Effects of Leaf Removal and Applied Water on Flavonoid

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Effects of Leaf Removal and Applied Water on Flavonoid Accumulation in Grapevine (Vitis vinifera L. cv. Merlot) Berry in a Hot Climate Runze Yu, Michael G. Cook, Ralph S. Yacco, Aude Annie Watrelot, Gregory Gambetta, James A. Kennedy, and S. Kaan Kurtural J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03748 • Publication Date (Web): 12 Oct 2016 Downloaded from http://pubs.acs.org on October 17, 2016

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 free 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 accessible to all readers and 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.

Journal of Agricultural and Food Chemistry 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 32

Journal of Agricultural and Food Chemistry

1

Effects of Leaf Removal and Applied Water on Flavonoid Accumulation in

2

Grapevine (Vitis vinifera L. cv. Merlot ) Berry in a Hot Climate

3

Runze Yu, Michael G. Cookǀ, Ralph S. Yacco▪, Aude A. Watrelot, Gregory Gambetta‡, James

4

A. Kennedy §,

5

and S. Kaan Kurtural*

6

Department of Viticulture and Enology Oakville Experiment Station University of California, Oakville, California, 94562

7 §

8

9

10

ǀ

Constellation Brands, Inc., 12667 Road 24, Madera, California, 93637

Texas A&M AgriLife Ext. Ser. 401 West Hickory Street, Denton, Texas, 76201 ▪

Gusmer Enterprises, Inc., 124 M Street, Fresno, California, 93721

11

‡ 3UMR EGFV ISVV, 210 Chemin de Leysotte - CS 50008 33882 Villenave d'Ornon Cedex, France

12

* Corresponding author telephone: +1 530 752-0380, fax: +1 530 752-0382

13

e-mail: [email protected]

14

1 ACS Paragon Plus Environment


15

ABSTRACT

16

The relationships between variations in grapevine (Vitis vinifera L. cv. Merlot) fruit zone light

17

exposure and water deficits and the resulting berry flavonoid composition were investigated in a

18

hot climate. The experimental design involved application of mechanical leaf removal (control,

19

pre-bloom, post-fruit set) and differing water deficits (sustained deficit irrigation and regulated

20

deficit irrigation). Flavonol and anthocyanin concentration was measured by C18 reversed-

21

phased HPLC and increased with pre-bloom leaf removal in 2013, but with post-fruit set leaf

22

removal in 2014. Proanthocyanidin isolates were characterized by acid-catalysis in the presence

23

of excess phloroglucinol followed by reversed-phase HPLC. Post-fruit set leaf removal

24

increased total proanthocyanidin concentration in both years while no effect was observed with

25

applied water amounts. Mean degree of polymerization of skin proanthocyanidins increased

26

with post-fruit set leaf removal compared to pre-bloom while water deficit had no effect.

27

Conversion yield was greater with post-fruit set leaf removal. Seed proanthocyanidin

28

concentration was rarely affected by applied treatments. The application of post fruit-set leaf

29

removal, regardless of water deficit increased the proportion of proanthocyanidins derived from

30

the skin, while no leaf removal or pre-bloom leaf removal regardless of water deficits increased

31

the proportion of seed-derived proanthocyanidins. The study provides fundamental information

32

to viticulturists and winemakers on how to manage red wine grape low molecular weight

33

phenolics and polymeric proanthocyanidin composition in a hot climate.

34

Keywords: Leaf removal; applied water amount; viticulture practices; water deficit;

35

flavonols; anthocyanins; flavanols.


Page 2 of 32

Page 3 of 32

36


INTRODUCTION

37

Wine grapes grown in the San Joaquin Valley (SJV) of California are generally used for

38

bulk wine production due to less than ideal flavonoid concentration at harvest. There have been

39

more efforts to apply principles of canopy management with vineyard mechanization and water

40

deficits to enhance the flavonoid composition of red wine grapes grown in the region in the

41

presence of persistent droughts 1, 2.

42

Products from the phenylpropanoid biosynthetic pathway, compounds resulting from

43

metabolism of phenylalanine and to a lesser extent, tyrosine are of interest in viticulture. This

44

pathway is integral to the biosynthesis of flavonoids, which includes three major classes of

45

compounds: flavonols, anthocyanins and proanthocyanidins (PAs) 3, as well as stilbenes and

46

hydroxycinnamic acids. Flavonols are thought to function as plant tissue UV protectants

47

whereas anthocyanins are found to provide protection from UV radiation, high temperature

48

extremes, and to aid in seed dispersal 4-6. Although anthocyanins are responsible for the color of

49

red wine, flavonols are believed to contribute to red wine color via copigmentation 7. PAs,

50

polymers of flavan-3-ol subunits found in grape skin and seed, contribute to astringency

51

(mouthfeel) and in-mouth tactile sensations associated with wine 8, 9, are thought to deter

52

herbivores and possess antifungal properties 10. These three groups of flavonoids are of interest

53

in wine grapes because of their contribution to wine sensory properties.

54

Previous studies have investigated the effect of solar radiation on flavonoid biosynthesis

55

particularly on anthocyanins 6, 11 and some have investigated the effects of varying temperature

56

regimes on PAs 4. Some studies have shown that when the light transmittance into grapevine

57

canopy increased or the temperature altered corresponding to the change in light amount, grape

58

berry anthoycanins, flavonols and PA concentration and composition would be affected. Leaf 3 ACS Paragon Plus Environment


59

removal is applied as a grapevine canopy management tool to influence the exposure of the

60

berries to solar radiation 2, 11. In previous research, pre-bloom leaf removal when applied to

61

Merlot grapevine in the hot SJV resulted in no effect on yield with minimal vegetative

62

compensation; but increased total skin anthocyanin (TSA) concentration 11. Cluster light

63

exposure could increase (-)-epigallocatechin (EGC) concentration and decrease dihydroxylated

64

PA subunit (-)-epicatechin-3-O-gallate (ECG) 12. However, there is a lack of knowledge on the

65

relationships between variable light environments with PA composition of red wine grape in a

66

hot climate.

67

Reductions in applied water amounts that resulted in water deficits were shown to

68

promote higher concentrations of anthocyanins and flavonols on a berry weight basis in red wine

69

grapes, while no difference was observed on a per berry basis 13. However, water deficits have

70

been reported to have milder effects on PA concentration in berry skins 13-15. Some gene

71

expression studies have shown that water deficits in wine grapes could regulate flavonoid

72

biosynthesis 14. Water deficits in grapevine also resulted in less basal leaves contributing to

73

greater solar exposure of the clusters. 2, 16.

74

Although canopy and crop load management studies 1, 17 and trials implementing water

75

deficits have been conducted in the coastal grape growing regions of California 18, 19, few such

76

studies have been conducted on wine grapes grown in the hot climate of the SJV of California.

77

The objective of this experiment was to manipulate Merlot berry flavonoid accumulation in order

78

to quantitatively increase flavonoid concentration and assess berry skin PA composition without

79

adversely affecting yield in hot climate.

80

MATERIALS AND METHODS


Page 4 of 32

Page 5 of 32


Vineyard. This study was conducted in 2013 and 2014 at a commercial vineyard planted

81 82

in 1998 with V. vinifera L. cv. Merlot (clone 1, grafted onto Freedom 27% V. vinifera hybrid

83

rootstock), located in the northern SJV of California. Vines were planted at 2.13 m × 3.35 m

84

(vine × row) spacing in north-south orientated rows. All the vines were head trained on a

85

California sprawl trellis with six canes that were six to eight nodes long.

86

Experimental Design. The study was designed as a factorial arrangement of treatments

87

in a split-plot experiment in a randomized complete block with four replications. The main plot

88

was leaf removal and the sub-plot was water deficits. Each experimental unit consisted of 48

89

vines.

90

Leaf Removal Treatments. In this study, three leaf removal treatments (one untreated

91

control) were established on the morning (East) side of the canopy based on different grape

92

growing cycles. Pre-bloom leaf removal treatment was established at 200 Growing Degree Days

93

(GDD) in both 2013 and 2014, post-fruit set leaf removal treatment was established at 540 GDD

94

in 2013 and 644 GDD in 2014. A mechanical leaf remover (Model EL-50, Clemens Vineyard

95

Equipment, Woodland, CA) was used to create a 50 centimeter opening of the fruiting zone on

96

only the East side of the canopy. The severity of leaf removal was set to remove two layers of

97

leaves on the outer periphery of the canopy with the goal of retaining two external leaf layers, as

98

the implement rolled over it and plucked the leaves.

99

Irrigation Treatments. Vineyard crop evapotranspiration (ETc) was calculated by

100

multiplying the reference evapotranspiration (ETo ,obtained from the Denair weather station of

101

California Irrigation Management System) and the weekly crop coefficients (Kc) 20. The weekly

102

crop coefficients (Kcs) used to calculate water deficits were developed as reported by Cook et al.



103

11

104

the region 21.

105

. All other cultural practices were carried out according to accepted commercial practices for

Two irrigation treatments were established. A control treatment of sustained deficit

106

irrigation (SDI) was 80% of estimated ETc and applied to maintain a mid-day leaf water potential

107

Ψl of -1.2 MPa, from fruit set to harvest. A regulated deficit irrigation (RDI) treatment was

108

applied at the same Ψl with SDI soon after bud break but was decreased to 50% ETc from fruit

109

set to veraison with a Ψl of -1.4 MPa and then reinstated to SDI from veraison until harvest.

110

Field Data Collection and Berry Sampling. External leaf numbers and number of

111

canopy gaps per 30 cm of row were measured three times during the season using standard

112

methodology as reported elsewhere 1,10. Yield component measurements were taken on a single

113

harvest date (August 26, 2013 and August 19, 2014) when the total soluble solids reached 24

114

Brix. 100 and 20 random berries were harvested as two sets of subsamples: the first set of 100

115

berries were taken for total soluble solids, pH, titratable acidity and berry mass. The berries

116

were taken at random from clusters on the grapevine. Since there were no treatment effects on

117

total soluble solids, pH and titratable acidity the results were not shown. The second set of 20

118

berries were taken for High-Performance Liquid Chromatography (HPLC) and for dry skin and

119

seed mass measurements after lyophilization.

120

Extraction of Flavonoids. The extraction procedure was previously reported 23. Briefly,

121

berry skins and seeds were manually separated and lyophilized (Triad Freeze Dry System,

122

Labconco, Kansas City, MO) within the same as day as harvest. Skin and seed dry masses were

123

recorded after lypholization. Dry tissues were then extracted in 20 mL 2:1 acetone:water for 24

124

h, in the dark. The samples were filtered to exclude solid tissues and then 1 mL of the liquid 6 ACS Paragon Plus Environment

Page 6 of 32

Page 7 of 32


125

sample was collected, acetone was evaporated from this 1 mL with a centrivap concentrator

126

(model: 7810010, Labconco, Kansas City, MO) attached to a -103 oC cold trap (model: 7385020,

127

Labconco, Kansas City, MO). Following evaporation of acetone, the residue was brought to a

128

volume of 5 mL with water. Samples were centrifuged for 15 min at 1400 g, and the supernatant

129

was analyzed. After centrifugation the supernatant was filtered by Teflon filters (0.45 μm,

130

Acrodisc CR) before injection.

131

Chemicals. All chromatographic solvents were HPLC grade. Acetonitrile, acetone,

132

ascorbic acid, ethanol (EtOH), glacial acetic acid, maleic acid, methanol (MeOH), potassium

133

metabisulfite, potassium bitartrate, potassium hydroxide, sodium chloride and sodium hydroxide

134

were purchased from Fisher Scientific (Santa Clara, CA). Phloroglucinol, (+)-catechin ((+)-

135

catechin hydrate, ≥ 98%) (C), (-)-epicatechin ((-)-epicatechin, ≥ 90%) (EC) and hydrochloric

136

acid were purchased from Sigma-Aldrich (St. Louis, MO). Malvidin-3-O-glucoside and

137

quercetin-3-O-rutinoside were purchased from Extrasynthese (Genay, France). Ammonium

138

phosphate monobasic and orthophosphoric acid were purchased from VWR (Visalia, CA).

139

Hydrochloric acid and sodium acetate anhydrous were purchased from E.M. Science

140

(Gibbstown, NJ) and Mallinckrodt (Phillipsburg, NJ) respectively. Bovine serum albumin (BSA,

141

Fraction V powder), sodium dodecyl sulfate (SDS; lauryl sulfate, sodium salt), triethanolamine

142

(TEA) were purchased from Sigma-Aldrich (St. Louis, MO), ferric chloride hexahydrate was

143

purchased from Fisher Scientific (Pittsburg, PA).

144

Instrumentation. An Agilent, Model 1100 HPLC (Palo Alto, CA) consisting of a

145

vacuum degasser, autosampler, quaternary pump, diode array detector with a column heater was

146

used for monomeric phenolic analysis. An Agilent, Model 1260 Infinity HPLC (Santa Clara,



147

CA) was used for phloroglucinolysis. Chemstation software was used for chromatographic

148

analysis.

149

HPLC Analysis of Flavan-3-ol, Flavonol Monomers and Total Skin Anthocyanins.

150

Monomeric flavan-3-ol, flavonol and total anthocyanin in grape skin were measured by C18

151

reversed-phase HPLC. The mobile phase flow rate was 0.5 mL/min, and three mobile phases

152

were used, that included Solvent A = 50 mM dihydrogen ammonium phosphate adjusted to pH

153

2.6 with o-phosphoric acid; Solvent B = 20% Solvent A + 80% acetonitrile (v/v); Solvent C =

154

0.2 M o-phosphoric acid adjusted to pH 1.5 with NaOH 23. Eluting flavan-3-ol, flavonol

155

monomers and anthocyanins were identified and quantified using (+)-catechin, malvidin-3-O-

156

glucoside and quercetin-3-O-rutinoside standards, respectively.

157

Phloroglucinolysis. PA isolates were characterized by acid-catalysis in the presence of

158

excess phloroglucinol followed by reversed-phase HPLC using a previously described method 22.

159

To isolate PAs, the crude PAs were purified using DSC-18 Solid Phase Extraction (SPE) Tube

160

(bed wt. 500 mg, volume 6 mL, Sigma-Aldrich, St. Louis, MO). SPE column was conditioned

161

with MeOH then water with 3 column volumes each and separately. 1 mL supernatant from the

162

samples mentioned above were then eluted 3× 3 mL MeOH to remove glycosides and low

163

molecular weight flavan-3-ol monomer material 22. Eluent was lyophilized to dryness and then

164

redissolved in 1 mL MeOH. Individual methanolic extracts were combined (equal volumes) with

165

phloroglucinolysis reagent (double strength) before reaction. To calculate conversion yield, the

166

mass of all PA subunits was summed and divided by the total PA mass 23 , which was obtained

167

by iron-reactive phenolics measurement 23, 24.


Page 8 of 32

Page 9 of 32

168


Iron-reactive Phenolics Measurement. Iron-reactive phenolic measurement (IRP) was

169

used to analyze the total PA content, as described previously 24. UV-visible absorbance values

170

were collected with a spectrophotometer (Lambda 25 UV/VIS; PerkinElmer, Waltham, MA).

171

The protein precipitation method utilized bovine serum albumin, ferric chloride. Four buffer

172

solutions were used: Buffer A = 200 mM acetic acid + 170 mM NaCl, adjusted to pH 4.9 with

173

NaOH; Buffer B = 5g/L potassium bitartrate + 12% EtOH (v/v), adjusted to pH 3.3 with HCl;

174

Buffer C = 5% TEA (v/v) + 5% SDS (w/v), adjusted to pH 9.4 with HCl; Buffer D = 200 mM

175

maleic acid + 170 mM NaCl , adjusted to pH 1.8 with NaOH 26, quantified from a standard

176

curve for (+)-catechin.

177

Statistical Analysis. Data was tested for normality using Shapiro-Wilk’s test and were

178

subjected to a two-way (leaf removal × irrigation) analysis of variance appropriate for a split-plot

179

using SAS version 9.3 (SAS Institute, Cary, NC). To determine treatment mean separation

180

Duncan’s new multiple range test was conducted after a prior analysis of variance indicated

181

statistical difference at 0.05 or less.

182

RESULTS

183

Weather, and Applied Water Amounts. In both years of the experiment the GDD

184

accumulation was greater than the 5 year cumulative GDD mean of 2037 (www.cimis.org). The

185

cumulative GDD in both 2013 and 2014 were 2242 and 2368 respectively. As presented in

186

Table 1, the research site was quite arid, only receiving precipitation between budbreak and fruit

187

set. The applied water amounts for SDI and RDI varied between years. Between fruit-set and

188

verasion the amount of water applied to RDI treatment was 58% and 55% of SDI in 2013 and

189

2014, respectively. The total applied water amount for the RDI treatment was 80% and 74% of



190

SDI in 2013 and 2014, respectively. The applied water amounts were successful in reaching the

191

Ψl target in both years. In 2013 there were 7 weeks of RDI and in 2014, 8 weeks of RDI applied

192

(Figure 1). The Ψl during these intervals were significantly different between the irrigation

193

treatments. Canopy assessment. We reported the photosynthetically active radiation transmittance

194 195

through the fruit zone in a companion paper 11. The external leaf layer number decreased and

196

number of canopy gaps per 30 cm of row increased in both years of the study by the application

197

of pre-bloom leaf removal treatment, regardless of measurement date (Figure 2.). Likewise, the

198

post-fruit set leaf removal reduced external leaf number after its application. However, number

199

of canopy gaps only increased by post-fruit set leaf removal soon after its application date.

200

Irrigation treatments did not have an effect on the canopy variables measured in either year of the

201

study.

202

Yield Components. In 2013, leaf removal reduced berry mass. However, there was no

203

effect of leaf removal on berry mass in 2014. Leaf removal affected berry skin mass. Post-fruit

204

set leaf removal reduced berry skin mass compared to pre-bloom leaf removal in 2013 (Table 2).

205

Berry skin mass with pre-bloom leaf removal performed similar to the control in both years. The

206

number of clusters harvested did not respond to leaf removal treatments consistently. In 2013,

207

post-fruit set leaf removal had the greatest yield when compared to control and pre-bloom.

208

Conversely, in 2014 post fruit-set leaf removal had the least clusters harvested when compared to

209

control and pre-bloom leaf removal. Yield at harvest was not affected by leaf removal

210

treatments in 2013. However, in 2014 post-fruit set leaf removal reduced yield by about 30%

211

compared to pre-bloom leaf removal. Water deficits consistently affected berry mass in both


Page 10 of 32

Page 11 of 32


212

years where RDI reduced it. Water deficits did not affect berry skin mass or clusters harvested

213

per vine.

214

Low Molecular Weight Phenolics. Leaf removal affected skin flavonol and

215

anthocyanin concentrations (Table 3). In 2013, total skin flavonol concentration was greater

216

with pre-bloom leaf removal. TSA concentration increased by pre-bloom leaf removal in 2013,

217

but by post fruit-set leaf removal in 2014. Water deficits had no effect on flavonol or TSA

218

concentration on a per berry basis in either year.

219

C monomer concentration was not affected by leaf removal in either year of the study

220

(Table 3). No effect was observed with leaf removal on EC monomer concentration in either

221

year. There was no effect of applied water amounts on monomeric flavan-3-ol concentration on a

222

per berry basis except in 2014 where more EC monomers per berry were observed with the SDI

223

treatment.

224

Skin Proanthocyanidin Composition and mDP. In 2013, post-fruit set leaf removal

225

increased EGC extension subunit in 2013 compared to control in skins. However, a similar

226

response was not evident in 2014. Water deficits only affected C proportion in 2013 where it

227

increased with the SDI treatment in 2013. EC extension subunits were consistently affected by

228

leaf removal treatments (Table 4). EC extension subunit concentration (% mol proportion) was

229

less with leaf removal treatments in 2013, but increased by 1.7% with post-fruit set leaf removal

230

in 2014.

231

C terminal subunits were the only subunit observed in both 2013 and 2014, EC and ECG

232

terminal subunits were not observed (Table 4, concentration was expressed in mg per kg). C

233

terminal subunit concentration was greater with post-fruit set leaf removal compared to control 11 ACS Paragon Plus Environment


234

and pre-bloom leaf removal. Water deficits did not affect terminal subunit composition in either

235

year.

236

The mDP was consistently affected by the leaf removal treatments (Table 4). Post-fruit

237

set leaf removal had the greatest mDP compared to pre-bloom leaf removal in both years. Water

238

deficits had no effect on mDP.

239

Total PA Concentration and Qualitative PA Composition of Berry Skin and Seed.

240

Total skin PA concentration was greater in 2013 compared to 2014 (Table 5). Nevertheless, leaf

241

removal treatments consistently affected total PA concentration (mg/L) in berry skin where post-

242

fruit set leaf removal had greater concentration compared to other treatments. There was no

243

effect of leaf removal or water deficits on total skin PAs when measured by IRP. The mol

244

proportion of trihydroxylated EGC extension subunits were generally less in 2014 compared to

245

2013 (Table. 4), and in 2013 EGC extension subunit concentration (mg/L) were greater with

246

post-fruit set leaf removal. For seed tissue, there was no effect of leaf removal except when

247

applied at pre-bloom that enhanced PA concentration in 2014. The water deficits did not affect

248

PA content in skin tissue in either year. However, in 2014 the PA content in seed was greater

249

with the RDI treatment.

250

Conversion Yield. In 2013, post-fruit set leaf removal had the greatest conversion yield

251

(28.3%) in skin, but same response was not evident in 2014 (Table 5). For seed, conversion yield

252

was observed to be greater without any leaf removal in 2013 (6.4%), but the same response was

253

not evident in 2014. Water deficits did not affect conversion yield in either year. Seed

254

conversion yield was generally lower in 2014 than in 2013, while skins had greater conversion

255

yield in 2014 than in 2013.


Page 12 of 32

Page 13 of 32

256 257


DISCUSSION Weather, and Applied Water Amounts. In both years of 2013 and 2014, an increase in

258

mean temperature, accumulation of GDD, and extreme temperatures were observed when

259

compared to the 5-year mean of the region. It was reported that increasing mean temperature

260

yearly may lead to an earlier bud break 25, as was the case in our study. Water deficits had no

261

consistent effect on canopy microclimate in either year, in contrast with other deficit irrigation

262

studies 2, 16.

263

Canopy assessment. Shade conditions may be managed by reducing leaf layers with a

264

proportional increase in number of canopy gaps, thus minimizing canopy density 1,16. Canopy

265

architecture was positively manipulated by reducing canopy density with of leaf removal.

266

Number of canopy gaps increased with pre-bloom leaf removal treatments when compared to

267

control 2,10,17. Terry and Kurtural 16 reported vine efficiency was optimized with a total leaf layer

268

number of 3.0 that consisted of two external leaf layers and an internal leaf layer for non-shoot

269

positioned grapevine canopies in central California. In 2013, both leaf removal treatments

270

achieved a more optimal external leaf layer number as compared to control. In 2014, however,

271

all treatments were found to be near optimum levels. This can be explained by a general decrease

272

in overall growth thought to have been moderated due to the worsening drought conditions in

273

central California as seen in the second year. Nevertheless, leaf removal, in particular pre-bloom

274

leaf removal treatment, consistently lowered vegetative growth indices resulting in a less dense

275

canopy when compared to control29.

276 277

Effects of Leaf Removal and Water Deficit on Yield Components. Carbohydrate supply during anthesis is the primary determinant of fruit-set and thus final yield at harvest 26.



278

Therefore, the extent to which yield is reduced by early leaf removal varies greatly due to the

279

magnitude of altering source-sink relationships 26. The principal factors involved in source

280

inhibition include variances in timing and severity of leaf removal, genotype, canopy size, and

281

growing conditions throughout season 2, 26. Percival et al. 27 stated that grapevines often produce

282

more leaves than required, especially in warm climates, and thus a reduction in external leaf

283

layers or an increase in canopy gaps may not be adequate in eliciting a negative response in

284

yield.

285

In previous work, post-bloom leaf removal typically did not decrease yield as source-sink

286

imbalance was often avoided 28, 29. Although yield was not affected by either leaf removal

287

treatment in 2013, berry mass was slightly reduced as previously reported 26 . This was attributed

288

to be the result of excessive solar radiation exposure 10,30 and with suggestions in lowering

289

cluster water potential of exposed berries 2. However, the effect of leaf removal on berry mass

290

was not repeatable in 2014.

291

Berry skin mass played an important role in phenolic accumulation and subsequent

292

protection of berry integrity 31, 32. The increase in skin mass of Merlot berries with pre-bloom

293

leaf removal compared to post fruit-set leaf removal was indicative of the long term adaptation

294

mechanism of berry thickening 31. Poni et al. 33 and Gatti et al. 34 reported that positive changes

295

in skin mass were affected by solar radiation and temperature, that prevailed over any deleterious

296

effect of source-sink imbalance. Kliewer 35 reported in Tokay that berry skin thickness was

297

reduced during fruit set when temperatures were maintained at 40°C compared to 25°C. Our

298

results indicated that the long-term adaptation mechanism of berry skin thickening was a

299

response to prolonged inflorescence and cluster exposure as a result of pre-bloom leaf removal

300

when compared to post-set leaf removal. 14 ACS Paragon Plus Environment

Page 14 of 32

Page 15 of 32

301


Reproductive growth was more sensitive to vine water status than that of vegetative

302

growth. The RDI treatment was successful in reducing berry mass as found in other studies 2, 18.

303

However, in our study, skin mass was unaltered due to phenological timing of water deficit

304

treatments 36. It could be conceived that the reduction in berry mass was due to a decline of

305

inner mesocarp cell sap 15 and inhibition of cell expansion over cell division 18, 37. Conversely,

306

yield was not significantly altered by irrigation treatments in 2013, possibly due to the mitigation

307

of vegetative compensation through water deficits invigorating lateral shoot regeneration just

308

enough to set similar berries per cluster 10,11. In 2014, a reduction in berry mass was more

309

pronounced with RDI, possibly as a result of lowered cluster water potential 2 and more a porous

310

canopy11.

311

Effects of Leaf Removal and Water Deficit on Low Molecular Weight Phenolics.

312

Kemp et al. 38 reported that flavan-3-ol monomers were affected by different timing of

313

leaf removal treatments in wine. In our study, pre-bloom leaf removal displayed a tendency to

314

have greater C monomer concentration between the two leaf removal treatments in both years.

315

Leaf removal generally provided greater flavonol concentration than control. Flavonol

316

concentration in berry skins seemed sensitive to light condition changes, as highly induced

317

accumulation of flavonols along with increasing expression of flavonol synthase (FLS), has been

318

reported by greater light exposure 39 attributed to greater canopy porosity.

319

Prolonged exposure to direct solar radiation may lower the anthocyanin accumulation.

320

Previous work indicated that vacuolar storage may be impeded as berry skin mass decreased by

321

post fruit-set leaf removal 40. This would provide explanation as to why TSA concentration in

322

2013 was greater with pre-bloom leaf removal. Conversely, in the following year, the TSA 15 ACS Paragon Plus Environment


323

concentration was greater with post fruit-set leaf removal. We attributed greater TSA

324

concentration in 2014 by post fruit-set leaf removal to less yield than other treatments. More

325

source material was available per sink unit during the season, thus promoting the accumulation

326

of anthocyanin content 41, and ultimately overriding the deleterious effects of diminished skin

327

mass in post fruit-set leaf removal treatment in 2014. These observations were similar to that of

328

pre-bloom defoliation in other studies where yield control occurred 26, 42.

Page 16 of 32

Water deficits had no effect on berry flavan-3-ol concentration in either year, which was

329 330

similar to Kennedy et al. 13 on a per berry basis. A minor effect was observed with early water

331

deficits that enhanced the expression of leucoanthocyanidin-reductase (LAR) before veraison in

332

Merlot 14, which can reduce leucoanthocyanidin into flavan-3-ol monomers. Ojeda et al. 36 reported water deficits influenced the biosynthesis and concentration of

333 334

flavonols in skin tissue. Similarly, Castellarin et al. 14 noted that water deficits affected flavonol

335

accumulation but not to the degree of anthocyanins. In contrast, Kennedy et al. 13 determined

336

with Cabernet Sauvignon that water deficits were inadequate in altering flavonol concentration.

337

In our study, water deficits had no effect on flavonol concentration in either year. There was no

338

effect of water deficits on TSA concentration observed, which was contrary to previous reports 43,

339

44

340

irrigation treatments, where timing and amount of irrigation applied between SDI and RDI was

341

insufficient at achieving a significant response at the berry level.

342

. The discrepancy between prior studies and ours could be attributed to the difference in

Skin PA Concentration, Composition and mDP. Flavan-3-ol free monomers and

343

polymeric PAs are relatively stable in both skin and seed 45. PAs consist of several constitutive

344

subunits which are able to be linked different locations on the three ring structure of flavonoid


Page 17 of 32


345

compounds 46, most through C4-C8 linkage in anthocyanin and flavanol units 47. PAs

346

accumulated in grape skin are not simple to investigate due to the ability of anthocyanins and

347

flavonols to form pigmented polymers with PAs 48 as well as their ability to interact with cell

348

wall material and to be oxidized when cells are disrupted. The concentration of PA subunits was

349

relatively high at fruit set either for free flavan-3-ol monomers or terminal subunits 49. In our

350

study, the EGC extension subunit concentration was greater by applying post-fruit set leaf

351

removal in 2013 but not in 2014, that was corroborated by previous works 12, 39 where the

352

exposure of clusters to sunlight has led to compositional shift to extension subunits. Similar

353

results were reported 50 where visible light, the primary inducing factor of PA biosynthesis,

354

increased the level of B-ring hydroxylation of EC extension subunits. There was an observation

355

of stable reduction of EC extension subunits in exposed berries 12. In Pinot noir, skin extension

356

subunits were found to be composed of about 63% EC and 34% EGC 23, showing a similar

357

proportion of about 50% EC, 46% EGC observed in our study with Merlot.

358

For skin PAs, only the C terminal unit was observed in our study, similar to previous

359

reports in Pinot noir 12. However, the C terminal subunit concentration was consistently affected

360

by leaf removal. Post-fruit set leaf removal enhanced C terminal because PA biosynthetic genes

361

may have been induced in the flavonoid pathway 51. In the flavonoid pathway, the PAs branch

362

from prodelphinidin and procyanidin prior to veraison, where LAR and anthocyanidin-reductase

363

(ANR) convert proanthocyanidins and anthocyanidins into C and EC monomers, the subunits of

364

skin PAs 14. During PA biosynthesis, it would be reasonable to assume that electrophilic

365

subunits combine with the nucleophilic 8 or 6 position of the starter unit 52. It was suggested that

366

encoding LAR in A. thaliana seed coat because of its similarity with dihydroflavonol-reductase

367

(DFR), the BANYULS gene could convert flavan-3,4-diols to the corresponding 2,3-trans17 ACS Paragon Plus Environment


Page 18 of 32

368

flavan-3-ols such as (+)-catechin 53. Our results suggest that BANYULS could be managed with

369

post-fruit leaf removal. Berry skin mDP was greater with the post-fruit set leaf removal compared to pre-bloom

370 371

leaf removal. Kennedy et al. 54 reported that bitterness was derived from lower molecular weight

372

flavan-3-ol compounds and astringency from higher molecular weight PA compounds. The mDP

373

of skin PA in Shiraz grapes increased with berry development 23 and skin PA mDP increased

374

during the early phase of berry development and decreased at the onset of veraison

375

concentration decreased 48, 56. The overall decline of skin PAs mDP between veraison and

376

harvest was explained by the formation of increasingly stable associations with other cellular

377

components 49. Downey et al. 39 reported that, exclusion of sunlight from berries would reduce

378

skin PA mDP. In our study, early cluster exposure as pre-bloom leaf removal resulted in lower

379

mDP than control in 2014. Post-fruit set skin mDP were greater in both years. The EGC

380

extension subunits had a greater proportion in 2013, total PA concentration was greater and mDP

381

was higher in skin of post fruit-set leaf removal. Cohen et al. 4 reported there was no consistent

382

relationship between temperature and total PA accumulation across three seasons, and

383

composition was affected because decreasing thermal time in degree-days and shift towards

384

trihydroxylated forms. Nonetheless, the mDP values we observed were much lower than that

385

found in previous studies 12, 23 which were conducted in cooler climates than SJV.

12, 55

as PA

The mDP values were not affected by water deficits, in agreement with previous studies

386 387

12, 57

388

mass was the greatest contributor to total skin PAs under water deficits. However, this was not

389

evident in our work 13. Although water deficits treatments in our study were not sufficient to

. Conversely, a greater total skin PA concentration was also reported where larger berry skin


Page 19 of 32


390

change PA concentration, the ability to apply less water to achieve similar total skin PA

391

concentration and mDP is important for viticulturists in a hot climate.

392

Total PA concentration and Qualitative PA composition. Total skin PA concentration

393

was affected by leaf removal when measured by phloroglucinolysis. Post-fruit set leaf removal

394

consistently increased total skin PA concentration compared to pre-bloom leaf removal that was

395

in contrast to TSA as being consistently greater with pre-bloom leaf removal. This suggested that

396

the PA and anthocyanin biosynthesis in skin were independent, as reported previously 39.

397

However, previous work 49 also suggested that the accumulation of PAs and anthocyanidins in

398

skin was a coordinated process and the EGC extension subunits increased as trihydroxylated

399

derivatives on the B-ring, which occured immediately prior to veraison when the anthocyanidins

400

accumulated in skin. Cortell et al. 12 reported the skin PA concentration was much lower with a

401

shading treatment, but others 39 reported even excluding solar radiation had no significant effect

402

on total skin PAs. Results suggest that typical vintage variability in total skin PA concentration

403

was due to timing of events such as low or high temperatures and to the integrated seasonal value

404

in addition to cluster exposure after fruit set. There was a decrease of total skin PAs with

405

phloroglucinolysis but an increase with IRP shown from 2013 to 2014. As reported previously 4,

406

there was no consistent relationship between seasonal temperature and total skin PA

407

accumulation.

408

In 2013, greater trihydroxylation was observed when post-fruit set leaf removal was

409

applied, as reported previously 12, 39. The phenyl constituent B-ring hydroxyl group configuration

410

is determined to be the primary determinant of scavanging of reactive oxygen species (ROS) 58, 59

411

and reactive nitrogen species (RNS) 60, 61. The number of hydroxyl groups on the B-ring was

412

shown to enhance the antioxidant activity based on the hydroxyl group redox petential of 19 ACS Paragon Plus Environment


413

donating hydrogen and an electron to hydroxyl, peroxyl, and peroxynitrite radicals 62, 63. Cook et

414

al. 11, reported the activity flavonoid 3',5'-hydroxylase (F3’5’H) or the corresponding enzyme

415

was upregulated by pre-bloom leaf removal, or the flavonoid 3'-hydroxylase (F3’H) side was

416

downregulated. The increasing concentration of trihydroxylated flavan-3-ols provided a more

417

detailed speculation that LAR or anthocyanidin-reductase (ANR) maybe upregulated by post-

418

fruit set leaf removal.

419

Conversion yield. Conversion yield indicates the effectiveness of phloroglucinolysis in the

420

conversion of PAs into their constitutive subunits 22 that can give an indication of oxidation

421

reaction. This provided an explanation as to why total PAs were greater with IRP but lower with

422

phloroglucinolysis in skin tissue. A steady decrease in conversion yield was shown to occur after

423

veraison, which suggested the PAs were being oxidized after veraison 22. The conversion yield

424

values in our study were lower than previous studies 22, 48. In our study, effects were only

425

observed in 2013 where post-fruit set leaf removal had the greatest conversion yield in skin.

426

Conversely, post-fruit set leaf removal had the lowest value in seeds. It was observed that

427

pigmented PAs had a lower conversion yield than unpigmented PAs 48, and TSA values in our

428

study were greatest with pre-bloom leaf removal in 2013. Therefore, by applying early leaf

429

removal, covalently associated pigmentation occurred with a greater TSA content resulting in the

430

PA conversion yield reduction in 2013. The worsening of drought conditions in 2014 more than

431

likely resulted in no response in 2014. Furthermore, PAs exposed to oxidation had lower

432

conversion yield 64, 65, forming more extensive intramolecular bonds which would be resistant to

433

acid-catalysed cleavage 46. McRae et al. 65, reported that with oxygen treatment, conversion yield

434

and mDP were lower but percent color was greater, which were consistent with our results.


Page 20 of 32

Page 21 of 32

435


In summary, leaf removal and water deficits resulted in manipulating not only the yield

436

component, canopy porosity and quantitative flavonoid concentration but also qualititive

437

responses of PA subunit accumulation. Total skin flavonols and anthocyanins were greater with

438

pre-bloom leaf removal in 2013 but with post-fruit set leaf removal in 2014; total skin PAs, mol

439

proportion of EGC subunits, mDP of skin tissues and PA conversion yields were enhanced by

440

post-fruit set leaf removal. In this study, an increasing understanding of the different responses of

441

the grapevine towards fruit zone light management and applied water were shown, which can

442

provid an insight into flavonoid accumulation in hot climate.

443

Uncategorized References

444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472

1. Kurtural, S. K.; Wessner, L. F.; Dervishian, G., Vegetative Compensation Response of a Procumbent Grapevine (Vitis vinifera cv. Syrah) Cultivar under Mechanical Canopy Management. HortScience 2013, 48, 576-583. 2. Williams, L. E., Interaction of applied water amounts and leaf removal in the fruiting zone on grapevine water relations and productivity of Merlot. Irrigation Science 2012, 30, 363-375. 3. Souquet, J.-M.; Cheynier, V.; Brossaud, F.; Moutounet, M., Polymeric Proanthocyanidins from Grape Skins. Phytochemistry 1996, 43, 509-512. 4. Cohen, S. D.; Tarara, J. M.; Gambetta, G. A.; Matthews, M. A.; Kennedy, J. A., Impact of diurnal temperature variation on grape berry development, proanthocyanidin accumulation, and the expression of flavonoid pathway genes. Journal of experimental botany 2012, 63, 2655-2665. 5. Dixon, R. A.; Achnine, L.; Kota, P.; Liu, C. J.; Reddy, M.; Wang, L., The phenylpropanoid pathway and plant defence—a genomics perspective. Molecular Plant Pathology 2002, 3, 371-390. 6. Winkel-Shirley, B., Biosynthesis of flavonoids and effects of stress. Current opinion in plant biology 2002, 5, 218-223. 7. Boulton, R., The copigmentation of anthocyanins and its role in the color of red wine: a critical review. American Journal of Enology and Viticulture 2001, 52, 67-87. 8. Vidal, S.; Courcoux, P.; Francis, L.; Kwiatkowski, M.; Gawel, R.; Williams, P.; Waters, E.; Cheynier, V., Use of an experimental design approach for evaluation of key wine components on mouth-feel perception. Food Quality and Preference 2004, 15, 209-217. 9. Gawel, R.; Iland, P.; Francis, I., Characterizing the astringency of red wine: a case study. Food quality and preference 2001, 12, 83-94. 10. Aerts, R. J.; Barry, T. N.; McNabb, W. C., Polyphenols and agriculture: beneficial effects of proanthocyanidins in forages. Agriculture, Ecosystems & Environment 1999, 75, 1-12. 11. Cook, M. G.; Zhang, Y.; Nelson, C. J.; Gambetta, G.; Kennedy, J. A.; Kurtural, S. K., Anthocyanin Composition of Merlot is Ameliorated by Light Microclimate and Irrigation in Central California. American Journal of Enology and Viticulture 2015, ajev. 2015.15006. 12. Cortell, J. M.; Kennedy, J. A., Effect of shading on accumulation of flavonoid compounds in (Vitis vinifera L.) pinot noir fruit and extraction in a model system. Journal of Agricultural and Food Chemistry 2006, 54, 8510-8520. 21 ACS Paragon Plus Environment


473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518

13. Kennedy, J. A.; Matthews, M. A.; Waterhouse, A. L., Effect of maturity and vine water status on grape skin and wine flavonoids. American Journal of Enology and Viticulture 2002, 53, 268-274. 14. Castellarin, S. D.; Matthews, M. A.; Di Gaspero, G.; Gambetta, G. A., Water deficits accelerate ripening and induce changes in gene expression regulating flavonoid biosynthesis in grape berries. Planta 2007, 227, 101-112. 15. Roby, G.; Harbertson, J. F.; Adams, D. A.; Matthews, M. A., Berry size and vine water deficits as factors in winegrape composition: anthocyanins and tannins. Australian Journal of Grape and Wine Research 2004, 10, 100-107. 16. Terry, D. B.; Kurtural, S. K., Achieving vine balance of Syrah with mechanical canopy management and regulated deficit irrigation. American Journal of Enology and Viticulture 2011, ajev. 2011.11022. 17. Wessner, L. F.; Kurtural, S. K., Pruning Systems and Canopy Management Practice Interact on the Yield, and Fruit Composition of Syrah. American Journal of Enology and Viticulture 2012, ajev. 2012.12056. 18. Matthews, M. A.; Anderson, M. M., Reproductive development in grape (Vitis vinifera L.): responses to seasonal water deficits. American Journal of Enology and Viticulture 1989, 40, 52-60. 19. Williams, L. E., Determination of evapotranspiration and crop coefficients for a Chardonnay vineyard located in a cool climate. American Journal of Enology and Viticulture 2014, ajev. 2014.12104. 20. Allen, R. G.; Pereira, L. S.; Raes, D.; Smith, M., Crop evapotranspiration-Guidelines for computing crop water requirements-FAO Irrigation and drainage paper 56. FAO, Rome 1998, 300, D05109. 21. Bettiga, L. J., Grape pest management. UCANR Publications: 2013; Vol. 3343. 22. Kennedy, J. A.; Jones, G. P., Analysis of proanthocyanidin cleavage products following acidcatalysis in the presence of excess phloroglucinol. Journal of Agricultural and Food Chemistry 2001, 49, 1740-1746. 23. Rio, J. L. P. d.; Kennedy, J. A., Development of Proanthocyanidins in Vitis Vinifera L. Cv. Pinot Noir Grapes and Extraction into Wine. American Journal of Enology and Viticulture 2006, 57, 125-132. 24. Harbertson, J. F.; Picciotto, E. A.; Adams, D. O., Phenolic and Anthocyanin assay for Use with Spectrophotometer. In University of California, Davis: 2005. 25. Poni, S.; Gatti, M.; Bernizzoni, F.; Civardi, S.; Bobeica, N.; Magnanini, E.; Palliotti, A., Late leaf removal aimed at delaying ripening in cv. Sangiovese: physiological assessment and vine performance. Australian Journal of Grape and Wine Research 2013, 19, 378-387. 26. Poni, S.; Casalini, L.; Bernizzoni, F.; Civardi, S.; Intrieri, C., Effects of early defoliation on shoot photosynthesis, yield components, and grape composition. American Journal of Enology and Viticulture 2006, 57, 397-407. 27. Percival, D.; Fisher, K.; Sullivan, J., Use of fruit zone leaf removal with Vitis vinifera L. cv. Riesling grapevines. II. Effect on fruit composition, yield, and occurrence of bunch rot (Botrytis cinerea Pers.: Fr.). American journal of enology and viticulture 1994, 45, 133-140. 28. Bledsoe, A.; Kliewer, W.; Marois, J., Effects of timing and severity of leaf removal on yield and fruit composition of Sauvignon blanc grapevines. American journal of enology and viticulture 1988, 39, 49-54. 29. Poni, S.; Bernizzoni, F.; Briola, G.; Cenni, A. In Effects of early leaf removal on cluster morphology, shoot efficiency and grape quality in two Vitis vinifera cultivars, VII International Symposium on Grapevine Physiology and Biotechnology 689, 2004; 2004; pp 217-226. 30. Bergqvist, J.; Dokoozlian, N.; Ebisuda, N., Sunlight exposure and temperature effects on berry growth and composition of Cabernet Sauvignon and Grenache in the Central San Joaquin Valley of California. American Journal of Enology and Viticulture 2001, 52, 1-7.


Page 22 of 32

Page 23 of 32

519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566


31. Diago, M. P.; Ayestarán, B.; Guadalupe, Z.; Poni, S.; Tardáguila, J., Impact of Prebloom and FruitSet Basal Leaf Removal on the Flavonol and Anthocyanin Composition of Tempranillo Grapes. American journal of enology and viticulture 2012, ajev. 2012.11116. 32. Palliotti, A.; Gatti, M.; Poni, S., Early leaf removal to improve vineyard efficiency: gas exchange, source-to-sink balance, and reserve storage responses. American Journal of Enology and Viticulture 2011, ajev. 2011.10094. 33. Poni, S.; Bernizzoni, F.; Civardi, S.; Libelli, N., Effects of pre‐bloom leaf removal on growth of berry tissues and must composition in two red Vitis vinifera L. cultivars. Australian Journal of Grape and Wine Research 2009, 15, 185-193. 34. Gatti, M.; Bernizzoni, F.; Civardi, S.; Poni, S., Effects of cluster thinning and pre-flowering leaf removal on growth and grape composition in cv. Sangiovese. American Journal of Enology and Viticulture 2012, ajev. 2012.11118. 35. Kliewer, W., Effect of high temperatures during the bloom-set period on fruit-set, ovule fertility, and berry growth of several grape cultivars. American Journal of Enology and Viticulture 1977, 28, 215222. 36. Ojeda, H.; Andary, C.; Kraeva, E.; Carbonneau, A.; Deloire, A., Influence of pre-and postveraison water deficit on synthesis and concentration of skin phenolic compounds during berry growth of Vitis vinifera cv. Shiraz. American Journal of Enology and Viticulture 2002, 53, 261-267. 37. Keller, M.; Tarara, J.; Mills, L., Spring temperatures alter reproductive development in grapevines. Australian Journal of Grape and Wine Research 2010, 16, 445-454. 38. Kemp, B.; Harrison, R.; Creasy, G., Effect of mechanical leaf removal and its timing on flavan‐ 3‐ol composition and concentrations in Vitis vinifera L. cv. Pinot Noir wine. Australian Journal of Grape and Wine Research 2011, 17, 270-279. 39. Downey, M. O.; Harvey, J. S.; Robinson, S. P., The effect of bunch shading on berry development and flavonoid accumulation in Shiraz grapes. Australian Journal of Grape and Wine Research 2004, 10, 55-73. 40. Pastore, C.; Zenoni, S.; Fasoli, M.; Pezzotti, M.; Tornielli, G. B.; Filippetti, I., Selective defoliation affects plant growth, fruit transcriptional ripening program and flavonoid metabolism in grapevine. BMC plant biology 2013, 13, 30. 41. King, P. D.; McClellan, D. J.; Smart, R. E., Effect of severity of leaf and crop removal on grape and wine composition of Merlot vines in Hawke’s Bay vineyards. American journal of enology and viticulture 2012, ajev. 2012.12020. 42. Intrieri, C.; Filippetti, I.; Allegro, G.; Centinari, M.; Poni, S., Early defoliation (hand vs mechanical) for improved crop control and grape composition in Sangiovese (Vitis vinifera L.). Australian Journal of Grape and Wine Research 2008, 14, 25-32. 43. Castellarin, S. D.; Pfeiffer, A.; Sivilotti, P.; Degan, M.; Peterlunger, E.; Di Gaspero, G., Transcriptional regulation of anthocyanin biosynthesis in ripening fruits of grapevine under seasonal water deficit. Plant, Cell & Environment 2007, 30, 1381-1399. 44. Romero, P.; Fernández-Fernández, J. I.; Martinez-Cutillas, A., Physiological thresholds for efficient regulated deficit-irrigation management in winegrapes grown under semiarid conditions. American Journal of Enology and Viticulture 2010, 61, 300-312. 45. Teixeira, A.; Eiras-Dias, J.; Castellarin, S. D.; Gerós, H., Berry phenolics of grapevine under challenging environments. International journal of molecular sciences 2013, 14, 18711-18739. 46. Cheynier, V.; Dueñas-Paton, M.; Salas, E.; Maury, C.; Souquet, J.-M.; Sarni-Manchado, P.; Fulcrand, H., Structure and properties of wine pigments and tannins. American Journal of Enology and Viticulture 2006, 57, 298-305. 47. Cheynier, V., Polyphenols in foods are more complex than often thought. The American journal of clinical nutrition 2005, 81, 223S-229S. 23 ACS Paragon Plus Environment


567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610

48. Kennedy, J. A.; Hayasaka, Y.; Vidal, S.; Waters, E. J.; Jones, G. P., Composition of grape skin proanthocyanidins at different stages of berry development. Journal of Agricultural and Food Chemistry 2001, 49, 5348-5355. 49. Downey, M. O.; Harvey, J. S.; Robinson, S. P., Analysis of tannins in seeds and skins of Shiraz grapes throughout berry development. Australian Journal of Grape and Wine Research 2003, 9, 15-27. 50. Koyama, K.; Ikeda, H.; Poudel, P. R.; Goto-Yamamoto, N., Light quality affects flavonoid biosynthesis in young berries of Cabernet Sauvignon grape. Phytochemistry 2012, 78, 54-64. 51. Mellway, R. D.; Constabel, C. P., Metabolic engineering and potential functions of proanthocyanidins in poplar. Plant signaling & behavior 2009, 4, 790-792. 52. Dixon, R. A.; Xie, D. Y.; Sharma, S. B., Proanthocyanidins–a final frontier in flavonoid research? New phytologist 2005, 165, 9-28. 53. Devic, M.; Guilleminot, J.; Debeaujon, I.; Bechtold, N.; Bensaude, E.; Koornneef, M.; Pelletier, G.; Delseny, M., The BANYULS gene encodes a DFR‐like protein and is a marker of early seed coat development. The Plant Journal 1999, 19, 387-398. 54. Kennedy, J. A.; Saucier, C.; Glories, Y., Grape and Wine Phenolics: History and Perspective. American Journal of Enology and Viticulture 2006, 57, 239-248. 55. Bogs, J.; Downey, M. O.; Harvey, J. S.; Ashton, A. R.; Tanner, G. J.; Robinson, S. P., Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves. Plant Physiology 2005, 139, 652-663. 56. Czochanska, Z.; Foo, L. Y.; Porter, L. J., Compositional changes in lower molecular weight flavans during grape maturation. Phytochemistry 1979, 18, 1819-1822. 57. Kennedy, J. A.; Matthews, M. A.; Waterhouse, A. L., Changes in grape seed polyphenols during fruit ripening. Phytochemistry 2000, 55, 77-85. 58. Pannala, A. S.; Chan, T. S.; O'Brien, P. J.; Rice-Evans, C. A., Flavonoid B-ring chemistry and antioxidant activity: fast reaction kinetics. Biochemical and Biophysical Research Communications 2001, 282, 1161-1168. 59. Burda, S.; Oleszek, W., Antioxidant and antiradical activities of flavonoids. Journal of Agricultural and Food Chemistry 2001, 49, 2774-2779. 60. Haenen, G. R.; Paquay, J. B.; Korthouwer, R. E.; Bast, A., Peroxynitrite scavenging by flavonoids. Biochemical and biophysical research communications 1997, 236, 591-593. 61. Kerry, N.; Rice‐Evans, C., Inhibition of peroxynitrite‐mediated oxidation of dopamine by flavonoid and phenolic antioxidants and their structural relationships. Journal of neurochemistry 1999, 73, 247-253. 62. Rice-Evans, C. A.; Miller, N. J.; Paganga, G., Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free radical biology and medicine 1996, 20, 933-956. 63. Heim, K. E.; Tagliaferro, A. R.; Bobilya, D. J., Flavonoid antioxidants: chemistry, metabolism and structure-activity relationships. The Journal of nutritional biochemistry 2002, 13, 572-584. 64. McRae, J. M.; Schulkin, A.; Kassara, S.; Holt, H. E.; Smith, P. A., Sensory properties of wine tannin fractions: implications for in-mouth sensory properties. Journal of agricultural and food chemistry 2013, 61, 719-727. 65. McRae, J. M.; Day, M. P.; Bindon, K. A.; Kassara, S.; Schmidt, S. A.; Schulkin, A.; Kolouchova, R.; Smith, P. A., Effect of early oxygen exposure on red wine colour and tannins. Tetrahedron 2015, 71, 3131-3137.

611


Page 24 of 32

Page 25 of 32


Figure 1. Effects of applied water amounts on Merlot grapevine mid-day leaf water potential in 2013 (A) and 2014 (B) in a commercial vineyard in the northern San Joaquin Valley of California. 612



Figure 2. Effect of leaf removal on external leaf numbers and number of canopy gaps per 30 cm of row in Merlot grapevine canopy in 2013 (A) and 2014 (B). 26 ACS Paragon Plus Environment

Page 26 of 32

Page 27 of 32

613


Table 1. Amount of precipitation received, irrigation water applied at research site in 2013 and 2014.

Growth stage

Applied water (L/vine) with sustained deficit irrigation treatment

Precipitation (mm)

Bud break-fruit set Fruit-set – veraison Veraison-harvest Sum

30.7 0.6 0 31.3

Bud break-fruit set Fruit-set – veraison Veraison-harvest Sum

47.3 0 0 47.3

2013 159.6 640.3 599.9 1399.8 2014 188.2 862.2 439.6 1490.0

614


Applied water (L/vine) with regulated deficit irrigation treatment 159.6 374.2 599.9 1133.7 188.2 477.7 439.6 1105.5


Table 2. Effects of leaf removal and applied water amounts on berry, berry skin mass and yield of Merlot grapevine in northern San Joaquin Valley of California in 2013 and 2014.

a

Berry mass (g)

Berry skin mass (mg)

Clusters/vine

Yield/vine (kg)

Leaf removal Control

1.36 a

55.0 a

86 b

13.9

Pre-bloom

1.27 b

51.7 a

93 ab

13.3 14.2

2013

Post-fruit set

1.28 b

45.0 b

102 a

Pr>F

0.0216

0.0020

0.0451

0.4996

Applied water SDI

1.34 a

51.3

98

14.4 13.2

RDI

1.26 b

47.8

90

Pr>F

0.0068

0.5103

0.0876

0.0748

LR × applied water

0.9004

0.9074

0.5855

0.8684

Leaf removal Control

2014 1.09

45.3 a

116 a

12.9 a

Pre-bloom

1.07

42.9 ab

113 a

12.8 a

Post-fruit set

1.11

39.5 b

94 b

9.4 b

Pr>F

0.5314

0.0310

0.0022

0.0016

Applied water SDI

1.14 a

42.7

110

12.8 a

RDI Pr>F LR × applied water

1.04 b 0.0021 0.4878

42.3 0.6963 0.5892

107

11.1 b 0.0003 0.0053

0.9589 0.0949

Anova to compare data (P indicated); n=4. Letters within columns indicate significant mean separation according to Duncan’s new multiple range test.


Page 28 of 32

Page 29 of 32


Table 3. Effects of leaf removal and applied water amounts on low molecular weight phenolics and total skin anthocyanins (mg per berry) of Merlot grapevine at harvest in northern San Joaquin Valley of California in 2013 and 2014 On per Berry Basis (mg/berry) EC

Total skin flavonols

Total skin anthocyanins

0.0209 0.0335 0.0242 0.0852

0.0280 0.0334 0.0238 0.2918

0.2405 b 0.3456 a 0.2676 b 0.0020

4.5162 b 6.0095 a 4.4101 b 0.0388

0.0294 0.0230 0.1811 0.7089

0.0323 0.0245 0.1171 0.3322 2014

0.2849 0.2842 0.9770 0.3999

5.1259 4.8313 0.5997 0.0790

0.0207 0.0198 0.0169 0.0891

0.0536 0.0627 0.0520 0.1271

0.1570 0.1726 0.1938 0.0661

2.7655 a 2.4290 b 2.8352 a 0.0199

0.0203 0.0178 0.0845 0.2285

0.0630 a 0.0490 b 0.0031 0.6796

0.1799 0.1692 0.4008 0.6236

2.6304 2.7261 0.3937 0.3512

C

2013 Leaf removal Control Pre-bloom Post-fruit set p valuea Applied water SDI RDI p valuea LR × applied water Leaf removal Control Pre-bloom Post-fruit set p valuea Applied water SDI RDI p valuea LR × applied water a




Table 4. Effects of leaf removal and applied water amounts on berry skin proanthocyanidin extension subunits of (-)- epigallocatechin (EGC),catechin (C), epicatechin (EC), (-)- epicatechin-3-O-gallate (ECG) and terminal subunits catechin and mean degree of polymerization (mDP) of Merlot at harvest in northern San Joaquin Valley of California in 2013 and 2014

EGC

% mol

mg/kg

Extension C EC

Terminal C

mDP

ECG 2013

Leaf removal Control Pre-bloom Post-fruit set p value Applied water SDI RDI p value LR × applied water Leaf removal Control Pre-bloom Post-fruit set p valuea Applied water SDI RDI p value LR × applied water a

40.2 b 42.1 b 42.3 a 0.0005

1.7 ab 1.8 a 1.6 b 0.0139

55.8 a 54.1 b 54.6 ab 0.0004

2.3 2 1.5 0.1033

19.9 b 19.3 b 24.8 a 0.0024

14.1 ab 13.9 b 15.9a 0.0172

41.5 41.5 0.8146 0.8669

1.8 a 1.6 b 0.0049 0.8098

54.8 54.8 0.7119 0.8172

1.86 2.03 0.5167 0.9073 2014

21.8 20.9 0.5579 0.8833

14.1 15.1 0.0854 0.4905

48.5 49.8 50.8 0.1010

1.5 1.4 1 0.3414

44.1 b 45.4 ab 47.1 a 0.0234

3.6 3.3 3.3 0.0646

29.5 b 30.7 b 38.1 a

20.2 a 17.9 b 18.6 a 0.0454

49.1 50.3 0.9677 0.5429

1.5 1.2 0.1825 0.9475

46 45 0.2666 0.4502

3.4 3.5 0.9678 0.6678

0.0223 34.6 30.9 0.1468 0.5774

18.4 19.4 0.2326 0.9236



Page 30 of 32

Page 31 of 32


Table 5. Effects of leaf removal and applied water amounts on berry skin and seed proanthocyanidin content and conversion yield of Merlot at harvest in northern San Joaquin Valley of California in 2013 and 2014 Skin

Leaf removal Control Pre-bloom Post-fruit set p valuea Applied water SDI RDI p valuea LR × applied water Leaf removal Control Pre-bloom Post-fruit set p valuea Applied water SDI RDI p valuea LR × applied water a

Total PAs (Phloro) (mg/L) 2013

Total PAs (IRP) (mg/L)

Tri-OH (mg/L)

Conversion Yield (%)

Total PAs (Phloro) (mg/L)

Seed Total PAs (IRP) (mg/L)

426.4 b 409.1 b 577.1 a 0.0004

1797.1 2082.4 2099.9 0.1001

164.5 b 165.2 b 234.9 a 0.0005

24.9 b 20.6 c 28.3 a 0.0001

302.7 295.0 265.7 0.2161

4862.6 4883.6 4941.3 0.9912

6.4 6.0 5.4 0.0664

466.7 475.0 0.8060 0.8453 2014

2062.2 1924.0 0.2016 0.8696

186.3 190.0 0.8146 0.8669

23.6 b 25.6 a 0.0427 0.3087

302.5 273.1 0.1172 0.2366

4907.3 4884.4 0.6945 0.3839

6.2 5.7 0.0769 0.1605

264.4ab 240.6 b 278.0 a 0.0566

1067.2 1038.0 1064.5 0.8276

127.6 112.7 131.9 0.1010

32.4 24.6 36.1 0.4241

228.4 b 268.79 a 247.44ab 0.1611

7317.2 7606.5 7835.3 0.3745

3.0 3.3 3.3 0.1874

265.7 257.5 0.5396 0.4595

1092.7 1019.5 0.3602 0.7499

123.8 124.3 0.9677 0.5429

30.1 32.0 0.9284 0.1942

237.9 b 258.5 a 0.0588 0.4580

7682.5 7490.2 0.6407 0.1048

3.1 3.3 0.0875 0.5447

Conversion Yield (%)




Leaf removal

Pre-bloom At 200 GDD

Page 32 of 32

Applied water amount

Berry mass

Total skin flavonols (Year 1) Total skin anthocyanins (Year 1)

SDI At 0.8 of estimated ETc from anthesis (EL-Stage 19) until harvest (EL-Stage 38)

Berry Skin Mass Total skin flavonols (Year 2) Berry mass

Total skin anthocyanins (Year 1)

Post-fruit set At 644 GDD

Mean Degree of polymerization

Yield (Year 2)

Total Skin PAs (by ploroglucinolysis)

RDI At 0.8 ETc from anthesis (EL-Stage 19) to fruit set (EL-Stage 28) with a Yl threshold of -1.2 MPa, 0.5 ETc from fruit set to veraison (EL-Stage 35)

Conversion yield (Skin) (Year 1) C terminal subunit

ACS Paragon Plus Environment

Effects of Leaf Removal and Applied Water on Flavonoid

Recommend Documents