Elisabete Carvalho

Study of carbohydrate influence on protein–tannin aggregation by nephelometry

Abstract The formation of polyphenol/BSA aggregates was measured by nephelometry; the amount of insoluble complexes increased with BSA concentration up to a maximum turbidity value but, in the presence of excess protein, a solubilization of the complexes was observed. The stoicheiometry of the complex at the point of maximum light scattering was calculated, yielding a molar ratio, tannin/BSA, of 7:1. Increase in ionic strength by NaCl addition decreased the amount of protein/tannin aggregates, suggesting that hydrophilic forces are the main driving forces in the complexation between BSA and condensed tannins. The influence of the following carbohydrate concentration on the interactions of BSA with procyanidin compounds was assayed using nephelometry: dextran, glucose, arabinogalactan, β-cyclodextrin, pectin, gum arabic, polygalacturonic acid and xanthan. Overall, carbohydrate concentration induced a solubilization of the protein/tannin complexes, with neutral and ionic polysaccharides displaying different behaviours in this process. Pectin, xanthan, polygalacturonic acid and gum arabic were much more effective in solubilizing the protein/tannin aggregates than glucose, dextran, β-cyclodextrin or arabinogalactan.

Carotenoids and tocopherols in yellow and red raspberries

The composition of carotenoids, chlorophyll derivatives and tocopherols in raspberries of different varieties, including yellow and red varieties, over different ripening stages has been studied. The profile of pigments in ripening raspberries changes drastically, with a dramatic decrease of β-carotene and chlorophyll derivatives, the xanthophyll lutein has also decreased but not to the same extent. In contrast esterified lutein increased and is present in ripe raspberries esterified with saturated fatty acids with C8-C16 chains. Ripe raspberries contain considerable amounts of free lutein, esterified lutein, and tocopherols (up to 20, 49 and 366 mg/kg dry weight, respectively). The different samples analysed show different contents of carotenoids and tocopherols. Whether the differences arise from the variety or other factors such as the environmental conditions needs to be ascertained but isoprenoids should not be neglected when considering raspberry antioxidant and nutraceutical composition.

Monochromatic light increases anthocyanin content during fruit development in bilberry.

BackgroundLight is one of the most significant environmental factors affecting to the accumulation of flavonoids in fruits. The composition of the light spectrum has been shown to affect the production of phenolic compounds during fruit ripening. However, specific information on the biosynthesis of flavonoids in fruits in response to different wavelengths of light is still scarce. In the present study bilberry (Vaccinium myrtillus L.) fruits, which are known to be rich with anthocyanin compounds, were illuminated with blue, red, far-red or white light during the berry ripening process. Following the illumination, the composition of anthocyanins and other phenolic compounds was analysed at the mature ripening stage of fruits.ResultsAll the three monochromatic light treatments had significant positive effect on the accumulation of total anthocyanins in ripe fruits compared to treatment with white light or plants kept in darkness. The elevated levels of anthocyanins were mainly due to a significant increase in the accumulation of delphinidin glycosides. A total of 33 anthocyanin compounds were detected in ripe bilberry fruits, of which six are novel in bilberry (cyanidin acetyl-3-O-galactose, malvidin acetyl-3-O-galactose, malvidin coumaroyl-3-O-galactose, malvidin coumaroyl-3-O-glucose, delphinidin coumaroyl-3-O-galactose, delphinidin coumaroyl-3-O-glucose).ConclusionsOur results indicate that the spectral composition of light during berry development has significant effect on the flavonoid composition of ripe bilberry fruits.

High-throughput carotenoid profiling using multivariate curve resolution

We present automated data analysis of high-throughput high-performance liquid chromatography with diode array detection (HPLC-DAD) data using multivariate curve resolution. This technique provides spectra and elution profiles of all UV-Vis active compounds present in the mixture. The specifics of using this method in noninteractive fashion are discussed. A case study on the stability of isoprenoids in grape extracts under two different experimental regimes serves to illustrate the potential of the method: quantitative results clearly show that the addition of triethylamine is beneficial in that carotenoid, chlorophyll, and tocopherol compounds are much more stable and in this way can be kept up to at least 30 days without any sign of degradation.

Carotenoid metabolism during bilberry (Vaccinium myrtillus L.) fruit development under different light conditions is regulated by biosynthesis and degradation

BackgroundCarotenoids are important pigments and precursors for central signaling molecules associated in fruit development and ripening. Carotenoid metabolism has been studied especially in the climacteric tomato fruit but the content of carotenoids and the regulation of their metabolism have been shown to be highly variable between fruit species. Non-climacteric berries of the genus Vaccinium are among the best natural sources of health-beneficial flavonoids but not studied previously for carotenoid biosynthesis.ResultsIn this study, carotenoid biosynthetic genes, PSY, PDS, ZDS, CRTISO, LCYB, LCYE, BCH and CYP450-BCH, as well as a carotenoid cleavage dioxygenase CCD1 were identified from bilberry (V. myrtillus L.) fruit and their expression was studied along with carotenoid composition during fruit development under different photoperiod and light quality conditions. Bilberry was found to be a good source of carotenoids among fruits and berries. The most abundant carotenoids throughout the berry development were lutein and β-carotene, which were accompanied by lower amounts of 9Z-β-carotene, violaxanthin, neoxanthin, zeaxanthin, antheraxanthin and β-cryptoxanthin. The expression patterns of the biosynthetic genes in ripening fruits indicated a metabolic flux towards β-branch of the carotenoid pathway. However, the carotenoid levels decreased in both the β-branch and ε,β-branch towards bilberry fruit ripening along with increased VmCCD1 expression, similarly to VmNCED1, indicating enzymatic carotenoid cleavage and degradation. Intense white light conditions increased the expression of the carotenoid biosynthetic genes but also the expression of the cleavage genes VmCCD1 and VmNCED1, especially in unripe fruits. Instead, mature bilberry fruits responded specifically to red/far-red light wavelengths by inducing the expression of both the carotenoid biosynthetic and the cleavage genes indicating tissue and developmental stage specific regulation of apocarotenoid formation by light quality.ConclusionsThis is the first report of carotenoid biosynthesis in Vaccinium berries. Our results indicate that both transcriptional regulation of the key biosynthetic genes and the enzymatic degradation of the produced carotenoids to apocarotenoids have significant roles in the determination of the carotenoid content and have overall effect on the metabolism during the bilberry fruit ripening.

Anthocyanin biosynthesis in gerbera cultivar ‘Estelle’ and its acyanic sport ‘Ivory’

AbstractMain conclusionIdentification of distinct allelic versions for dihydroflavonol 4-reductase in gerbera cultivars reveals that gerbera DFR enzymes have strong substrate preference in vivo that is not reflected to the activity in vitro. Flavonoids in the model ornamental plant Gerbera hybrida consist of flavones, flavonols and anthocyanins. Anthocyanins accumulate in the adaxial epidermis of petals and give the different cultivars their characteristic red and violet colour. Both pelargonidin and cyanidin derivatives are found in gerbera, but none of the cultivars contain delphinidin. ‘Ivory’, a cultivar with white petals, is a sport of the pelargonidin-containing pink cultivar ‘Estelle’, i.e. it originates from an acyanic branch of ‘Estelle’. In this work, four different alleles encoding dihydroflavonol 4-reductase (DFR) were identified in gerbera cultivars. We found that, in contrast to ‘Estelle’ with the functional allele GDFR1-2, ‘Ivory’ carries a mutation in this gene that results in an inactive enzyme. Interestingly, ‘Ivory’ also expresses a second, nonmutated allele (GDFR1-3) in petal epidermi, leading to extractable DFR activity but not to anthocyanin biosynthesis. The second allele encodes a protein identical in amino acid sequence to the DFR of the cyanidin-containing variety ‘President’. Pelargonidin-containing cultivars do not react to the flavonoid 3′-hydroxylase inhibitor tetcyclacis, but cyanidin-containing cultivars lose their colour, instead of starting to synthesise pelargonidins, indicating the specificity of GDFR1-3 for the cyanidin pathway. This explains why petals of ‘Ivory’ are white, even when it has lost only one of the two enzymatically functional DFR forms, and shows that anthocyanin biosynthesis in gerbera is under more complex regulation than earlier thought.

Metabolite profiling in LC-DAD using multivariate curve resolution: the alsace package for R

For those chemical compounds absorbing in the UV–Vis region and not readily applicable to routine mass spectrometry ionisation methods, liquid chromatography coupled to diode array detection is a convenient platform to perform metabolite profiling. Data processing by hand is labour-intensive and error prone. In the present study a strategy based on multivariate curve resolution, and its implementation in an R package called alsace are described. The final result of an analysis is a table containing peak heights or peak areas for all features of the individual injections. The capabilities of the software, providing elements such as splitting the data into separate, possibly overlapping time windows, merging the results of the individual time windows, and parametric time warping to align features, are illustrated using a cassava-derived data set.

The R2R3MYB VvMYBPA1 from grape reprograms the phenylpropanoid pathway in tobacco flowers

AbstractMain ConclusionThis work shows that, in tobacco, the ectopic expression ofVvMYBPA1, a grape regulator of proanthocyanidin biosynthesis, up- or down-regulates different branches of the phenylproanoid pathway, in a structure-specific fashion. Proanthocyanidins are flavonoids of paramount importance for animal and human diet. Research interest increasingly tilts towards generating crops enriched with these health-promoting compounds. Flavonoids synthesis is regulated by the MBW transcriptional complex, made of R2R3MYB, bHLH and WD40 proteins, with the MYB components liable for channeling the complex towards specific branches of the pathway. Hence, using tobacco as a model, here, we tested if the ectopic expression of the proanthocyanidin regulator VvMYBPA1 from grape induces the biosynthesis of these compounds in not-naturally committed cells. Here, we show, via targeted transcriptomic and metabolic analyses of primary transgenic lines and their progeny, that VvMYBPA1 alters the phenylpropanoid pathway in tobacco floral organs, in a structure-specific fashion. We also report that a modest VvMYBPA1 expression is sufficient to induce the expression of both proanthocyanidin-specific and early genes of the phenylpropanoid pathway. Consequently, proanthocyanidins and chlorogenic acids are induced or de novo synthetised in floral limbs, tubes and stamens. Other phenylpropanoid branches are conversely induced or depleted according to the floral structure. Our study documents a novel and distinct function of VvMYBPA1 with respect to other MYBs regulating proanthocyanidins. Present findings may have major implications in designing strategies for enriching crops with health-promoting compounds.

Nonsense Mutation Inside Anthocyanidin Synthase Gene Controls Pigmentation in Yellow Raspberry (Rubus idaeus L.)

Yellow raspberry fruits have reduced anthocyanin contents and offer unique possibility to study the genetics of pigment biosynthesis in this important soft fruit. Anthocyanidin synthase (Ans) catalyzes the conversion of leucoanthocyanidin to anthocyanidin, a key committed step in biosynthesis of anthocyanins. Molecular analysis of the Ans gene enabled to identify an inactive ans allele in a yellow fruit raspberry (“Anne”). A 5 bp insertion in the coding region was identified and designated as ans+5. The insertion creates a premature stop codon resulting in a truncated protein of 264 amino acids, compared to 414 amino acids wild-type ANS protein. This mutation leads to loss of function of the encoded protein that might also result in transcriptional downregulation of Ans gene as a secondary effect, i.e., nonsense-mediated mRNA decay. Further, this mutation results in loss of visible and detectable anthocyanin pigments. Functional characterization of raspberry Ans/ans alleles via complementation experiments in the Arabidopsis thaliana ldox mutant supports the inactivity of encoded protein through ans+5 and explains the proposed block in the anthocyanin biosynthetic pathway in raspberry. Taken together, our data shows that the mutation inside Ans gene in raspberry is responsible for yellow fruit phenotypes.

Discovery of A-type procyanidin dimers in yellow raspberries by untargeted metabolomics and correlation based data analysis

IntroductionRaspberries are becoming increasingly popular due to their reported health beneficial properties. Despite the presence of only trace amounts of anthocyanins, yellow varieties seems to show similar or better effects in comparison to conventional raspberries.ObjectivesThe aim of this work is to characterize the metabolic differences between red and yellow berries, focussing on the compounds showing a higher concentration in yellow varieties.MethodsThe metabolomic profile of 13 red and 12 yellow raspberries (of different varieties, locations and collection dates) was determined by UPLC–TOF-MS. A novel approach based on Pearson correlation on the extracted ion chromatograms was implemented to extract the pseudospectra of the most relevant biomarkers from high energy LC–MS runs. The raw data will be made publicly available on MetaboLights (MTBLS333).ResultsAmong the metabolites showing higher concentration in yellow raspberries it was possible to identify a series of compounds showing a pseudospectrum similar to that of A-type procyanidin polymers. The annotation of this group of compounds was confirmed by specific MS/MS experiments and performing standard injections.ConclusionsIn berries lacking anthocyanins the polyphenol metabolism might be shifted to the formation of a novel class of A-type procyanidin polymers.