Phenotypic changes associated with RNA interference silencing of chalcone synthase in apple (Malus × domestica).

We have identified in apple (Malus × domestica) three chalcone synthase (CHS) genes. In order to understand the functional redundancy of this gene family RNA interference knockout lines were generated where all three of these genes were down-regulated. These lines had no detectable anthocyanins and radically reduced concentrations of dihydrochalcones and flavonoids. Surprisingly, down-regulation of CHS also led to major changes in plant development, resulting in plants with shortened internode lengths, smaller leaves and a greatly reduced growth rate. Microscopic analysis revealed that these phenotypic changes extended down to the cellular level, with CHS-silenced lines showing aberrant cellular organisation in the leaves. Fruit collected from one CHS-silenced line was smaller than the 'Royal Gala' controls, lacked flavonoids in the skin and flesh and also had changes in cell morphology. Auxin transport experiments showed increased rates of auxin transport in a CHS-silenced line compared with the 'Royal Gala' control. As flavonoids are well known to be key modulators of auxin transport, we hypothesise that the removal of almost all flavonoids from the plant by CHS silencing creates a vastly altered environment for auxin transport to occur and results in the observed changes in growth and development.

Fecal microbial metabolism of polyphenols and its effects on human gut microbiota.

We investigated the biotransformation of four common dietary polyphenols, rutin, quercetin, chlorogenic acid and caffeic acid, in an in vitro mixed culture model of human intestinal microbiota, to determine effects on human gut bacteria. All four compounds were biotransformed rapidly, disappearing from the medium within 0.5 h and later replaced by known phenolic acid breakdown products, at concentrations up to hundreds of micromolar, much higher than in no-polyphenol control experiments. Quantitative PCR was used to measure effects of the polyphenols on the balance between the major groups of intestinal bacteria that are known to influence gut health, i.e., Bifidobacterium spp., Bacteroidetes, and Firmicutes. Fermentation of polyphenols stimulated proliferation of bifidobacteria and decreased the ratio of Firmicutes to Bacteroidetes, relative to controls. Polyphenols also stimulated short chain fatty acid production by the bacteria. Pure bifidobacterial cultures were treated separately with either fermented media isolated from the incubations, the pure test polyphenols, or the biotransformation products detected in the fermentations. Growth stimulation was observed only with fermented polyphenol media and the pure biotransformation products. It appears that dietary polyphenols may have the ability to modify the gut microbial balance, but this effect is indirect, i.e., it is mediated by biotransformation products, rather than the original plant compounds.

Apple skin patterning is associated with differential expression of MYB10.

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.

The potential influence of fruit polyphenols on colonic microflora and human gut health.

The effect of common dietary polyphenols on growth of human gut bacteria and their adhesion to enterocytes was investigated. The influence on the growth of a probiotic (Lactobacillus rhamnosus), a commensal (Escherichia coli) and two pathogenic bacteria (Staphylococcus aureus, Salmonella typhimurium) was determined, together with effects on adhesion of pathogenic and probiotic bacteria to cultured Caco-2 cells. All polyphenols, except rutin, were found to affect the viability of representative gut flora in vitro, at doses likely to be present in the gastrointestinal tract, but to differing degrees. Naringenin and quercetin were the most active with the lowest minimum inhibitory concentrations for all the four bacteria tested. The remaining polyphenols had the most marked effect on the Gram positive enteropathogen S. aureus. Naringenin and phloridzin were the most effective inhibitors of S. typhimurium adherence to Caco-2 enterocytes while phloridzin and rutin enhanced the adherence of the probiotic L. rhamnosus. Polyphenols appear to have potential to alter gut microecology and, by affecting the total number of beneficial microflora in the gut, may confer positive gut health benefits.

Comparison of enzymically glucuronidated flavonoids with flavonoid aglycones in an in vitro cellular model of oxidative stress protection.

This study modeled, in vitro, the potential effect of conjugative (phase II) metabolism on the cytoprotective capacity of fruit flavonoids against oxidative stress. Flavonoid aglycones were compared with their corresponding isomeric mixtures of glucuronides for their ability to enhance the survival of cultured human Jurkat T and neuroblastoma cells stressed with hydrogen peroxide. Various polyphenolic compounds were tested as substrates in vitro for an ovine liver glucuronyl transferase preparation. Flavonoids and their glycoside derivatives were found to be good substrates, whereas phenolic acids were either poor or nonsubstrates. Five common flavonoids were glucuronidated to prepare mixtures for bioassay testing. Glucuronidation generally weakened the cytoprotective capacities of flavonoids (in the presence of H(2)O(2)), but some compounds were weakened much more than others. The concentration that halved cell death was well below 0.5 microM for most flavonoids tested, but glucuronidation increased median effective concentration values to a range of 1-16 microM. This compares with the generally accepted physiological range (0.1-10 microM) for circulating dietary polyphenolics detected in the body. Therefore, some flavonoids may retain a reduced cytoprotective capacity in vitro, after glucuronidation, whereas others may be effectively inactivated.

Preparative enzymatic synthesis of glucuronides of zearalenone and five of its metabolites.

The resorcylic acid lactones zearalenone ( 1), alpha-zearalenol ( 2), beta-zearalenol ( 3), alpha-zearalanol (zeranol) ( 4), beta-zearalanol (taleranol) ( 5), and zearalanone ( 6) were converted to their glucuronides on a preparative scale in good yields. Reactions were conducted with bovine uridine 5'-diphosphoglucuronyl transferase (UDPGT) as catalyst and uridine 5'-diphosphoglucuronic acid (UDPGA) as cofactor. The glucuronides were isolated by column chromatography and characterized by NMR spectroscopy and mass spectrometry. Although the principal products were 4- O-glucuronides (i.e., linkage through a phenolic hydroxyl), significant quantities of the 6'- O-glucuronides (i.e., linkage through the aliphatic hydroxyl) of alcohols 2, 4, and 5 were also isolated. In the case of 3, the 2- O-glucuronide was isolated as the minor product. Overall isolated yields of glucuronides, performed on a 20-50 mg scale, were typically ca. 80% based on the resorcylic acid lactone starting material. LC-UV-MS (2) analysis of purified specimens revealed MS (2) fragmentations useful for defining the point of attachment of the glucuronide moiety to the zearalenone nucleus.

Comparison of the relative recovery of polyphenolics in two fruit extracts from a model of degradation during digestion and metabolism.

To simulate the effects of digestion and metabolism on the survival of different polyphenolic compounds, extracts of blueberry and apple were deglycosylated by acid hydrolysis, followed by enzymic glucuronidation under neutral conditions, yielding approximately 5% overall recovery of polyphenolics. The major polyphenolics before and after the treatment were compared, to estimate which species are likely to be present in the intestinal lumen, undegraded and available for absorption, after consumption of the fruit. Whereas blueberry extract consisted predominantly of anthocyanins, epicatechin and caffeoyl quinate esters, the major components of the treated extract were quercetin glucuronides and (unglucuronidated) caffeoyl quinates, with only traces of anthocyanidin derivatives. In apple extract, compositional changes were less marked, but caffeoyl quinates, procyanidins and quercetin were enriched at the expense of caffeic acid, epicatechin and catechin. Hydrophobic compounds like phloretin and quercetin were extensively glucuronidated, whereas caffeic acid and caffeoyl quinate were not. These results suggest that the major polyphenolic components of a fruit are not necessarily the most important contributors to any health benefits because the polyphenolic composition in the intestinal lumen and consequently, in the circulation, may be considerably different.

Methylated polyphenols are poor "chemical" antioxidants but can still effectively protect cells from hydrogen peroxide-induced cytotoxicity.

Several polyphenolic compounds, including flavonoids and phenolic acids, were compared with their per-methylated forms in both chemical and cell-based assays for antioxidant capacity. Methylation largely eliminated "chemical" antioxidant capacity, according to ferric reducing antioxidant power and oxygen radical absorbance capacity assays. Methylation, however, only moderately reduced protection of human Jurkat cells in culture, from hydrogen peroxide-mediated cytotoxicity, at physiologically relevant concentrations. Neither methylated nor un-methylated compounds were detectably metabolized by the cells. It appears that the protective mechanism of polyphenolic antioxidants against high concentrations of hydrogen peroxide in human cells may be largely unrelated to chemical antioxidant capacity.

Structural analysis of carrageenans from the red alga, Callophyllis hombroniana Mont. Kütz (Kallymeniaceae, Rhodophyta).

The use of a range of modern analytical techniques has facilitated the structural characterisation of the polysaccharide from the New Zealand endemic red alga, Callophyllis hombroniana. The native polysaccharide contains a number of structural units with the largest proportion consisting of 3-linked beta-D-galactopyranosyl 2-sulfate units, alternating with 4-linked 3,6-anhydro-alpha-D-galactopyranosyl 2-sulfate units, that is, theta-carrageenan (36 mol%). C. hombroniana is the first red seaweed reported to naturally contain such a large proportion of theta-carrageenan.

Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10.

Anthocyanin concentration is an important determinant of the colour of many fruits. In apple (Malus x domestica), centuries of breeding have produced numerous varieties in which levels of anthocyanin pigment vary widely and change in response to environmental and developmental stimuli. The apple fruit cortex is usually colourless, although germplasm does exist where the cortex is highly pigmented due to the accumulation of either anthocyanins or carotenoids. From studies in a diverse array of plant species, it is apparent that anthocyanin biosynthesis is controlled at the level of transcription. Here we report the transcript levels of the anthocyanin biosynthetic genes in a red-fleshed apple compared with a white-fleshed cultivar. We also describe an apple MYB transcription factor, MdMYB10, that is similar in sequence to known anthocyanin regulators in other species. We further show that this transcription factor can induce anthocyanin accumulation in both heterologous and homologous systems, generating pigmented patches in transient assays in tobacco leaves and highly pigmented apple plants following stable transformation with constitutively expressed MdMYB10. Efficient induction of anthocyanin biosynthesis in transient assays by MdMYB10 was dependent on the co-expression of two distinct bHLH proteins from apple, MdbHLH3 and MdbHLH33. The strong correlation between the expression of MdMYB10 and apple anthocyanin levels during fruit development suggests that this transcription factor is responsible for controlling anthocyanin biosynthesis in apple fruit; in the red-fleshed cultivar and in the skin of other varieties, there is an induction of MdMYB10 expression concurrent with colour formation during development. Characterization of MdMYB10 has implications for the development of new varieties through classical breeding or a biotechnological approach.