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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

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Journal of Agricultural and Food Chemistry

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Effects of Leaf Removal and Applied Water on Flavonoid Accumulation in

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

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

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A. Kennedy §,

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and S. Kaan Kurtural*

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

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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

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

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

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e-mail: [email protected]

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1 ACS Paragon Plus Environment


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ABSTRACT

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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phenolics and polymeric proanthocyanidin composition in a hot climate.

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

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flavonols; anthocyanins; flavanols.


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INTRODUCTION

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

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

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

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

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presence of persistent droughts 1, 2.

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Products from the phenylpropanoid biosynthetic pathway, compounds resulting from

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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hot climate.

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

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

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

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

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

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

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greater solar exposure of the clusters. 2, 16.

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

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

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

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

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

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adversely affecting yield in hot climate.

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MATERIALS AND METHODS


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

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

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

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

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

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

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

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

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vines.

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

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

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

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

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

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

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

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

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the implement rolled over it and plucked the leaves.

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

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

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

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



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11

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the region 21.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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seed mass measurements after lyophilization.

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

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

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

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

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

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

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

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

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

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

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Acrodisc CR) before injection.

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

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

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

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

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

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

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

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

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

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

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

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

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purchased from Fisher Scientific (Pittsburg, PA).

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

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

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



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

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analysis.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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by iron-reactive phenolics measurement 23, 24.


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

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

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

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

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

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

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

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

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curve for (+)-catechin.

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

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

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

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

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statistical difference at 0.05 or less.

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RESULTS

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

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

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

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

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

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

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



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

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

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

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

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

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

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

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

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

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

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study.

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

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

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

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

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

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

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

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

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

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


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

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per vine.

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Low Molecular Weight Phenolics. Leaf removal affected skin flavonol and

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

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

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

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concentration on a per berry basis in either year.

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

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

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

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

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treatment.

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

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

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

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

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

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

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in 2014.

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

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

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


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

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year.

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

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

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deficits had no effect on mDP.

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

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

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

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

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

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

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

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

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

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

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with the RDI treatment.

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

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

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

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

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

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yield in 2014 than in 2013.


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

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

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

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

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

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studies 2, 16.

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

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

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

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

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

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

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

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

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

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

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

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

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canopy when compared to control29.

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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.



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

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

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

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

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

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

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yield.

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

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

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

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

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

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was not repeatable in 2014.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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of vegetative compensation through water deficits invigorating lateral shoot regeneration just

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

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

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canopy11.

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

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

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

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

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Leaf removal generally provided greater flavonol concentration than control. Flavonol

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

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

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

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

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

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

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


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

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

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

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

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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

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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.


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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


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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


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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

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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

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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



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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

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