From Flavanols Biosynthesis to Wine Tannins - American Chemical

Subscriber access provided by United Arab Emirates University | Libraries Deanship

Review

From flavanols biosynthesis to wine tannins: what place for grape seeds? Pauline ROUSSERIE, Amélie RABOT, and Laurence GENY-DENIS J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05768 • Publication Date (Web): 11 Jan 2019 Downloaded from http://pubs.acs.org on January 11, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 73

Journal of Agricultural and Food Chemistry

From flavanols biosynthetis to wine tannins: what place for grape seeds? Pauline ROUSSERIE *, Amélie RABOT, Laurence GENY-DENIS Université de Bordeaux, Unité de recherche Œnologie, EA 4577, USC 1366 INRA, ISVV, 33882 Villenave d’Ornon Cedex, France Pauline ROUSSERIE : [email protected] - +(33) 6.31.93.57.46

1 ACS Paragon Plus Environment


Page 2 of 73

1

ABSTRACT

2

Phenolic compounds are among the most important quality factors of wines. They contribute

3

to the organoleptic characteristics of wine such as colour, astringency and bitterness

4

Although tannins found in wine can come from microbial and oak sources, the main sources

5

of polyphenols are grape skins and seeds 8. Since the 1960s 9, this subject has been widely

6

studied by a large number of researchers covering different types of wine, climate conditions,

7

growing practices, grape varieties

8

conditions, the data collected can be conflicting. Moreover, even though the biosynthesis of

9

the major proanthocyanidins units (+)-catechin and (-)-epicatechin is well known, the

10

mechanism of their polymerization remains unexplained. This is why the question remains:

11

what factors influence the biosynthetis, the quantity and the distribution of tannins in grape

12

seeds and how can winemaking processes impact the extractability of seed tannins in wine?

13

Keywords: Grape, Vitis vinifera, seed, tannin, proanthocyanidin, phenolic metabolism, wine

10.

1–7.

As these works have been conducted under different

14


Page 3 of 73


15

INTRODUCTION

16

Tannins, also called condensed tannins or proanthocyanidins are polyphenolic compounds

17

widely found in the plant kingdom. They are essentially secondary metabolites produced by

18

plant for their adaptation and protection to biotic and abiotic stresses

19

phenolics contributes to wine quality and have beneficial effects of human health many

20

studies have attempted to improve knowledge of their chemistry and biosynthesis in order to

21

better understand their roles in vine physiology and wine quality.

22

In wine, the main sources of tannins are skin and seed berry. Initially, berry skins have greater

23

practical importance because the skin tannins appeared to be more easily extracted in the must

24

during the winemaking than the seeds one. Despite this fact, and even though seeds represent

25

only 0 to 6% of the berry weight, they are a large source of phenolic compounds, from 20 to

26

55% of the total grape polyphenol content

27

projects have been conducted on the composition and the biosynthesis of skins tannins, this

28

review focuses on seeds tannins 16–28.

29

Depending on grape tissue and developmental stage of the berry, the quantity and the

30

structure of proanthocyanidins (PA) differ

31

more polymerized and less galloylated than the seed one 30,31,33–40.

32

To go further, berry development can be divided into two successive sigmoidal growth period

33

separated by a lag phase

34

approximatively 60 days later. During this period, the berry is formed and the seed embryos

35

are produced. Tannins are accumulated; they are present in skins and seeds and reach

36

maximum quantity at veraison. The second period begins at the onset of ripening or veraison

37

and finishes at harvest time. This period is characterized by the softening and coloring of the

41.

14,15.

29–34.

11–13.

Since berry

As a large number of studies and research

Indeed, generally skin tannins appear to be

The first growth period begins at bloom and finishes



38

berry. Generally, there is a decline in seed tannins during ripening that accompanies seed

39

browning due to tannin oxidation.

40

The importance of grape seed in wine is related to their ability to improve wine structure and

41

to enhance ageing potential, but also to their ability to protect wine against oxidation and to

42

stabilize wine colour 42–46. This is why, an optimal extraction of those compounds is crucial to

43

ensure wine quality. The diffusion of seed tannins from grape to must, their extractability and

44

final concentration largely depends on their tissue location and the processes used during the

45

vinification 15,47–58.

46

Despite the importance of grape seed tannins in wine, at this time no reliable tools exist to

47

determine the phenolic maturity of grape seeds. In this context, it appears necessary to gain

48

more insight on the contribution of grape seeds tannins in wine. The answer of this question

49

obviously requires an improvement of knowledges on (i) the phenolic metabolism of grape

50

seeds, in other words the impact of phenolic metabolism on the seed tannins composition,

51

structure and extractability, and (ii) the impact of winemaking processes on seed tannins

52

extraction. For that, a literature review has been conducted on the grape berry tannins

53

structure at harvest and the biosynthetic pathway of PAs. Secondly, studies of the evolution of

54

grape seeds tannins among berry development and of winemaking processes known to

55

enhance their wine concentration are highlighted.

56

GRAPE BERRY TANNINS STRUCTURE: A FOCUS AT HARVEST

57

TIME

58

Chemically, polyphenols can be defined by the composite parts of the word itself: “phenol”,

59

which means that these compounds have a single aromatic ring containing one or more

60

hydroxyl groups, and “poly”, which means that they have multiple rings in the structure.


Page 4 of 73

Page 5 of 73


61

Phenolic compounds are classified into four major classes: stilbenes, lignans, phenolic acids

62

and flavonoids. We focused here on this last class of polyphenols compounds: the flavonoids.

63

The general structure of flavonoids structure is defined by a specific three-ring system: a

64

central oxygen-containing pyran ring (C ring) fused to an aromatic ring (A ring) along one

65

bond and attached to another aromatic ring with a single bond (B ring)

66

than 6000 different flavonoids have been identified and listed. Each of these flavonoids is

67

classified into groups. Within these groups, flavonoids differ by the number and the position

68

of the substitution groups (hydroxylation, hydrogenation, methylation, glycosylation,

69

malonylation and sulfation). The main flavonoid classes are flavonols, flavones, flavanones,

70

flavanols, anthocyanidins, isoflavones, dihydroflavonols and chalcones 60.

71

Flavanols, also named flavan-3-ols because of the presence of a hydroxyl group at 3-position

72

of the C ring, are the most reduced form of flavonoids

73

according to the stereochemistry and the hydroxylation of the C ring as well as the number of

74

hydroxyl groups on the B ring. The presence of two chiral centres on the molecule (C2 and

75

C3) allows a single flavan-3-ols to have four possible configurations, thus four possible

76

diastereoisomers (figure 2).

77

In wine as well as in grapes, flavan-3-ols are found in monomeric form, dimeric form,

78

oligomeric form (3 to 10 units of flavan-3-ols) and polymeric form (more than 10 units of

79

flavan-3-ols). The principal forms of flavan-3-ols found in wine and grapes are summarised in

80

table 1.

81

Tannins, also named condensed tannins or proanthocyanidins, result from the polymerisation

82

of flavan-3-ols units. Their structures depend on the flavan-3-ols starter and extension units,

83

the position and the stereochemistry of the linkage to the lower units, the degree of

84

polymerisation, and the presence or absence of modifications of the 3-hydroxyl group. The

59.


59

(figure 1). More

Structurally, flavan-3-ols differ


Page 6 of 73

85

most commonly found monomeric units in PAs are (2,3-trans)-(±)-catechin and (2,3-cis)-(±)-

86

epicatechin. The PA classification is based on the nature of monomeric derivatives released

87

after acid-catalysed reaction upon heating in an alcohol solution. In wine and grapes, two

88

predominant classes of PAs are found: prodelphinidin, composed of (2-3-trans)-(+)-

89

gallocatechin and (2-3-cis)-(-)-epigallocatechin subunits, and procyanidin, composed of (2-3-

90

trans)-(+)-catechin and (2-3-cis)-(-)-epicatechin subunits.

91

Phenol represents the third most abundant constituent in grapes and wines after carbohydrates

92

and fruit acids

93

tissue location and the developmental stage of the berry, the quantity, the structure and the

94

degree of polymerisation and galloylation of grape PAs differ

95

extractable phenolics in grape are distributed as follow: 10% or less in pulp, 28 to 35% in the

96

skins and 60 to 70% in the seeds

97

varieties, the terroir and the vintage, grape seed extractable tannins can reach 15 times that of

98

the skin (table 2). Furthermore, several authors have noted that unlike skins, which contain

99

both procyanidins and prodelphinidins, only procyanidins are found in seeds. Besides, the

100

extension and terminal subunits of PAs in seeds and skins are not the same. While (+)-

101

catechin appears to be the terminal unit and (-)-epicatechin appears to be the extension unit in

102

skins, in seeds there is no preferred flavan-3-ols for the terminal and extension subunits of

103

PAs 30. Additionally, although differences are observed across varieties and vintages, it seems

104

that at maturity, skin PAs present a significantly higher degree of polymerisation (from 2.1 to

105

85.7) than those of seeds (from 2.3 to 30.3) (table 3). However, this is not always true: this is

106

the case of Agiorgitiko grape where the average mDP of tannin seeds (8) is higher than that of

107

skins (2,8) 63. Moreover, for the same grape variety, considerable differences are seen. Indeed,

108

according to studies conducted on Cabernet Sauvignon grape, mDP seeds tannins and skins

109

tannins can be respectively ranged between 3 and 16,1 and 3,4 and 85,7. Due to the high

61.

They are broadly distributed inside grapes and depending on the grape

61.

29–34,62.

Indeed, the total of

Moreover, at harvest time, depending on the grape


Page 7 of 73


110

heterogeneity of mDP values reported in literature, this parameter could not be considered as

111

an index able to characterize and classify the different grape varieties

112

galloylation, differences between skin PAs and seed PAs at maturity have also been observed:

113

seed PAs seem to present a higher percentage of galloylation (G%) (from 13.1 to 32.2%) than

114

skin PAs (from 1.4 to 19%) (table 4). Since that bitterness and astringency is strongly

115

positively correlated with the total concentration of tannins, the percent of galloylation and

116

the mean degree of polymerisation, grape PAs composition will greatly affect wine quality

117

1,6,64,65.

118

PAs and how those mechanisms are regulated by the vine.

119

THE

120

PROANTHOCYANIDINS

121

63.

In terms of

This is why, it is important to understand the mechanisms leading to the formation of

TANNINS

BIOSYNTHESIS:

FROM

FLAVANOLS

TO

The phenylpropanoid pathway

122

Phenolic secondary metabolites are derived from two primary metabolism pathways: the

123

acetate pathway and the shikimate pathway. In the acetate pathway, acetyl-CoA is

124

transformed into malonyl-CoA under the action of acetyl-CoA carboxylase. The shikimate

125

pathway provides carbon skeletons for the production of aromatic amino acids including L-

126

phenylalanine, which is the starting point of the phenylpropanoid pathway 66,67. The synthesis

127

of flavonoids, including PAs and anthocyanins, is cytosolic and is managed by a cytosolic

128

multienzyme complex, known as flavonoid metabolon which is associated with the

129

cytoplasmic face of the endoplasmic reticulum 68.

130

Flavonoid biosynthesis begins with the conversion of phenylalanine into p-coumaroyl CoA by

131

the phenylalanine ammonia lyase (PAL). Then, under the action of cinnamate 4-hydroxylase

132

(C4H), p-coumaroyl CoA is transformed into p-coumaric acid which will itself be converted



Page 8 of 73

133

into coumaroyl-CoA by the 4-coumarate-CoA ligase (4CL). The reaction of the malonoyl-

134

CoA, coming from the acetate pathway, and the coumaroyl-CoA can be catalysed by the

135

stilbene synthase, which will lead to the formation of stilbenes, or can be catalysed by the

136

chalcone synthase (CHS) which will lead to the formation of flavonoids. Tannin precursors

137

are then formed by the chalcone synthase from the phenylpropanoid pathway.

138

Dihydroflavonol-4-reductase (DFR) will act on the dihydroflavonols such as dihydroquercetin

139

or dihydromyricetin to form leucoanthocyanidin such as leucocyanidine or leucodelphinidin.

140

Subsequently, two paths, each involving one known enzyme, can lead to the formation of the

141

principal PAs monomers: the path of the leucoanthocyanidine reductase (LAR) which

142

catalyses the formation of catechin, and the path of the anthocyanidin reductase (ANR) which

143

catalyses the formation of epicatechin 16 (figure 3).

144

In both grape skins and grape seeds, the phenylpropanoid pathway is active

145

complexity makes it difficult to establish a link between gene level expression, enzyme

146

activity and tannin composition and quantity.

147

16,

but its

Focus on two key enzymes for flavanol biosynthesis

148

The conversion of leucocyanidine to (+)-catechin by LAR was the first reaction identified in

149

proanthocyanidin biosynthesis

150

with the accumulation of proanthocyanidins 69–71.

151

Leucoanthocyanidin reductase is a cytosolic NADPH-dependent enzyme, which catalyses the

152

reduction of (2R, 3S, 4S)-flavan-3,4-diols to the corresponding 2,3-trans-(2R,3S)-flavan-3-

153

ols. While (2R,3S,4S)-leucocyanidin is the preferred flavan-3,4-diols substrate, (2R,3S,4S)-

154

leucodelphinidin and (2R,3S,4S)-leucoperlargonidin can also act as substrates, but with low

155

affinity. NADH can also be used by the enzyme, at 30% of the rate of NADPH leading to a

156

slower reaction

72.

69.

Subsequently, many works have revealed its correlation

Consequently, in the reaction catalysed by LAR, the benzylic hydroxyl


Page 9 of 73


157

group of leucoanthocyanidins is eliminated in association with the oxidation of one NADPH

158

molecule.

159

After LAR purification and cloning from leaves of Desmodium uncinatum (Jacq.) DC, Tanner

160

showed that LAR is a 42.7 kDa monomer protein containing 382 amino acids 72. Even though

161

LAR is an enzyme shared by several organisms, it seems that each organism has its own

162

enzyme characteristics. In Vitis vinifera, LAR is a 45,4 kDa monomer protein containing 331

163

to 346 amino acids 73.

164

LAR belongs to the PIP enzyme family of the short chain dehydrogenase/reductase (SDR)

165

superfamily. The PIP enzyme family has been named thus beacause of its thre initially

166

identified members: Pinoresinol-Lariciresinol Reductase (PLR), Isoflavone Reductase (IFR)

167

and Phenylcoumaran Benzylic Reductase (PCBER)

168

structure shared by most of the SDR protein superfamily is the presence of a glycine-rich

169

Rossman-fold scaffold which allows

170

composed of two domains separated by a cleft: an N-terminal domain which adopts a

171

Rossman fold motif, and a smaller C-terminal domain which contains five alpha helices.

172

The NADPH binding site is located in the cleft between the N-terminal and C-terminal

173

domains. The side chains of four amino acids could be involved in the catalytic mechanism:

174

His122, Tyr137, which is the only one in direct contact with the substrate, Lys140 and

175

Ser161. The enzymatic mechanism appears to be a two-step mechanism. The first step

176

consists in dehydration via a Lys140-catalyzed deprotonation of the phenolic OH7 and a

177

protonation of His122-catalyzed protonation of the leaving hydroxide group at C4. The

178

second step begins with the creation of a quinone methide intermediate which serves as an

179

electrophilic target for the NADPH with the help of the ammonium form of the Lys140. Then,

74–77.

One of the common criterion

the cofactor NADPH to bind


78.

Indeed, LAR is


Page 10 of 73

180

a hydrogen from NADPH is transferred to the C4 of the phenolic structure which leads to the

181

formation of the (+)-catechin 73 (figure 4).

182

In Vitis vinifera, two homologous genes encoding LAR have been identified: VvLAR1 and

183

VvLAR2. Unlike VvLAR2, VvLAR1 is expressed in neither skin nor leaves and so appears to be

184

a seed-specific gene. In terms of gene expression, VvLAR1 and VvLAR2 seem to follow

185

different patterns: VvLAR1 reaches its highest expression two weeks after flowering while the

186

maximum expression of VvLAR2 occurs at veraison 16.

187

Like LAR, ANR is known to be a cytosolic NADPH-dependent enzyme. This enzyme

188

catalyses the double NADPH reduction of anthocyanidins which lead to the production of C3

189

epimers (2S, 3R) and (2S, 3S)-flavan-3-ols, i.e the naturally rare (+)-epicatechin and (-)-

190

catechin. The production of these two epimers raised two major issues concerning (i) the

191

production of two different products by the same enzyme and (ii) the production of two rare

192

flavan-3-ols which have, to our knowledge, never been found in grape. The scientific

193

explanation for the production of this epimer mixture has been discussed. Some authors

194

assume that this is the result of a spontaneous epimerisation at C3

195

possible C3 epimerase activity of ANR, alongside the reductase activity

196

fact that (-)-catechin and (+)-epicatechin have never been found in grape, it is noteworthy that

197

in most of grape flavan-3-ols investigations, identification are achieved thanks to the retention

198

times using reverse-phase HPLC, a technology able to separate cis and trans isomers, such as

199

catechin and epicatechin, but not able to discriminate enantiomers such as (-)-catechin and

200

(+)-catechin or (-)-epicatechin and (+)-epicatechin 80.

201

As LAR, ANR belongs to the SDR superfamily, and presents two domains: an N-terminal

202

which adopts a glycine-rich Rossman fold motif, and a shorter C-terminal domain which is

203

composed of six alpha-helices and five beta-strands 80.


79

while others envision a 80.

Concerning the

Page 11 of 73


204

The NADPH binds the enzyme in a glycine-rich loop located at the N-term domain. The ANR

205

catalytic site is composed of the triad Ser131, Tyr168 and Lys172. The enzymatic scheme can

206

be divided into three parts. It begins with the transfer of a NADPH hydride to the C2 position

207

of anthocyanidins, leading to the production of an enolic form which adopts a 2S-aryl

208

configuration. It is worth noting that this hydride transfer to C2 appears to be irreversible,

209

which explains why VvANR exclusively converts anthocyanidins to 2S,3R-trans-flavan-3-ol

210

and 2S,3S-cis-flavan-3-ol

211

the side chain of Lys172 or Tyr168, leads to the production of quinone methide intermediate.

212

The enolic form created undergoes an enzyme-catalysed tautomerization leading to the

213

formation of two quinone methide epimer intermediates. At this stage of the reaction, the 2,3-

214

stereochemistry of the final products is in place. Next, another hydride transfer from the

215

second NADHP to the anthocyanidin C4 is observed. Finally, a third proton, coming from the

216

aqueous medium, is transferred to C3 and either 3R-OH or 3S-OH can be produced

217

5).

218

In the presence of NADP+, VvANR can convert (-)-epicatechin (2R,3R-cis-flavan-3-ol) to

219

(+)-catechin (2R,3S-trans-flavan-3-ol) or vice versa. The epimerization pathway might not

220

require catalytic assistance. Indeed, in the event of an equilibrium between the

221

ANR/NADP+/flavan-3-ol complex and the ANR/NADPH/quinone methide intermediate

222

complex, the latter would slowly epimerize through interconversion with its enolic tautomer

223

81,82

224

In the same way as with LAR, Bogs identified the gene coding for ANR

225

LAR, only one known gene is coding for ANR in Vitis vinefera: VvANR. This gene is

226

expressed at similar levels in leaves and flowers as well as in the skin and the seeds of the

227

berries.

80.

Next, an enzyme-assisted deprotonation of C5 or C7 involving

81

(figure

(figure 6).


16.

In contrast to


228

The fate of flavanols

229

In grapevines as in other plants (tea, alfafa, Arabidopsis thalania…), the biosynthesis of

230

flavan-3-ols occurs on the cytosolic side of the reticulum endoplasmic surface. However, the

231

mechanism of flavan-3-ols condensation into PA oligomers and polymers still remains

232

unknown 83.

233

Nevertheless, recent new insights into PA polymerisation in Camellia sinensis and Medicago

234

truncatula have contributed to a greater understanding of flavan-3-ols polymerization.

235

One of the first question which can be asked about the PAs polymerisation mechanism is the

236

question of the units used to build the PAs, is it native flavan-3-ols such as epicatechin or

237

catechin, or other molecular specie(s)? In 2008, Dixon et al., have discovered a

238

glucosyltransferase named UGT72L1 which catalyses the formation of epicatechin 3’-O-

239

glucoside in the cytoplasm. Interestingly, an over expression of this enzyme leads to an

240

accumulation of PA compounds-like in the vacuole 84. The glycosylation of epicatechin was

241

postulated to be an important step in both PAs precursors transport and assembly in Medicago

242

truncatula

243

glucosyl-(+)-catechin and 7- O-β-glucosyl-(+)-catechin in grape and wine, raising the

244

legitimate question of their involvement of in PAs biosynthesis in grape 85.

245

Another question which can be asked concerning the PAs polymerization is the question

246

about the mechanism itself: is it an enzymatic, a non-enzymatic mechanism or both?

247

Surprisingly, Liu et al., report a possible LAR role in the extension of PAs. Indeed, in

248

Medicago truncatula LAR mutant, a loss of epicatechin-derived PAs, contributing to an

249

increase in insoluble PAs and an accumulation of 4β-(S-cysteinyl)-epicatechin has been

250

noted. Using

251

commencement of PA polymerization occurs between an epicatechin starter unit and an

84.

Recently, Zerbib et al., have found two glucosylated flavan-3-ols: 4’-O-β-

13C

labelled 4β-(S-cysteinyl)-epicatechin, Liu et al., relate that the


Page 12 of 73

Page 13 of 73


252

epicatechin carbon unit formed by a facile nucleophilic displacement of the Cys of 4β-(S-

253

cysteinyl)-epicatechin

254

remains to be determined. In Camellia sinensis, Jiand et al., speculate that the formation of

255

trimeric procyanidins is resulting from an auto-condensation involving the release of

256

carbocations through certain cleavage reaction of the procyanidin B2. As electrophilic units,

257

these carbocations would attack the nucleophilic procyanidin, leading to the formation of a

258

trimer. Nevertheless, in in vitro assays, the conversion rate of auto-condensation of

259

procyanidin appeared to be very low, which did not correspond to the concentration or PAs in

260

tea plant

261

enzymatic condensation. In Arabidopsis thaliania, Pourcel et al., demonstrate the existence of

262

a laccase like flavonoid oxidase named TT10 is able to catalyse the oxidative polymerisation

263

of epicatechin 88. In the same way, the polyphenol oxidase from Capsicum annum appears to

264

be able to catalyse the condensation of epicatechin into dimers, trimers, tetramers and even

265

larger polymers 89.

266

In summary, at this time we still don’t know if the flavan-3-ols polymerisation is resulting

267

from a flavan-3-ols auto-condensation mechanism, or if it is resulting from an enzymatic

268

mechanism, or a combination of them.

269

The type of flavan-3-ols and PAs found in grape berry is variable between species,

270

developmental stage and tissue types. These concentration and composition differences are

271

determined by genetic factors, and are affecting by environmental factors and viticultural

272

practices. Furthermore, even though the flavonoid biosynthetic pathway takes place in the

273

cytoplasm, most of the products are delivered and stored in different compartments such as

274

cell walls and vacuoles. Thus, after their cytosolic synthesis, an efficient transport mechanism

275

is required.

87.

86.

It is noteworthy that the origin of 4β-(S-cysteinyl)-epicatechin

This observation leads us to think about a more efficient reaction such as



276

PA BIOSYNTHESIS REGULATION AND TRANSPORT

277

As mentioned earlier, the PA biosynthesis pathway is relatively well known in terms of the

278

genes and enzymes involved. Nevertheless, the regulation of this pathway has principally

279

been considered through studies of the transcription factors, which might be also modulated

280

by endogenous and exogenous parameters.

281

In plants, three major families of protein involved in the regulation of PA biosynthesis are

282

identified: MYB (MyeloBlastome), bHLH (basic Helix-Loop-Helix), also called MYC, and

283

WD40 proteins (tryptophan-aspartic acid repeat), also called WDR. These three families of

284

proteins can interact together to form the MBW (MYB-bHLH-WD40) complex which acts as

285

a major transcriptional complex. De facto, they must be mobilized into the nucleus to exert

286

their regulatory action. In Arabidopsis thaliana the complex MBW, composed of the proteins

287

TT2 (Transparent Testa 2, MYB), TT8 (Transparent Testa 8, bHLH) and TTG1 (Transparent

288

Testa Glabra3, WD40) activates the BANYULS gene which is encoding ANR 90.

289

In Vitis vinifera, all these MYB, bHLH and WD40 proteins have been identified as regulators

290

of flavonoid biosynthesis, while to our knowledge, no MBW complex has been described 91–95

291

(figure 7).

292

To date, whether in Vitis vinifera or in other plants, no precise mechanism or comprehensive

293

PA transport model has been proposed. Nevertheless, the different evidence which has been

294

highlighted leads us to think that several mechanisms could coexist. Three main synergic and

295

non-exhaustive transport mechanisms have been proposed: transport mediated by vesicle

296

trafficking, Glutathione S transferase-mediated transport (GST), and transport by membrane

297

transporters 96 (figure 8).


Page 14 of 73

Page 15 of 73

298


Vesicle trafficking

299

In plants in general, phytochemicals products can be transported by at least two distinct

300

vesicle trafficking pathways, addressed either to the cell wall or to the vacuole

301

one is a trans-Golgi network and the second one leads to the accumulation of phenolic

302

compounds in anthocyanin vacuolar inclusion (AVIs) 98.

303

In Arabidopsis thaliana, Poustak et al., demonstrated that cells utilize components of the

304

protein secretory trafficking pathway, also called the trans-Golgi network, for direct transport

305

of anthocyanins from endoplasmic reticulum to vacuole 99. Anthocyanins have been found in

306

cytoplasmic vesicles called anthocyanoplasts of protoplasts from grape cell cultures. In the

307

vacuole, where polyphenolic compounds are found, no anthocyanoplasts have been observed,

308

but AVIs have. In Lisanthius, Zhang et al., demonstrate that ACPs are transported to the

309

vacuoles, where they merge with AVIs by cytoplasmic vesicles derived from ER membranes

310

called anthocyanin-containing pre-vacuolar compartments (PVCs)

311

such PVCs have also been found to be filled with PAs and transported to the central vacuole

312

in Arabidopsis thaliana seed coat cells 101.

313

100.

97.

The first

It is worth noting that

GST-flavonoid complex

314

Plant GSTs are considered as enzymes of the secondary metabolism and are typically

315

associated with xenobiotic detoxification, while other basic functions are less understood.

316

GSTs can bind to anthocyanin or flavonol to form GST-anthocyanin or GST-flavonol

317

complexes, which prevent flavonoids from oxidising and guide them to the central vacuoles

318

102,103.

319

vinifera involves the participation of more than one GST in the tissues where flavonoids are

320

accumulated. Thus, VviGST3 seems to play a predominant role in the accumulation of PAs in

321

seeds, whereas VviGST4 acts as an anthocyanin transporter in berry skin and could transport

322

PAs in skin and seeds 104.

Recently, Perez-Diaz et al., posited that flavonoid transport mediated by GSTs in Vitis



323

Page 16 of 73

Membrane transporters

324

Three different categories of membrane transporters have been shown to be involved in

325

flavonoid transport: ATP Binding Cassette (ABC) transporters (primary active transport),

326

Multidrug and Toxic Compound Extrusion (MATE) transporters (secondary active transport),

327

and mammalian bilitranslocase (BLT) transporters (secondary active transport).

328

ABC transporters cover a large spectrum of substrates, from small inorganic compounds to

329

larger organic compounds including polyphenolic compounds. Several authors have

330

suggested the involvement of plant ABC transporters, in particular from the ABCC subfamily,

331

also called multidrug resistance proteins, in vacuolar flavonoid sequestration

332

vinifera, Francisco et al., demonstrate that ABCC-type proteins can actively transport

333

anthocyanidin 3-O-glucosides and that glutathione S-transferase is essential for this ABCCC-

334

type mediated transport

335

transporters in grapevines have been carried out.

336

Unlike ABC transporters, MATE transporters, called secondary transporters, are driven by the

337

H+ or Na+ electrochemical gradient of a “helper” molecule to transport molecules across the

338

membrane. They are widely distributed in all kingdoms of living organisms, and are

339

responsible for multidrug resistance through the extrusion of xenobiotics and toxic

340

metabolites from cells

341

metabolites, iron translocation, plant hormone signalling and aluminium resistance

342

Grapevine VvMATE1 and VvMATE2-GFP are respectively located on the tonoplast and the

343

Golgi complex. Their expression at the early stages of seed development is concomitant with

344

PA accumulation, suggesting putative functions in PA transport 109,110.

345

Besides these two transporters families, in grapevines, flavonoid transport could be

346

accomplished by the activity of a putative flavonoid carrier protein, similar to mammalian

106.

107.

105.

In Vitis

To our knowledge, no investigations on PA transport by ABCC

In plants, they are also involved in the accumulation of secondary


108.

Page 17 of 73


347

BLT. In 2008, Braidot et al., found BLT proteins in the skins of white and red grape varieties,

348

while at subcellular level BLT expression is associated with the cell wall and vacuolar

349

compartments. Moreover, the expression pattern of the potential carrier protein is correlated

350

with flavonoid accumulation, supporting the involvement of the grape BLT homologue in

351

flavonoid accumulation inside the vacuole

352

existence of BLT protein in grape seeds.

353

These mechanisms of regulation and transport are active all along the berry development, and

354

thus they might module the concentration, the composition and the location of grape seeds

355

PAs during grape development.

356

THE SEED PROANTHOCYANIDINS: FROM GREEN STAGE TO

357

HARVEST

358

To date, several studies have investigated the effect of fruit maturity on grape seed

359

polyphenols in terms of amount, composition and molecular weight 30,35–37,47,112–116 (table 2).

360

Seed development can be divided into three phases which can be referred to

361

histodifferenciation (phase I), expansion or reserve disposition (phase II) and maturation

362

drying (phase III)

363

end of the phase all seed structures are formed. Phase II corresponds to the linear phase of

364

development. During this period seeds accumulate reserve materials and even though no cell

365

divisions occur during this period, a rise of seed volume is observed. This volume

366

enhancement is due to the increase in seed water content leading to cell expansion. Finally

367

phase III corresponds to the end of seed growth, the seeds lose water, desiccate and “mature”.

368

Near the end of reserve food accumulation, vascular connections between the mother plant

369

and the seed become non-functional. As a result, the maturing seeds lose water and desiccate,

117.

12,111.

To date, no studies have reported the

Phase I includes fertilization and begins with rapid cell division, at the



370

the dry weight stays content but the fresh weight decrease 15,117–120. It may be noted that some

371

authors identified a fourth growing phase which corresponds to the germination of the mature

372

seed leading to the establishment of a new plant

373

between berry growth and seed development: the seed number and the seed weight are both

374

related to berry weight and ripening

375

is divided in two growth phases separated by a lag phase 41,123,124. During the first phase of

376

rapid growth the rate of pericarp cell divisions is correlated with the rate of developing seeds.

377

Moreover, the second berry growth phase which begins with veraison seems to be associated

378

with the cessation of seed growth. Finally, the maximum embryo length and maximum seed

379

dry weight coincided with maximum berry weight. Even though the relationship between seed

380

and pericarp development in a berry are highly variable, there is evidence that the number and

381

the weight of seeds are linked to the final berry size, fresh and dry berry weight 118,122,125.

382

Even though it is generally accepted that PA seeds are accumulated during the first berry

383

growth phase, and decrease during the second growth phase, opinion on the evolution of the

384

amount of seed tannins during ripening is divided. On one side, some authors report a

385

significant decrease in the amount of PAs during the berry development, in some cases

386

reaching 60% of the start amount 30,112,113,115,126. On the other side, a minority of authors have

387

not found significant differences in PA seed content all along berry development 35,37. In cases

388

where differences in polyphenol content are observed during berry maturation, observations

389

show the same pattern consisting of two distinct periods, one of accumulation (from pea sized

390

stage to veraison) and one of decline (from veraison to maturity). To describe the pattern of

391

PA seed content all along berry development, Kennedy went further, proposing four distinct

392

stages. The two first stages consist of a period of polyphenol synthesis while the two last

393

stages correspond to a period of decline. The first stage, which begins at anthesis and finishes

394

after the first month of berry development, corresponds to a period of procyanidin

118,122.

121.

Correlations have been attempted

As said before, berry development and formation


Page 18 of 73

Page 19 of 73


395

biosynthesis. The second one, which commences after the first month of berry development

396

and ends at the beginning of veraison, corresponds to the synthesis of flavan-3-ols monomers.

397

At the end of this phase, the seed tannins content reached its peak. The third stage, which

398

starts at veraison, corresponds to a programmed oxidation of polyphenols, coinciding with the

399

change of seed colour from green to brown. During this stage, the polyphenol biosynthesis is

400

more or less stopped, and the decrease in polyphenol content due to oxidation is more

401

noteworthy in the flavan-3-ols monomers than in the procyanidins. The end of this stage is

402

generally associated with the maximum berry weight and the completion of seed desiccation.

403

The fourth and last stage, which begins when seed desiccation is complete, corresponds to

404

non-programmed polyphenol oxidation. Small changes in the amount and composition of

405

extracted polyphenol are observed at this stage 126. The same pattern of seed tannins evolution

406

along berry development has been observed by Ristic et al., who proposed three phases model

407

by coupling the two last phase of the model suggested by Kennedy et al.,. During berry

408

development, changes in seed colour coat (from green to brown) partly due to tannin

409

oxidation are observed. These colour modifications are linked to changes in seed phenolic

410

composition and extractable seed tannins

411

may be used as an additional indicator of seed ripeness 128,129.

118,127.

Then, the external appearance of seed coats

412

In terms of PA composition, isolated seed polyphenols consist of flavan-3-ol

413

monomers (+)-catechine, (-)-epicatechine and (-)-epicatechin-3-O-gallate) and procyanidins.

414

As with seed PA content, opinion on the evolution of seed PA composition during berry

415

development is divided and seems to be varietal-dependent.

416

Generally, (-)-epicatechin appears to be the most abundant PA extension unit,

417

followed by the epicatechin gallate and the catechin. It is worth noting that this observation is

418

made for each stage of berry development, proving that there is limited variation in the

419

composition of PA extension units during growth and ripening 30,36,47,115. Unlike the extension 19 ACS Paragon Plus Environment


Page 20 of 73

420

units, the composition of PA terminal units varies according to the developmental stage of the

421

berry, to the grape variety and probably to environmental factors. These observed variations

422

make it difficult to propose a general pattern of the evolution of PA terminal units throughout

423

berry development. Nevertheless, in most cases, it seems that the terminal units’ percentages

424

of C and ECG are decreasing from the first developmental stages to harvest, and conversely,

425

the EC percentage tends to increase in the same time frame 30,36,47,115.

426

The evolution of the mean degree of PA polymerization (mDP) along the berry

427

development also appears to be controversial. In the literature, three major evolution types are

428

described: a constant rate, a decrease, and an increase in mDP through berry development

429

30,35–37,47,130.

430

grape variety, dissimilarities are observed. This is the case for Cabernet Sauvignon, where

431

Obreque

432

and Bordiga observed a decline, leading us to believe that environmental factors can impact

433

upon mDP values

434

environmental factors on the mDP value. During berry development, the percentage of

435

galloylation of flavan-3-ol appears relatively constant, with some exceptions in terms of the

436

grape variety

437

percentage is seen throughout development 37. Concerning the evolution of the PAs molecular

438

weight during fruit ripening, in 2010, Obreque-Slier observed on Carménère and Cabernet

439

Sauvignon seeds, a diminution from veraison to harvest followed by an increase until

440

overmaturity

441

noticed an increase in the molecular weight of PAs all along berry development

442

researches have to be conducted in order to understand the influence of external factors on

443

tannin biosynthesis along berry development. This knowledge might be used to manage and

35

These profile differences could be varietal-dependent, but even within the same

did not observe significant differences in the mDP rate along berry development,

36,37

35.

36

but so far no investigations have been conducted on the impact of

notably with Merlot seeds where a diminution of the galloylation

Surprisingly, in 2012 on seeds of the same grape varieties, Obreque-Slier


37.

Thus,

Page 21 of 73


444

manipulate the tannin composition of seed at harvest, which will influence the final tannin

445

composition of wine and consequently impact wine quality.

446

MANAGEMENT OF VINE AND WINE PRACTICES: FROM SEED TO

447

WINE

448

The impact of vine practices on grape seeds tannins

449

Phenolic compound synthesis is an inducible mechanism that plays a major role in plant

450

responses to environmental signals and in particular to biotic and abiotic stresses. In this

451

respect, flavonoids could be seen as marks of an adaptive metabolism which exert protective,

452

antibiotic and modulatory effects 12. As no studies have highlighted the potential connection

453

between biotic stress and the PA composition and content of grape seed, we will first focus

454

here on the effect of abiotic stresses (temperature, light and water status) on grape phenolic

455

compounds. Secondly we will focus on the know impact of viticultural practices such as

456

pruning, thinning, leaf removal and irrigation on the PAs seed biosynthesis and content.

457

In warm climates, high light exposure can increase the concentration of phenolic compounds

458

by inducing the activity of the PAL enzyme

459

exposure treatment to Pinot Noir grapes, Cortell and Kennedy found no significant differences

460

in seed PAs at harvest

461

have a minimal influence on the condensed tannin content of seeds. However, some studies

462

have suggested a positive correlation between temperature and the number of seeds and total

463

proanthocyanidin levels per berry at harvest. Indeed, the number of seeds per berry for vines

464

grown at 25 °C can reach twice that of vines grown at 15 °C

465

impact of temperature on grape seed tannin composition has been proved so far, an increase in

466

the seed number per berry will improve the quantity of seed-derived PAs in wines.

132.

131.

Nevertheless, after having applied a light

This observation leads us to think that shade or light treatments


133.

De facto, even though no


Page 22 of 73

467

Due to the global warming, vine might be confronted to water deficit which could impact

468

tannins content. Indeed, grapevine berries respond to drought by modulating, among other

469

pathways, the phenylpropanoid pathways which directly or indirectly impact the berry’s

470

tannin composition and consequently the wine’s tannic characteristics

471

grapes, it has been shown that water deficit leads to an upregulation of most of the structural

472

flavonoid genes encoding for enzymes related to flavan-3-ols biosynthesis: three CHS, two

473

CHI, one F3’5’H, two F3H and one DFR

474

principal enzymes responsible for flavan-3-ols synthesis, LAR1, LAR2 and ANR, seems to

475

respond differently to water deficits. Indeed, Savoi et al., suggest that VvLAR1 seems to be

476

upregulated at 41 days after flowering (DAF) while VvLAR2 seems to be alternatively

477

downregulated and upregulated later in the berry development. Concerning VvANR, an

478

upregulation of genes has been observed at 41 DAF, and a downregulation has been noted at

479

68 DAF

480

highlighted.

481

In red grapes, Roby et al., found that water deficits increased the amount of skin tannin but

482

did not significantly affect the content or the concentration of seed tannins in Merlot and

483

Shiraz grapes

484

Tempranillo seeds and Cabernet Sauvignon seeds respectively, which lead them to conclude

485

that water stressed had no impact on seed PAs content

486

deficits on genes encoding for enzymes of the phenylpropanoid pathway, it seems that most of

487

the genes of the anthocyanin biosynthesis pathway are upregulated: F3H, DFR, and UFGT,

488

leading to an increase in berry anthocyanidins. Despite the fact that these enzymes are part of

489

the phenylpropanoid pathway, only limited effects have been observed on PAs concentration.

490

In the same way, Genedra et al., observed that water deficit enhances the expression of the

491

genes VvLAR1, VvLAR2 and VvANR encoding for flavonoid biosynthetic enzymes. Despite

136.

136.

20,134,135.

On white

To go further, the gene encoding the three

The impact of those regulations on PA concentration in seeds has not been

137.

Genedra et al., and Koundouras et al., have made the same observation on

134,138.Concerning


the impact of water

Page 23 of 73


492

this fact, water stressed seeds do not present higher PAs content at full maturation stages

493

suggesting the occurrence of other mechanisms, namely, oxidation and/or degradation of PAs

494

at late stages of maturation resulting from the impact of watering on seed ripening

495

Furthermore, Braidot et al., have shown that a water shortage can boost the expression of the

496

grape BLT homologue, suggesting that water-stressed conditions affect not only the flavonoid

497

biosynthesis pathway, but also the expression of proteins involved in flavonoid transport and

498

accumulation 111.

499

In general, it is recognized that water deficits promote the synthesis and increase the

500

concentration of flavonoids, specifically anthocyanins in grapes

501

sensory attributes of wines. This observation is most often due to a reduction in the berry

502

volume and consequently an increase in the relative skin masses 140.

503

Cortell et al., found that generally there were greater numbers of seeds per berry in low-vigor

504

zones compared to high-vigor zones. Nevertheless, although an overall reduction in total

505

flavan-3-ols monomers per seed has been seen with a reduction in vigor, on a per berry basis

506

no significant differences have been observed. In the same way, the amount of grape seed PAs

507

appears to be independent of vine vigor 141.

508

Viticultural management practices such as pruning, thinning, leaf removal and irrigation are

509

here exanimated as a possible source of variability in PAs seed biosynthesis and PAs seed

510

content.

511

Regarding the effect of spur or cane pruning on fruit composition of Cabernet Sauvignon

512

grapes, Peppi and Kania found no significant differences in terms of both total phenol content

513

and phenol composition of seeds. This observation lead us to think that the pruning technique

514

had only a limited effect on grape seed tannins content and composition 142. Furthermore, PAs

515

seed tannins content of Corot noir grapes seems to be not affected by cluster or shoot thinning 23 ACS Paragon Plus Environment

139,

138.

often leading to better


Page 24 of 73

516

143.

517

Chorti et al., have detected no significant differences in seed tannin content

518

knowledge, no published studies are treated about the impact of viticultural practices on seed

519

tannin biosynthetic pathway.

520

In spite of the major lack of knowledges on the impact of vine practices on grape seed PAs

521

biosynthesis and content, the winemaker will take the seed tannins content into account when

522

estimating the best date for the grape harvest by the Glories method. Indeed, the Glories

523

method is used for the assessment of the phenolic maturity during berry maturation. This

524

method delivered estimation on the total anthocyanins content (mg/L), the extractable

525

anthocyanins (mg/L), the extractability of anthocyanins (%), and the seed maturity index (%)

526

also called MP %. With this estimation, the winemaker will manage the first step of

527

vinification to either reduce or enhance the seed tannins content in the produced wine.

528

Concerning the impact of leaf removal and irrigation on Agiorgitiko grape composition, 144.

To our

The impact of winemaking processes on seed tannins extractability

529

A relatively poor number of published studies treat about the link between grape phenolic

530

concentration and wine composition. Nevertheless, some studies have estimated the

531

percentage of grape tannins incorporated in wine between 9% and50 %

532

estimation can be driven by the difference of methods used to obtain data’s, or by the

533

difference of grape varieties used to conduct the experiments. Studies which have explored

534

the relationship between grape phenolics concentration and the corresponding wines have

535

shown a positive correlation between grape anthocyanin and wine colour indices, but the

536

relationship between grape and wine tannins have been found to be weak or absent

537

Explanations for this observation are numerous; it can be due to the limited solubility of

538

phenolic compounds, to the nature of cells within constituents are located, to their cross-

539

linking with carbohydrates or proteins through oxidations process and to their binding with

540

insoluble matrix of the grape berry 28,56,58,64,126,146–150. 24 ACS Paragon Plus Environment

8,56–58.

This broad

145.

Page 25 of 73


541

Furthermore, because of the disruption of the tissues (de-stemming, crushing) prior to

542

vinification, the relative contribution of anatomically distinct berry tissues (skin, seed and

543

pulp) to wine tannins remains difficult to establish. Nevertheless, there are a lot of common

544

thoughts about the grape tannins extraction regarding to their tissue location. The first one is

545

probably that grape skin tannins are extracted faster than seed tannins and that because of a

546

difference of extraction kinetics. Indeed, a considerable number of studies have shown that

547

skin tannins concentration tends to reach a maximum in the first days of fermentative

548

maceration, while seed tannins are extracted later, during the maceration 149,151–154. Yet, it has

549

also been proved that skin tannins extraction can increase continuously during fermentation

550

and maceration 24,145,155,156. Another common thought is that seeds tannins are extracted in the

551

late phase of fermentation thanks to the apparition of ethanol in the wine. Although ethanol

552

leads to a more intense and a faster extraction of seed tannins, the key parameter of this

553

extraction seems to be the hydration of seed cells, under meaning the extraction time 24,145,157–

554

159.

555

independently of alcohol content 157. Then, ethanol would not act as a crucial factor but as an

556

“extraction catalyser” by helping to disorganize the outer lipidic layer that protects seeds. A

557

last common thought is that as tannins are more easily extracted from skins than from seeds,

558

skins tannins are present in higher quantity in wine than the seeds one. Although it is true that

559

the extractibility of seed tannins is lower than the skin one, the concentration of seed tannins

560

can be ten-fold higher than the skin one. Consequently, during normal winemaking practices

561

seed tannins are present in higher proportion in wine than the skin one (around 60% of seed

562

tannins for 40% of skin tannins) 8,61,160. Furthermore, Kovac et al., found that the presence of

563

higher quantities of seeds in contact with the must during fermentation resulted in wines with

564

higher content of phenolics, especially (+)-catechin and PAs. Moreover, the addition of seeds

565

appears to increase colour intensity and free anthocyanins proving there the ability of seed

Indeed, once the level of seed cell hydration is reached, tannins are extracted



566

phenolics to stabilize wine colour 161. Interestingly, Lee et al., who have studied the effect of

567

early seed removal during fermentation on PAs extraction, have found no significant

568

differences in wines PAs content between the control and the seeds removed wine. They have

569

also found an improvement of colour intensity in the case of seed removed wine which is

570

conflicting with the previous work of Kovac et al 161,162.

571

Because of the significant influence of tannins on red wine quality, many winemaking

572

techniques have been developed as an alternative to the traditional elaboration of red wines.

573

We outline here three different maceration techniques, one fermentative technique and three

574

innovative processes (thermovinification, flash release and pulsed electric field) known to

575

enhance the contribution of grape seed tannins to wine.

576

Maceration

577

Fermentative maceration consist to let the must in contact with the raw material (skins and

578

seeds) which enhance the extraction of phenolics, polysaccharides, nitrogen compounds,

579

minerals and volatile compounds. Since this technique is generally improving wine structure,

580

it is considered as one of the most important step of winemaking process. Several maceration

581

techniques have been developed in order to improve phenolic extraction from red grapes,

582

skins and seeds. Among these techniques, we found the cold maceration also called cold soak,

583

the post fermentative maceration, the enzymatic maceration and the carbonic maceration.

584

To optimize the transfer of phenolic compounds into must, one of the most applied practices

585

is the cold soak or cold maceration. During this pre-fermentative maceration, the solid parts of

586

the berry are in contact with the must under low temperature, to prevent the start of

587

fermentation, and in an alcohol free environment. This technique allows improving the

588

transfer of phenolic compounds into the must and, in theory, into the wine.


Page 26 of 73

Page 27 of 73


589

Roughly speaking, essentially two approaches can be followed when performing cold soak.

590

The first one consists to applied low temperature ranged between 10 and 15 °C to the must

591

during 3 to 5 days. The second one, more extreme consist to applied temperatures around 4 °C

592

for periods around 10 days 163. When wines made with the first approach of cold soak present

593

no difference of phenolic composition 55, it seems that wines produced using the second and

594

more extreme approach present a higher proportion of seed tannins

595

can be explained by the fact that longer maceration time allows a more important hydration of

596

seeds cells which leads to a better extraction of grape seed tannins.

597

Enzyme addition during winemaking is a common practice that can be used to improve the

598

extraction of free-run juice during maceration, to aid clarification and filtration and to

599

facilitate the processes 166. Generally, the commercial enzyme preparations are a complex mix

600

of enzymes with different activities such as pectinases, cellulases, hemicellulases and

601

glycosidases

602

on grape skin wall degradation, wine anthocyanins and color characteristics with a lot less

603

attention payed to their impact on wine PAs. Yet, in 2010 Ducasse et al., reported that the use

604

of enzyme cocktail can enhance PAs extraction in wine. Moreover, by estimated the

605

percentage of epigallocatechin (flavan-3-ol present only in grape skin) in the resulted wines,

606

the increase of PAs in wine appeared to be more relative to an increase in seed derived PAs.

607

Comparable results have also been found suggesting that the enzyme facilitates the seed PAs

608

extraction

609

solution can lead to an augmentation of 400% of seeds derived PAs in aqueous conditions and

610

an augmentation of 700% in alcoholic conditions 165.

611

There are essentially two explanations possible for the enhancement of seed tannins extraction

612

by maceration enzyme. On one hand, as the seed cell walls are composed of cellulose,

613

hemicellulose, pectins, proteins lignin, mucilage and gums, the use of a cocktail enzyme as

167.

163–165.

This observation

The first studies on maceration enzymes were focused on the enzyme effect

49,50,149,168,169.

As a proof, a commercial enzyme addition in a synthetic model



Page 28 of 73

614

described above, may be able to disrupt the cellular and subcellular organization of seed

615

tissues, which could be responsible of the release of seed tannins. On the other hand we know

616

that seed derived tannins have a strong affinity for cell wall material

617

Sparrow et al., have noticed that the presence of pulp material can reduce wine-extractable

618

tannin by a factor of 3 in Pinot Noir wines. They have also noticed that seed tannins were

619

more strongly adsorbed by the pulp cell material than skin tannins 58. The use of maceration

620

enzymes allow the degradation of cell wall pectic fractions which promotes a lower PAs

621

adsorption on cell wall material, enhancing the PAs concentration in the final wine 170.

622

Post fermentative maceration consists to extend skin and seed contact after the must has

623

fermented for a duration comprised between 5 and 44 days 55. The maceration time affects the

624

phenolic composition of wines: the total proanthocyanidins content increases with the

625

maceration. The augmentation of phenolic extraction into wine is the result of an

626

improvement of skin-derived and seed-derived phenolic compounds

627

example, for a 20 days post fermentative maceration , an augmentation of almost 20% of the

628

skin derived PA extraction and 10% of seed derived PA can be observed 24.

629

Fermentative Temperature

630

During fermentative maceration, temperature plays a crucial role in the extraction of phenolic

631

compounds.

632

High fermentation temperatures have been reported to enhance and increase phenolic

633

extraction. Indeed, Ough and Amerine proved that wines produced from Pinot noir and

634

Cabernet Sauvignon grapes under high fermentation temperatures made more coloured wines

635

172,

636

temperatures. It seems that the increase in phenolic extraction at higher fermentation

637

temperatures is due to an increase in the permeability of the hypodermal cells which will then

57,58,64,146,147.

Indeed,

24,48,55,154,159,171.

As an

meaning that anthocyanidins are extracted more readily under high fermentation


Page 29 of 73


638

more easily release anthocyanidins. Nevertheless, it appears that high fermentation

639

temperatures increase the wine’s total phenolic content, including tannins which are more

640

easily extracted 55.

641

Innovative processes

642

Thermovinification

643

Although several variations on the thermovinification procedure exist, the basic one consists

644

in heating the harvest at a temperature generally set between 60 and 70 °C for a short time.

645

This rapid temperature rise leads to significant damage to the cell membrane, leading to easy

646

and rapid anthocyanin extraction compared to the usual vinification processes

647

Nevertheless, the effect of thermovinification on tannin extraction appears to be variable, or

648

unknown 55,176.

649

Flash release

650

The flash release process consists in rapidly warming up the grapes and then applying a

651

powerful vacuum. This technique is used to boost the polyphenolic content of wine, but

652

appears to be more effective on tannins from skins than those from seeds 177.

653

Pulsed electric field (PEF)

654

The brief application of electric field pulses to living cells induces a transmembrane potential

655

difference across the cell membranes. When this difference in potential reaches a critical

656

value called the breakdown potential, a membrane electroporation phenomena appears

657

This electroporation enhances cell permeability, and consequently improves the extraction of

658

plant metabolites such as polyphenols.

659

PEF treatment is usually applied during traditional maceration and fermentation of red must

660

53,54,179–184.

661

dependent upon the physico-chemical composition of the grape. This observation implies that

173–175.

178.

These studies have demonstrated that the efficacy of cell electroporation by PEF is



662

the efficacy of the PEF treatment depends upon the grape variety, the harvesting period and

663

the vintage. Additionally, the choice of treatment modality (strength, duration and energy)

664

will modify the kinetics of polyphenol extraction 182.

665 666

Wine quality depends on numerous factors, and among them grape and wine phenolics are of

667

great importance. Due to their chemical complexity and sensory attributes, considerable

668

research has been conducted on phenolic compounds regarding their chemical structure, their

669

biosynthesis and their extractability potential (figure 9). Despite the fact that seed tannins

670

chemical structure have become well know, no strong consensus has emerged about the

671

mechanism of their polymerization nor the units used to build the PAs. Furthermore, even if

672

the flavonoids biosynthetic pathway itself begins to be quite well understood, its regulation

673

appears to be under a hierarchy of complex events. Indeed, environmental factors are known

674

to impact the tannins content of the berry, yet their impacts on the biosynthesis pathway

675

remain unclear. Moreover, the site of seed flavan-3-ol biosynthesis and storage is known to

676

differ at subcellular, cell and even tissue levels meaning that efficient flavonoid transport

677

system are required all along berry and seed development. So far, although hypothesis for

678

flavonoid transport came out, a complete understanding of flavonoid transport mechanisms is

679

far from being achieved. In addition, as flavonoids transport and location may be partly

680

correlated with the extractability potential of tannins, an improvement of knowledges could be

681

helpful in enabling new strategies for the management of seed tannins extractability during

682

winemaking process.

683

To conclude, due to a lack of knowledge, it remains difficult to estimate the seed phenolic

684

maturity, even though the winemaker will take into account this maturity to determine the best

685

harvest date. Indeed, no reliable, simple tool exists to determine the phenolic maturity of the


Page 30 of 73

Page 31 of 73


686

seed, which is why winemakers often delay harvest time until the seeds turn uniformly brown.

687

The consequence of this delay is an increase in the Brix level, leading to undesirably high

688

ethanol levels during maceration. To get a better idea of the phenolic maturity of grape seeds

689

and its impact on the sensory properties of wine, a robust study of the phenolic metabolism of

690

seeds should be conducted. Knowledge of how tannins are metabolised by the grape seed will

691

lead to the discovery of strong maturity markers which can be used to create a reliable tool to

692

determine the phenolic maturity of seeds.



693

ABBREVIATIONS

694

4CL: 4-coumarate :CoA ligase

695

ANR: Anthocyanidin Reductase

696

BLT: Bilitranslocase

697

C4H: Cinnamate 4-hydroxylase

698

CHI: Chalcone isomerase

699

CHS: Chalcone synthase

700

DAF: Day After Flowering

701

DFR: Dihydroflavonol 4-reductase

702

F3’5’H: Flavonoid 3’5’-hydroxylase

703

F3’H: Flavonoid 3’5’-hydroxylase

704

F3H: Flavonoid 3-hydroxylase

705

G% : Percentage of Galloylation

706

GST: Glutathione S Transferase

707

LAR: Leucoanthocyanidin Reductase

708

LDOX: Leucoanthocyanidin dioxygenase

709

mDP: mean Degree of Polymerization

710

NADH: Nicotinamide Adenine Dinucleotide

711

NADPH: Nicotinamide Adenine Dinucleotide Phosphate 32 ACS Paragon Plus Environment

Page 32 of 73

Page 33 of 73


712

PA : Proanthocyanidins

713

PAL: Phenylalanine Ammonia Lyase

714

PEF: Pulsed Electric Field

715

PVC: Pre-Vacuolar Compartment

716

ACKNOWLEDGEMENT

717

We thank the Maison de la Traduction for its English corrections.



718

REFERENCES

719

1.

Gawel, R. Red wine astringency: a review. Aust. J. Grape Wine Res. 4, 74–95 (1998).

720

2.

Vidal, S., Francis, L., Noble, A., Kwiatkowski, M., Cheynier, V. & Waters, E. Taste and

721

mouth-feel properties of different types of tannin-like polyphenolic compounds and

722

anthocyanins in wine. Anal. Chim. Acta. 513, 57–65 (2004).

723

3.

Cheynier, V., Dueñas-Paton, M., Salas, E., Maury, C., Souquet, J. M., Sarni-Manchado,

724

P. & Fulcrand, H. Structure and properties of wine pigments and tannins. Am. J. Enol.

725

Vitic. 57, 298 (2006).

726

4.

Mercurio, M. D., Dambergs, R. G., Cozzolino, D., Herderich, M. J. & Smith, P. A.

727

Relationship between red wine grades and phenolics. 1. tannin and total phenolics

728

concentrations. J. Agric. Food Chem. 58, 12313–12319 (2010).

729

5.

Teissedre, P.-L. & Jourdes, M. Tannins and anthocyanins of wine: phytochemistry and

730

organoleptic properties. in Natural Products (eds. Ramawat, K. G. & Mérillon, J.-M.)

731

2255–2274 (Springer Berlin Heidelberg, 2013).

732

6.

733 734

bitterness perception of tannins in wine. Trends Food Sci. Technol. 40, 6–19 (2014). 7.

735 736

739

Soares, S., Brandão, E., Mateus, N. & de Freitas, V. Sensorial properties of red wine polyphenols: astringency and bitterness. Crit. Rev. Food Sci. Nutr. 57, 937–948 (2017).

8.

737 738

Ma, W., Guo, A., Zhang, Y., Wang, H., Liu, Y. & Li, H. A review on astringency and

Kennedy, J. A. Grape and wine phenolics: observations and recent findings. Cienc. E Investig. Agrar. 35, 107–120 (2008).

9.

Ribéreau-Gayon, P. Les composés phénoliques du raisin et du vin. Ann. Physiol. Veg. 6, 211–242 (1964).

740

10. Ortega-Regules, A., Romero-Cascales, I., Ros García, J. M., Bautista-Ortín, A. B.,

741

López-Roca, J. M., Fernández-Fernández, J. I. & Gómez-Plaza, E. Anthocyanins and


Page 34 of 73

Page 35 of 73


742

tannins in four grape varieties (Vitis vinifera L.). Evolution of their content and

743

extractability. OENO One. 42, 147–156 (2008).

744

11. Quideau, S., Deffieux, D., Douat-Casassus, C. & Pouységu, L. Plant polyphenols:

745

chemical properties, biological activities, and synthesis. Angew. Chem. Int. Ed. 50, 586–

746

621 (2011).

747

12. Petrussa, E., Braidot, E., Zancani, M., Peresson, C., Bertolini, A., Patui, S. & Vianello,

748

A. Plant flavonoids: biosynthesis, transport and involvement in stress responses. Int. J.

749

Mol. Sci. 14, 14950–14973 (2013).

750 751 752 753

13. Xia, E.-Q., Deng, G.-F., Guo, Y.-J. & Li, H.-B. Biological activities of polyphenols from grapes. Int. J. Mol. Sci. 11, 622–646 (2010). 14. Ribéreau-Gayon, P., Glories, Y., Maujean, A. & Dubourdieu, D. Traité d’oenologie Tome 2, Chimie du vin, Stabilisation et traitements. (DUNOD, 2017).

754

15. Cadot, Y., Miñana-Castelló, M. T. & Chevalier, M. Anatomical, histological, and

755

histochemical changes in grape seeds from Vitis vinifera L. cv Cabernet franc during

756

fruit development. J. Agric. Food Chem. 54, 9206–9215 (2006).

757

16. Bogs, J., Downey, M. O., Harvey, J. S., Ashton, A. R., Tanner, G. J. & Robinson, S. P.

758

Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin

759

reductase and anthocyanidin reductase in developing grape berries and grapevine leaves.

760

Plant Physiol. 139, 652–663 (2005).

761 762 763 764

17. Pirie, A. J. G. & Mullins, M. G. Concentration of phenolics in the skin of grape berries during fruit development and ripening. Am. J. Enol. Vitic. 31, 34–36 (1980). 18. Souquet, J. M., Cheynier, V., Brossaud, F. & Moutounet, M. Polymeric proanthocyanidins from grape skins. Phytochemistry. 43, 509–512 (1996).



765

19. Kennedy, J. A., Hayasaka, Y., Vidal, S., Waters, E. J. & Jones, G. P. Composition of

766

grape skin proanthocyanidins at different stages of berry development. J. Agric. Food

767

Chem. 49, 5348–5355 (2001).

768 769

20. Kennedy, J. A., Matthews, M. A. & Waterhouse, A. L. Effect of maturity and vine water status on grape skin and wine flavonoids. Am. J. Enol. Vitic. 53, 268–274 (2002).

770

21. Hochberg, U., Degu, A., Cramer, G. R., Rachmilevitch, S. & Fait, A. Cultivar specific

771

metabolic changes in grapevines berry skins in relation to deficit irrigation and hydraulic

772

behavior. Plant Physiol. Biochem. 88, 42–52 (2015).

773

22. Matus, J. T., Loyola, R., Vega, A., Peña-Neira, A., Bordeu, E., Arce-Johnson, P. &

774

Alcalde, J. A. Post-veraison sunlight exposure induces MYB-mediated transcriptional

775

regulation of anthocyanin and flavonol synthesis in berry skins of Vitis vinifera. J. Exp.

776

Bot. 60, 853–867 (2009).

777

23. Azuma, A., Yakushiji, H., Koshita, Y. & Kobayashi, S. Flavonoid biosynthesis-related

778

genes in grape skin are differentially regulated by temperature and light conditions.

779

Planta. 236, 1067–1080 (2012).

780

24. Busse-Valverde, N., Bautista-Ortín, A. B., Gómez-Plaza, E., Fernández-Fernández, J. I.

781

& Gil-Muñoz, R. Influence of skin maceration time on the proanthocyanidin content of

782

red wines. Eur. Food Res. Technol. 235, 1117–1123 (2012).

783

25. Bindon, K. A., Madani, S. H., Pendleton, P., Smith, P. A. & Kennedy, J. A. Factors

784

affecting skin tannin extractability in ripening grapes. J. Agric. Food Chem. 62, 1130–

785

1141 (2014).

786 787

26. Gagné, S., Saucier, C. & Gény, L. Composition and cellular localization of tannins in Cabernet Sauvignon skins during growth. J. Agric. Food Chem. 54, 9465–9471 (2006).


Page 36 of 73

Page 37 of 73


788

27. Hanlin, R. L. & Downey, M. O. Condensed tannin accumulation and composition in skin

789

of Shiraz and Cabernet Sauvignon grapes during berry development. Am. J. Enol. Vitic.

790

60, 13–23 (2009).

791

28. Fournand, D., Vicens, A., Sidhoum, L., Souquet, J. M., Moutounet, M. & Cheynier, V.

792

Accumulation and extractability of grape skin tannins and anthocyanins at different

793

advanced physiological stages. J. Agric. Food Chem. 54, 7331–7338 (2006).

794

29. de Freitas, V., Glories, Y., Bourgeois, G. & Vitry, C. Characterisation of oligomeric and

795

polymeric procyanidins from grape seeds by liquid secondary ion mass spectrometry.

796

Phytochemistry. 49, 1435–1441 (1998).

797

30. Downey, M. O., Harvey, J. S. & Robinson, S. P. Analysis of tannins in seeds and skins

798

of Shiraz grapes throughout berry development. Aust. J. Grape Wine Res. 9, 15–27

799

(2003).

800

31. Monagas, M., Gómez-Cordovés, C., Bartolomé, B., Laureano, O. & Ricardo da Silva, J.

801

M. Monomeric, oligomeric, and polymeric flavan-3-ol composition of wines and grapes

802

from Vitis vinifera L. Cv. Graciano, Tempranillo, and Cabernet Sauvignon. J. Agric.

803

Food Chem. 51, 6475–6481 (2003).

804

32. Vivas, N., Nonier, M. F., de Gaulejac, N., Absalon, C., Bertrand, A. & Mirabel, M.

805

Differentiation of proanthocyanidin tannins from seeds, skins and stems of grapes (Vitis

806

vinifera) and heartwood of Quebracho (Schinopsis balansae) by matrix-assisted laser

807

desorption/ionization time-of-flight mass spectrometry and thioacidolysis/liquid

808

chromatography/electrospray ionization mass spectrometry. Anal. Chim. Acta. 513, 247–

809

256 (2004).

810

33. Hernández-Jiménez, A., Gómez-Plaza, E., Martínez-Cutillas, A. & Kennedy, J. A. Grape

811

skin and seed proanthocyanidins from Monastrell × Syrah grapes. J. Agric. Food Chem.

812

57, 10798–10803 (2009).



813

34. Lorrain, B., Chira, K. & Teissedre, P. L. Phenolic composition of Merlot and Cabernet-

814

Sauvignon grapes from Bordeaux vineyard for the 2009-vintage: Comparison to 2006,

815

2007 and 2008 vintages. Food Chem. 126, 1991–1999 (2011).

816

35. Obreque-Slier, E., Peña-Neira, Á., López-Solís, R., Zamora-Marín, F., Ricardo-da Silva,

817

J. M. & Laureano, O. Comparative study of the phenolic composition of seeds and skins

818

from Carménère and Cabernet Sauvignon grape varieties (Vitis vinifera L.) during

819

ripening. J. Agric. Food Chem. 58, 3591–3599 (2010).

820

36. Bordiga, M., Travaglia, F., Locatelli, M., Coïsson, J. D. & Arlorio, M. Characterisation

821

of polymeric skin and seed proanthocyanidins during ripening in six Vitis vinifera L. cv.

822

Food Chem. 127, 180–187 (2011).

823

37. Obreque-Slier, E., López-Solís, R., Castro-Ulloa, L., Romero-Díaz, C. & Peña-Neira, Á.

824

Phenolic composition and physicochemical parameters of Carménère, Cabernet

825

Sauvignon, Merlot and Cabernet Franc grape seeds (Vitis vinifera L.) during ripening.

826

LWT - Food Sci. Technol. 48, 134–141 (2012).

827

38. Kyraleou, M., Kallithraka, S., Theodorou, N., Teissedre, P.-L., Kotseridis, Y. &

828

Koundouras, S. Changes in tannin composition of Syrah grape skins and seeds during

829

fruit ripening under contrasting water conditions. Molecules. 22, 1453 (2017).

830

39. Mattivi, F., Vrhovsek, U., Masuero, D. & Trainotti, D. Differences in the amount and

831

structure of extractable skin and seed tannins amongst red grape varieties. Aust. J. Grape

832

Wine Res. 15, 27–35 (2009).

833

40. Ćurko, N., Kovačević Ganić, K., Gracin, L., Đapić, M., Jourdes, M. & Teissedre, P. L.

834

Characterization of seed and skin polyphenolic extracts of two red grape cultivars grown

835

in Croatia and their sensory perception in a wine model medium. Food Chem. 145, 15–

836

22 (2014).


Page 38 of 73

Page 39 of 73

837 838


41. Coombe, B. G. Research on development and ripening of the grape berry. Am. J. Enol. Vitic. 43, 101–110 (1992).

839

42. Neves, A. C., Spranger, M. I., Zhao, Y., Leandro, M. C. & Sun, B. Effect of addition of

840

commercial grape seed tannins on phenolic composition, chromatic characteristics, and

841

antioxidant activity of red wine. J. Agric. Food Chem. 58, 11775–11782 (2010).

842

43. Yilmaz, Y. & Toledo, R. T. Major flavonoids in grape seeds and skins: antioxidant

843

capacity of catechin, epicatechin, and gallic acid. J. Agric. Food Chem. 52, 255–260

844

(2004).

845

44. Spranger, I., Sun, B., Mateus, A. M., Freitas, V. de & Ricardo-da-Silva, J. M. Chemical

846

characterization and antioxidant activities of oligomeric and polymeric procyanidin

847

fractions from grape seeds. Food Chem. 108, 519–532 (2008).

848

45. Mirbagheri, V. sadat, Alizadeh, E., Yousef Elahi, M. & Esmaeilzadeh Bahabadi, S.

849

Phenolic content and antioxidant properties of seeds from different grape cultivars

850

grown in Iran. Nat. Prod. Res. 32, 425–429 (2018).

851

46. Bozan, B., Tosun, G. & Özcan, D. Study of polyphenol content in the seeds of red grape

852

(Vitis vinifera L.) varieties cultivated in Turkey and their antiradical activity. Food

853

Chem. 109, 426–430 (2008).

854

47. Bautista-Ortín, A. B., Rodríguez-Rodríguez, P., Gil-Muñoz, R., Jiménez-Pascual, E.,

855

Busse-Valverde, N., Martínez-Cutillas, A., López-Roca, J. M. & Gómez-Plaza, E.

856

Influence of berry ripeness on concentration, qualitative composition and extractability

857

of grape seed tannins: composition and extractability of grape seed tannins. Aust. J.

858

Grape Wine Res. 18, 123–130 (2012).

859

48. del Llaudy, M. C., Canals, R., Canals, J. M. & Zamora, F. Influence of ripening stage

860

and maceration length on the contribution of grape skins, seeds and stems to phenolic



861

composition and astringency in wine-simulated macerations. Eur. Food Res. Technol.

862

226, 337–344 (2008).

863

49. Bautista-Ortín, A. B., Jiménez-Pascual, E., Busse-Valverde, N., López-Roca, J. M., Ros-

864

García, J. M. & Gómez-Plaza, E. Effect of wine maceration enzymes on the extraction of

865

grape seed proanthocyanidins. Food Bioprocess Technol. 6, 2207–2212 (2013).

866

50. Busse-Valverde, N., Gómez-Plaza, E., López-Roca, J. M., Gil-Muñoz, R., Fernández-

867

Fernández, J. I. & Bautista-Ortín, A. B. Effect of different enological practices on skin

868

and seed proanthocyanidins in three varietal wines. J. Agric. Food Chem. 58, 11333–

869

11339 (2010).

870

51. Cerpa-Calderón, F. K. & Kennedy, J. A. Berry integrity and extraction of skin and seed

871

proanthocyanidins during red wine fermentation. J. Agric. Food Chem. 56, 9006–9014

872

(2008).

873

52. Koyama, K., Goto-Yamamoto, N. & Hashizume, K. Influence of maceration temperature

874

in red wine vinification on extraction of phenolics from berry skins and seeds of grape

875

(Vitis vinifera). Biosci. Biotechnol. Biochem. 71, 958–965 (2007).

876

53. Delsart, C., Ghidossi, R., Poupot, C., Cholet, C., Grimi, N., Vorobiev, E., Milisic, V. &

877

Mietton Peuchot, M. Enhanced extraction of phenolic compounds from Merlot grapes by

878

pulsed electric field treatment. Am. J. Enol. Vitic. 63, 205–211 (2012).

879

54. Delsart, C., Cholet, C., Ghidossi, R., Grimi, N., Gontier, E., Gény, L., Vorobiev, E. &

880

Mietton-Peuchot, M. Effects of pulsed electric fields on Cabernet Sauvignon grape

881

berries and on the characteristics of wines. Food Bioprocess Technol. 7, 424–436

882

(2014).

883 884

55. Sacchi, K. L., Bisson, L. F. & Adams, D. O. A review on the effect of winemaking techniques on phenolic extraction in red wines. Am. J. Enol. Vitic. 56, 197–206 (2005).


Page 40 of 73

Page 41 of 73


885

56. Hanlin, R. L., Hrmova, M., Harbertson, J. F. & Downey, M. O. Review: condensed

886

tannin and grape cell wall interactions and their impact on tannin extractability into

887

wine. Aust. J. Grape Wine Res. 16, 173–188 (2010).

888

57. Bindon, K. A., Smith, P. A., Holt, H. & Kennedy, J. A. Interaction between grape-

889

derived proanthocyanidins and cell wall material. 2. Implications for vinification. J.

890

Agric. Food Chem. 58, 10736–10746 (2010).

891

58. Sparrow, A. M., Dambergs, R. G., Bindon, K. A., Smith, P. A. & Close, D. C.

892

Interactions of grape skin, seed, and pulp on tannin and anthocyanin extraction in Pinot

893

noir wines. Am. J. Enol. Vitic. 66, 472–481 (2015).

894

59. Waterhouse, A. L. Wine Phenolics. Ann. N. Y. Acad. Sci. 957, 21–36 (2002).

895

60. Panche, A. N., Diwan, A. D. & Chandra, S. R. Flavonoids: an overview. J. Nutr. Sci. 5,

896 897 898

(2016). 61. Shi, J., Yu, J., Pohorly, J. E. & Kakuda, Y. Polyphenolics in grape seeds—biochemistry and functionality. J. Med. Food. 6, 291–299 (2003).

899

62. Chira, K., Lorrain, B., Ky, I. & Teissedre, P.-L. Tannin composition of Cabernet-

900

Sauvignon and Merlot Grapes from the Bordeaux area for different vintages (2006 to

901

2009) and comparison to tannin profile of five 2009 vintage mediterranean grapes

902

varieties. Molecules. 16, 1519–1532 (2011).

903

63. Petropoulos, S., Kanellopoulou, A., Paraskevopoulos, I., Kotseridis, Y. & Kallithraka, S.

904

Characterization of grape and wine proanthocyanidins of Agiorgitiko (Vitis vinifera L.

905

cv.) cultivar grown in different regions of Nemea. J. Food Compos. Anal. 63, 98–110

906

(2017).

907

64. Harrison, R. Practical interventions that influence the sensory attributes of red wines

908

related to the phenolic composition of grapes: a review. Int. J. Food Sci. Technol. 53, 3–

909

18 (2018).



910

65. Preys, S., Mazerolles, G., Courcoux, P., Samson, A., Fischer, U., Hanafi, M., Bertrand,

911

D. & Cheynier, V. Relationship between polyphenolic composition and some sensory

912

properties in red wines using multiway analyses. Anal. Chim. Acta. 563, 126–136

913

(2006).

914 915 916 917

66. Herrmann, K. M. & Weaver, L. M. The shikimate pathway. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 473–503 (1999). 67. Maeda, H. & Dudareva, N. The shikimate pathway and aromatic amino acid biosynthesis in plants. Annu. Rev. Plant Biol. 63, 73–105 (2012).

918

68. Stafford, H. A. Possible multienzyme complexes regulating the formation of C6-C3

919

phenolic compounds and lignins in higher plants. in Recent Advances in Phytochemistry.

920

8, 53–79 (Elsevier, 1974).

921

69. Stafford, H. A. Flavonoid metabolism. (CRC Press, 1990).

922

70. Joseph, R., Tanner, G. & Larkin, P. Proanthocyanidin synthesis in the forage legume

923

Onobrychis viciifolia. A study of chalcone synthase, dihydroflavonol 4-reductase and

924

leucoanthocyanidin 4-reductase in developing leaves. Aust. J. Plant Physiol. 25, 271–

925

278 (1998).

926 927

71. Marles, M. A. S., Ray, H. & Gruber, M. Y. New perspectives on proanthocyanidin biochemistry and molecular regulation. Phytochemistry. 64, 367–383 (2003).

928

72. Tanner, G. J., Francki, K. T., Abrahams, S., Watson, J. M., Larkin, P. J. & Ashton, A. R.

929

Proanthocyanidin biosynthesis in plants: purification of legume leucoanthocyanidin

930

reductase and molecular cloning of its cDNA. J. Biol. Chem. 278, 31647–31656 (2003).

931

73. Maugé, C., Granier, T., Langlois d’Estaintot, B., Gargouri, M., Manigand, C., Schmitter,

932

J. M., Chaudière, J. & Gallois, B. Crystal structure and catalytic mechanism of

933

leucoanthocyanidin reductase from Vitis vinifera. J. Mol. Biol. 397, 1079–1091 (2010).


Page 42 of 73

Page 43 of 73


934

74. Min, T., Kasahara, H., Bedgar, D. L., Youn, B., Lawrence, P. K., Gang, D. R., Halls, S.

935

C., Park, H., Hilsenbeck, J. L., Davin, L. B., Lewis, N. G. & Kang, C. Crystal structures

936

of pinoresinol-lariciresinol and phenylcoumaran benzylic ether reductases and their

937

relationship to isoflavone reductases. J. Biol. Chem. 278, 50714–50723 (2003).

938

75. Koeduka, T., Louie, G. V., Orlova, I., Kish, C. M., Ibdah, M., Wilkerson, C. G.,

939

Bowman, M. E., Baiga, T. J., Noel, J. P., Dudareva, N. & Pichersky, E. The multiple

940

phenylpropene synthases in both Clarkia breweri and Petunia hybrida represent two

941

distinct protein lineages. Plant J. 54, 362–374 (2008).

942

76. Gang, D. R., Kasahara, H., Xia, Z. Q., Vander Mijnsbrugge, K., Bauw, G., Boerjan, W.,

943

Van Montagu, M., Davin, L. B. & Lewis, N. G. Evolution of plant defense mechanisms

944

relationships of phenylcoumaran benzylic ether reductases to pinoresinol-lariciresinol

945

and isoflavone reductases. J. Biol. Chem. 274, 7516–7527 (1999).

946

77. Wang, X., He, X., Lin, J., Shao, H., Chang, Z. & Dixon, R. A. Crystal structure of

947

isoflavone reductase from alfalfa (Medicago sativa L.). J. Mol. Biol. 358, 1341–1352

948

(2006).

949

78. Kavanagh, K. L., Jörnvall, H., Persson, B. & Oppermann, U. Medium- and short-chain

950

dehydrogenase/reductase gene and protein families: The SDR superfamily: functional

951

and structural diversity within a family of metabolic and regulatory enzymes. Cell. Mol.

952

Life Sci. 65, 3895–3906 (2008).

953

79. Xie, D. Y., Sharma, S. B. & Dixon, R. A. Anthocyanidin reductases from Medicago

954

truncatula and Arabidopsis thaliana. Arch. Biochem. Biophys. 422, 91–102 (2004).

955

80. Gargouri, M., Manigand, C., Maugé, C., Granier, T., Langlois d’Estaintot, B., Cala, O.,

956

Pianet, I., Bathany, K., Chaudière, J. & Gallois, B. Structure and epimerase activity of

957

anthocyanidin reductase from Vitis vinifera. Acta Crystallogr. D Biol. Crystallogr. 65,

958

989–1000 (2009).



959

81. Gargouri, M., Chaudière, J., Manigand, C., Maugé, C., Bathany, K., Schmitter, J. M. &

960

Gallois, B. The epimerase activity of anthocyanidin reductase from Vitis vinifera and its

961

regiospecific hydride transfers. Biol. Chem. 391, 219–227 (2010).

962

82. Gargouri, M., Gallois, B. & Chaudière, J. Binding-equilibrium and kinetic studies of

963

anthocyanidin reductase from Vitis vinifera. Arch. Biochem. Biophys. 491, 61–68 (2009).

964

83. Dixon, R. A., Xie, D. Y. & Sharma, S. B. Proanthocyanidins - a final frontier in

965

flavonoid research?: Tansley Review. New Phytol. 165, 9–28 (2004).

966

84. Pang, Y., Peel, G. J., Sharma, S. B., Tang, Y. & Dixon, R. A. A transcript profiling

967

approach reveals an epicatechin-specific glucosyltransferase expressed in the seed coat

968

of Medicago truncatula. Proc. Natl. Acad. Sci. 105, 14210–14215 (2008).

969

85. Zerbib, M., Mazauric, J.-P., Meudec, E., Le Guernevé, C., Lepak, A., Nidetzky, B.,

970

Cheynier, V., Terrier, N. & Saucier, C. New flavanol O -glycosides in grape and wine.

971

Food Chem. 266, 441–448 (2018).

972 973

86. Liu, C., Wang, X., Shulaev, V. & Dixon, R. A. A role for leucoanthocyanidin reductase in the extension of proanthocyanidins. Nat. Plants. 2, 16182 (2016).

974

87. Jiang, X., Liu, Y., Wu, Y., Tan, H., Meng, F., Wang, Y. S., Li, M., Zhao, L., Liu, L.,

975

Qian, Y., Gao, L. & Xia, T. Analysis of accumulation patterns and preliminary study on

976

the condensation mechanism of proanthocyanidins in the tea plant Camellia sinensis.

977

Sci. Rep. 5, (2015).

978

88. Pourcel, L., Routaboul, J.-M., Kerhoas, M., Caboche, M., Lepiniec, L. & Debeaujon, I.

979

Transparent testa 10 encodes a laccase-like enzyme involved in oxidative polymerization

980

of flavonoids in Arabidopsis seed coat. Plant Cell Online. 17, 2966–2980 (2005).

981

89. Sancher-Mundo, M. L., Escobedo-Crisantes, V. M., Mendoza-Arvizu, S. & Jramillo-

982

Flores, M. E. Polymerization of phenolic compounds by polyphenol oxidase from bell

983

pepper with increase in their antioxidant capacity. CyTA - J. Food. 14, 594–603 (2016).


Page 44 of 73

Page 45 of 73


984

90. Lepiniec, L., Debeaujon, I., Routaboul, J. M., Baudry, A., Pourcel, L., Nesi, N. &

985

Caboche, M. Genetics and biochemistry of seed flavonoids. Annu. Rev. Plant. Biol. 57,

986

405–430 (2006).

987

91. Deluc, L., Barrieu, F., Marchive, C., Lauvergeat, V., Decendit, A., Richard, T., Carde, J.

988

P., Mérillon, M. & Hamdi, S. Characterization of a grapevine R2R3-MYB transcription

989

factor that regulates the phenylpropanoid pathway. Plant Physiol. 140, 499–511 (2006).

990

92. Deluc, L., Bogs, J., Walker, A. R., Ferrier, T., Decendit, A., Merillon, J.-M., Robinson,

991

S. P. & Barrieu, F. The transcription factor VvMYB5b contributes to the regulation of

992

anthocyanin and proanthocyanidin biosynthesis in developing grape berries. Plant

993

Physiol. 147, 2041–2053 (2008).

994

93. Terrier, N., Torregrosa, L., Ageorges, A., Vialet, S., Verries, C., Cheynier, V. &

995

Romieu, C. Ectopic expression of VvMybPA2 promotes proanthocyanidin biosynthesis

996

in grapevine and suggests additional targets in the pathway. Plant Physiol. 149, 1028–

997

1041 (2008).

998

94. Koyama, K., Numata, M., Nakajima, I., Goto-Yamamoto, N., Matsumura, H. & Tanaka,

999

N. Functional characterization of a new grapevine MYB transcription factor and

1000

regulation of proanthocyanidin biosynthesis in grapes. J. Exp. Bot. 65, 4433–4449

1001

(2014).

1002

95. Huang, Y. F., Vialet, S., Guiraud, J. L., Torregrosa, L., Bertrand, Y., Cheynier, V., This,

1003

P. & Terrier, N. A negative MYB regulator of proanthocyanidin accumulation, identified

1004

through expression quantitative locus mapping in the grape berry. New Phytol. 201, 795–

1005

809 (2014).

1006 1007

96. Zhao, J., Pang, Y. & Dixon, R. A. The mysteries of proanthocyanidin transport and polymerization. Plant Physiol. 153, 437–443 (2010).



1008 1009

97. Lin, Y., Irani, N. G. & Grotewold, E. Sub-cellular trafficking of phytochemicals explored using auto-fluorescent compounds in maize cells. BMC Plant Biol. 3, (2003).

1010

98. Braidot, E., Zancani, M., Petrussa, E., Peresson, C., Bertolini, A., Patui, S., Macrì, F. &

1011

Vianello, A. Transport and accumulation of flavonoids in grapevine (Vitis vinifera L.).

1012

Plant Signal. Behav. 3, 626–632 (2008).

1013

99. Poustka, F., Irani, N. G., Feller, A., Lu, Y., Pourcel, L., Frame, K. & Grotewold, E. A

1014

Trafficking pathway for anthocyanins overlaps with the endoplasmic reticulum-to-

1015

vacuole protein-sorting route in Arabidopsis and contributes to the formation of vacuolar

1016

inclusions. Plant Physiol. 145, 1323–1335 (2007).

1017

100. Zhang, H., Wang, L., Deroles, S., Bennett, R. & Davies, K. New insight into the

1018

structures and formation of anthocyanic vacuolar inclusions in flower petals. BMC Plant

1019

Biol. 6, 29 (2006).

1020

101. Kitamura, S., Shikazono, N. & Tanaka, A. Transparent Testa 19 is involved in the

1021

accumulation of both anthocyanins and proanthocyanidins in Arabidopsis. Plant J. 37,

1022

104–114 (2004).

1023

102. Mueller, L. A., Goodman, C. D., Silady, R. A. & Walbot, V. AN9, a Petunia glutathione

1024

S-transferase required for anthocyanin sequestration, is a flavonoid-binding protein.

1025

Plant Physiol. 123, 1561–1570 (2000).

1026 1027

103. Zhao, J. & Dixon, R. A. The ‘ins’ and ‘outs’ of flavonoid transport. Trends Plant Sci. 15, 72–80 (2010).

1028

104. Pérez-Díaz, R., Madrid-Espinoza, J., Salinas-Cornejo, J., González-Villanueva, E. &

1029

Ruiz-Lara, S. Differential roles for VviGST1, VviGST3, and VviGST4 in

1030

proanthocyanidin and anthocyanin transport in Vitis vinífera. Front. Plant Sci. 7, 1166

1031

(2016).


Page 46 of 73

Page 47 of 73


1032

105. Klein, M., Burla, B. & Martinoia, E. The multidrug resistance-associated protein

1033

(MRP/ABCC) subfamily of ATP-binding cassette transporters in plants. FEBS Lett. 580,

1034

1112–1122 (2006).

1035

106. Francisco, R. M., Regalado, A., Ageorges, A., Burla, B. J., Bassin, B., Eisenach, C.,

1036

Zarrouk, O., Vialet, S., Marlin, T., Chaves, M. M., Martinoia, E. & Nagy, R. ABCC1, an

1037

ATP Binding Cassette Protein from grape berry, transports anthocyanidin 3-O-

1038

glucosides. Plant Cell 25, 1840–1854 (2013).

1039

107. Diener, A., Gaxiola, R. & Fink, G. Arabidopsis ALF5, a multidrug efflux transporter

1040

gene family member, confers resistance to toxins. Plant Cell 13, 1625–1637 (2001).

1041

108. Takanashi, K., Shitan, N. & Yazaki, K. The multidrug and toxic compound extrusion

1042

(MATE) family in plants. Plant Biotechnol. 31, 417–430 (2014).

1043

109. Pérez-Díaz, R., Ryngajllo, M., Pérez-Díaz, J., Peña-Cortés, H., Casaretto, J. A.,

1044

González-Villanueva, E. & Ruiz-Lara, S. VvMATE1 and VvMATE2 encode putative

1045

proanthocyanidin transporters expressed during berry development in Vitis vinifera L.

1046

Plant Cell Rep. 33, 1147–1159 (2014).

1047 1048

110. Zhao, J. Flavonoid transport mechanisms: how to go, and with whom. Trends Plant Sci. 20, 576–585 (2015).

1049

111. Braidot, E., Petrussa, E., Bertolini, A., Peresson, C., Ermacora, P., Loi, N., Terdoslavich,

1050

M., Passamonti, S., Macrì, F. & Vianello, A. Evidence for a putative flavonoid

1051

translocator similar to mammalian bilitranslocase in grape berries (Vitis vinifera L.)

1052

during ripening. Planta. 228, 203–213 (2008).

1053

112. Romeyer, F. M., Macheix, J. J. & Sapis, J. C. Changes and importance of oligomeric

1054

procyanidins during maturation of grape seeds. Phytochemistry. 25, 219–221 (1985).

1055

113. Prieur, C., Rigaud, J., Cheynier, V. & Moutounet, M. Oligomeric and polymeric

1056

procyanidins from grape seeds. Phytochemistry. 36, 781–784 (1994).



1057

114. de Freitas, V., Glories, Y. & Monique, A. Developmental changes of procyanidins in

1058

grapes of red Vitis vinifera varieties and their composition in respectives wines. Am. J.

1059

Enol. Vitic. 51, 397–403 (2000).

1060 1061

115. Kennedy, J. A., Matthews, M. A. & Waterhouse, A. L. Changes in grape seed polyphenols during fruit ripening. Phytochemistry. 55, 77–85 (2000).

1062

116. Jordao, A. M., Ricardo-da Silva, J. M. & Laureano, O. Evolution of catechins and

1063

oligomeric procyanidins during grape maturation of Castelão Francês and Touriga

1064

Francesa. Am. J. Enol. Vitic. 52, 230–234 (2001).

1065 1066

117. Srivastava, L. M. Seed Development and Maturation. in Plant Growth and Development 431–446 (Elsevier, 2002).

1067

118. Ristic, R. & Iland, P. G. Relationships between seed and berry development of Vitis

1068

Vinifera L. cv Shiraz: Developmental changes in seed morphology and phenolic

1069

composition. Aust. J. Grape Wine Res. 11, 43–58 (2005).

1070 1071 1072 1073

119. Kennedy, J. A. Understanding grape berry development. Pract. Winery Vineyard 1–5 (2002). 120. Pratt, C. Reproductive Anatomy of cultivated grapes - A review. Am. J. Enol. Vitic. 22, 92–109 (1971).

1074

121. Adams, C. A. & Rinne, R. W. Moisture content as a controlling factor in seed

1075

development and germination. in International Review of Cytology 68, 1–8 (Elsevier,

1076

1980).

1077 1078 1079 1080

122. Wang, L., Zhou, Y., Duan, B., Jiang, Y. & Xi, Z. Relationship between seed content and berry ripening of wine grape (Vitis vinifera L.). Sci. Hortic. 243, 1–11 (2019). 123. Coombe, B. G. Growth Stages of the Grapevine: Adoption of a system for identifying grapevine growth stages. Aust. J. Grape Wine Res. 1, 104–110 (1995).


Page 48 of 73

Page 49 of 73


1081

124. Conde, C., Silva, P., Fontes, N., Dias, A. C. P., Tavares, R. M., Sousa, M. J., Agasse, A.,

1082

Delrot, S. & Gerós, H. Biochemical changes throughout grape berry development and

1083

fruit and wine quality. Food 1, 1–22 (2007).

1084 1085

125. Ebadi, A., Sedgley, M., May, P. & Coombe, B. G. Seed development and abortion in Vitis vinifera L., cv. Chardonnay. Int. J. Plant Sci. 157, 703–712 (1996).

1086

126. Kennedy, J. A., Troup, G. J., Pilbrow, J. R., Hutton, D. R., Hewitt, D., Hunter, C. R.,

1087

Ristic, R., Iland, P. G. & Jones, G. P. Development of seed polyphenols in berries from

1088

Vitis vinifera L. cv. Shiraz. Aust. J. Grape Wine Res. 6, 244–254 (2000).

1089

127. Ferrer-Gallego, R., García-Marino, M., Miguel Hernández-Hierro, J., Rivas-Gonzalo, J.

1090

C. & Teresa Escribano-Bailón, M. Statistical correlation between flavanolic

1091

composition, colour and sensorial parameters in grape seed during ripening. Anal. Chim.

1092

Acta. 660, 22–28 (2010).

1093

128. Fredes, C., Von Bennewitz, E., Holzapfel, E. & Saavedra, F. Relation between Seed

1094

Appearance and phenolic maturity: a case study using grapes cv. Carménère. Chil. J.

1095

Agric. Res. 70, 381–389 (2010).

1096

129. Rabot, A., Rousseau, C., Li-Mallet, A., Antunes, L., Osowski, A. & Geny, L. Using of a

1097

combined approach by biochemical and image analysis to develop a new method to

1098

estimate seed maturity stage for Bordeaux area grapevine. OENO One. 51, 29–35

1099

(2017).

1100

130. Kennedy, J. A., Troup, G. J., Pilbrow, J. R., Hutton, D. R., Hewitt, D., Hunter, C. R.,

1101

Ristic, R., Iland, P. G. & Jones, G. P. Development of seed polyphenols in berries from

1102

Vitis vinifera L. cv. Shiraz. Aust. J. Grape Wine Res. 6, 244–254 (2000).

1103

131. Roubelakis-Angelakis, K. A. & Kliemer, W. M. Effects of exogenous factors on

1104

phenylalanine ammonia-lyase activity and accumulation of anthocyanins and total

1105

phenolics in grape berries. Am. J. Enol. Vitic. 37, 275–280 (1986).



1106

132. Cortell, J. M. & Kennedy, J. A. Effect of shading on accumulation of flavonoid

1107

compounds in Vitis vinifera L. Pinot Noir fruit and extraction in a model system. J.

1108

Agric. Food Chem. 54, 8510–8520 (2006).

1109

133. Pastor del Rio, J. L. & Kennedy, J. A. Development of proanthocyandins in Vitis

1110

vinifera L. cv. Pinot noir grapes and extraction into wine. Am. J. Enol. Vitic. 57, 125–

1111

132 (2006).

1112

134. Koundouras, S., Marinos, V., Gkoulioti, A., Kotseridis, Y. & van Leeuwen, C. Influence

1113

of vineyard location and vine water status on fruit maturation of nonirrigated Cv.

1114

Agiorgitiko (Vitis vinifera L.). Effects on wine phenolic and aroma components. J.

1115

Agric. Food Chem. 54, 5077–5086 (2006).

1116

135. Castellarin, S. D., Matthews, M. A., Di Gaspero, G. & Gambetta, G. A. Water deficits

1117

accelerate ripening and induce changes in gene expression regulating flavonoid

1118

biosynthesis in grape berries. Planta. 227, 101–112 (2007).

1119

136. Savoi, S., Wong, D. C. J., Arapitsas, P., Miculan, M., Bucchetti, B., Peterlunger, E., Fait,

1120

A., Mattivi, F. & Castellarin, S. D. Transcriptome and metabolite profiling reveals that

1121

prolonged drought modulates the phenylpropanoid and terpenoid pathway in white

1122

grapes (Vitis vinifera L.). BMC Plant Biol. 16, 67 (2016).

1123

137. Roby, G., Harbertson, J. F., Adams, D. A. & Matthews, M. A. Berry size and vine water

1124

deficits as factors in winegrape composition: Anthocyanins and tannins. Aust. J. Grape

1125

Wine Res. 10, 100–107 (2004).

1126

138. Genebra, T., Santos, R., Francisco, R., Pinto-Marijuan, M., Brossa, R., Serra, A., Duarte,

1127

C., Chaves, M. & Zarrouk, O. Proanthocyanidin accumulation and biosynthesis are

1128

modulated by the irrigation regime in Tempranillo seeds. Int. J. Mol. Sci. 15, 11862–

1129

11877 (2014).


Page 50 of 73

Page 51 of 73


1130

139. Bucchetti, B., Matthews, M. A., Falginella, L., Peterlunger, E. & Castellarin, S. D.

1131

Effect of water deficit on Merlot grape tannins and anthocyanins across four seasons.

1132

Sci. Hortic. 128, 297–305 (2011).

1133

140. Roby, G. & Matthews, M. A. Relative proportions of seed, skin and flesh, in ripe berries

1134

from Cabernet Sauvignon grapevines grown in a vineyard either well irrigated or under

1135

water deficit. Aust. J. Grape Wine Res. 10, 74–82 (2008).

1136

141. Cortell, J. M., Halbleib, M., Gallagher, A. V., Righetti, T. L. & Kennedy, J. A. Influence

1137

of vine vigor on grape (Vitis vinifera L. Cv. Pinot Noir) and wine proanthocyanidins. J.

1138

Agric. Food Chem. 53, 5798–5808 (2005).

1139 1140

142. Peppi, M. C. & Kania, E. Effects of spur or cane pruning on fruit composition of ‘Cabernet Sauvignon’ grapes. Acta Hortic. 1157, 17–20 (2017).

1141

143. Sun, Q., Sacks, G. L., Lerch, S. D. & Vanden Heuvel, J. E. Impact of shoot and cluster

1142

thinning on yield, fruit composition, and wine quality of Corot noir. Am. J. Enol. Vitic.

1143

63, 49–56 (2012).

1144

144. Chorti, E., Kyraleou, M., Kallithraka, S., Pavlidis, M., Koundouras, S. & Kotseridis, Y.

1145

Irrigation and leaf removal effects on polyphenolic content of grapes and wines

1146

produced from cv. ‘Agiorgitiko’ (Vitis vinifera L.). Not. Bot. Horti Agrobot. Cluj-

1147

Napoca 44, 133–139 (2016).

1148

145. Bindon, K. A., Kassara, S., Cynkar, W. U., Robinson, E. M. C., Scrimgeour, N. &

1149

Smith, P. A. Comparison of extraction protocols to determine differences in wine-

1150

extractable tannin and anthocyanin in Vitis vinifera L. cv. Shiraz and Cabernet

1151

Sauvignon grapes. J. Agric. Food Chem. 62, 4558–4570 (2014).

1152

146. Hazak, J. C., Harbertson, J. F., Lin, C. H., Ro, B. H. & Adams, D. O. The phenolic

1153

components of grape berries in relation to wine composition. Acta Hortic. 689, 189–196

1154

(2005).



1155

147. Bindon, K. A., Smith, P. A. & Kennedy, J. A. Interaction between grape-derived

1156

Proanthocyanidins and cell wall material. 1. Effect on proanthocyanidin composition and

1157

molecular mass. J. Agric. Food Chem. 58, 2520–2528 (2010).

1158

148. Renard, C. M. G. C., Watrelot, A. A. & Le Bourvellec, C. Interactions between

1159

polyphenols and polysaccharides: Mechanisms and consequences in food processing and

1160

digestion. Trends Food Sci. Technol. 60, 43–51 (2017).

1161

149. Bautista-Ortín, A. B., Busse-Valverde, N., Fernández-Fernández, J. I., Gómez-Plaza, E.

1162

& Gil-Muñoz, R. The extraction kinetics of anthocyanins and proanthocyanidins from

1163

grape to wine in three different varieties. OENO One. 50, 91–100 (2016).

1164

150. Zhu, W., Khalifa, I., Peng, J. & Li, C. Position and orientation of gallated

1165

proanthocyanidins in lipid bilayer membranes: influence of polymerization degree and

1166

linkage type. J. Biomol. Struct. Dyn. 36, 2862–2875 (2017).

1167

151. Pascual, O., González-Royo, E., Gil, M., Gómez-Alonso, S., García-Romero, E., Canals,

1168

J. M., Hermosín-Gutíerrez, I. & Zamora, F. Influence of Grape Seeds and Stems on

1169

Wine Composition and Astringency. J. Agric. Food Chem. 64, 6555–6566 (2016).

1170 1171

152. Peyrot des Gachons, C. & Kennedy, J. A. Direct method for determining seed and skin proanthocyanidin extraction into red wine. J. Agric. Food Chem. 51, 5877–5881 (2003).

1172

153. Amrani Joutei, K. & Glories, Y. Etude en conditions modèles de l’extractibilité des

1173

composés phénoliques des pellicules et des pépins de raisins rouges. OENO One. 28,

1174

303–317 (1994).

1175

154. González-Manzano, S., Rivas-Gonzalo, J. C. & Santos-Buelga, C. Extraction of flavan-

1176

3-ols from grape seed and skin into wine using simulated maceration. Anal. Chim. Acta.

1177

513, 283–289 (2004).

1178

155. Gil, M., Kontoudakis, N., González, E., Esteruelas, M., Fort, F., Canals, J. M. &

1179

Zamora, F. Influence of Grape Maturity and Maceration Length on Color, Polyphenolic


Page 52 of 73

Page 53 of 73


1180

Composition, and Polysaccharide Content of Cabernet Sauvignon and Tempranillo

1181

Wines. J. Agric. Food Chem. 60, 7988–8001 (2012).

1182

156. Aron, P. M. & Kennedy, J. A. Compositional investigation of phenolic polymers isolated

1183

from Vitis vinifera L. Cv. Pinot Noir during fermentation. J. Agric. Food Chem. 55,

1184

5670–5680 (2007).

1185

157. Hernandez-Jimenez, A., Kennedy, J. A., Bautista-Ortin, A. B. & Gomez-Plaza, E. Effect

1186

of ethanol on grape seed proanthocyanidin extraction. Am. J. Enol. Vitic. 63, 57–61

1187

(2012).

1188

158. Canals, R., Llaudy, M. C., Valls, J., Canals, J. M. & Zamora, F. Influence of ethanol

1189

concentration on the extraction of color and phenolic compounds from the skin and the

1190

seeds of Tempranillo grapes at different stages of ripening. J. Agric. Food Chem. 53,

1191

4019–4025 (2005).

1192

159. Federico Casassa, L., Beaver, C. W., Mireles, M. S. & Harbertson, J. F. Effect of

1193

extended maceration and ethanol concentration on the extraction and evolution of

1194

phenolics, colour components and sensory attributes of Merlot wines: Extended

1195

maceration and ethanol concentration. Aust. J. Grape Wine Res. 19, 25–39 (2013).

1196 1197

160. Nel, A. P. Tannins and anthocyanins: from their origin to wine analysis – a review. South Afr. J. Enol. Vitic. 39, 1–17 (2018).

1198

161. Kovac, V., Alonso, E. & Revilla, E. The effect of adding supplementary quantities of

1199

seeds during fermentation on the phenolic composition of wines. Am. J. Enol. Vitic. 46,

1200

363–367 (1995).

1201

162. Lee, J., Kennedy, J., Devlin, C., Redhead, M. & Rennaker, C. Effect of early seed

1202

removal during fermentation on proanthocyanidin extraction in red wine: A commercial

1203

production example. Food Chem. 107, 1270–1273 (2008).



1204 1205

163. Aleixandre-Tudo, J. L. & du Toit, W. Cold maceration application in red wine production and its effects on phenolic compounds: A review. LWT 95, 200–208 (2018).

1206

164. Panprivech, S., Lerno, L., Brenneman, C., Block, D. & Oberholster, A. Investigating the

1207

effect of cold soak duration on phenolic extraction during Cabernet Sauvignon

1208

fermentation. Molecules. 20, 7974–7989 (2015).

1209

165. Busse-Valverde, N., Gómez-Plaza, E., López-Roca, J. M., Gil-Muñoz, R. & Bautista-

1210

Ortín, A. B. The extraction of anthocyanins and proanthocyanidins from grapes to wine

1211

during fermentative maceration is affected by the enological technique. J. Agric. Food

1212

Chem. 59, 5450–5455 (2011).

1213

166. Ducasse, M.-A., Canal-Llauberes, R.-M., de Lumley, M., Williams, P., Souquet, J.-M.,

1214

Fulcrand, H., Doco, T. & Cheynier, V. Effect of macerating enzyme treatment on the

1215

polyphenol and polysaccharide composition of red wines. Food Chem. 118, 369–376

1216

(2010).

1217

167. Fia, G., Canuti, V. & Rosi, I. Evaluation of potential side activities of commercial

1218

enzyme preparations used in winemaking. Int. J. Food Sci. Technol. 49, 1902–1911

1219

(2014).

1220

168. Moreno-Pérez, A., Fernández-Fernández, J. I., Bautista-Ortín, A. B., Gómez-Plaza, E.,

1221

Martínez-Cutillas, A. & Gil-Muñoz, R. Influence of winemaking techniques on

1222

proanthocyanidin extraction in Monastrell wines from four different areas. Eur. Food

1223

Res. Technol. 236, 473–481 (2013).

1224

169. Li, S., Bindon, K., Bastian, S. E. P., Jiranek, V. & Wilkinson, K. L. Use of winemaking

1225

supplements to modify the composition and sensory properties of Shiraz wine. J. Agric.

1226

Food Chem. 65, 1353–1364 (2017).


Page 54 of 73

Page 55 of 73


1227

170. Castro-López, L. del R., Gómez-Plaza, E., Ortega-Regules, A., Lozada, D. & Bautista-

1228

Ortín, A. B. Role of cell wall deconstructing enzymes in the proanthocyanidin–cell wall

1229

adsorption–desorption phenomena. Food Chem. 196, 526–532 (2016).

1230

171. Gambuti, A., Capuano, R., Lecce, L., Fragasso, M. G. & Moio, L. Extraction of phenolic

1231

compounds from ‘Aglianico’ and ‘Uva di Troia’ grape skins and seeds in model

1232

solutions: Influence of ethanol and maceration time. VITIS - J. Grapevine Res. 48, 193–

1233

200 (2009).

1234 1235 1236 1237

172. Ough, C. S. & Amerine, M. A. Studies with controlled fermentation V. Effects on color, composition and quality of red wines. Am. J. Enol. Vitic. 12, 9–19 (1961). 173. Coffelt, R. J. & Berg, H. W. Color extraction by heating whole grapes. Am. J. Enol. Vitic. 16, 117–128 (1965).

1238

174. He, F., Liang, N. N., Mu, L., Pan, Q. H., Wang, J., Reeves, M. J. & Duan, C. Q.

1239

Anthocyanins and their variation in red wines I. Monomeric anthocyanins and their color

1240

expression. Molecules. 17, 1571–1601 (2012).

1241

175. Smith, P. A., McRae, J. M. & Bindon, K. A. Impact of winemaking practices on the

1242

concentration and composition of tannins in red wine: Impact of winemaking practices

1243

on tannins. Aust. J. Grape Wine Res. 21, 601–614 (2015).

1244 1245

176. Wagener, G. W. W. The effect of diffferent thermovinification systems on red wine quality. Am. J. Enol. Vitic. 32, 179–184 (1981).

1246

177. Morel-Salmi, C., Souquet, J. M., Bes, M. & Cheynier, V. Effet of flash release treatment

1247

on phenolic extraction and wine composition. J. Agric. Food Chem. 54, 4270–4276

1248

(2006).

1249 1250

178. Zimmermann, U., Pilwat, G. & Riemann, F. Dielectric breakdown of cell membranes. Biophys. J. 14, 881–899 (1974).



1251

179. López, N., Puértolas, E., Condón, S., Álvarez, I. & Raso, J. Effects of pulsed electric

1252

fields on the extraction of phenolic compounds during the fermentation of must of

1253

Tempranillo grapes. Innov. Food Sci. Emerg. Technol. 9, 477–482 (2008).

1254

180. Boussetta, N., Vorobiev, E., Le, L. H., Cordin-Falcimaigne, A. & Lanoisellé, J.-L.

1255

Application of electrical treatments in alcoholic solvent for polyphenols extraction from

1256

grape seeds. LWT - Food Sci. Technol. 46, 127–134 (2012).

1257

181. El Darra, N., Grimi, N., Vorobiev, E., Maroun, R. G. & Louka, N. Pulsed electric field

1258

assisted cold maceration of Cabernet Franc and Cabernet Sauvignon grapes. Am. J. Enol.

1259

Vitic. 64, 476–484 (2013).

1260

182. Cholet, C., Delsart, C., Petrel, M., Gontier, E., Grimi, N., L’Hyvernay, A., Ghidossi, R.,

1261

Vorobiev, E., Mietton-Peuchot, M. & Gény, L. Structural and biochemical changes

1262

induced by pulsed lectric field treatments on Cabernet Sauvignon grape berry skins:

1263

impact on cell wall total tannins and polysaccharides. J. Agric. Food Chem. 62, 2925–

1264

2934 (2014).

1265

183. El Darra, N., Rajha, H. N., Ducasse, M. A., Turk, M. F., Grimi, N., Maroun, R. G.,

1266

Louka, N. & Vorobiev, E. Effect of pulsed electric field treatment during cold

1267

maceration and alcoholic fermentation on major red wine qualitative and quantitative

1268

parameters. Food Chem. 213, 352–360 (2016).

1269

184. Saldaña, G., Cebrián, G., Abenoza, M., Sánchez-Gimeno, C., Álvarez, I. & Raso, J.

1270

Assessing the efficacy of PEF treatments for improving polyphenol extraction during red

1271

wine vinifications. Innov. Food Sci. Emerg. Technol. 39, 179–187 (2017).

1272

185. Sun, B., Leandro, C., Ricardo da Silva, J. M. & Spranger, I. Separation of grape and

1273

wine proanthocyanidins according to their degree of polymerization. J. Agric. Food

1274

Chem. 46, 1390–1396 (1998).


Page 56 of 73

Page 57 of 73


1275

186. Kyraleou, M., Kallithraka, S., Koundouras, S., Chira, K., Haroutounian, S.,

1276

Spinthiropoulou, H. & Kotseridis, Y. Effect of vine training system on the phenolic

1277

composition of red grapes (Vitis vinifera L. cv. Xinomavro). OENO One. 49, 71–84

1278

(2015).

1279

FUNDING

1280

We thank the CIVB (Conseil Interprofessionnel des Vins de Bordeaux) for its financial

1281

support.



Page 58 of 73

1283

FIGURE CAPTIONS

1284

Figure 1: Chemical structure of flavonoids

1285

Figure 2: Chemical structure of flavan-3-ols

1286

Figure 3: Flavonoid biosynthetic pathway (PAL: Phenylalanine ammonia lyase ; C4H:

1287

Cinnamate 4-hydroxylase ; 4CL: 4-coumarate :CoA ligase ; CHS: Chalcone synthase ; CHI:

1288

Chalcone isomerase ; F3H: Flavonoid 3-hydroxylase ; F3’H: Flavonoid 3’5’-hydroxylase ;

1289

F3’5’H:

1290

Leucoanthocyanidin

1291

Anthocyanidin reductase)

1292

Figure 4: Catalytic mechanism of LAR 73

1293

Figure 5: Catalytic mechanism of ANR 81

1294

Figure 6: Mechanism of ANR reverse epimerization 81

1295

Figure 7: Gene transcription factors of PAs biosynthetis

1296

Figure 8: Hypothetic scheme of flavonoid transport in grapevine cells 12

1297

Figure 9: Global pattern of grape seed metabolism and how winemaking processes impact the

1298

extractability of seed tannins (1: Berry integrity, 2: Maceration time, 3: PEF, 4: Cold soak, 5:

1299

Maceration enzyme, 6: High fermentation temperature, 7: Thermovinification and Flash

1300

release)

Flavonoid

3’5’-hydroxylase ; dioxygenase ;

DFR:

LAR:

Dihydroflavonol

4-reductase ;

LDOX:

reductase ;

ANR:

Leucoanthocyanidin


Page 59 of 73


TABLES Table 1: Chemical Structure of the Principal Flavan-3-ols Monomers Structure

Flavan-3-ols

R1

R2

C-2

C-3

(+)-Catechin

H

H

R

S

(+)-Gallocatechin

OH

H

R

S

R

S

(+)-Gallocatechin OH gallate

(-)-Epicatechin

H

H

R

R

(-)-Epigallocatechin

OH

H

R

R

(-)-Epicatechin gallate

H

R

R



Page 60 of 73

Table 2: Percentage of Extractable Tannins from Seeds and Skins in Grape – Influence of Variety, Vintage, Maturity Stages, and Vineyard

Variety

Vintage

1999 Shiraz 2000 2004 Pinot Noir 2004 2004 Cabernet Sauvignon

2004 2008 2004

Merlot 2004

Maturity Stage Harvest time Harvest time Harvest time Harvest time Harvest time Harvest time Harvest time Harvest time Harvest time

Grape polyphenols sources Reference Seed Skin (%) (%)

Vineyard

° Brix

-

23,5

59

41

30

-

29

75

25

30

Montalto

18-19

84,8

15,2

39

Montalto

18-19

81

19

39

18-19

96

4

39

18-19

94,2

5,8

39

20

75

25

36

Avio

18-19

72,8

27,2

39

Arco

18-19

77,8

22,2

39

Roverè della Luna Roverè della Luna Grinzane Cavour


Page 61 of 73


Table 3: Mean Degree of Polymerisation (mDP) of Seed and Skin Tannins at Harvest Time: Influence of Variety, Vintage, and Vineyard Variety Tinta Miuda Cabernet Sauvignon Cabernet Sauvignon Cabernet Sauvignon Cabernet Sauvignon Cabernet Sauvignon Cabernet Sauvignon Cabernet Sauvignon Malvasia bianca Moscato bianco Nascetta Nebbiolo Pinot bianco Shiraz Shiraz Monastrell Syrah Syrah Syrah Merlot Merlot Merlot Carménère Carménère Carménère Cabernet Franc Alicante Bouschet Plavac mali Babić Agiorgitiko

Vintage 1994 2008 2009 2008 2008 2004 2000 2008 2008 2008 2008 2008 2000 2000 2007 2007 2004 2009 2008 2004 2008 2008 2004 2008 2010 2010 2012

Vineyard Dois Portos, Portugal Piemonte, Italy Davis, USA Bordeaux, France Chili Chili Italy Navarra, Spain Piemonte, Italy Piemonte, Italy Piemonte, Italy Piemonte, Italy Piemonte, Italy South Australia South Australia Southeastern Spain Southeastern Spain Italy Epanomi, Greece Bordeaux, France Chili Italy Chili Chili Italy Chili Var, France Dalmatia, Croatia Dalmatia, Croatia Nemea, Greece

Seed mDP 14 10,3 5,63 16,1 2,7 5,5 3 6,4 11,6 9,3 8,9 9,5 10,3 4 5,6 8,3 3,7 3,5 15 11,5 4,2 2,3 3,2 4,5 2,8 5 30,3 7 8 8,15

Skin mDP 36,6 21,3 7,1 3,4 85,7 28,9 35,7 31,3 50,2 33,2 28,5 27,5 13,9 3,4 33 37,8 4,2 3,8 2,1 30 40 2,8


Analytical Method Thiolysis Phloroglucinolysis Thiolysis Phloroglucinolysis Phloroglucinolysis Phloroglucinolysis Thiolysis Phloroglucinolysis Phloroglucinolysis Phloroglucinolysis Phloroglucinolysis Phloroglucinolysis Phloroglucinolysis Phloroglucinolysis Phloroglucinolysis Phloroglucinolysis Phloroglucinolysis Thiolysis Phloroglucinolysis Phloroglucinolysis Phloroglucinolysis Thiolysis Phloroglucinolysis Phloroglucinolysis Thiolysis Phloroglucinolysis Thiolysis Phloroglucinolysis Phloroglucinolysis Phloroglucinolysis

Reference 185 36 115 34 35 37 39 31 36 36 36 36 36 30 126 157 157 39 38 34 37 39 35 37 39 37 113 40 40 63


Agiorgitiko Xinomavro

2012 2010

Koutsi, Greece Naoussa, Greece

7,20 8


2,8 8

Page 62 of 73

Phloroglucinolysis Phloroglucinolysis

63 186

Page 63 of 73


Table 4: Percentage of Galloylation (G%) of Seed and Skin Tannins : Influence of Variety, Vintage and Vineyard

Variety

Vintage

Vineyard

° Brix

G% (seed)

G% (skin)

Cabernet Sauvignon Cabernet Sauvignon Cabernet Sauvignon Cabernet Sauvignon Cabernet Sauvignon Malvasia bianca Moscato bianco Nascetta Nebbiolo Pinot bianco Merlot Merlot Merlot Carménère Carménère Cabernet Franc Alicante Bouschet

2008 2009 2008 2008 2000 2008 2008 2008 2008 2008 2009 2008 2008 2008 2008 -

Piemonte, Italy Bordeaux, France Chili Chili Navarra, Spain Piemonte, Italy Piemonte, Italy Piemonte, Italy Piemonte, Italy Piemonte, Italy Bordeaux, France Chili France Chili Chili Chili Var, France

18-19 25,3 24,5 18-19 18-19 18-19 18-19 18-19 24,5 23,7 25,7 26,5 -

13,6 32,2 16,3 23 12,9 20,3 18,7 21,2 13,1 14,2 22,4 25 27,5 28 30 30,3

1,2 16,7 19 3,8 2,9 4,1 3,1 1,4 2,0 16,9 3,95 12,5 -


Analytical method Phloroglucinolysis Phloroglucinolysis Phloroglucinolysis Phloroglucinolysis Phloroglucinolysis Phloroglucinolysis Phloroglucinolysis Phloroglucinolysis Phloroglucinolysis Phloroglucinolysis Phloroglucinolysis Phloroglucinolysis Thiolysis Phloroglucinolysis Phloroglucinolysis Phloroglucinolysis Thiolysis

Reference 36 34 35 37 31 36 36 36 36 36 34 37 18 35 37 37 113


FIGURE GRAPHICS

Figure 1


Page 64 of 73

Page 65 of 73


Figure 2



Figure 3


Page 66 of 73

Page 67 of 73


Figure 4



Figure 5


Page 68 of 73

Page 69 of 73


Figure 6



Figure 7


Page 70 of 73

Page 71 of 73


Figure 8



Figure 9


Page 72 of 73

Page 73 of 73


TOC Graphic


From Flavanols Biosynthesis to Wine Tannins - American Chemical

Recommend Documents