AtMYBL2 coincided with HTLL-induced down-regulation of anthocyanin biosynthesis. * HTLL treatment offers a model system with which to explore anthocyanin catabolism and to discover novel genes involved in the environmental control of anthocyanins.
Asunto(s)
Arabidopsis/fisiología , Ambiente , Pigmentación , Hojas de la Planta/fisiología , Factores de Transcripción/metabolismo , Antocianinas/química , Antocianinas/metabolismo , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis , Biomasa , Análisis por Conglomerados , Flavonoles/química , Flavonoles/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Genes Reguladores , Glicósidos/química , Glicósidos/metabolismo , Luz , Proteínas Asociadas a Pancreatitis , Pigmentación/efectos de la radiación , Hojas de la Planta/efectos de la radiación , Temperatura , Factores de Transcripción/genéticaRESUMEN
Apple extract powders from three different manufacturers were investigated for their anti-inflammatory activity, their total phenolic content, and their chemical composition. The samples represented two production batches for two products and a single batch of a third. The samples showed similar, but clearly different, anti-inflammatory activities, and had substantially different total phenolic contents, and different chemical compositions. Differences in chemical composition for batches of the same product were significant, although not as great as differences between products. The samples were fractionated into chemical classes. The most active fractions were those that contained epicatechin, catechin with phloridzin and quercetin glycosides, or those that contained procyanidin polymers. It was not possible to link activity to the presence of individual components or combinations of these. If fruit extracts are to be reliably linked to validated health benefits, then the source materials, the extraction processes, and the final composition of such products need to be more clearly defined than at present.
Asunto(s)
Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Frutas/química , Malus/química , Fenoles/análisis , Extractos Vegetales/química , Extractos Vegetales/farmacología , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Flavonoides/análisis , Flavonoides/química , Flavonoides/farmacología , Glicósidos/análisis , Glicósidos/química , Glicósidos/farmacología , Inflamación/tratamiento farmacológico , Mediadores de Inflamación/metabolismo , Concentración 50 Inhibidora , Lipopolisacáridos/toxicidad , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Fenoles/química , Fenoles/farmacología , Fitoterapia , Polvos , Reproducibilidad de los ResultadosRESUMEN
Anthocyanins are a group of secondary metabolites that colour fruit and flowers orange, red, purple or blue depending on a number of factors, such as the basic structure, co-pigmentation, metal ion complexation and vacuolar pH. The biosynthesis of anthocyanin is regulated at the transcriptional level by a group of transcription factors, the MYB-bHLH-WD40 (MBW) complex. In this study, the purple colouration in several kiwifruit (Actinidia) species was identified and characterised as red cyanidin-based and blue delphinidin-based anthocyanins. The differential pigmentation in the skin and flesh can be attributed to the differential ratio of cyanidin and delphinidin derivatives accumulated in the total anthocyanin profile. The expression of anthocyanin biosynthetic genes chalcone synthase (CHS), flavonoid 3-O-glucosyltransferase (F3GT), flavonoid 3'-hydroxylase (F3'H) and flavonoid 3'5'-hydroxylase (F3'5'H) is crucial for anthocyanin accumulation. However, the balance of expression of the F3'H and F3'5'H genes appears responsible for the ratio of cyanidin and delphinidin derivatives, while a lack of CHS, F3GT and MYB110 expression is responsible for a lack of total anthocyanins. The transcriptional regulation of the F3'H and F3'5'H promoters by the R2R3 MYB transcription factor MYB110 is markedly different in tobacco transient assays. When kiwifruit MYB10 or MYB110 are over-expressed in Actinidia chinensis both cyanidin-based and delphinidin-based anthocyanins are elevated, but F3'H and F3'5'H genes are not strongly correlated with MYB expression. These results suggest that the core kiwifruit anthocyanin pathway genes are dependent on characterised MYB transcription factors, while other regulatory proteins are more directly responsible for the expression of the F3'H and F3'5'H genes.
RESUMEN
Aim: To evaluate blackcurrant anthocyanin-rich extract (BAE) consumption on time- and dose-dependent plasma anthocyanin bioavailability and conduct a pilot study to explore the potential effect of BAE in promoting recovery from exercise-induced oxidative stress, and maintenance of circulating neutrophil function. Methods: Time- and dose-dependent blackcurrant anthocyanin bioavailability was assessed using LC-MS in 12 participants over 6 h after the ingestion of a placebo or BAE containing 0.8, 1.6, or 3.2 mg/kg total anthocyanins. In a separate pilot intervention exercise trial, 32 participants consumed either a placebo or 0.8, 1.6, or 3.2 mg/kg BAE (8 individuals per group), and then 1 h later performed a 30 min row at 70% VO2max. Blood was collected during the trial for oxidative, antioxidant, inflammatory, and circulating neutrophil status. Results: Consumption of BAE caused a time- and dose-dependent increase in plasma anthocyanins, peaking at 2 h after ingestion of 3.2 mg/kg BAE (217 ± 69 nM). BAE consumed 1 h prior to a 30 min row had no effect on plasma antioxidant status but hastened the recovery from exercise-induced oxidative stress: By 2 h recovery, consumption of 1.6 mg/kg BAE prior to exercise caused a significant (P < 0.05) 34 and 32% decrease in post-exercise plasma oxidative capacity and protein carbonyl levels, respectively, compared to placebo. BAE consumption prior to exercise dose-dependently attenuated a small, yet significant (P < 0.01) transient 13 ± 2% decline in circulating neutrophils observed in the placebo group immediately post-exercise. Furthermore, the timed consumption of either 1.6 or 3.2 mg/kg BAE attenuated a 17 ± 2.4% (P < 0.05) decline in neutrophil phagocytic capability of opsonised FITC-Escherichia coli observed 6 h post-exercise in the placebo group. Similarly, a dose-dependent increase in neutrophil surface expression of complement receptor-3 complex (CR3, critical for effective phagocytosis of opsonised microbes), was observed 6 h post-exercise in both 1.6 and 3.2 mg/kg BAE intervention groups. Conclusions: Consumption of BAE (>1.6 mg/kg) 1 h prior to exercise facilitated recovery from exercise-induced oxidative stress and preserved circulating neutrophil function. This study provides data to underpin a larger study designed to evaluate the efficacy of timed BAE consumption on post-exercise recovery and innate immunity.
RESUMEN
BACKGROUND: Affective responses experienced during exercise are a significant determinant on exercise adherence. We have previously demonstrated that consumption of New Zealand (NZ) blackcurrants preserves cognition by attenuating the feeling of fatigue. This positive affective response correlated with the ability of blackcurrant polyphenols to support monoamine neurotransmission via inhibition of monoamine oxidase-B (MAO-B) activity. Here we explore how the consumption of a NZ blackcurrant juice (BJ) influenced affective responses and potential ergogenic action on the motivation to adhere to a low impact walking exercise. METHODS: In a parallel randomized controlled study (Trial registration #: ACTRN12617000319370p, registered 28th February 2017, http://www.anzctr.org.au/ ), 40 healthy sedentary male and female participants drank a BJ or matched placebo (PLA) (n = 20 per group), 1 h prior to a self-motivated treadmill walk, where heart rate and affective responses (exertion [ES] or feeling / mood [FS]) scores) were recorded at 3 or 5 min intervals. Blood glucose, lactate, malondialdehyde (MDA) and platelet MAO-B activity were measured pre- and post-exercise and comparisons were conducted using with Student's t-tests. Subjective data were analysed using 2-way ANOVA with appropriate post hoc tests. RESULTS: Consuming a BJ 1 h prior to exercise caused a 90% decline in platelet MAO-B activity. The exercise had no significant (p > 0.05) effect on blood lactate, glucose or plasma MDA levels. Assessment of affective responses over the first 60 mins (adjusting for participant drop-out) revealed a time-dependent ES increase in both groups, with ES reported by participants in the BJ group consistently lower than those in the PLA group (p < 0.05). FS declined in PLA and BJ groups over 60 mins, but an inverse relationship with ES was only observed within the PLA group (r2 = 0.99, p = 0.001). Whilst the average time walked by participants in the BJ group was 11 mins longer than the PLA group (p = 0.3), and 30% of the BJ group achieving > 10 km compared to only 10% for the PLA group (p = 0.28), statistical significance was not achieved. CONCLUSION: Our findings demonstrate that drinking a polyphenolic-rich NZ blackcurrant juice 1 h prior to exercise supports positive affective responses during a self-motivated exercise.