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Characterization of gene expression profile, phenolic composition and antioxidant capacity in red-fleshed grape berries and their wines Qianyu Yue, Lili Xu, Guangqing Xiang, Xin Yu, and Yuxin Yao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01323 • Publication Date (Web): 19 Jun 2018 Downloaded from http://pubs.acs.org on June 20, 2018

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Journal of Agricultural and Food Chemistry

Characterization of gene expression profile, phenolic composition and antioxidant capacity in red-fleshed grape berries and their wines Qianyu Yue, Lili Xu, Guangqing Xiang, Xin Yu and Yuxin Yao*

State Key Laboratory of Crop Biology, Key Laboratory of Biology and Genetic Improvement of Horticultural Crops in Huang-Huai Region, Ministry of Agriculture, College of Horticulture Science and Engineering, Shandong Agricultural University, Tai-An 271018, China

*

Corresponding author

Tel: +86-538-8246258. E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT: Gene expression profile, phenolic composition and antioxidant capacity

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were evaluated in red-fleshed berries and their wines (RF berries and wines) from new grape

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genotypes. Transcriptomic analysis revealed that ten metabolic pathways involved in

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polyphenol synthesis and catabolism were significantly altered, and 13 genes related to the

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biosynthesis and transport of phenolics were largely up-regulated in RF berries compared to

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that of Cabernet Sauvignon (CS). Expression of MybA1 was associated with anthocyanin

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accumulation in red flesh. Additionally, RF berries and wines contained higher concentrations

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of total anthocyanins, phenols, flavonoids and proanthocyanidins than that in CS berries and

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wine. Particularly, diglucosides of malvidin, peonidin, delphinidin and cyanidin were present

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in red flesh and RF wines, but they were undetectable or present at very low concentrations in

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CS flesh and wine. Cinnamic acid and ferulic acid were clearly increased in the RF wines

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compared to that in the CS wine. Additionally, the RF wines had higher antioxidant capacity

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than that in the CS wine, and total anthocyanin content was significantly correlated to

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antioxidant capacity. This research provides insight into the mechanisms underlying grape

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flesh coloration and the composition of phenolic compounds in RF berries and wines.

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KEYWORDS: Red-fleshed grapes, wine, gene expression, phenolic compound, antioxidant

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activity

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1. Introduction

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Red-fleshed (RF) grapes are known as teinturier cultivars and produce wines with deeper

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color. Several RF grape cultivars, such as Morrastrel Bouschet and Jacquez, were produced 2


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via so-called DPHs (direct producer hybrids).1 RF grapes are commonly used for blending to

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give a very dense color to red wine. Recently, RF fruits including grape, apple and peach have

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attracted increasingly more attention from breeders, researchers and consumers because of a

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high amount of anthocyanins with strong antioxidant activity, making them potential

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functional foods.

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Large amounts of phenolic compounds are present in the skin, flesh and seeds of grapes,

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including flavonoids, such as anthocyanins, flavanols and proanthocyanidins, and

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non-flavonoid compounds, such as phenolic acids, stilbenes and resveratrol.2 Anthocyanins

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and other antioxidant phenolic compounds are known to be biologically active and play a key

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role in preventing oxidative damage; nutritionally, they have important roles in human health,

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protecting against cancers, cardiovascular diseases and many other disorders.3,4 High amounts

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of phenolics, particularly anthocyanins, confer grape products and wine high antioxidant

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capacity and nutritional value.4 A great degree of variation in phenolic compound composition

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exists among different grape cultivars; e.g., malvidin derivatives are the most abundant

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anthocyanins in some grape germplasms, whereas cyaniding and delphinidin derivatives are

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the primary components of some other cultivars;5 (+)-catechin and epicatechin are the most

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abundant phenols in Chardonnay grapes,6 whereas Sauvignon Blanc is characterized by high

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levels of gallic and caftaric acid.7 In general, the berry skin provides anthocyanin pigments,

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the flesh provides sugars and acids, and the seed provides condensed tannins; all contribute to

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the formation of the organoleptic characteristics of red wine.8 In most red wine cultivars,

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anthocyanins are present only in the skin, and only low levels accumulate in the flesh. In

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contrast, anthocyanins accumulate in both the flesh and skin of RF grapes. It has been 3



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reported that anthocyanin profiles are very similar in the skin and flesh of Lacryma grapes;9

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the flesh of Garnacha Tintorera almost exclusively accumulates peonidin-3-glucoside;9 the

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total concentrations of anthocyanins in the skin and flesh of Yan-73 are almost equal, but

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there are significant differences in individual anthocyanin compounds.8 No other phenolic

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compounds apart from anthocyanins have been studied in RF grapes. It has been reported that

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significant variation in the content of phenolic compounds is found among RF apple

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cultivars;10 it is worth noting that the flesh of the RF apples contains high amounts of

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chlorogenic acid and phloretin xylosyl glucoside in addition to cyanidin-3-galactoside,

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suggesting that both anthocyanins and some specific phenolic compounds might be regulated

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by the same mutation in RF flesh.10

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Anthocyanins and other antioxidant phenolic compounds, such as chlorogenic acid,

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(+)-catechin, and phloridzin, are biosynthesized through the phenylpropanoid pathway.11 It

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has been shown that Myb transcription factors control fruit skin coloration by regulating

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genes in the phenylpropanoid pathway.12,13 The coloration of grape skin is controlled by

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MybA1 and MybA2, which are closely clustered in a single locus (berry color locus); rare

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mutations in both MybA1 and MybA2 are essential for the genesis of white grapes.14

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Additionally, several other Myb transcription factors participate in the regulation of the

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flavonoid biosynthetic pathway in grapevine. For example, MybF1 promotes flavanol

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production in berry by regulating FLS1 expression, and MybPA1, MybPA2 and Myb5a are

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associated with regulation of proanthocyanidin biosynthesis.13 Recently, it was reported that

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Myb4-like may play a key role in regulating anthocyanin biosynthesis in grapevine by acting

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as a transcriptional repressor of flavonoid structural genes.15 In contrast, the mechanism 4


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underlying the coloration of grape flesh remains largely unknown, although the a comparison

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between the expression of some specific genes, including MybA1, AM3, OMT and others, in

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white vs. red flesh has been done.9,16 It has been shown that the red-flesh trait in apples is

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controlled by Myb10 and Myb110a in type 1 and type 2 RF apples, respectively;17,18

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transcriptomic analysis revealed some novel genes involved in anthocyanin biosynthesis in

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peach flesh19 and elucidated that a key structural gene, AaLDOX, is involved in anthocyanin

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biosynthesis in all RF kiwifruit.19,20

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We obtained six RF candidate grape cultivars through a cross between Yan-73 and

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Cabernet Sauvignon in our previous work. In this study, RNA-Seq was employed to reveal

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comprehensive changes in polyphenol metabolism and identify key genes controlling the

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coloration of berry flesh. The composition of anthocyanin and non-anthocyanin phenolic

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compounds were characterized in RF grape berries as well as in six wines made from RF

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grapes (RF wines). Additionally, the antioxidant activities of the RF wines were evaluated.

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This research provides insight into the underlying mechanisms of berry flesh coloration and

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may promote the application of RF grapes to make red wine with high antioxidant activity.

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2. Materials and methods

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2.1. Grapevine materials. The experiments were performed at an experimental vineyard in

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Tai-An City, Shandong Province, China. Four-year-old Cabernet Sauvignon (CS) and six RF

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candidate cultivars grafted onto rootstock of 101-14M were used in 2016. All the vines were

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planted at a row × vine spacing of 2.5 m × 1.0 m. The yield was controlled to be

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approximately 5.6 ton/acre by limiting the number of fruiting shoots and clusters on each 5



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shoot. Each vine had seven vertical fruiting shoots on the horizontal cordon. Each fruiting

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shoot was controlled to produce two clusters. The experiment was designed with three

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replicates. Each replicate consisted of 12 vines. Berry flesh at 70 days after bloom (DAB) was

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frozen in liquid nitrogen and used for RNA-Seq analysis. When the Total Soluble Solids (TSS)

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of the berry reached 20 °Brix, the berries were harvested for extracting polyphenols and

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small-scale winemaking.

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2.2. Extractions of polyphenols and phenolic compounds. Total anthocyanins, phenols,

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flavonoids, proanthocyanidin and phenolic compounds were extracted using previously

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published method21 with some modifications. One gram of ground skin, flesh, or seeds or one

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milliliter of wine was mixed with 8 ml acidified methanol (0.1% HCl, v/v), sonicated in an

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ultrasonic bath for 15 min, and centrifuged at 8,000 rpm for 15 min. The supernatant was

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collected, and the precipitate was re-extracted with 8 ml of the same solvent two more times.

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The supernatants were combined and then filtered through filter paper. The filtrate was

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evaporated to dryness at 30°C in a rotary evaporator. The residue was dissolved in 5 ml

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chromatographic grade methanol. Part of each extract was directly used for determination of

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total anthocyanins, phenols, flavonoids and proanthocyanidins. The remainder was purified

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through a C18 solid phase extraction (SPE) cartridge (ProElutTM, DIKMA, China) according to

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the manufacturer’s guidelines. Briefly, the cartridges were rinsed with one cartridge filling of

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methanol immediately before use. The samples were passed through the cartridges with the

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help of a vacuum pump at flow rates not exceeding 0.7 ml. s-1. The samples were collected

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into the sample bottles attached to the SPE cartridges. The purified extracts were used for the

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determinations of phenolic compounds. Extractions were performed in three replicates. 6


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2.3. Photometric determination of total anthocyanins, phenols, flavonoids, and

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proanthocyanidins. The total anthocyanin content was determined by the formula OD =

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A530-0.25*A657, and the results were calculated by means of a calibration curve with the

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standard malvidin-3-monoglucoside (Extrasynthese, Lyon, France).22 The total phenol content

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was spectrophotometrically determined using the Folin-Ciocalteu method as previously

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described.23 The results were calculated using a calibration curve made with gallic acid. Total

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flavonoids were determined using a colorimetric method as previously reported;24 rutin was

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used as standard, and the results were calculated using the rutin calibration curve. Total

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proanthocyanidin content was determined using a vanillin assay25 with vanillin as the

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standard.

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2.4. UHPLC-MS analysis of phenolic compounds. Analyses of anthocyanin

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compounds were carried out using ultra-high performance liquid chromatography (UHPLC)

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coupled to quadrupole-time-of-flight mass spectrometry (Q-ToF-MS; Waters Corp., Milford,

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MA, United States). HPLC separation was performed on a reversed-phase C18 analytical

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column (100 mm × 2.1 mm and 1.7 µm particle size, Acquity UPLC BEH C18) at 35°C. The

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parameters and conditions were set according to the method described by Machado et al

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(2015)26 with some modifications. The injection volume was 5 µl, and the detection

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wavelength of the UV detector was set to 530 nm. The mobile phase consisted of A (water:

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formic acid, 100: 3, v/v) and B (acetonitrile: formic acid, 100: 3, v/v) with the following

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gradient: 0–30 min, 0–50% B; 30–35 min, 50–100% B; 35–37 min, 100–0% B. The solvent

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flow rate was set to 0.3 ml/min. Quantitative analysis was carried out using an electrospray

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source operated in positive ionization mode with a scanning range from m/z 50-2000, and the 7



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parameters were set as follows: desolvation gas flow, 12 L/min; desolvation temperature,

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400°C; cone gas flow, 1.2 L/min; source temperature, 100°C; capillary voltage, 3.0 kV; cone

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voltage, 30 V; collision energy, 25.0 eV. The amount of anthocyanins was calculated using an

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external calibration curve made with malvidin-3-monoglucoside (Extrasynthese, Lyon,

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France). The calibration curve equation was listed in Supporting information.

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Analyses of non-anthocyanin compounds were carried out using ultra-high performance

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liquid chromatography (UHPLC) coupled to ESI-triple quadrupole mass spectrometry

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(Dionex Ultimate 3000; Thermo Fisher Scientific, San Jose, CA, USA). HPLC separation was

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performed on a reversed-phase C18 analytical column (100 mm × 2.1 mm and 1.9 µm particle

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size, Thermo Scientific) at 30°C. The main parameters and conditions was set according to

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the method of Nováková et al (2010)27 with some modifications. Injection volume was 5 µl.

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Gradient elution was done using a mobile phase consisting of acetonitrile (solvent A) and 5

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mM ammonium acetate (solvent B) at a flow-rate of 0.3 ml/min: 0-1 min, 0-10% A; 1-3.5 min,

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10-40% A; 3.5-5min, 40-65% A; 5-11 min, 65-90% A; 11-12 min, 90% A; 12-12.1 min, 90-10%

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A; 12.1-14min, 10% A. Negative ion mode was used for the ESI (capillary voltage 3.0 kV, ion

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source temperature 300°C), desolvation temperature was 400°C, cone gas flow was 0.2 L/min,

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source temperature was 100°C, cone voltage was 35 V, and the collision energy was 25.0 eV.

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The amount of seven compounds (gallic acid, (+)-catechin, epicatechin, caffeic acid, coumaric

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acid, myricetin and isoquercetin) was calculated using external calibration curves of the

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corresponding standards (ChromaDex, Irvine, CA, USA). The calibration curve equations

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were listed in Supporting information. The results of chlorogenic acid, syringic acid, ferulic

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acid and cinnamic acid were expressed as gallic acid equivalents by means of the calibration 8


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curve with standard gallic acid.

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2.5. Assays of antioxidant capacities. Free radical scavenging capacity of DPPH was

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determined according to the method of Katalinić et al. (2010).28 The amount of antioxidant

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necessary to decrease the initial DPPH concentration by 50% was defined as EC50, which

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was calculated using a gallic acid calibration curve. Antiradical activity was defined as

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1/EC50. For the ABTS assay, a previously reported procedure was used.29 The radical

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scavenging activity was expressed as Trolox equivalent antioxidant capacity. Ferric reducing

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antioxidant power (FRAP) was determined according to the method of Sun et al. (2011).30

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The radical scavenging activities were also expressed as Trolox equivalent antioxidant

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capacity.

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2.6. Small-scale wine making. Small-scale winemaking was performed in triplicate (35 kg

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of berries for each replicate). CS and RF1-6 berries were squeezed in a squeezing roller, and

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the grape must was transferred to 50-liter stainless steel fermentation tanks. The must was

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treated with sulfur dioxide (80 mg/L) prior to undergoing fermentation at 25°C. The wine cap

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was punched down twice daily until it remained submerged. After 10 days of maceration

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when fermentation was finished, the wine residue was pressed. Free-run and press wines were

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combined and stored in vessels at 15°C. After one month of storage, sulfur dioxide (30 mg/L)

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was added a second time, and the wines were cold-stabilized for one month at 4°C. Finished

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wines were filtered and bottled for subsequent storage.

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2.7. RNA-Seq and Quantitative Real-time PCR (qRT-PCR). Total RNA of flesh was

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extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, United States), and mRNA was

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purified using poly-T oligo-attached magnetic beads. Sequencing libraries were constructed 9



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using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (#7530L, NEB, United States).

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Then, RNA concentration and insert size in the sequencing library were assessed, and

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clustering of the index-coded samples was performed on a cBot cluster generation system

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using HiSeq PE Cluster Kit v4-cBot-HS (Illumina). After cluster generation, the libraries were

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sequenced on an Illumina Hiseq 4000 platform, and 150-bp paired-end reads were produced.

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Clean reads were assembled into transcripts using Cufflinks referencing the grape genome

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(http://genomes.cribi.unipd.it/grape/). The unigene expression levels were quantified using

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reads per fragments per kilobase of transcript per million mapped reads (RPKM). Unigenes

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that were differentially expressed between two samples were screened using false discovery

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rate

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