Preveraison Leaf Removal Changes Fruit Zone Microclimate And

AJEV Papers in Press. Published online March 19, 2019.

American Journal of Enology and Viticulture (AJEV). doi: 10.5344/ajev.2019.18052 AJEV Papers in Press are peer-reviewed, accepted articles that have not yet been published in a print issue of the journal or edited or formatted, but may be cited by DOI. The final version may contain substantive or nonsubstantive changes.

1 Research Article 2 Preveraison Leaf Removal Changes Fruit Zone 3 Microclimate and Phenolics in Cold Climate Interspecific 4 Hybrid Grapes Grown under Cool Climate Conditions

5 Jacob Scharfetter,1 Beth Ann Workmaster,1 and Amaya Atucha*1

6 1Department of Horticulture, University of Wisconsin-Madison, 1575 Linden Drive, Madison, WI 53706. 7 *Corresponding author ([email protected]) 8 Acknowledgments: The authors acknowledge Nick Smith and the University of Wisconsin-Madison Food 9 Sciences Department for their assistance in the winemaking process. The authors also thank the West 10 Madison Agricultural Research staff, and in particular, Janet Hedtcke, the undergraduate interns, and the 11 graduate students who assisted in the daily and seasonal tasks of vineyard management and data 12 collection. This work was supported by a grant from the Wisconsin Department of Agriculture, Trade and 13 Consumer Protection Specialty Crop Block Grant Program (award no. 10-16). 14 Manuscript submitted Jun 7, 2018, revised Oct 9, 2018, Nov 12, 2018, Nov 20, 2018, Jan 20, 2019, Feb 15 20, 2019, accepted Feb 27, 2019 16 Copyright © 2019 by the American Society for Enology and Viticulture. All rights reserved. 17 By downloading and/or receiving this article, you agree to the Disclaimer of Warranties and Liability. 18 The full statement of the Disclaimers is available at http://www.ajevonline.org/content/proprietary-rights- 19 notice-ajev-online. If you do not agree to the Disclaimers, do not download and/or accept this article. 20 21 Abstract: Changes in grape cluster microclimate during ripening have been extensively explored

22 across V. vinifera cultivars, but there is limited information about its effect on cold climate

23 interspecific hybrid grape (CCIHG) cultivars grown under cool climate conditions. The influence

24 of a single preveraison leaf and lateral shoot removal (“exposed”) treatment on fruit zone

25 microclimate and juice total phenolic concentration (TPC), monomeric anthocyanin

26 concentration (MAC), and percent polymeric color was evaluated in the white cultivars Brianna

27 and La Crescent and the red cultivars Frontenac, Marquette, and Petite Pearl over three

28 consecutive growing seasons and their respective wines in the last year of the study. Treatment

29 effect on juice TPC in the white cultivars was cultivar dependent and there was no detectable

30 treatment effect on wine TPC. For the red cultivars, Frontenac, Marquette, and Petite Pearl, the 1

31 exposed treatment increased the TPC, MAC, and increased the percent polymeric color in the

32 wines. The beneficial influence of the exposed treatment on MAC, TPC, percent polymeric

33 color, and titratable acidity was associated with an increase in berry temperature and an increase

34 in photosynthetically active radiation in the fruiting zone. The cultural practice of a single,

35 preveraison leaf and lateral shoot removal may be an effective strategy to improve red wine color

36 stability of CCIHG cultivars grown under cool climate conditions.

37 Key words: anthocyanin, cold-climate grapes, copigmentation, polymeric pigment

38 Introduction

39 Cold climate interspecific hybrid grapes (CCIHG) have backgrounds consisting of

40 primarily Vitis riparia, V. vinifera, and V. labrusca heritage, which confer distinct fruit chemical

41 composition relative to V. vinifera (Slegers et al. 2015, Pedneault et al. 2013, Manns et al. 2013).

42 CCIHG cultivars have more rapid berry development, superior mid-winter cold tolerance, higher

43 berry acid concentration, less available free tannins within the fruit, and poorer color stability in

44 red wines and juice products than V. vinifera (Riesterer-Loper 2018, Rice et al. 2017, Slegers et

45 al. 2015, Pedneault et al. 2013, Manns et al. 2013). The phenolic compounds, such as

46 anthocyanins, flavanols, and tannins in CCIHG cultivars differ notably from V. vinifera cultivars,

47 which contributes to the poor color stability of CCIHG red wines (Rice et al. 2017, Sun et al.

48 2011a).

49 Anthocyanins, free tannins, and flavonols interact to influence color and color and

50 stability through two main processes, polymeric pigment formation and copigmentation (Manns

51 et al. 2013, Boulton 2001). Polymeric pigment formation results from linkages achieved between

52 free tannins and monomeric anthocyanins, while copigmentation occurs when monomeric

53 anthocyanins π-stack between one another or between small monomeric flavonoid and planar

54 compounds, in particular flavonols (Boulton 2001). In most wines, the bulk of the color stability

55 is derived from polymeric pigment formation, but copigmentation can account for 30-50% of the

56 pigments within a wine (Boulton 2001). Many red CCIHG wines have poor color stability,

57 primarily due to the lack of polymeric pigment formation with free tannins (Manns et al. 2013,

58 Sun et al. 2011b). CCIHG cultivars have low levels of unbound, free tannins when compared to

59 many red V. vinifera cultivars (Springer and Sacks 2014, Pedneault et al. 2013, Manns et al.

60 2013, Sun et al. 2011b). A high percentage of the tannins in the fruit and wines of CCIHG

61 cultivars are bound to protein complexes, which precipitates the tannins out of the wine or juice

62 matrix, leaving a limited concentration of free tannin available for polymeric pigment formation

63 (Springer and Sacks 2014). This results in red wines with less stable color over time, and as the

64 wine ages it will not develop the characteristic brick-red color of V. vinifera red wines that

65 consumers favor.

66 The majority of anthocyanins in red V. vinifera cultivars are 3-monoglucosides

67 (Ribéreau-Gayon 1982), while in red CCIHG cultivars 3,5-diglucoside anthocyanins

68 predominate (Manns et al. 2013). A diglucoside anthocyanin carries a lower ionic charge within

69 the anthocyanidin backbone than a monoglucoside, resulting in a less vibrant, more stable

70 pigment than a monoglucoside anthocyanin (García-Viguera and Bridle 1999, Robinson et al.

71 1966). From the enhanced molecular stability, diglucosides have the capacity to more readily

72 form copigmentation complexes with cofactor compounds, such as monomeric flavan-3-ols,

73 hydroxycinnimates, and in particular, flavonols (Cheynier et al. 2006, Boulton 2001, Davies and

74 Mazza 1993), which can ultimately increase the color stability of red wines (Boulton, 2001). The

75 color stability in red CCIHG wines could be improved if the concentration of copigmentation

76 reactants, such as monomeric anthocyanins and monomeric flavonols, were increased to

77 encourage a greater amount of copigmentation.

78 The concentration of monomeric anthocyanin and total phenolics in the berry vary by

79 cultivar and are influenced by temperature and incident light exposure during berry development

80 (Chorti et al. 2010, Spayd et al. 2002). Berry anthocyanin content increases with increasing light

81 exposure, provided the concomitant increase in berry temperatures does not inhibit anthocyanin

82 anabolism (Kotseridis et al. 2012, Chorti et al. 2010). A practical, non-labor-intensive cultural

83 practice that facilitates ripening by increasing sunlight and temperature in the fruit zone could

84 beneficially increase the amount of anthocyanin and anthocyanin precursor molecules that can

85 develop during the short growing seasons characteristic to cool climate regions. We

86 hypothesized that a single preveraison leaf and lateral shoot removal (“exposed”) treatment could

87 alter the fruit zone microclimate to increase the total phenolic concentration (TPC) in both the

88 red and white CCIHG cultivars and the monomeric anthocyanin concentration (MAC) in red

89 CCIHG berries. Furthermore, we hypothesized that increased MAC in CCIHG berries would

90 also increase the percentage of copigmentation in red wine, which could improve the color

91 stability of red wines. The main objective of the present study was to evaluate the effect of the

92 exposed treatment on TPC, MAC, and percent polymeric color in five CCIHG cultivars. In

93 addition, this is the first study to report the phenolic composition and maturity indices for

94 Brianna and Petite Pearl cultivars. This study was done in conjunction with an additional canopy

95 management study (Riesterer-Loper, 2018).

96 Materials and Methods

97 Site Description. The trial was conducted over three consecutive growing seasons, 2015

98 to 2017, at a research vineyard block at the University of Wisconsin-Madison West Madison

99 Agricultural Research Station (WMARS) in Verona, WI (43°03’37”N; 89°31’54”W). Soil

100 conditions were deep silt Griswold loam soil (fine loamy, mixed mesic, Typic Argiudoll), with a

101 2 to 6% slope (USDA-NRCS 2017). The 30-year average annual ambient temperature,

102 precipitation, number of frost-free days, and growing degree day (GDD) accumulation at

103 WMARS was 8.5°C, 907 mm, 174 days, and 1425 GDD (°C), respectively (Figure 1A; NOAA

104 2018). For the 2015-2017 seasons, cumulative precipitation (mm) and GDDs were recorded from

105 April 1 until September 30 (Figure 1B). GDDs were calculated from the daily maximum and

106 minimum using a base temperature of 10°C and a daily maximum temperature threshold of

107 30°C.

108 Vineyard design and vine material. All of the cultivars were planted as one-year-old bare

109 rooted vines. Brianna, Frontenac, La Crescent, and Marquette were planted in 2008 and grown

110 on a vertical shoot positioned (VSP) trellis system with double trunks trained into unilateral

111 cordons one meter above ground. Petite Pearl was planted in 2011 and was trained to a high

112 cordon system with double trunks trained into unilateral cordons 1.5 meters above ground. The

113 experimental design was a randomized complete block with four replications. Each block

114 contained two rows of vines with six, four-vine panels per row. Seven of the 12 panels within

115 each block contained cultivars that were not included as a part of this study. Row orientation was

116 north-south with 3.4 m between rows and 2.1 m between vines, for a total density of 1398

117 vines/ha (566 vines/acre).

118 Cultural and commercial practices typical for Midwestern vineyards were implemented

119 (Wolf 2008, Dami et al. 2005). Permanent sod alleyways and intra-row strips were maintained

120 with post-emergence herbicide and with wood chip mulch placed beneath the vines for weed

121 control. Drip irrigation was installed at the time of planting but was utilized only during the

122 establishment years.

123 Vineyard management. In early spring (March-April), all vines were spur-pruned to

124 approximately 45 nodes per vine. The pruning level was determined using the Ravaz index

125 (Table 1), which was calculated as the ratio of winter pruning weight/vine to vine yield (Ravaz

126 1903). Canes were pruned in March and April of each year and the weight per vine was

127 recorded. Yield per vine was recorded at harvest. To maintain vine balance, La Crescent shoots

128 were thinned to 23 shoots per meter-length of trellis, while the remainder of the cultivars were

129 thinned to 20 shoots per meter-length of trellis. All cultivars were shoot thinned at the E-L 12 to

130 14 stages of phenological development (Lorenz et al. 1977). To ensure optimal canopy airflow

131 and maintain shoot spacing, shoots were positioned and tucked weekly into a trellis system with

132 multiple pairs of catch wires. VSP shoots ≥ 30 cm above the top catch wire and high cordon

133 shoots touching the ground were hedged and skirted periodically.

134 Treatments. The treatments consisted of either a single preveraison leaf and lateral shoot

135 removal (hereafter referred to as “exposed”), or a control that had no leaf or lateral shoot

136 removal. The exposed treatment was applied a single time at two weeks post fruit set at E-L

137 stage 28-29 (Lorenz et al. 1977). The exposed treatment consisted of removing 2 to 3 leaves per

138 shoot, which were shading clusters, as well as removing lateral shoots from the first three node

139 positions. No leaves or lateral shoots were removed from the shoots of the control vines. In 2015,

140 the exposed treatment was applied to one vine per panel and one vine per panel served as a

141 control. In 2016 and 2017, the exposed treatment was applied to two adjacent vines and the other

142 two adjacent vines served as controls.

143 Canopy light and temperature measurements. In 2016 and 2017, photosynthetically

144 active radiation (PAR), ambient air temperature in the fruiting zone, and berry skin temperatures

145 were continuously monitored on the exposed and control vines in three out of the four replicate

146 blocks of the cultivar Frontenac from two weeks post-fruit set (June 23 both years) until the date

147 of harvest (Table 1). No measurements were made in 2015. PAR SQ-311 light bars (Apogee

148 Instruments, Logan, UT, USA) equipped with ten inline sensors were connected to CR10X or

149 CR1000 data loggers (Campbell Scientific, Inc., Logan, UT, USA). The light bars were placed

150 parallel to vine cordons at the fruit zone level and on both sides of the canopy in each treatment.

151 PAR was measured as photosynthetic photon flux density (PPFD, μmol/m2/s) and logged as the

152 average of the ten sensors on each bar.

153 Ambient air temperature and berry skin temperature were monitored using copper-

154 constantan (Type T) thermocouples (22 AWG for air, 36 AWG for berries; Omega Engineering,

155 Norwalk, CT, USA) connected to the data loggers as described above via an AM16/32B

156 multiplexer (Campbell Scientific, Inc., Logan, UT, USA). Within each treatment, separate micro-

157 thermocouples were inserted just beneath the skin of two berries from different clusters, on each

158 side of the canopy.

159 The panels that were monitored with sensors were evenly distributed throughout the

160 vineyard, being located in two panels on the periphery at the north and south ends, and within a

161 third panel in the relative center of the vineyard block. Temperatures and PAR levels were

162 measured each minute and data were recorded as ten-minute average values from two weeks

163 post-fruit set (June 23 both years) until harvest (Table 1).

164 Grape sampling. Beginning at veraison (~898 GDD°C, July 30, 2015; ~1015 GDD°C,

165 August 5, 2016; ~1018 GDD°C August 11, 2017), three to five berries were randomly sampled

166 from ten representative clusters (five clusters from each side of the canopy) to monitor fruit

167 maturity. The berries collected were pooled (total berries per sample 30 to 50) to obtain one juice

168 sample per treatment per block. Only berries with fully intact skins and no visible defects were

169 selected. Samples were processed within two hours post-sampling and were kept on ice for

170 transport to the laboratory, where they were crushed into juice using Wear-Ever aluminum hand

171 presses (Millville, NJ). The presses were lined with 300 micron nylon mesh screening to provide

172 resistance against which to tear the skins, as well as to hold the berries for crushing. The ~40 mL

173 juice samples were analyzed within 24 hours of pressing for basic fruit composition metrics

174 (TSS, TA, and pH). Remaining samples were placed into cold storage at -20°C for further

175 chemical analyses within three months.

176 Winemaking and storage. Wine was produced from fruit harvested in the 2017 season.

177 Grapes used for winemaking were hand harvested by cultivar on different dates, based upon

178 standard maturity indicators (Tables 1 and 2). Winemaking commenced the day of harvest. Once

179 harvested, grapes were temporarily stored in a 4°C cooler before transport to the winemaking

180 facility. After recording replicate cluster counts and yield weights, harvested grapes were pooled

181 by cultivar and treatment, with approximately 30 kg of grapes from each cultivar’s treatment

182 groups used to produce multiple wine batches.

183 Clusters were mechanically destemmed and crushed in a Cantinetta C.D.A.

184 crusher/destemmer (Zambelli Enotech, Camisano Vicentino, Italy), and 25 ppm of potassium

185 metabisulfite (K2S2O5) was added to each crush lot. Each batch per cultivar and treatment

186 yielded approximately five gallons of juice. Fermentation replications (n = 3 to 5) were created

187 from the juice of the pooled fruit of each vineyard treatment and cultivar combination, due to the

188 relatively limited amount of fruit.

189 The red cultivars’ fermentations were done in five-gallon HDPE plastic drums (M&M

190 Industries, Chattanooga, TN) at room temperature (19°C). Enologica Vason VP5 yeast

191 (Enolgoica Vason, Cariano, Italy) was added to the red cultivars, per manufacturer’s instructions.

192 All cultivars received a 1:1 (w/w) ratio of yeast to BSG Superferm (BSG, Napa, CA, USA)

193 startup. The red cultivars’ must fermented for one week and were punched down once daily.

194 Following primary fermentation, the red wines were pressed under 1.5 psi in a 20 L Speidel

195 Stainless Steel Bladder Press (Ofterdingen, Germany), placed into new containers, and allowed

196 to settle for one day at 19°C. Subsequently, red wines were racked and placed into new one-

197 gallon glass carboys, which were retained for chemical analyses, and stored at 4°C after sitting

198 for one day at room temperature.

199 Following the initial crush, white cultivars were immediately pressed in the Speidel

200 Bladder Press at 1.5 psi, placed into one-gallon carboys, and allowed to ferment for one week

201 before being filtered. ERSA 1376 yeast (Enolgoica Vason, Cariano, Italy) was added to the white

202 cultivars, per manufacturer’s instructions. All cultivars received a 1:1 (w/w) ratio of yeast to

203 BSG Superferm (BSG, Napa, CA, USA) startup. Subsequently, white wines were racked into

204 new one-gallon glass carboys and placed in 4°C storage. Analytical samples of both red and

205 white wines were collected one week post-fermentation and stored at -20°C. Samples were

206 processed for chemical analyses within three months of collection.

207 Juice and wine TSS, TA, pH, and CIELAB. Total soluble solids (TSS, in Brix) was

208 measured using a digital refractometer (HI 96801 model, Hanna Instruments, Woonsocket, RI,

209 USA) (OIV, 2009). Titratable acidity (g/L tartaric acid equiv) by endpoint titration at pH 8.2 and

210 pH measurements were taken on juice and wine samples using an automatic titrator system (HI

211 902C model, Hanna Instruments, Woonsocket, RI, USA) (Iland et al. 2004). CIELAB

212 measurements were taken on a Genesys 5 spectrophotometer (Thermo Electron Corp., Milton

213 Roy, NY, USA) and were conducted as described in Han et al. (2008).

214 Total phenolics. TPC were acquired using a modified microplate-adapted Folin-Ciocalteu

215 method (Ainsworth and Gillespie 2007, Singleton and Rossi 1965). In brief, juice and wine

216 samples were diluted into 95% (v/v) methanol and then centrifuged at 13,000 rpm to remove any

217 excess particulate. Then, 100 μL of diluted sample was added to 200 μL of a 10% dilution of

218 Folin-Ciocalteu reagent, and 800 μL of a 7.5% (w/v) sodium carbonate (Na2CO3) solution in

219 water. Samples were incubated in the dark at room temperature for 120 minutes and then

220 aliquoted into microplate wells (200 μL). Methanol (95% v/v) was used as the blank. Sample

221 absorbance readings were taken at 765 nm using a μQuant microplate reader (BioTek

222 Instruments, Winooski, VT, USA). All samples were run in triplicate. Standard curves were

223 made using 0-1000 μg gallic acid/L and results were reported in mg/L gallic acid equivalents

224 (GAE). Folin-Ciocalteu reagent and gallic acid standard were purchased from Sigma Aldrich (St.

225 Louis, MO, USA).

226 Monomeric and Percent Polymeric Color. Anthocyanins were extracted from juice and

227 wine samples using 95:5 (v/v) methanol to 2 M HCl. Samples were centrifuged at 10,000 rpm for

228 5 min and kept in the dark at 20°C for 24 h before spectrophotometric analysis began.

229 Monomeric anthocyanin concentration (MAC) were measured using the pH differential method

230 (Giusti and Wrolstad 2001). Following the appropriate dilution factor, wine and juice samples

231 were diluted using pH 1.0 (0.03 M KCl) and pH 4.5 (0.4 M CH3CO2Na · 3H2O) buffers. Samples

232 were diluted to a final volume of 3.25 mL and run in duplicate. Absorbance was measured at 520

233 nm against a distilled water blank using a Genesys 5 spectrophotometer. Anthocyanin

234 concentration (mg/L) were calculated according to the following formula and were expressed as

235 Malvidin-3-glucoside equivalents (Mv-3-glc equiv):

× × × 10 236 = × 1 3 𝐴𝐴 𝐷𝐷𝐷𝐷 𝑀𝑀𝑀𝑀 𝑀𝑀𝑀𝑀𝑀𝑀 𝜀𝜀 237 where A is the absorbance differential ((Aλvis-max)pH 1.0 − (Aλvis-max)pH 4.5), DF is the dilution factor

238 (DF=20-100), MW is the molecular weight (g/mol) for Mv-3-glc (493.441), and ε is the

239 extinction coefficient (L / cm / mol) for Mv-3-glc (28,000), where L (pathlength) = 1cm.

240 Percent polymeric color was estimated using the method described by Giusti and

241 Wrolstad (2001). Following the previously determined dilution factor, wine and juice samples

242 were diluted with water to a volume of 1.85 mL in two separate cuvettes, after which 0.15 mL of

243 potassium metabisulfite solution or water was added, respectively. Samples were allowed to

244 equilibrate for 15 min before reading at 420, 520, and 700 nm on the Genesys 5

245 spectrophotometer. Analyses were conducted in duplicate for each sample. Percent polymeric

246 color was calculated using the equations provided (Giusti and Wrolstad, 2001).

247 Statistical Analysis. Statistical analyses for normality (Shapiro-Wilks test) and

248 homogeneity were conducted for all comparisons. For wine data, Welch’s t-tests were used for

249 treatment comparisons. All juice, yield, and pruning weight data were analyzed by cultivar using

250 repeated measures models to account for the effect of the treatments over multiple years (2015-

251 2017). The model consisted of four parameters: treatment, block, year, and treatment by year.

252 For juice total phenolics analysis, assay duplicates were nested within block, and block was

253 treated as a random variable (n = 14-16). For MAC and percent polymeric color analyses, assay

254 averages were taken, but were not nested (n = 4). All repeated measures models utilized Tukey’s

255 HSD tests to make multiple comparisons, and main effects were only assessed when there was

256 not a statistically significant interaction effect between treatment by year. P-values were assessed

257 to be significant at α = 0.05. Statistical tests were performed using Minitab 18 (State College,

258 PA, USA). Graphical representations were made using Sigma Plot version 13.0 (Systat Software

259 Inc, San Jose, CA, USA).

260

261 Results

262 Weather. The 2015, 2016, and 2017 seasons accumulated 1449, 1510, and 1580 GDD°C

263 (April 1- Sep. 30), respectively (Figure 1A). During the 2017 season, July had high precipitation

264 followed by a dry mid-late veraison period (Figure 1B). Overall, total rainfall accumulations

265 across all three seasons were comparable. Temperatures in the 2015 and 2016 seasons were

266 comparable. However, August 2016 was warmer than August 2015, which resulted in an earlier

267 ripening for 2016. The 2017 season had a cooler August compared to the 30-year average and an

268 unseasonably warm mid-late September, which resulted in a ripening lag phase for August and

269 then rapid ripening in September.

270 Canopy temperature and PAR. The exposed treatment increased the amount of PAR in

271 the morning on the east side of the canopy and in the afternoon on the west side of the canopy

272 (Figure 2). For the post-fruit set to harvest periods documented, average daily PAR (average of

273 2016 and 2017) from 05:00 to 21:00 h on the east-facing side of the canopy was 103 µmol/m2/s

274 and 52 µmol/m2/s in the exposed and control treatments, respectively. On the west-facing side of

275 the canopy the average daily PAR was 99 µmol/m2/s and 44 µmol/m2/s for the exposed and

276 control treatments, respectively.

277 During the period from July 7 to August 31, averaged across the 2016 and 2017 seasons,

278 the maximum hourly average berry skin temperatures recorded on the west side of the canopy

279 was 28.0°C and 26.1°C in the exposed treatment and control, respectively (Figure 3). While on

280 the east side of the canopy, maximum berry temperatures were 26.2°C and 25.6°C in the exposed

281 treatment and control, respectively. During portions of days under sunny conditions and low

282 wind speeds (<16 km/hr), internal berry skin temperatures in the exposed treatment were 14 to

283 5○C higher than those in the control treatment on the east and west side of the canopy,

284 respectively (data not shown). Some 10-minute average berry skin temperature measurements

285 were above 30°C. The 2016 cumulative 10-minute average berry skin temperature above 30°C,

286 from July 7 to August 31, were 3.5 and 0.9 days for the west side of the canopy in the exposed

287 and control treatments, respectively; and 2.8 and 1.5 days for the east side of the canopy in the

288 exposed and control treatments, respectively. The 2017 cumulative 10-minute average berry

289 average berry skin temperature above 30°C, from July 7 to August 31, were 2.3 and 1.1 days for

290 the west side of the canopy in the exposed and control treatments, respectively; and 2.1 and 1.1

291 days for the east side of the canopy in the exposed and control treatments, respectively. Berry

292 internal temperatures of 38°C or greater were not observed at 10-minute intervals. There were no

293 differences in nocturnal (21:00-05:00 h) ambient air and berry temperatures between treatments.

294 Juice and Wine TSS, TA, and pH. Juice and wine TSS concentration did not differ

295 between treatments for either red or white cultivars. Across the 2015-2017 seasons, the pattern of

296 lower juice TA values for the exposed treatment persisted across cultivars (Table 2). Juices from

297 Frontenac, Marquette, and Petite Pearl for the exposed treatment exhibited TA values 6.1%,

298 5.9%, and 13.9% lower than the control, while Brianna and La Crescent were 14.4% and 7.4%

299 lower than the control, respectively. Both red and white wines from exposed treatment grapes

300 had lower TA than the control (Table 3). During vinification TA reduction from the juice ranged

301 between 30.5%-66.5% depending on the cultivar and treatment. The reduction in TA during

302 vinification was similar between both treatments. Similar to TSS, pH differed little among

303 treatments for juice and wine samples in red and white cultivars, with only minor differences

304 between treatments observed in Brianna juice (Table 2 and 3).

305 Total Phenolic Concentration: Within a particular cultivar, TPC varied from year to year.

306 Among the white cultivars, La Crescent juice TPC was higher in the exposed treatment than the

307 control, over the three growing seasons, by an average of 15%. However, La Crescent wine did

308 not demonstrate TPC differences (Table 4). Differences in TPC for Brianna were not detected in

309 either the juice or wine (Table 4).

310 Accounting for all three years of juice data, the red cultivars Frontenac and Marquette

311 had higher TPC in the exposed treatment than in the control (Table 4). TPC levels in the exposed

312 treatment for Frontenac and Marquette averaged 10.4% and 26.4% greater than the control

313 (Table 4). Petite Pearl juice had no differences in TPC between treatments (Table 4). Frontenac

314 and Petite Pearl wines made from the exposed treatment grapes had higher TPC than the control

315 (Table 4). However, there were no differences between treatments for Marquette wines (Table

316 4).

317 Monomeric Anthocyanin Concentration and Percent Polymeric Color: Frontenac,

318 Marquette, and Petite Pearl exposed treatments had higher juice and wine MAC, and higher wine

319 polymeric color than the controls (Table 5). There were no differences in juice polymeric color

320 between treatments.

321

322 Discussion

323 The impact of fruit zone leaf and lateral shoot removal on juice and wine chemical

324 composition of V. vinifera cultivars has been extensively explored, however there is limited

325 information for CCIHG cultivars. In our study, the exposed treatment changed juice and wine

326 TA, TPC, MAC, and percent polymeric color, without influencing yield and winter pruning

327 weights (Table 1). Across all cultivars, fruit TA levels at harvest were lower in the exposed

328 treatment compared to the control, but no differences between treatments were observed in TSS

329 and pH, which is consistent with other reports in the literature (Riesterer-Loper 2018, Feng et al.

330 2015, Spayd et al. 2002, Bergqvist et al. 2001).

331 CCIHG juice and wine from grapes grown under the increased sunlight and temperature

332 conditions of the exposed treatment had an increase in TPC and MAC (Tables 4 and 5). In the

333 case of red CCIHG cultivars, the increase in juice and wine TPC in the exposed treatment can be

334 explained through the increase in MAC (Table 4 and 5). Within the V. vinifera literature, sunlight

335 exposure on berry clusters has been reported to increase anthocyanin concentration (Feng et al.

336 2015, Song et al. 2015, Spayd et al. 2002, Morrison and Noble 1990), while in several other

337 studies, manipulation of grape cluster sunlight exposure had a neutral or negative effect on the

338 total anthocyanin concentration (Chorti et al. 2010, Downey et al. 2004, Berqvist et al. 2001,

339 Haselgrove et al. 2000, Dokoozlian and Kliewer 1996, Price et al. 1995). This discrepancy in the

340 literature is in part explained by the effect of increased cluster sunlight exposure on berry

341 temperature (Lee and Skinkis 2013, Mori et al. 2005, Spayd et al. 2002). The increased sunlight

342 exposure stimulates flavonol production, a vital precursor of anthocyanin synthesis; however,

343 sustained high berry temperatures (>30 °C) may inhibit the activity of anthocyanin synthesis

344 enzymes, thus resulting in lower anthocyanin concentration (Chorti et al. 2010, Tarara et al.

345 2008, Yamane et al. 2006, Spayd et al. 2002). In cold climate regions, such as the Northeast and

346 upper Midwest of the United States where most of the CCIHG are grown, the number of days in

347 which berry temperatures are over 30 °C is limited. In our study, for 2016 and 2017 growing

348 seasons we observed that the hourly mean internal berry temperature, although higher in the

349 exposed treatment, never exceeded 30 °C, and an increase in juice and wine MAC was observed

350 for all cultivars (Figure 2, Table 5).

351 In the white cultivar La Crescent, the increased sunlight and temperature conditions of

352 the exposed treatment resulted in higher juice TPC in all years compared to the control (Table 4).

353 Even though the higher juice TPC did not carry over to the wine (possibly due to the high

354 variance associated with the assay), we observed that wine made from the exposed treatment had

355 a more intense yellow hue and a significant CIELAB b value (p = 0.02) compared to wines made

356 from the control treatment (CIELAB Mean ± SEM: Control: b = 67.5±7.8, Exposed: b = 98±5.5).

357 This intensification in yellow coloration may be due to increases in either or both carotenoid

358 degradation products and monomeric flavonol concentration (Reshef et al. 2018). We did not

359 measure flavonol concentration directly, however, analyses on Riesling in upstate New York

360 (Kwasniewski et al. 2010) and preliminary SPME GC-MS data from our laboratory both suggest

361 carotenoid degradation products differed little between treatments. Therefore, we infer the

362 increased TPC levels in La Crescent juice from the exposed treatment could be the result of an

363 increase in monomeric flavonols. Monomeric flavonols, such as quercetin, myricetin, and

364 kaempferol, have a yellow pigmentation (Castillo-Muñoz et al. 2008), and are a source of

365 increased TPC in juice and wine from grapes provided higher sunlight interception (Reshef et al.

366 2018, Price et al. 1995).

367 Copigmentation and free tannin polymeric pigment formation are both chemical

368 processes that contribute to color stability (Boulton 2001). Free tannin polymeric pigment

369 formation is likely limited in CCIHG wines as there is low availability of free tannins, thus

370 leaving copigmentation as the primary route to color stability (Pedneault et al. 2013, Manns et al.

371 2013). The stabilization of monomeric anthocyanin pigments through copigmentation occurs

372 through the interaction of planar molecules such as flavonols, hydroxycinnamates, and even

373 monomeric anthocyanin themselves (Boulton 2001). The exposed treatment wines from all red

374 cultivars had higher MAC values, as well as higher percent polymeric color, when compared to

375 the control treatment (Table 5). Additionally, TPC in these red wines were slightly higher than

376 the control, although not statistically different for Marquette (Table 4). The increase in wine

377 percent polymeric color from the exposed treatment could be the result of increased flavonol

378 concentration (see above discussion on observed TPC increases) and the observed increased in

379 MAC, all of which contribute to enhance copigmentation (Song et al. 2015, Manns et al. 2013,

380 Spayd et al. 2002, Price et al. 1995).

381 In our study, juice percent polymeric color did not differ between treatments (Table 5),

382 minimizing the possibility that higher percent polymeric color in wine was the result of higher

383 percent polymeric color in the juice in the exposed treatment (Table 5). Higher percent

384 polymeric color values can also result from undesirable browning reactions involving citric acid

385 and reducing sugars (Boulton 2001). The rates of enzymatic and non-enzymatic browning are

386 likely to be similar for both treatment groups as reducing sugar and citrate levels did not differ

387 between treatments for both juices and wines, regardless of cultivar (Table 2 and 3; Riesterer-

388 Loper 2018). Lastly, while copigmentation stabilizes anthocyanin pigments, it can also result in

389 an undesirable spectral shift of wines to more intense blue hues (Boulton 2001). However, the

390 red wines’ peak absorbances did not shift more than 5 nm between treatments after three months

391 post-fermentation (data not shown).

392 Conclusion

393 In southern Wisconsin, a region with short cool growing seasons, increasing light

394 exposure of grape clusters through a single preveraison leaf and lateral shoot removal

395 (“exposed”) resulted in an increase in phenolic content of both red and white CCIHG berries. In

396 Frontenac, Marquette, and Petite Pearl, higher light interception and moderately increased berry

397 temperatures in the BBL treatment increased wine TPC, MAC, and percent polymeric color, this

398 latter possibly a result of enhanced copigmentation. Despite the less vibrant nature of diglucoside

399 anthocyanins and low tannin extractability of red CCIHG cultivars, higher light interception and

400 moderately increased berry temperatures through preveraison leaf and lateral shoot removal can

401 increase color stability and wine quality in red CCIGH wines.

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513

Table 1 Cumulative growing degree days (GDD, base temperature 10°C with an upper limit of 30°C) from 1 April until the date of harvest, date of harvest, harvest yield (kg/vine), and winter pruning weights (kg/vine) in 2015, 2016, and 2017 seasons for the single preveraison leaf and lateral removal (Exposed) and control treatments at the University of Wisconsin-Madison West Madison Agricultural Research Station located in Verona, WI, for the cold climate interspecific hybrid grape cultivars Brianna, Frontenac, La Crescent, Marquette, and Petite Pearlab. Yield (kg/vine) Pruning Weight (kg/vine) Ravaz Index Dates Treatment Treatment (kg fruit/kg Cultivar Harvested GDD Control Exposed p-value Control Exposed p-value prunings)

Brianna 09/06/2015 1304 9.3 7.0 2.2 2.7 3.3 08/30/2016 1297 3.7 4.5 2.6 2.8 1.5 09/05/2017 1223 6.2 4.0 2.6 2.5 2 Treatment 0.154 Treatment 0.227 Year <0.001* Year 0.447 Block 0.307 Block 0.022*

La Crescent 4.7 5.0 2.5 2.9 2.1 09/06/2015 1304 5.0 5.6 1.9 1.7

08/30/2016 1297 8.5 7.6 1.9 1.8 2.8 09/20/2017 1340 8.2 6.2 3.9 Treatment 0.538 Treatment 0.135 Year 0.003* Year 0.044* Block 0.141 Block 0.062

Frontenac 10/02/2015 1514 8.3 6.5 2.4 1.6 3.7 09/02/2016 1320 8.5 7.6 1.9 1.8 4.4 09/27/2017 1436 8.4 7.7 2.1 2.1 3.8 Treatment 0.149 Treatment 0.097 Year 0.758 Year 0.433 Block 0.724 Block 0.019*

Marquette 09/28/2015 1492 4.4 3.6 2.6 2.1 1.7 09/12/2016 1421 3.9 3.7 2.2 2.2 1.7 09/25/2017 1412 2.0 1.9 2.7 3.0 0.7 Treatment 0.154 Treatment 0.470 Year <0.001* Year <0.001* Block 0.307 Block <0.001* 24

Petite Pearl 10/02/2015 1514 11.7 11.6 0.9 0.8 13.7 09/12/2016 1421 6.2 5.9 1.3 1.1 5.0 09/27/2017 1436 7.8 10.2 1.3 1.1 7.5 Treatment 0.597 Treatment 0.122 Year <0.001* Year 0.007* Block 0.389 Block <0.001* aYield and pruning data were analyzed using a repeated measures model to account for the treatment’s effect over multiple years. Statistical significance was reported among treatments with means separated using Tukey’s HSD test. *Denotes statistical significance between the exposed and control treatments at α = 0.05. Treatment by Year interactions were not found to be significant among any of the cultivars at α = 0.05. bRavaz index was calculated from average vine yield/average pruning weight.

Table 2 Juice total soluble solids (TSS, Brix), titratable acidity (TA, g/L tartaric acid equiv), and pH at harvest (means ± SEM) in 2015, 2016, and 2017 for the single preveraison leaf and lateral shoot removal (Exposed) and control treatments (n = 4) in the cold climate interspecific hybrid grapes Brianna, Frontenac, La Crescent, Marquette, and Petite Pearla. TSS (Brix) TA (g/L tartaric acid equiv) pH

Cultivar Treatment 2015 2016 2017 2015 2016 2017 2015 2016 2017 Brianna Control 16.7 17.0 17.5 6.3 7.3 10.1 3.29 3.22 3.37 Exposed 17.4 16.1 18.0 5.6 6.2 8.7 3.34 3.24 3.41 p-value Treatment 0.682 0.004* 0.013* Year 0.010* <0.001* <0.001* Block 0.768 0.868 0.876

La Crescent Control 17.1 17.1 20.3 19.0 15.2 17.6 2.93 2.99 3.20 Exposed 17.8 16.4 20.4 17.4 14.5 16.2 2.97 2.96 3.22 p-value Treatment 0.937 0.011* 0.466 Year <0.001* <0.001* <0.001* Block 0.032* 0.912 0.019*

Frontenac Control 20.1 16.2 22.7 15.8 16.4 18.7 3.21 2.96 3.27 Exposed 20.9 16.3 22.5 14.3 15.2 18.4 3.23 2.98 3.25 p-value Treatment 0.579 0.014* 0.692 Year <0.001* <0.001* <0.001* Block 0.805 0.040* 0.005*

Marquette Control 21.9 23.3 21.4 11.9 12.4 16.6 3.06 3.08 3.23 Exposed 23.3 23.2 21.4 11.0 11.6 16.0 3.08 3.11 3.21 p-value Treatment 0.206 0.012* 0.519 Year 0.023* <0.001* <0.001* Block <0.001* 0.370 0.158

Petite Pearl Control 18.4 20.4 20.8 8.9 9.2 11.0 3.30 3.36 3.45 Exposed 15.9 20.6 20.5 6.8 9.1 9.5 3.33 3.41 3.45 p-value Treatment 0.092 <0.001* 0.205 Year <0.001* <0.001* <0.001* Block 0.021* 0.252 0.158 aJuice data was analyzed using a repeated measures model to account for the treatment’s effect over multiple years. Statistical significance was reported among treatments with means separated using Tukey’s HSD test. *Denotes statistical significance between the exposed and control treatments at α = 0.05. Treatment by Year interactions were not found to be significant among any of the cultivars at α = 0.05.

Table 3 Wine total soluble solids (TSS, in Brix), titratable acidity (TA, g/L tartaric acid equiv), and pH one-week postfermentation (means ± SEM) in 2017 for the single preveraison leaf and lateral shoot removal (Exposed) and control treatments (n = 3-5) in the cold climate interspecific hybrid grapes Brianna, Frontenac, La Crescent, Marquette, and Petite Pearl. TSS (Brix) TA (g/L tartaric equiv) pH Cultivar Control Exposed p-value Control Exposed p-value Control Exposed p-value Brianna 5.3 5.3 0.098 4.33 4.17 *0.032 3.30 3.28 0.091 La Crescent 6.8 6.3 0.292 12.13 10.02 *0.001 3.18 3.19 0.680 Frontenac 8.4 8.0 *0.033 13.36 13.10 0.595 3.30 3.32 0.123 Marquette 9.6 9.2 0.205 11.87 9.24 *0.043 3.40 3.43 0.734 Petite Pearl 7.3 7.3 0.920 7.77 6.99 *0.028 3.62 3.59 0.485 *Denotes statistical significance between the exposed and control treatments; Welch’s t-test at α = 0.05.

Table 4 Total phenolics (mg/L gallic acid equiv) for the cold climate interspecific hybrid grape cultivars Brianna, Frontenac, La Crescent, Marquette, and Petite Pearl with or without the single preveraison leaf and lateral shoot removal (Exposed) treatment for juice at harvest (means ± SEM) in 2015, 2016, and 2017, and in wine one-week postfermentation (means ± SEM) for the 2017 seasonab. Total Phenolic Concentration (mg/L) Juice Wine Cultivar Treatment 2015 2016 2017 2017 Brianna Control 738 664 783 726 Exposed 822 781 633 739 p-value Treatment 0.556 0.719 Year 0.522 Block 0.007*

La Crescent Control 776 1088 960 592 Exposed 841 1348 1090 625 p-value Treatment 0.001* 0.515 Year <0.001* Block 0.532

Frontenac Control 1311 1864 1534 1753 Exposed 1549 1975 1701 1907 p-value Treatment 0.011* <0.001* Year <0.001* Block 0.633

Marquette Control 1149 1246 1112 1678 Exposed 1534 1680 1358 1783 p-value Treatment <0.001* 0.239 Year 0.015* Block 0.021*

Petite Pearl Control 1494 1392 1252 1439 Exposed 1465 1364 1442 1645 p-value Treatment 0.451 <0.001* Year 0.318 Block 0.058 a Juice data was analyzed using a repeated measures model to account for the treatment’s effect over multiple years. Statistical significance was reported among treatments with means separated using Tukey’s HSD test. *Denotes statistical significance between the exposed and control treatments. Treatment by Year interactions were not found to be significant among any of the cultivars at an α = 0.05. b Wine data was analyzed using Welch’s t-test, at α = 0.05.

Table 5 Monomeric anthocyanin concentration (mg/L MV-3-glc. equiv) and percent polymeric color in three red cold climate interspecific hybrid grape cultivars, Frontenac, Marquette, and Petite Pearl, with or without the single preveraison leaf and lateral shoot removal (Exposed) treatment for harvest juice in 2015, 2016, and 2017, and in wine one week postfermentation in 2017ab.

Juice Wine Cultivar Treatment 2015 2016 2017 2017 Monomeric Anthocyanins (mg/L) Frontenac Control 311 272 313 974 Exposed 485 314 395 1165 p-value Treatment <0.001* 0.029* Year 0.033* Block 0.212

Marquette Control 78.1 82.3 99 733 Exposed 106 109 122 833 p-value Treatment 0.010* 0.001* Year 0.235 Block 0.168

Petite Pearl Control 64.7 146 139 797 Exposed 125 219 162 904 p-value Treatment 0.002* 0.009* Year 0.001* Block 0.292 Percent Polymeric Color Frontenac Control 8.59 11.10 4.76 12.13 Exposed 6.36 9.51 6.88 15.70 p-value Treatment 0.438 0.008* Year <0.001* Block 0.528

Marquette Control 29.48 36.20 22.70 26.4 Exposed 33.69 36.45 22.06 33.0 p-value Treatment 0.312 0.013* Year <0.001* Block 0.519

Petite Pearl Control 17.65 9.35 8.03 13.1 Exposed 16.67 8.15 5.96 15.9 p-value Treatment 0.312 0.011* Year <0.001* Block 0.685 a Juice data was analyzed using a repeated measures model to account for the treatment’s effect over multiple years. Statistical significance was reported among treatments with means separated using Tukey’s HSD test. *Denotes statistical significance between the exposed and control treatments. Treatment by Year interactions were not found to be significant among any of the cultivars at an α = 0.05. b Wine data was analyzed using Welch’s t-test, at α = 0.05. *Denotes statistical significance between the exposed and control treatments.

Figure 1 Precipitation (mm) (A) and growing degree day (GDD) (B), base temperature 10°C with an upper limit of 30°C) for the 2015, 2016, and 2017 growing seasons and the 30-year average. Meteorological data was acquired from the nearest National Weather Service weather station, approximately 5 km from the University of Wisconsin-Madison West Madison Agricultural Research Station (Charmany Farm, WI, USA; NOAA, 2018; www.ncdc.noaa.gov).

Figure 2 Hourly average photosynthetically active radiation (PAR) (μmol/m2/s) in the fruiting zone of the east and west side of the canopy of six Frontenac grapevines for the single preveraison leaf and lateral shoot removal (Exposed) and control treatments measured from 7 July to 31 August 2016 (A) and 2017 (B) at the University of Wisconsin-Madison West Madison Agricultural Research Station located in Verona, WI. Bars indicate the standard error among three replicates.

Figure 3 Hourly average berry skin temperature (°C) of 12 Frontenac grapevine berries located in clusters on the east and west sides of the canopy for the single preveraison leaf and lateral shoot removal (Exposed) and control treatments measured from 7 July to 31 August 2016 (A) and 2017 (B) at the University of Wisconsin-Madison West Madison Agricultural Research Station located in Verona, WI. Bars indicate the standard error among three replicates.