Article pubs.acs.org/JAFC
Tracing Phenolic Metabolism in Vitis vinifera Berries with 13 C6‑Phenylalanine: Implication of an Unidentified Intermediate Reservoir Alexander W. Chassy,† Douglas O. Adams, and Andrew L. Waterhouse* Department of Viticulture and Enology, University of California, Davis, California 95616, United States S Supporting Information *
ABSTRACT: Understanding the regulation of phenolic compounds in agricultural products has been a topic of great interest. In V. vinifera berries, phenolics are responsible for important sensory and functional characteristics. To elucidate the ripening profile of phenolic compounds in Cabernet Sauvignon berries, the stable-isotope tracer L-phenyl-13C6-alanine (Phe13) was incorporated in situ, and the development of labeled and unlabeled phenolics was tracked in the vineyard at different stages of maturity over two vintages. Phenolic profiles during ripening were consistent with previous research. However, individual anthocyanins accumulated with different profiles during ripening; malvidin species continually climbed in concentration, whereas other anthocyanins tended to plateau or drop near the end of the growing season. The isotopic label was predominantly incorporated into anthocyanins, presumably because of their dominant accumulation during ripening. Notably, the incorporation of label continued long after levels of Phe13 had dropped to below 1 nmol/berry, preventing an accurate assessment of the hypothesized turnover of anthocyanins. Although our tracer did not perform exactly as we had expected, the results of this study suggest the presence of a previously unreported pool of substrate in the phenolic pathway. KEYWORDS: phenolamides, Cabernet Sauvignon, stable isotope, phenolic metabolism, anthocyanin, catabolism
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Whereas flavonoids are produced throughout the flowering and growth of red grapes, anthocyanins begin to accumulate only at the onset of ripening, known as veraison. At this time, the enzyme UDP glucose:flavonoid-3-O-glucosyltransferase (UFGT) is expressed, attaching glucose to anthocyanidins. Prior to veraison, all of the anthocyanin pathway genes except UFGT are highly expressed, and it is only after veraison that UFGT is also induced and anthocyanins begin to accumulate. The preveraison activity of anthocyanin pathway genes is reflected by the production of polymeric flavan-3-ols as well as flavonols prior to the onset of ripening.11 Total grape anthocyanin concentrations tend to climb steadily throughout ripening and either plateau or drop slightly near harvest;7,10,12 however, grape anthocyanin concentrations and profiles are dependent upon genotype (cultivar) as well as environmental conditions such as climate and agricultural conditions.13 In particular, temperature plays an important role in both the quantity and composition of grape anthocyanins. High-temperature climates have long been associated with fewer pigments; however, the impact and mechanisms are poorly understood.14 Originally, researchers hypothesized that temperature was negatively correlated with transcription of anthocyanin biosynthetic genes. Although higher temperatures were associated with less anthocyanin pathway gene expression, detectable levels of mRNA were still present, whereas anthocyanin concentrations would cease to accumulate.11
INTRODUCTION Phenolic compounds are ubiquitous in flora. Despite diverse structure and function, virtually all phenolic metabolites stem from phenylalanine. This single carbon backbone presents an ideal experimental system for the introduction of isotopic label because it will be incorporated into a multitude of metabolites. Indeed, this branch point has been vital in understanding the network as a whole.1,2 With advancements in technology has come unprecedented ability to track isotopic labels, particularly using mass spectrometry. Phe-derived Vitis vinifera grape berry phenolics include anthocyanins, hydroxycinnamates, flavonols, flavan-3-ols (proanthocyanidins and condensed tannins), and stilbenes (the last two mentioned will not be discussed). Phenylpropanoid classes are uniquely regulated throughout maturation. Early in berry development, following flowering, comes the production of hydroxycinnamates in the pulp present as tartaric acid esters. These phenolics are reported to accumulate early and then drop precipitously in concentration at the onset of ripening. 3−5 The final fate of the hydroxycinnamate carbon remains unknown. Grape flavonol regulation is specific for the degree of hydroxylation as well as glycosylation. Flavonols in grapes are produced in response to light, particularly the UV spectrum, as well as other environmental factors.6−8 Flavonols do not seem to amass in large quantities until after the onset of ripening;9 however, individual flavonol glycosides may be differentially regulated during maturation.10 For example, the glucoside and glucuronide conjugates of quercetin had distinct, but consistent, profiles during ripening; the quercetin-3-glucoside gradually rose after the onset of ripening while quercetin-3-glucuronide slowly declined.10 © 2014 American Chemical Society
Received: Revised: Accepted: Published: 2321
August 19, 2013 February 25, 2014 February 25, 2014 March 10, 2014 dx.doi.org/10.1021/jf402229u | J. Agric. Food Chem. 2014, 62, 2321−2326
Journal of Agricultural and Food Chemistry
Article
to separate phenolics on an Agilent Zorbax SB-C18 Rapid Resolution HT small-particle column (4.6 mm × 100 mm, 1.8 μm) with a precolumn filter. The diode array detector (DAD) monitored four wavelenths: 280, 316, 365, and 520 nm. The mass spectrometer acquired data using “dynamic MRM” mode so it was possible to measure positively and negatively ionized isotopomers during the same run. Specific retention times and MRM transitions for each isotopomer can be found in our previous publication.19 Quantitation of total phenolic levels was achieved through external standardization by UV−vis absorbance using representative standards for each class of compounds (caffeic acid for hydroxycinnamates, rutin for flavonols, and malvidin-3-glucoside for anthocyanins). The total concentration was then multiplied by the ratio of labeled to unlabeled phenolics to determine their respective concentrations. External standards of Phe and Phe13 were used for quantitation by LC-MRM-MS (166.3 → 120 and 172.3 → 126, respectively). The graphical heatmaps in Figures 2 and 3 were created using the open source Excel add-in imDev.23 Statistical Analysis. Comparisons of each metabolite over time were analyzed using R (version 3.0.2). A one-way ANOVA with p values adjusted for multiple hypothesis testing at q = 0.05 was used to determine significant changes over time for each metabolite in red and green berries from 2010 and 2011.24
This led researchers to monitor stable isotope labeled anthocyanins in grapes cultured in vitro at high and low temperatures, demonstrating that high temperatures resulted in anthocyanin degradation.15 Increased temperature has been shown to alter the profile of anthocyanins, resulting in an increase of trihydroxylated as well as acetylated and coumarylated moiety proportions.16,17 At present, a clear understanding of temperature’s influence on individual anthocyanin regulation and/or degradation is still lacking. The perceived quality of red grapes for winemaking is highly dependent on the resulting pigmentation of the final wine.18 Although color is not the only factor, red grapes grown in warm growing regions such as the central valley of California, Riverlands in Australia, or Languedoc in France have only moderate color and command prices that are significantly lower than those grown in cooler regions. With an increased understanding of how warm climates affect anthocyanin concentrations, it may be possible to improve the pigmentation of warm-region grapes. An understanding of the mechanisms that control color loss as suggested below may lead to treatments to preserve color in warmer climates. To address the need for following the metabolic pathway of anthocyanins in grape berries, a method was recently reported that utilizes a distinct isotopic label.19 Tracing this label can provide a means to follow the appearance and loss of substances in the phenylpropanoid pathway as well as how environmental factors may influence the pathway. Anthocyanin turnover has been shown in Brunfelsia calycina flowers and in mustard seedlings;20,21 however, the prevention of anthocyanin degradation has only recently been targeted for color improvement in agricultural crops.22 Anthocyanin degradation in grapes has been demonstrated, but only using an in vitro model on Petri dishes.15 The current study utilizes Lphenyl-13C6-alanine (Phe13) as a tracer to investigate flavonoid metabolism and catabolism in V. vinifera cv. Cabernet Sauvignon berries on the vine throughout maturation during two growing seasons to begin to address the color issue in warm-climate grapes.
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RESULTS AND DISCUSSION Using our recently developed technique to incorporate stable isotopic label into grape flavonoids in situ, we sought to probe the evolution and turnover of grape phenolics throughout maturation.19 Multiple classes of Phe-derived phenolics were monitored throughout the maturation process over two vintages, providing a rare glimpse of overall phenolic metabolism. Furthermore, using the Phe13 tracer, we hoped to shed light on several facets of grape phenolic metabolism: the disappearance of hydroxycinnamates, the different patterns of flavonol biosynthesis, and the turnover or degradation of anthocyanins. Although our tracer did not perform exactly as we had expected, the deviations from our expectations suggest there are even more mysteries in phenolic substrate regulation. Levels of grape phenolics were monitored throughout the 2010 and 2011 growing seasons starting on July 29 and July 7, respectively. In addition, the stable isotopic tracer Lphenyl-13C6-alanine was introduced to berry clusters on the vine two times during each season to observe the incorporation of label at two stages of maturity: green preripened and red ripening grapes. Following each of the four treatments, the concentrations of both unlabeled and labeled phenolic metabolites were monitored throughout the ripening season. Berry variability was generally below 15, 20, or 15% (relative standard error) for unlabeled hydroxycinnamates, flavonols, and anthocyanins at concentrations above 10 nmol/berry, respectively (Supporting Information file 1). Phenylalanine Tracer. Phenylalanine levels ranged from 7.3 to 57.2 nmol/berry in 2010 and from 12.0 to 112.2 nmol/ berry in 2011 in the red and green grape trials (Figure 1b). After the treatment of the clusters, Phe13 was readily found in the grape berries, and its gradual decline typically followed within 2 weeks. However, in all four trials a steady, but very low, level of Phe13 persisted until the end of the season, in one case 89 days after the treatment was concluded (Figure 1b). In both years more Phe13 was absorbed into red berries, although we cannot explain why this occurred. Phenolic Metabolites during Maturation. Hydroxycinnamate tartrate esters were already present in green grapes prior to treatment and declined precipitously after the onset of ripening. Caftaric, cis-coutaric, and trans-coutaric acids dropped to
