EFFECT OF SUN EXPOSURE ON THE EVOLUTION AND DISTRIBUTION OF ANTHOCYANINS IN INTERSPECIFIC RED HYBRID WINEGRAPES

A Thesis Presented to the Faculty of the Graduate School of Cornell University

in Partial Fulfillment of the Requirements for the Degree of Master of Science

by Catherine Hope Dadmun August 2020

© 2020 Catherine Hope Dadmun

ABSTRACT

Interspecific hybrid winegrapes are economically important in areas where environmental pressures inhibit traditional Vitis vinifera production. To clarify the effect of vine microclimate on red hybrid wine color, skin extract anthocyanins were characterized via

HPLC for shaded and unshaded fruit from three economically significant cool-climate hybrid cultivars (Vitis spp): Corot noir, Maréchal Foch, and Marquette. Light exposure and berry and air temperature were monitored in Corot noir to represent generalized vine microclimate.

Across all cultivars, the samples that underwent the leaf-pulling treatment (exposed samples) did not have significantly different concentrations of total anthocyanins compared to the control

(shaded samples). However, certain individual anthocyanins within each cultivar demonstrated different concentrations with the exposure treatment. This work is the first step in defining the evolution of anthocyanin profiles during interspecific hybrid grape ripening to allow cool- climate wine grape growers to optimize viticultural production methods for high-quality red hybrid wines.

Keywords: anthocyanin, interspecific hybrid, ripening, sunlight exposure, viticultural practice, leaf removal

BIOGRAPHICAL SKETCH

Catherine Dadmun joined Anna Katharine Mansfield’s group in the Department of Food

Science and Technology at Cornell University in August 2018. She studies grape and wine chemistry, primarily focusing on hybrid Vitis spp. and the chemical color composition of grapes. Beyond academics, Catherine was heavily involved in the Food Science Graduate

Student Organization (FSGSO), the Graduate and Professional Women’s Network

(GPWomeN), and tutoring students at Beverly J. Martin Elementary School and later virtually through Weill Cornell Tutors. She served as a member of the Social Committee for FSGSO and the Communications Chair for GPWomeN. Prior to her graduate studies, Catherine received her B.S. in Chemistry and B.A. in French and Francophone Studies from the College of

Charleston in Charleston, South Carolina. She participated in a Chemistry Undergraduate

Research Program funded by the National Science Foundation at Virginia Tech over the summer of 2017, where her work in Susan Duncan’s Lab introduced her to Food Science. Upon completion of her Master’s in Science, Catherine will be continuing to study the chemistry of grapes and wine in Anna Katharine Mansfield’s lab at Cornell University for her Ph.D. and hopes to become even further involved in science communication and Extension.

iii

To my parents, Mark and Jayne Dadmun, who taught me to think critically, question everything, and find passion in everything I do.

iv ACKNOWLEDGMENTS

I would like to acknowledge Prejean Winery, Stever Hill Vineyards, and the Finger

Lakes Teaching and Demonstration Vineyard for their generous donation of grape samples and

The Cornell Agritech Venture Grant for funding this research. I’d also like to thank Don

Caldwell, who did all of the hard work leaf-pulling, monitored the vineyards, and helped with sample collection.

I am so thankful for my advisor, Dr. Anna Katharine Mansfield. Thank you for welcoming me into your lab with open arms and not only being an academic advisor, but a true mentor. Thank you for listening to my many questions and ideas with an open mind and responding truthfully from your wealth of knowledge and experience. Thank you for your support and trust in my ambitions, flexibility, genuine kindness, adventures in Italy, recipe exchanges, and laughter over Zoom calls. I look forward for more to come.

I’d also like to thank my Committee member, Dr. Gavin L. Sacks, for his patience, open door, and inquisitive interest in any question I could bring his way. Your wisdom and advice, in addition to your invaluable class, have been crucial in the progression of my studies and my understanding of wine chemistry.

I want to acknowledge Dwayne Bershaw and Patrick Gibney, the two of whom inspired me by their passion in the classes they taught. Not only did I learn an expanse of knowledge from their expertise, but I am extremely grateful for their commitment to finding new ways to ensure the understanding of their students, their sense of humor, and the amount of hard work they put into their teaching. I’d also like to thank Erin Atkins, whose door was always open and

v whose face was always smiling. She is an invaluable member of the Food Science and

Technology Graduate Program and her endless hard work should never go unnoticed.

Of course, I’d like to thank Demi Perry, who began as my lab technician and manager and after transitioning out of that position into a full-time graduate student, became purely a friend and mentor. Thank you for your patience, honest criticism, wisdom, response to my late- night HPLC questions (even after you were no longer working in my lab), 80’s dance parties as we packed up the lab, and sunny adventures. Who knows where I would be without you.

I’d also like to extend the deepest gratitude to my family, whose unconditional support in any and every way has given me the confidence and security to pursue my ambitions: my mom and dad, Jayne and Mark Dadmun, whose rock-solid support and love has never once wavered, and to my sister and brothers, Maggie, Ryan, and Ben, who keep me smiling and inspired to the be the best I can be.

I also need to thank my friends here in Ithaca, in the department and beyond, who have formed an unbreakable network of supportive, smiling, resilient confidants. I’m glad we’re on this journey together.

Finally, I want to thank Dr. John Turner, who explained the chemistry of the crème brûlée we were making in Oxford when I was 14. I didn’t know it then, but your passion for food chemistry was contagious.

vi TABLE OF CONTENTS Biographical Sketch...... iii Acknowledgements ...... v List of Figures...... ix List of Tables ...... xi CHAPTER 1: LITERATURE REVIEW 1 I. Anthocyanin Chemistry ...... 1 II. Interspecific Hybrid Grape Cultivars ...... 19 III. Anthocyanins and Wine Quality ...... 29 IV. Unique Qualities of Anthocyanins in Hybrid Grapes ...... 32 V. Viticultural and Winemaking Practices: Influence on Anthocyanins ...... 35 VI. References ...... 42 CHAPTER 2: EFFECT OF SUN EXPOSURE ON THE EVOLUTION AND DISTRIBUTION OF ANTHOCYANINS IN INTERSPECIFIC RED HYBRID WINEGRAPES 61 I. Introduction ...... 61 II. Materials and Methods ...... 64 a. Sample Collection ...... 64 b. Solvents and Instrumentation ...... 65 c. Counting and Weighing Samples ...... 66 d. Separating Skin from Pulp...... 66 e. Freeze-Drying Skins ...... 66 f. Grinding Skins ...... 67 g. Methanol Extraction ...... 67 h. Solid-Phase Extraction ...... 68 i. HPLC Analysis ...... 69 j. Statistical Analysis ...... 69 III. Results ...... 70 a. Corot noir...... 70 b. Maréchal Foch ...... 79 c. Marquette ...... 87 d. Viticulture ...... 94 IV. Discussion...... 95 a. Corot noir...... 96 b. Maréchal Foch ...... 97 c. Marquette ...... 98 d. Viticulture ...... 99 V. Conclusion ...... 100 VI. References ...... 102 CHAPTER 3: RESEARCH CHALLENGES AND FUTURE WORK 107 I. Research Challenges ...... 107 II. Future Work...... 108

vii LIST OF FIGURES

Figure 1.1. Anthocyanin forms and their corresponding colors. 4 Figure 1.2. Visible color range of anthocyanidins (Ananga et al. 2013). 5 Figure 1.3. Anthocyanidins found in wine. 6 Figure 1.4. Evolution of anthocyanins in red wine (He et. al 2012b). 9 Figure 2.1. Representative HPLC chromatogram for Corot noir (Year 1, Timepoint 1 71 Treatment (Exposed), Panel 2). Figure 2.2. Representative HPLC chromatogram for Corot noir (Year 1, Timepoint 4 71 Control (Shaded), Panel 2). Figure 2.3. Individual peak means over the course of ripening for Corot noir (Year 1). 74 Figure 2.4. Total anthocyanin content means over the course of ripening for 75 Corot noir (Year 1). Figure 2.5. Anthocyanin composition during ripening for Corot noir (Year 1). 76 Figure 2.6. Individual peak means over the course of ripening for Corot noir (Year 2). 77 Figure 2.7 Total anthocyanin content means over the course of ripening for 77 Corot noir (Year 2). Figure 2.8. Anthocyanin composition during ripening for Corot noir (Year 2). 78 Figure 2.9. Representative HPLC chromatogram for Maréchal Foch (Year 2, Timepoint 80 1, Control (Shaded), Panel 2). Figure 2.10. Representative HPLC chromatogram for Maréchal Foch (Year 2, Timepoint 80 3, Control (Shaded), Panel 3). Figure 2.11. Individual peak means over the course of ripening for 82 Maréchal Foch (Year 1). Figure 2.12. Total anthocyanin content means over the course of ripening for 82 Maréchal Foch (Year 1). Figure 2.13. Anthocyanin composition during ripening for Maréchal Foch (Year 1). 83 Figure 2.14. Individual peak means over the course of ripening for 84 Maréchal Foch (Year 2). Figure 2.15. Total anthocyanin content means over the course of ripening for 85 Maréchal Foch (Year 2). Figure 2.16. Anthocyanin composition during ripening for Maréchal Foch (Year 2). 86 Figure 2.17. Representative HPLC chromatogram for Marquette (Year 1, 87 Timepoint 2, Treatment (Exposed), Panel 2). Figure 2.18. Representative HPLC chromatogram for Marquette (Year 2, Timepoint 2, 87 Control (Shaded), Panel 1). Figure 2.19. Individual peak means over the course of ripening for Marquette (Year 1). 89 Figure 2.20. Total anthocyanin content means over the course of ripening for 89 Marquette (Year 1).

viii Figure 2.21. Anthocyanin composition during ripening for Marquette (Year 1). 90 Figure 2.22. Individual peak means over the course of ripening for Marquette (Year 2). 91 Figure 2.23. Total anthocyanin content means over the course of ripening for 92 Marquette (Year 2). Figure 2.24. Anthocyanin composition during ripening for Marquette (Year 2). 93 Figure 2.25. Light exposure (PAR) in exposed (E) and shaded (S) fruit zones of 94 Corot noir.

ix LIST OF TABLES

Table 2.1. Dates of veraison (50%), sampling, and harvest for each grape cultivar. 65 Table 2.2. Number of appearances of extra peaks in Corot noir at each 71 sampling (Year 1). Table 2.3. Anthocyanins with accumulation significance between E and S 72 samples for Corot noir (Year 1). Table 2.4. Tentative identifications based on retention factor and their relative 73 distributions in Corot noir. Table 2.5. Number of appearances of extra peaks in Maréchal Foch at each sampling. 79 Table 2.6. Tentative identifications based on retention factor and their relative 81 distributioins in Maréchal Foch. Table 2.7. Tentative identifications based on retention factor and their relative 88 distributions in Marquette. Table 2.8. Average berry temperature difference (°C) between exposed and 95 shaded samples. Table 2.9. Average berry temperature (°C). 95

x CHAPTER 1

LITERATURE REVIEW

I. Anthocyanin Chemistry

Despite being a small proportion of the complex wine matrix, phenolics play a critical role in determining wine quality. The core structure of phenolics is the phenol, which is a benzene ring with a single hydroxyl group (Mazza and Francis 1995, Ribéreau-Gayon et al.

2006). There are two categories of phenolics: non-flavonoids and flavonoids. Non-flavonoids are either grape-derived (hydroxycinnamates, stilbenes, and gallic acid) or oak-derived

(ellagitannins and vanillin) (Harbertson 2016). There are four classes of flavonoids: flavan-3- ols (catechins), flavonols, tannins, and anthocyanins (Harbertson 2016, Perry 2018). Flavan-3- ols, the building blocks of tannins, have properties of bitterness (Aleixandre-Tudó et al. 2015,

Cheynier et al. 2006, Lawless and Heymann 2010, Moreno-Arribas and Polo 2009, Somers and

Evans 1974)(Harbertson 2016, Perry 2018). Catechin is the most abundant flavan-3-ol in grapes and is very reactive (Perry 2018). Flavonols are yellow pigments that also have properties of bitterness (Casassa and Harbertson 2014). There are two types of tannins, both of which contribute astringency to wine: condensed tannins or proanthocyanidins, which are polymers of flavan-3-ol subunits, and hydrolysable tannins, which are non-flavonoid ellagitannins derived from oak barrels (Harbertson 2016). Anthocyanins are water-soluble molecules that occur in nature and can contribute red, purple, and blue colors but have no organoleptic properties (Formaker 2015, Harbertson 2016, Heredia et al. 1998, Mazza and Francis 1995,

Perry 2018). They are the major natural pigments in plant-derived foods and are the principal component of color in red wines (Formaker 2015, Winkel-Shirley 2001, Wrolstad et al. 2005).

1 The typical concentration of free anthocyanins in full-bodied red wines is 500 mg/L, but they can be as high as 2000 mg/L (Burns et al. 2002, He et al. 2012a, Mulinacci et al. 2008,

Nikfardjam et al. 2006). During ripening, anthocyanins accumulate in grape skin cell vacuoles, and in the pulp of certain teinturier cultivars (Cheynier et al. 2006a, Formaker 2015).

Anthocyanins are synthesized in grapes via the phenylpropanoid pathway (Downey et al. 2006). Initially, only dihydroxylated anthocyanins glucosides cyanidin and peonidin accumulate, followed by the trihydroxylated anthocyanins delphinidin, petunidin, and malvidin

(Boss et al. 1996a). Expression of the gene encoding the final step in anthocyanin biosynthesis,

UDP glucose-flavonoid 3-O-glucosyl transferase (UFGT), results in the accumulation of anthocyanins in the skin of red grapes (Boss et al. 1996b). In white grapes, there are no anthocyanins in the skin because the gene encoding UFGT is not expressed (Boss et al. 1996a,

Downey et al. 2006). Anthocyanin biosynthesis is greatly regulated based on internal and external disruptions from physiological and environmental conditions (Downey et al. 2006,

Mazzuca et al. 2005). Anthocyanins also have properties beneficial to human health including free radical scavenging, antioxidant activity, protective effects against UV irradiation, and anticancer and antimutagenic activity (Downey et al. 2006, Koide et al. 1996, Lapidot et al.

1999, Maletić et al. 2009, Muñoz-Espada et al. 2004, de Pascual-Teresa et al. 2010, de Pascual-

Teresa and Sanchez-Ballesta 2008, Saint-Cricq de Gaulejac et al. 1999, Singletary et al. 2007,

Tomaino et al. 2006, Yun et al. 2010). Anthocyanins are glycosides and acylglycosides of anthocyanidins, which are made of a simple flavonoid ring system with a C6-C3-C6 skeleton

(Cheynier et al. 2006a, Formaker 2015, Perry 2018, Waterhouse et al. 2016). The skeleton consists of one heterocyclic benzopyran ring (C ring), one fused aromatic ring (A ring) and one phenyl constituent (B ring) (He et al. 2012a). Anthocyanin color comes from the fully-

2 conjugated ten electron A-C aromatic flavonoid ring system, which is cross conjugated with the B ring (Waterhouse et al. 2016). Monomeric anthocyanins occur in equilibrium as five major molecular forms: the bisulfite addition flavene compound, the quinoidal base, the flavylium cation, the hemiketal or carbinol pseudobase, and the chalcone (Cheynier et al. 2006a,

Jackson 2008, Perry 2018, Ribéreau-Gayon et al. 2006) (Figure 1.1). These different forms can be distinguished with high-resolution proton NMR (Ribéreau-Gayon et al. 2006). The flavylium cation goes through two main reactions: an acid-base reaction to the quinoidal base, or a hydration reaction to the colorless carbinol form (He et al. 2012a, Perry 2018). The ring- opening and rearrangement of carbinol will form the chalcone, but this transformation occurs slowly (Brouillard and Delaporte 1977, Perry 2018). The distribution of anthocyanin forms in acidic conditions in young red wine can be calculated using the thermodynamic constants of hydration and of proton transfer (Brouillard and Delaporte 1977). At red wine pH (3.3-3.5), equilibrium is largely toward the colorless hemiketal state (Cheynier et al. 2006a, Lee et al.

2005). The pKa of the flavylium–pseudobase equilibrium is 2.7, so at a typical wine pH of 3.7,

90% of the anthocyanins are in a colorless state (Brouillard and Delaporte 1977). The quinone form has a violet hue and a pKa of 4.7, so it is only present in small amounts at common wine pH values (Waterhouse et al. 2016). Many factors affect the distribution of anthocyanin forms, including temperature, pH, and amount of free SO2 (He et al. 2012a, Torskangerpoll and

Andersen 2005). Sulfur dioxide “bleaches” free monomeric anthocyanins in the flavylium state by nucleophilic addition at C4 in the C ring, producing the colorless flavene compound

(Cheynier et al. 2006a, He et al. 2012a, Jackson 2017). At low pH, the concentration of the flavylium cation form increases because hydrolysis is slowed; at a higher pH the flavylium cation, and therefore color density, decreases (He et al. 2012a, Jackson 2008). When pH is less

3 than 2, the eight conjugated double bonds carrying a positive charge in the flavylium ion give an intense red/orange color (Formaker 2015, Torskangerpoll and Andersen 2005).

Anthocyanins vary from mauve to blue at a pH above 4 and then turn yellow and colorless in natural or alkaline solutions (Formaker 2015, He et al. 2012a, Jackson 2017).

Figure 1.1. Anthocyanin forms and their corresponding colors.

Of the 23 anthocyanidins reported in vascular plants, only 6 anthocyanidins have been identified in Vitis vinifera: pelargonidin, cyanidin, delphinidin, peonidin, petunidin and malvidin (Casassa and Harbertson 2014, Cheynier et al. 2006a, Formaker 2015, Jackson 2017)

(Figures 1.2 and 1.3). Malvidin-3-O-glucoside is the most abundant anthocyanin in V. vinifera, making up roughly 40% of the anthocyanins depending on cultivar (Casassa and Harbertson

2014, Formaker 2015, Harbertson 2016, He et al. 2012a, Jackson 2017). Anthocyanins are classified by the amount and position of hydroxyl and methyl groups on the second phenyl ring, the B ring (“Anthocyanins in Wine”, He et al. 2012a, Jackson 2017). The number of hydroxyl groups on the B ring of the flavonoid skeleton affects the hue of the anthocyanin due to the effect on the delocalized electrons’ path length in the molecule (He et al. 2012a). With more hydroxyl groups the blueness of the anthocyanin increases; with more methylation the redness of the anthocyanin increases (“Anthocyanins in Wine”, He et al. 2012a, Jackson 2017). Due to

4 its high level of methylation, malvidin-3-O-glucoside has the reddest hue of the anthocyanins

(“Anthocyanins in Wine”, Jackson 2008).

Figure 1.2. Visible color range of anthocyanidins (Ananga et al. 2013).

The anthocyanidins peonidin and cyanidin have more red hues than delphinidin, petunidin, and malvidin, which have more violet hues (Robinson et al. 1966) (Figure 1.2).

Anthocyanins can be further diversified through the esterification of the glucoside group to either an acetyl, coumaroyl, or caffeoyl group (Cheynier et al. 2006a, Waterhouse et al. 2016).

Acylation of the anthocyanin can increase its stability and solubility, but the amount of acylated anthocyanins is dependent on cultivar (He et al. 2012a). Non-vinifera grapes, including interspecific hybrids and native varieties, can include mono- and diglucosides, including pelargonidin-3,5-O-diglucoside (pelargonin), cyanidin-3,5-O-diglucoside (cyanin), delphinidin-3,5-O-diglucoside, peonidin-3,5-O-diglucoside, petunidin-3,5-O-diglucoside and malvidin-3,5-O-diglucoside (malvin) and their corresponding acylated anthocyanins (He et al.

5 2012a, Waterhouse et al. 2016). Normally, diglucosides are more stable than monoglucosides but are more susceptible to browning and are less colored (Hrazdina et al. 1970, Mazza and

Brouillard 1987, Robinson et al. 1966). Overall, the type, proportion, and concentration of anthocyanins is dependent on cultivar and environmental conditions, including climate and viticultural techniques (“Anthocyanins in Wine”, Cheynier et al. 2006a, He et al. 2012a,

Jackson 2017). Although anthocyanin profiles are a varietal characteristic, distinguishing between varietals based solely on anthocyanin profile is not always possible due to effects of winemaking techniques and similarities between varietal profiles (Cheynier et al. 2006a,

Waterhouse et al. 2016).

Research on anthocyanins is often performed in model solutions, which have simple and defined chemical compositions Figure 1.3. Anthocyanidins found in wine. unlike the complex matrix of wine

(Jackson 2008, Ribéreau-Gayon et al.

2006). While more complex model solutions can be used, they make studies more difficult (He et al. 2012b). Many methods have been established to separate anthocyanin derivatives in wine using solid phase extraction (SPE), high performance liquid chromatography

(HPLC), high-speed countercurrent chromatography (HSCCC), and electrophoresis (Dugo et al. 2004, Jeffery

6 et al. 2008, Sun et al. 2006, Vergara et al. 2010). Methods have also been established to identify anthocyanin derivatives using electrospray ionization mass spectrometry (ESI-MS), matrix- assisted laser desorption/ionization mass spectrometry (MALDI-MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), atmospheric pressure photo ionization quadrupole time-of-flight mass spectrometry (APPI-QTOF MS), and nuclear magnetic resonance (NMR) (Castañeda-Ovando et al. 2009, He et al. 2012b, Mateus et al. 2002a, Mazzuca et al. 2005, Wang and Sporns 1999, Wrolstad et al. 2005).

Free monomeric anthocyanins are the principal source of color in young red wines, but they are unstable and decline constantly after fermentation (Cheynier et al. 2006a, Formaker

2015, He et al. 2012a). This decline is often due to yeast adsorption; degradation and oxidation; precipitation with proteins, polysaccharides, or condensed tannins; or progressive and irreversible formation of more complex and stable anthocyanin-derived pigments including pyranoanthocyanins, polymeric anthocyanins, and further derivatives (Casassa and Harbertson

2014, He et al. 2012a). Color is lost if monomeric anthocyanins react with water, bisulfite, or other nucleophiles (Waterhouse et al. 2016). The stability of monomeric anthocyanins depends on structure, concentration, solution composition, wine pH, storage temperature and time, oxidation status, light exposure, and the presence of other substances such as ascorbic acid, sugars, sulfites, cofactors and metallic ions (Abyari et al. 2006, Bordignon-Luiz et al. 2007,

Brenes et al. 2005, He et al. 2012a, Hillmann et al. 2011, Hrazdina et al. 1970, Mazza and

Brouillard 1987, Ribéreau-Gayon et al. 2006, Tseng et al. 2006). In general, monomeric anthocyanins are more stable at lower pH and lower temperatures, and less stable in the presence of ascorbic acid and sugars (Abyari et al. 2006, Bordignon-Luiz et al. 2007, Brenes et al. 2005, He et al. 2012a, Hillmann et al. 2011, Tseng et al. 2006).

7 Several processes result in anthocyanin degradation, such as light exposure or the presence of ketones (Bordignon-Luiz et al. 2007, Ribéreau-Gayon et al. 2006). Anthocyanins heated to a high temperature, such as during the process of thermovinification, can lose color quickly and irreversibly (Bordignon-Luiz et al. 2007, Hillmann et al. 2011). Oxygen and light can catalyze a reaction between o-diquinones (which are generated by oxidation) and anthocyanins in their hydrated forms (chalcone or carbinol) to produce unstable and colorless phenolic acids and aldehydes. A higher pH environment can facilitate this reaction (Abyari et al. 2006, Bordignon-Luiz et al. 2007, Kader et al. 1999, Lopes et al. 2007).

Despite the loss of monomeric anthocyanins, aging wine remains red due to the gradual formation of polymeric pigments and modified anthocyanins (Jackson 2008, Ribéreau-Gayon et al. 2006) (Figure 1.4). Anthocyanin-derived pigments can be relatively short-term, such as self-association and cofactor copigmentation, or longer-term, such as polymeric anthocyanins and pyranoanthocyanins (Formaker 2015, He et al. 2012a, Jackson 2017). Copigmentation enhances color by forming complexes between anthocyanins and colorless cofactors

(Harbertson 2016). However, this copigmentation occurs primarily in young red wines because as monomeric anthocyanins, the precursors for copigmentation, decrease over time, the rate of

8 Figure 1.4. Evolution of anthocyanins in red wine (He et. al 2012b).

copigmentation also decreases (Harbertson 2016). Two types of copigmentation can occur: intramolecular copigmentation (also known as self-association), in which two anthocyanin molecules are held together by - stacking with hydrophobic interactions of the polarizable planar nuclei of the flavylium ion and the quinoidal base, or intermolecular copigmentation, which is held by - stacking between the anthocyanin B ring and the planar B ring of a cofactor

(Dangles and Brouillard 1992, Dangles et al. 1993, Eiro and Heinonen 2002, Figueiredo et al.

1996, Malien-Aubert et al. 2001). Other copigmentation complexes can be formed by covalent bonds between aromatic acyl groups linked to the sugar moieties of anthocyanins and cofactors

(Figueiredo et al. 1996, George et al. 2001, He et al. 2012a). The best cofactors for copigmentation are planar structures, as non-planar structures are disfavored due to steric effects (Waterhouse et al. 2016). Major cofactors include flavonols, flavan-3-ols, oligomeric

9 proanthocyanidins, cinnamic acids, and hydroxycinnamoyl derivatives (Boulton 2001, He et al.

2012a, Mirabel et al. 1999).

In intramolecular copigmentation, or self-association, anthocyanin molecules stack in vertical complexes promoted by hydrophilic interactions of the glucose components of anthocyanin molecules and hydrophobic repulsion of aromatic nuclei and water (Boulton 2001,

Cavalcanti et al. 2011, Gonzalez-Manzano et al. 2008, He et al. 2012a, Jackson 2008, Kunsági-

Máté et al. 2008). This association has a hyperchromic effect, increasing color density, and can also affect color tint with a bathochromic effect towards more purple hues common in young red wines (Baranac et al. 1997, Mirabel et al. 1999). More self-association is seen with a greater degree of methoxylation in the B ring (Gonzalez-Manzano et al. 2008). In fact, self-association of malvidin-3-O-glucoside is thermodynamically favored over intermolecular interactions with other cofactors (Lambert et al. 2011). For self-association to take place, there must be a minimum concentration of 1 mmol/L of anthocyanins in solution (Gonzalez-Manzano et al.

2008). In rosé wines, neither self-association nor copigmentation occurs because the anthocyanin content is too low. For this reason, blue and purple tones are often absent from these wines (He et al. 2012a). Stacking of anthocyanins in copigmentation complexes produces a sandwich conformation, physically limiting access to the chromophore by water, and therefore limits the formation of colorless hydrated forms (chalcone or carbinol) (Brouillard et al. 2003,

Gonzalez-Manzano et al. 2009). This is why copigmentation can lead to greater color intensity

(Boulton 2001, Jensen et al. 2008). Ethanol can limit self-association; the organic solvent weakens intermolecular hydrophobic interaction by disrupting the lattice-like interaction of water molecules and destroying the molecular stacking of anthocyanins (Casassa and

Harbertson 2014, Gonzalez-Manzano et al. 2008, He et al. 2012a). However, ethanol can

10 facilitate the extraction of anthocyanins and cofactors from grapes (Cheynier et al. 2006a,

Kunsági-Máté et al. 2008).

Intermolecular copigmentation, or copigmentation with other cofactors, can also stabilize color. Compared to self-association, intermolecular copigmentation has a larger impact in modifying the color of young wines through an increase in maximum absorption wavelength (bathochromic effect) and a shift towards higher intensities (hyperchromic effect)

(Boulton 2001, Dangles et al. 1993, Mirabel et al. 1999). The interaction between anthocyanins and cofactors can occur with either colored anthocyanin form, the flavylium cation or the quinoidal base, but at wine pH copigmentation primarily involves monomeric anthocyanins in the flavylium state; this is because cofactors have electron-rich pi systems, and are therefore able to more easily associate with the comparatively electron-poor flavylium pi system

(Dangles et al. 1993, Figueiredo et al. 1996, Malien-Aubert et al. 2001, Parisa et al. 2007).

When flavylium ions form copigments, they no longer participate in overall anthocyanin equilibrium and remain in a colored state, increasing color stability in the wine (Gonzalez-

Manzano et al. 2009, Jackson 2008). When hydrated, anthocyanins can be broken down into anthocyanidin and glucose components and are then susceptible to irreversible oxidative color loss and browning. Copigmentation prevents this hydration, and therefore protects and stabilizes color of anthocyanins (Malien-Aubert et al. 2001). Copigmentation can contribute

30-50% of color in young red wines (Jackson 2008, Monagas and Bartolomé 2009). Generally,

(+)-catechin and (-)-epicatechin are powerful cofactors that can form intensely colored complexes easily (Gonzalez-Manzano et al. 2009, Kunsági-Máté et al. 2011). However, the variation in relative proportion and concentrations of cofactors from different grape varieties with different vintages and winemaking processes can cause distinct color differences and

11 copigmentation (He et al. 2012a, Schwarz et al. 2005). Alkaloids, amino acids (proline and arginine), some organic acids, polysaccharides, purines and metal cations can also participate in copigmentation (Dangles and Brouillard 1992, He et al. 2012a, Jackson 2008, Ribéreau-

Gayon et al. 2006). Anthocyanins with ortho-dihydroxyl arrangement in the B ring (including cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, and petunidin-3-O-glucoside) can form colored complexes with Mg, Al, Fe, Sn, and Cu at certain concentrations, but it is uncertain if this plays a significant role in color (Boulton 2001, Cavalcanti et al. 2011, He et al. 2012a).

Temperature and pH can affect the rate of copigmentation; the optimal pH for copigmentation complex is around 3.5, and cool temperatures during fermentation and storage can favor copigmentation and slow the dissociation of colored complexes (Abyari et al. 2006, Gauche et al. 2010, Jackson 2008, Parisa et al. 2007). High temperatures, such as during the process of thermovinification, can destabilize the formation of copigments (Abyari et al. 2006, He et al.

2012a, Parisa et al. 2007).

While self-association and copigmentation can provide short-term stability for anthocyanins, anthocyanins can react with other compounds in the wine matrix to form copigments with longer-term stability, including pyranoanthocyanins and polymeric anthocyanins (He et al. 2012b, Jackson 2017). These pigments are more stable because they are less pH dependent and less susceptible to bleaching by bisulfite (Cheynier et al. 2006a,

Harbertson 2016).

Pyranoanthocyanins are formed by free anthocyanins and yeast byproducts such as acetaldehyde, pyruvic acid, and vinylphenols via a cycloaddition where an additional pyran ring is formed between the C4 position in the C ring and the hydroxyl group on the C5 position in the A ring (Casassa and Harbertson 2014, de Freitas and Mateus 2011, Jackson 2017, Rentzsch

12 et al. 2007, Schwarz et al. 2003, Wrolstad et al. 2005). This reaction can occur between an anthocyanin and any compound with a polarizable double bond (von Baer et al. 2008). There are many types of pyranoanthocyanins, including carboxy-pyranoanthocyanins (A type vitisins), B type vitisins, methylpyranoanthocyanins, hydroxyphenyl-pyranoanthocyanins

(pinotins), and flavanyl-pyranoanthocyanins. Following the formation of pyranoanthocyanins, secondary generated pigments can form, include flavanyl/phenyl-vinylpyranoanthocyanins

(portisins), pyranone-anthocyanins (oxovitisins), pyranoanthocyanin dimers, and others

(Casassa and Harbertson 2014, He et al. 2012b). Pyranoanthocyanins are highly stable because they are resistant to sulfite bleaching and oxidative degradation (de Freitas and Mateus 2011).

Most pyranoanthocyanins have a yellow or orange color, and their formation contributes to the shift in hue to tawny or brown in aged red wines (Cheynier et al. 2006a, de Freitas and Mateus

2011, Rentzsch et al. 2007).

Vitisins are usually the most abundant pyranoanthocyanins (He et al. 2012b). Their precursors are most often secondary metabolites derived from yeast glycolysis during alcohol fermentation (He et al. 2012b). For example, vitisin A is formed by malvidin-3-O-glucoside and pyruvic acid, and vitisin B is formed by malvidin-3-O-glucoside and acetaldehyde (Romero and Bakker 2001). Acetone, acetoin, oxalacetic acid, acetoacetic acid and diacetyl can also react with anthocyanins to form pyranoanthocyanins (de Freitas and Mateus 2011, He et al. 2012b).

Vitisins appear orange and are formed during early to mid-alcoholic fermentation when the concentration of pyruvic acid is high; their production increases with low pH and high temperature (Morata et al. 2006, Romero and Bakker 2000, 2001). These pigments can also form polymers with tannins, likely via the cycloaddition to the anthocyanin moiety of a catechin-anthocyanin adduct (Nave et al. 2010).

13 When hydroxycinnamic acids and 4-vinylphenols react with free anthocyanins they produce hydroxyphenyl-pyranoanthocyanins, some of which are called pinotins because they were first isolated from Pinotage wine (He et al. 2012b, Schwarz et al. 2002). Hydroxycinnamic acids that can undergo this reaction include p-coumaric acid, caffeic acid, ferulic acid, and sinapic acid. 4-vinylphenol and 4-vinylguaiacol, which arise from the decarboxylation of p- coumaric acid and ferulic acid, can also form hydroxyphenyl-pyranoanthocyanins (He et al.

2012b, Morata et al. 2007). Hydroxyphenyl-pyranoanthocyanins appear red-orange and tend to accumulate post-alcoholic fermentation (de Freitas and Mateus 2011, He et al. 2012b, Schwarz et al. 2004).

Flavanyl-pyranoanthocyanins, also known as vinylflavanol-pyranoanthocyanins or pyranoanthocyanin-flavanols, are formed via the acetaldehyde-mediated reaction between anthocyanins and flavanols (de Freitas and Mateus 2011, He et al. 2012b, Mateus et al. 2002b).

Flavanyl-pyranoanthocyanins possess a hypsochromically shifted maximum of absorption in the visible region, resulting in an orange color, and are more stable than their precursors at varying pH values (Francia-Aricha et al. 1997). They are also more stable against degradation and bleaching by sulfur dioxide than free anthocyanins (Chinnici et al. 2009, He et al. 2012b).

Vinylflavanol adducts, which are critical to this reaction, do not occur naturally in grapes and likely arise from the dehydration of flavanol-ethanol adducts and the decomposition of methylmethine-linked flavanol adducts (Cruz et al. 2008, Es-Safi et al. 1999).

Portisins, also known as flavanyl/phenyl-vinylpyranoanthocyanins, were first isolated in port (de Freitas and Mateus 2011, Mateus et al. 2003). These compounds bear a pyranoanthocyanin moiety linked through a vinyl bridge to a flavanol or phenol unit (Mateus et al. 2002a, 2003). With a longer maximum light absorption wavelength, they emit a blue-

14 violet hue (Mateus et al. 2003, Oliveira et al. 2006). Portisins also have a higher resistance to the attack of water or sulfite dioxide than monomeric anthocyanin precursors, likely due to the higher protection of the chromophore groups against nucleophilic attack (Mateus and de Freitas

2001, Oliveira et al. 2006). They are derived through the condensation of anthocyanin-pyruvic acid adducts (vitisin A type pyranoanthocyanins) and vinylphenolic compounds (Mateus and de Freitas 2001, Mateus et al. 2003, Oliveira et al. 2007).

Oxovitisins, also called pyranone-anthocyanins, contribute a yellow color and are direct derivatives of carboxypyranoanthocyanins, especially vitisin A (de Freitas and Mateus 2011,

He et al. 2010). Pyranoanthocyanin dimers present a rare turquoise color (with a maximum visible absorption wavelength at ~730 and ~680 nm) (Oliveira et al. 2010). They are formed by a reaction between carboxy-pyranoanthocyanins and methyl-pyranoanthocyanins (Oliveira et al. 2010). Both oxovitisins and pyranoanthocyanin dimers were initially detected in aged port wine (He et al. 2010, Oliveira et al. 2010).

Polymeric pigments, the other form of stable wine pigments, arise from phenolic or polyphenolic compounds that come from berry skins and seeds or oak wood (Castañeda-

Ovando et al. 2009, Jackson 2008, Ribéreau-Gayon et al. 2006). This often occurs through direct polymerization of anthocyanins and flavan-3-ols or proanthocyanidins (He et al. 2012b,

Jackson 2008, Ribéreau-Gayon et al. 2006). Polymeric pigments are more stable than monomeric pigments and stabilize wine color by protecting the chromophore of the anthocyanin from water and nucleophilic attack, oxidation, or other chemical modifications such as bleaching with SO2 (He et al. 2012b, Jackson 2008, Somers 1971). In addition, polymerization increases the number of anthocyanins in the colored flavylium and quinoidal forms. At a pH of

3.4, about 60% of anthocyanin-tannin polymers are colored, while at the same pH only about

15 20% of equivalent free anthocyanins are colored (Jackson 2017). It is estimated that 25% of anthocyanins may have polymerized with flavonoid compounds by the end of alcoholic fermentation, and likely 40% may have polymerized after just one year aging (He et al. 2012b,

Jackson 2008, 2017). With time, polymerization shifts the absorption properties of the anthocyanin chromophore from red to brown, or even to orange and yellow (He et al. 2012b,

Santos-Buelga et al. 1999). The condensation between anthocyanins and tannins (primarily flavan-3-ols and oligomeric proanthocyanidins) can be promoted by acidic pH and high temperature (He et al. 2012b). The neutral pseudobase form of anthocyanin – which account for about 90% of monomeric anthocyanins in wine – can act as a nucleophile and attack the electrophilic carbocation on a flavonoid, creating a T-A adduct (Brouillard and Delaporte 1977,

Waterhouse et al. 2016). The flavylium form of anthocyanin can act electrophilic and react with the nucleophilic A ring of a proanthocyanidin to make an A-T adduct, creating a colorless flavene compound (Waterhouse et al. 2016). Subsequent oxidation can rearrange the molecule to the colored flavylium state (He et al. 2012b). Diglucosidic anthocyanins can also participate in these polymerizations (Bishop and Nagel 1984). However, anthocyanins in the bisulfite- addition compound state cannot undergo this A-T polymerization because the C4 position is blocked (Berké et al. 1998, He et al. 2012b). Anthocyanin-anthocyanin dimers can also be formed; they can have structures similar to B-type proanthocyanidins or can form A-type proanthocyanidin-like structures with an additional intramolecular bond following an additional dehydration reaction (Alcalde‐Eon et al. 2007, Pati et al. 2009, Salas et al. 2005, Vidal et al.

2004). Anthocyanins can also react with non-flavonoids to form complicated pigments such as anthocyanoellagitannin, which has a deep red-purple color (Quideau et al. 2005).

16 Reddish or violet polymeric pigments can be formed via the mediation of acetaldehyde or glyoxylic acid (Casassa and Harbertson 2014, Dallas et al. 1996a, 1996b, Drinkine et al.

2007, He et al. 2012b). Acetaldehyde is the most abundant aldehyde in red wine because it arises from yeast metabolism and the oxidation of ethanol (He et al. 2012b). At wine pH, some acetaldehyde is in a protonated form, which can react with the nucleophilic position on the A ring of a flavan-3-ol molecule or a terminal unit of proanthocyanidins. After dehydration, this adduct can react with the nucleophilic A ring of an anthocyanin, and after deprotonation, the resulting compound can form a violet quinoidal base of the cross-linked flavanol-ethyl- anthocyanin adduct (Casassa and Harbertson 2014, Drinkine et al. 2007, He et al. 2012b,

Jackson 2017). These ethylene-bridged pigments can be between two flavonoid A rings, including 2 flavanols, a flavanol and an anthocyanin, or two anthocyanins (Waterhouse et al.

2016). This reversible reaction leads to a bathochromic shift and less susceptibility to nucleophilic addition (and color loss) by SO2 and water (Waterhouse et al. 2016). Ethylene- bridged pigments can undergo further polycondensation, resulting in a higher degree of polymerization (Es-Safi et al. 1999). However, anthocyanins are only present at the terminal points of the linear oligomers linked by ethyl bridging, and therefore there are no ethyl-linked pigments containing more than two anthocyanin units (Es-Safi et al. 1999). Low aging temperatures can slow the aldehyde-mediated polymerization between anthocyanins and flavan-3-ols and can also limit the fast formation of excessively large pigments, which will precipitate and cause color loss (Baranowski and Nagel 1983, He et al. 2012b, Rivas-Gonzalo et al. 1995). An environment with oxygen and lower pH can promote polymerization through the formation of acetaldehyde and its protonated form (He et al. 2012b). Other minor aldehydes in wine, including propionaldehyde, isovaleraldehyde, isobutyraldehyde, benzaldehyde,

17 formaldehyde, 2-methybutyraldehyde, vanillin, furfural and hydroxymethylfurfural can also mediate a condensation reaction (Es-Safi et al. 2000a, Pissarra et al. 2003, Sousa et al. 2007).

Glyoxylic acid comes from the iron-catalyzed oxidation of tartaric acid (Fulcrand et al. 1997).

Glyoxylic acid bearing an aldehyde moiety can also mediate the indirect condensation of anthocyanins and flavan-3-ols, which is thought to influence color change during wine aging

(Drinkine et al. 2005, Es-Safi et al. 2000b, He et al. 2012b).

Xanthylium pigments are polyphenolic pigment derived by direct or indirect condensation of flavonoids and can be a range of colors from yellow to red (He et al. 2012b,

Jackson 2017, Jurd and Somers 1970). For example, A-T adducts can form yellow- orange xanthylium pigments (Es‐Safi et al. 2000, He et al. 2012b). A glyoxylic acid-mediated dimer of flavan-3-ols and anthocyanins can also generate xanthylium pigments (Dallas et al.

1996b, Es-Safı et al. 1999). In aging, anthocyanins also play a large role in tannin retention by binding with proanthocyanidins (“Anthocyanins in Wine”, Casassa and Harbertson 2014).

Many aspects of anthocyanin chemistry specific to grapes and wine remain under investigation, including the influence of viticultural practices on the monomeric anthocyanin profiles of young red wines made from different cultivars, the anthocyanin profiles of red wines made from non-vinifera varieties, identification of trace monomeric anthocyanins in red wines, assessment of efficient cofactors and enhancement practices for copigmentation, and enology practices to improve anthocyanin stability, formation of stable pigments, and total wine color

(He et al. 2012a). The identification of new second generation pigments of carboxy- or methyl- pyranoanthocyanins and more complicated polymeric anthocyanins with higher degrees of polymerization will further understanding of color evolution (He et al. 2012b). The contribution of such pigments to wine color and enology practices to enhance the formation and stability of

18 these anthocyanin derivatives is also critical in our understanding of wine color chemistry (He et al. 2012b).

II. Interspecific Hybrid Grape Cultivars

Viticulture is expanding rapidly in regions of the United States and the world that previously were considered poorly suited to wine grape production (Atucha et al. 2018). Of the

50 United States, only Alaska does not grow grapes for wine production (Sabbatini and Howell

2014). However, severe winter freezing temperatures, late spring frosts, low numbers of frost- free days, and high inter-seasonal variation can be limiting factors in producing consistently high yields of quality wine grapes in these challenging regions (Atucha et al. 2018). In many northern temperate zones in the United States, consistency is key in the wine grape industry in order to secure supplies of local and regional fruit. The development of new interspecific hybrid cultivars has made commercial wine grape growing possible in states with hot summers, frigid winters, or high humidity (Sabbatini and Howell 2014).

These interspecific hybrid grape cultivars have been developed specifically to combat environmental challenges and the weaknesses of traditional wine grape vines, such as susceptibility to cold temperatures, rot, or mildew (Burgess 2017). These hybrids are produced by simple crossbreeding, which happens in nature; they are not genetically modified organisms

(Burgess 2017). Hybrids that are cold-hardy and resistant to disease are sometimes the only vines that can survive in places that are too humid or too frigid for V. vinifera production

(Burgess 2017). Cold-hardy cultivars must be able to withstand low winter temperature extremes while producing consistent and reliable crops by growing with moderate vigor, producing substantial yields, and possessing good fruit quality (Atucha et al. 2018). Wine grape

19 crops can be susceptible to many diseases, especially when the climate is not optimal for wine grape production. Powdery mildew and downy mildew can infect both the leaves and the developing fruit, therefore causing severe losses of crop (Tassoni et al. 2019). According to

Olmo et al., downy mildew is by far the most damaging and the most expensive fungus disease to control; it affects all major grape-growing areas of the world with summer rainfall, regardless of temperate or tropical zone, and the traditional V. vinifera wine cultivars have no resistance

(Olmo 1971). Phylloxera and nematodes can be somewhat controlled by using resistant rootstock, but this adds to the cost of establishing vineyards (Olmo 1971). Grape phylloxera can cause damage on foliage, resulting in a reduction of photosynthesis and impacts on fruit yield and juice quality; historically, it has caused devastating damage on European V. vinifera because this species lacks disease resistance (Yin et al. 2019).

Interspecific hybrid cultivars were first developed as a response to diseases like phylloxera that ravaged V. vinifera vines in the 1800s (Burgess 2017). The phylloxera outbreak in France started in the 1860s and lasted for 20 years, destroying 90% of French vineyards

(Stafne 2019). In response to this epidemic, cultivars derived from phylloxera-resistant

American species were developed and eventually 400,000 hectares were planted in France

(Martinson and Reisch 2018, Stafne 2019). In 1876, it was discovered that V. vinifera cultivars could be grafted onto American grape vines, and as this solution took hold, French-American hybrids were phased out in France (Stafne 2019). By the early 1970s, France effectively prohibited replanting French-American hybrids. This was mainly due to the perceived inferior quality of these early hybrids because they were developed using lower-quality V. vinifera parents (Martinson and Reisch 2018). By 2010, only 0.8% of the French vineyard land (in hectares) were French-American hybrids (Martinson and Reisch 2018). In contrast, hybrids

20 were widely planted in the Eastern United States by the 1970s (Martinson and Reisch 2018).

Prior to this, Eastern producers had to mostly use Vitis labrusca and native cultivars for winemaking (Martinson and Reisch 2018). However, Ontario’s Vintner’s Quality Alliance

(VQA) followed France’s example and prohibited the use of French-American hybrids in VQA- labeled wines, with the exception of a few cultivars (Martinson and Reisch 2018).

The parents of these early hybrids of France were lower quality V. vinifera used to make bulk wine, setting the reputation of hybrid wines as “low quality” as well (Martinson and Reisch

2018). In addition, older hybrids were known to have “intrusive flavors” from being crossed with American native grapes that have a characteristic “musky” or “foxy” aroma, which furthered their bad reputation (Stafne 2019). The history of V. vinifera in the United States and other New World wine regions also contributes to the current perception of hybrid grapes. Early settlers in North America found that the native grapes, such as Concord and Catawba, made unfamiliar (and therefore “unpleasant”) wines (Staff 2017). These settlers then brought over cuttings of V. vinifera vines, such as Merlot and Cabernet, from Europe rather than continuing to experiment with the native grapes (Staff 2017). New World winemakers have focused mostly on non-native grapes since, and native grapevines retained a suboptimal reputation (Staff 2017).

It is important to note that although V. vinifera cultivars are used to make high quality wines in some locations, this does not mean they will make good wines everywhere (Stafne 2019).

Hybrids make up less than 5 percent of global vineyards, perhaps because they are still generally believed to lack depth and nuance (Weltman 2018). The ways in which some hybrids, especially the older ones, can seem suboptimal for wine production varies; some lack complex flavors or contain inherent off-flavors and intense aromatics (Burgess 2017). Now, breeding materials are selected to combine desirable polyphenolic profiles of many species (Liang et al. 2013). Liang

21 et al. are establishing a comprehensive database of phytochemicals in the Vitis germplasm, as many hybrids have diverse genetic backgrounds and the polyphenolic profiles of each must be characterized (Liang et al. 2013). More recent hybrid grapes have been bred to combine traits that enhance wine quality from V. vinifera with traits such as root phylloxera resistance, cold- hardiness, and fungal disease resistance from native North American Vitis species (Yin et al.

2019). Modern hybrid grapes are capable of producing high quality wines that do not have off- aromas characteristic of some of the older hybrids (Stafne 2019).

In the wine industry, a dichotomy is created by producers and consumers alike to divide

“hybrid” cultivars from V. vinifera cultivars (Martinson and Reisch 2018). However, such a dichotomy is not used for other fruits that rely on hybrids for production, such as apples or strawberries. This distinction for wine grapes is likely due to initial reputation of hybrid cultivars in winemaking (Martinson and Reisch 2018). There is a stigma still associated with hybrid wines that needs to be overcome due to the perceived inferior quality of early hybrids of the late 1800’s (Martinson and Reisch 2018, Pellechia 2018). However, newer hybrid varieties are becoming more “vinifera-like,” meaning they make wines of excellent quality that are less distinct from V. vinifera wines than early hybrids (Martinson and Reisch 2018). In fact,

Germany classifies certain varieties as V. vinifera, despite the fact that these cultivars have

North American Vitis spp. germplasm incorporated; they can be legally classified as such if the vines are deemed to be indistinguishable from V. vinifera and the wines taste like those produced from V. vinifera cultivars (Martinson and Reisch 2018). Perhaps one way to re-market hybrid wines is to stop calling these cultivars “hybrids” and begin to call them “disease-resistant varieties.” This places a reasonable emphasis on the strengths of these cultivars to reduce pesticide use and still make a high-quality crop (Martinson and Reisch 2018). Rombough wrote

22 that hybrid grapes can be as successful as V. vinifera grapes: “The question is one of marketing, and nothing else. Most wineries make their money from the walk-in trade. And each and every walker-in is amenable to hand-selling…it doesn’t matter what name is on the label, so long as there is quality in the bottle” (Rombough 2002, Stafne 2019). Some hybrids, like Vidal blanc, successfully retain strong aromatics and complex flavors and are therefore more favorable than some that do not (Staff 2017). The challenge of grape breeding is to strike an ideal balance between hardiness and delicate flavors (Staff 2017). The Bordeaux producer Vignobles Ducourt manages 450 hectares of classified vineyards and recently planted a 3.5-hectare vineyard of hybrid grapes. The company states on its website: “We can tell you the results are amazing. We spray the very minimum in this vineyard, once or twice a year, using only copper and sulfur.

The vines are healthy, the resistance stable, and the wines are good.” (Martinson and Reisch

2018). As climate change intensifies, vines that are more resistant to intense weather are necessary, although the past poor reputation and lack of presence in the wine world makes hybrid wines struggle in the wine industry (Burgess 2017, Puckette 2016).

Environmental and cost concerns now cause producers to reconsider the use of hybrids in winemaking. The most critical of these concerns is the use of pesticides to control diseases

(Martinson and Reisch 2018). Intensive agricultural protection, often in the form of sprays, are needed to control insects and mildews. In non-organic farming, these sprays often include petrochemicals (Pellechia 2018). In 2008, a well-publicized event of Bordeaux schoolchildren falling ill due to suspected pesticide poisoning led to legislation that allowed local authorities to restrict spraying at certain times near housing and public buildings (Martinson and Reisch

2018, Pellechia 2018). This also led the French government to make a plan titled “EcoPhyto” in the same year that aimed to reduce pesticide use by 50% over the course of ten years

23 (Martinson and Reisch 2018, Pellechia 2018). Major climate shifts also increased disease stress, which therefore increased the need and cost of spraying (Pellechia 2018). The cost of pesticides, fuel, and labor needed to apply up the 15 sprays during the growing season is another factor that is pushing Europeans to reconsider hybrids (Martinson and Reisch 2018). In 2009, the

European Union revised the regulations that prohibited planting hybrids and in 2016 France started allowing experimental use for “distinct, uniform, and stable” hybrids (Martinson and

Reisch 2018, Pellechia 2018). In 2017 the French National Research Institute (INRA) released four new disease resistant varieties (Martinson and Reisch 2018, Pellechia 2018). Some of these hybrids require only 2-3 fungicide sprays per year while 10-15 sprays are often used for V. vinifera crops (Martinson and Reisch 2018, Pellechia 2018). European viticulture research facilities now work to find new cultivars with resistance to powdery and downy mildew, black rot, phylloxera, nematodes, and other vine pests (Sabbatini and Howell 2014) The second highest classification of French wines (Protected Geographical Indication, the highest level being AOC) allows hybrids crossed with V. vinifera to be used (Martinson and Reisch 2018).

Although these are progressive changes to improve the sustainability of the wine grape industry, it is only a small start for the large wine industry in Europe. As of 2018, a little more than 1% of the 2.7 million hectares of wine grapes planted in Western Europe are hybrids (Martinson and Reisch 2018, Pellechia 2018). In Eastern Europe, wine grapes are not as widely planted, but an estimated 22% of Bulgaria’s production and 47% of Romania’s are hybrids (Martinson and Reisch 2018, Pellechia 2018). Current programs are creating new hybrid cultivars that are increasingly more “vinifera-like” (Pellechia 2018). As the environmental and economic benefits of disease-resistance hybrids are realized and wine quality from these cultivars increases, there is renewed hope that industry acceptance will rise (Pellechia 2018).

24 Due to the high cost of vineyard establishment, there is a need for more standardized information in regard to hybrid grape cultivars to help growers select cultivars appropriate for their conditions, especially because much of cultivar selection is based on anecdotal information that can be outdated or inapplicable (Smiley et al. 2016). Cold-climate wine grape cultivars can vary in vigor, yield, and fruit composition traits (Atucha et al. 2018). Iowa State

University Extension and Outreach has an extensive resource titled “A Review of Cold Climate

Cultivars” that specifies characteristics and limitations of 74 cold-climate varieties, especially those which are grown in the Midwestern United States (Smiley et al. 2016). These cultivars must be able to withstand severe winters and mature in short growing seasons. Winter cold hardiness is determined by the ability of the cultivar to withstand the lowest expected temperature in the area (Smiley et al. 2016). Cold hardiness can be affected by the vine’s suitability to the site, the general health of the vine, crop load, and pest and disease control

(Smiley et al. 2016). Many V. vinifera cultivars, such as Chardonnay, Merlot, and Cabernet

Sauvignon, are only hardy down to between -5 and -10° F (“Growing Grapes”). Riesling is somewhat hardier, but is susceptible to rot in climates with heat and humidity (“Growing

Grapes”). Hybrids tend to set near-full crops on secondary shoots and are therefore more frost tolerant than V. vinifera varieties (“Growing Grapes”). However, some hybrid cultivars that experience early bud break can be susceptible to late frosts (Smiley et al. 2016). It is also critical that selected cultivars can adequately ripen in a specific area given the length and relative warmth of the growing season (Smiley et al. 2016). Cost for lab, labor, equipment, and materials can vary within a region and between regions, and therefore vineyard establishment and operating costs vary greatly. (“Growing Grapes”) Approximately, it takes $10,000 to plant an acre of grapevines (Davis 2018). In the Finger Lakes Region of New York State, the estimated

25 cost of establishing and maintaining a hybrid planting to production, which takes three years, on a commercial scale on a prime site in 2013 was $30,732 per acre (Tang et al. 2014). In this study land was valued at $6,000 per acre, vineyard size was 50 acres, varieties planted were

Corot Noir, Cayuga White, and Vidal Blanc, harvest method was by machine, and skilled labor was valued at $20 per hour (Davidhizar 2014, Tang et al. 2014).

Corot noir, Maréchal Foch, and Marquette are three economically significant cultivars of interest that represent the three major categories of interspecific hybrids: neo-American,

French-American, and riparia-based cold hardy, respectively.

The first, Corot noir, originated in Geneva, New York from the New York State

Agricultural Experiment Station at Cornell University (“Grape Variety: Corot noir”, Ogrodnick

2006, Reisch et al. 2006, Reisch and Luce 2013). This mid- to late-season red wine grape is a cross between Seyve-Villard 18-307 and Steuben (“Grape Variety: Corot noir”, Reisch et al.

2006, Reisch and Luce 2013). This interspecific hybrid includes V. vinifera, V. labrusca, V. rupestris, V. lincecumii, V. riparia, and V. berlandieri in its genome and produces large clusters of medium-sized black berries (“Grape Variety: Corot noir”, Reisch et al. 2006). It often experiences late bud break, so it is not susceptible to early spring frosts (Reisch et al. 2006,

Reisch and Luce 2013). Corot noir is rated as slightly susceptible to powdery mildew, black rot, and Botrytis, and moderately susceptible to downy mildew (Reisch et al. 2006, Reisch and

Luce 2013). Overall, the disease resistance observed is greater than other interspecific hybrid grapes and much better than V. vinifera grapes (Reisch et al. 2006). This cultivar can be excessively vegetative and sometimes requires cluster thinning (“Grape Variety: Corot noir”,

Reisch et al. 2006, Reisch and Luce 2013, Smiley et al. 2016). Wine produced from Corot noir has a deep red color and attractive cherry and berry fruit aromas and has a round and heavy

26 mouthfeel with big tannins, depending on winemaking temperature and yeast selection (“Grape

Variety: Corot noir”, Ogrodnick 2006, Reisch et al. 2006). It can be used for varietal wine production or for blending because it does not contain off-aromas typical to many red hybrid grapes and produces “vinifera-type” wine (“Grape Variety: Corot noir”, Ogrodnick 2006,

Reisch et al. 2006, Reisch and Luce 2013). However, if winemaking decisions are not optimal for the grape, wines can be somewhat thin with green aromas (Reisch et al. 2006). The pH and titratable acidity tend to be lower than many of the common red hybrid grapes (Reisch et al.

2006, Reisch and Luce 2013). Corot noir is considered moderately winter hardy (-10 to -15° F), ranked among the better French-American hybrids (Reisch et al. 2006, Reisch and Luce 2013).

It is considered to be hardier than V. vinifera cultivars and some interspecific hybrids such as

Chambourcin and Cayuga White, but not as hardy as riparia-based hybrids such as Maréchal

Foch and Frontenac (Reisch et al. 2006).

The second, Maréchal Foch, was bred in 1910 in Alsace, France and was introduced in the United States in 1946 (Aspler 2003, “Maréchal Foch Wine” 2015, Smiley et al. 2016). This cultivar, also known as Kuhlmann-188-2, includes V. riparia, V. rupestris, and V. vinifera and is a cross between Millardet et Degrasset 101-14 OP and Goldriesling (Bulas 2001, “Grape

Variety: Maréchal Foch”, “Maréchal Foch Wine” 2015, Pool et al. 1979, Reisch et al. 1993,

Robinson et al. 1967). It produces short tight clusters with small highly-pigmented black berries that can be attractive to birds (Bowen et al. 1972, Bulas 2001, “Grape Variety: Maréchal Foch”,

Hawkins 2007, Pool et al. 1979, Reisch et al. 1993, Robinson et al. 1967). Maréchal Foch is a very early-ripening grape, making it an ideal choice in regions where V. vinifera struggles to ripen (Bulas 2001, “Grape Variety: Maréchal Foch”, “Maréchal Foch Wine” 2015, Pool et al.

1979, Reisch et al. 1993). It is cold hardy up to -15° to -20°C and the vine is hardy and disease

27 resistant (Bowen et al. 1972, Bulas 2001, Domoto et al. 2008, “Grape Variety: Maréchal Foch”,

“Maréchal Foch Wine” 2015, Hawkins 2007, Pool et al. 1979, Reisch et al. 1993, Robinson et al. 1967). Vines are medium in vigor, and pruning is sometimes necessary to get sufficient yields (Pool et al. 1979, Reisch et al. 1993, Smiley et al. 2016). High quality red wines can be made from Maréchal Foch; it is very versatile and can produce light fruity wine with carbonic maceration or earthy, full-bodied reds (Bulas 2001, “Grape Variety: Maréchal Foch”,

“Maréchal Foch Wine” 2015, Hawkins 2007, Reisch et al. 1993, Smiley et al. 2016). It is known as a red interspecific hybrid with “vinifera quality” and can produce wine with dark red color, fruity aromas, full body, and an excellent bouquet that has been rated “good to excellent” by tasting panels (Bowen et al. 1972, Pool et al. 1979, Robinson et al. 1967). The herbaceous aroma and purplish color cause some to compare it to Burgundy wines (Bulas 2001, “Maréchal

Foch Wine” 2015, Smiley et al. 2016). This teinturier grape is also often used in blends to enhance color (“Maréchal Foch Wine” 2015).

The final grape cultivar of this study is Marquette. It is a cross between a complex hybrid of V. riparia, V. vinifera, and other Vitis species and Ravat 262, an offspring of Pinot noir (Cold

Hardy Wine Grapes: University of Minnesota 2011, “Grape Variety: Marquette”, Hemstad and

Luby 2008, MacGregor 2016, Smiley et al. 2016). It originates from the University of

Minnesota Horticultural Research Center in 1989 and was released in 2006 (Hemstad and Luby

2008). Marquette berries are black with a light pink pulp (“Grape Variety: Marquette”, Smiley et al. 2016). It has moderate vigor and can sometimes be vulnerable to frost due to somewhat early budbreak (Clark 2019, MacGregor 2016, Smiley et al. 2016). It has low susceptibility to black rot, bunch rot, downy mildew and powdery mildew, and its open, orderly growth makes vine canopy management efficient (Cold Hardy Wine Grapes: University of Minnesota 2011,

28 “Grape Variety: Marquette”, “Minnesota Hardy: Grapes” 2015, Wine Grape Comparisons:

University of Minnesota, Haun, Hemstad and Luby 2008, MacGregor 2016, Smiley et al. 2016).

It is considered very cold-hardy (-20° F to -30° F) (Hemstad and Luby 2008, Wine Grape

Comparisons: University of Minnesota). Marquette typically produces complex red wines with

V. vinifera-like color, moderate tannins, and cherry and black currant aromas (Cold Hardy Wine

Grapes: University of Minnesota 2011, “Minnesota Hardy: Grapes” 2015, Haun, Hemstad and

Luby 2008, MacGregor 2016). It can also contain complex aromas including notes of blackberries, pepper, plum, tobacco, leather, and spice (Cold Hardy Wine Grapes: University of Minnesota 2011, “Minnesota Hardy: Grapes” 2015, Haun, Hemstad and Luby 2008,

MacGregor 2016, Smiley et al. 2016). In some tastings it has been rated better than pure V. vinifera wines (MacGregor 2016). Its high sugar, medium acidity, pronounced tannins, and ruby color make it often best utilized as a medium-bodied red table wine (Cold Hardy Wine

Grapes: University of Minnesota 2011, Haun, MacGregor 2016, Smiley et al. 2016).

III. Anthocyanins and Wine Quality

Anthocyanins are essential to wine quality perception. Whether consciously or unconsciously, the consumer first analyzes wine according to its color. Prior to any taste, odor, or mouthfeel evaluation, color imparts an impression on the consumer of what to expect of the wine (Lawless and Heymann 2010). Anthocyanins define the color parameters of wine and therefore are key compounds in wine quality determination (Aleixandre-Tudó et al. 2015,

Cheynier et al. 2006b, Moreno-Arribas and Polo 2009, Somers and Evans 1974). Color can be used as a tool by consumers to determine wine quality (Charters and Pettigrew 2007). Lightly colored wine is sometimes perceived as a result of grape immaturity or poor winemaking

29 practices, although depth of color can also be due to the cultivar used (Jackson 2017). The key color features in wine analysis include hue (the shade or tint of the wine) and depth (intensity of color) (Jackson 2017). These aspects give hints to the consumer about grape maturity as well as vinification techniques used, such as skin contact, oak exposure, and wine aging (Jackson

2017). Three separate studies by Somers et al., Sáenez-Navajas et al., and Parpinello et al. indicated that more deeply colored wines scored highest by experienced wine judges (Parpinello et al. 2009, Sáenz-Navajas et al. 2011, Somers and Evans 1974).

Color can also spur “olfactory bias;” for example, a study by Parr et al. demonstrated that aroma perceptions during wine evaluation were affected in a white wine spiked with red coloring (Parr et al. 2003). The red color serves as a visual misinformation cue, skewing what the consumer expects of the wine prior to tasting. Morrot et al. demonstrated the psychological illusion color can play on olfactory perception. Tasters, consisting of 54 undergraduates from the Faculty of Oenology of the University of Bordeaux, created a list of descriptors for red and white wines using aromas of objects that have the same color of the wine: odor descriptors for red wines were mostly represented by red or dark objects, while those that described white wines were yellow or clear. In a secondary test, the panelists described a white wine spiked with red coloring with descriptors commonly associated with red wines, demonstrating olfactory bias (Morrot et al. 2001).

In addition to initial color, the quality of red wines is often determined by aging capacity

(Henry et al. 2006, Jackson 2016, 2014, Sáenz-Navajas et al. 2013, Verdú Jover et al. 2004).

Aging potential can be determined by the wine’s alcohol, sugar, and phenolic content (Jackson

2017). Aging capacity of traditional V. vinifera wines follow a hue change from red to brick red to brown (Kennedy et al. 2006, Moreno-Arribas and Polo 2009). This change in hue comes

30 from the replacement of anthocyanins with more stable polymeric pigments (Somers 1971). A study by Burtch et. al demonstrated that in single anthocyanin trials, monoglucosides showed greater changes in color than their diglucosides counterparts, except in the case of petunidin.

Therefore monoglucosides have a greater capacity to change color, and wines containing higher concentrations of monoglucosides will change color more over the course of aging (Burtch et al. 2017). There is also slower and lower polymeric pigment formation with diglucosides as compared to monoglucosides. The higher amount of diglucosides in hybrid cultivars leads to slower formation of polymeric pigments and therefore hybrid wines age differently than vinifera wines (Burtch et al. 2017). Without “ageability” that follows the expected pattern of

V. vinifera, the wines could be considered lower quality (Jackson 2014).

Wine quality is sometimes also correlated to varietal authenticity. The anthocyanin profile can be used to differentiate between V. vinifera and hybrid grapes, and potentially to verify the validity of varietal wine (Jackson 2008, Picariello et al. 2012).

Several methods have been proposed as standardized color measurement for wines. One of the most common color analysis methods is CIE-L*a*b*, which expresses color with three values: lightness (L*), green to red (a*) and blue to yellow (b*). CIE-L*a*b* attempts to quantify color with a numerical value that corresponds to the amount of perceived change. The equations used in this method were designed to account for the fact that the human eye is more sensitive to small color differences in some parts of the color wheel than others (Habekost

2013). However, a study by Fairchild et al. found that different spectrophotometric measurement techniques, different illumination scenarios, and different viewing geometries can all lead to different measured color of the wine (Fairchild 2018). The CIE-L*a*b* system does not give a fully accurate description of color, but is effective for measuring color differences

31 and color changes over processing and storage (Wrolstad et al. 2005). Further standardization of color measuring techniques is needed for the analysis of wine color and its impact on perceived wine quality.

IV. Unique Qualities of Anthocyanins in Hybrid Grapes

Anecdotally, interspecific hybrid wine grapes can produce wines with unusual or unstable color. New York winemakers have observed marked varietal differences in pigment stability and susceptibility to browning in hybrids since the 1960’s (Robinson et al. 1966). Cool- climate areas like New York that utilize disease-resistant and cold-hardy hybrid grapes generally also don’t have the climate conditions conducive to producing dark-colored red wines

(Jackson 2017). However, this “unusual” hybrid color has yet to be defined. Some hybrids have deeper color than that which is expected of traditional V. vinifera wines, while others have trouble with full color extraction (Burgess 2017). Literature on the color chemistry of interspecific hybrids is sparse. It is well understood that many interspecific hybrids contain diglycosylated anthocyanins that do not appear in V. vinifera grapes or wine; however, this is not true of all such hybrids. A further understanding of the color composition of, and reactions between, components in interspecific hybrid wines is necessary to optimize their production.

Buren et al. found a large variation in anthocyanin content among 151 hybrid grape cultivars examined by paper chromatography (Buren et al. 1970), and relative abundance of individual anthocyanins is cultivar-dependent (Balik et al. 2013). In a study comparing nine V. vinifera and ten interspecific hybrid cultivars grown in the Czech Republic, malvidin-3-O- glucoside was found as the most abundant anthocyanin, ranging from 30-64% of total content

(Balik et al. 2013). However, two hybrids showed very low levels of malvidin-3-O-glucoside

32 (5.49 % and 7.03%). Two interspecific hybrids also showed levels of cyanidin greater than 10% of the total content, while all other V. vinifera and hybrid cultivars showed low levels of cyanidin (Balik et al. 2013). In this study, no acylated anthocyanins were identified among the diglycosylated anthocyanins (Balik et al. 2013). The ratio of coumarylated anthocyanins to acetylated anthocyanins in the V. vinifera varieties and some hybrid cultivars favored coumarylated compounds, but other hybrids showed either the opposite ratio or equal amounts of coumarylated and acetylated anthocyanins (Balik et al. 2013). Normally, diglucosides are more stable than monoglucosides but are more susceptible to browning and are less colored (He et al. 2012a). At wine pH, diglucosides are less ionized compared to their monoglucoside counterparts, and therefore less colorful (Boulton 2001, Manns et al. 2013). Diglycosylated anthocyanins appear at a longer wavelength than the corresponding monoglucoside; however, diglycosylated anthocyanins do not have a more violet hue than the corresponding monoglycosylated anthocyanins because this shift in wavelength is balanced by an increase in absorption (Robinson et al. 1966). Anecdotally, the hue of some red hybrid wines tends toward blue or purple rather than red or brick, likely due to the relatively high concentrations of delphinidin, petunidin, and malvidin in hybrid cultivars (Manns et al. 2013, Romero et al. 2008).

All of these factors play a major role in the color of the final hybrid wine and can result in different color challenges depending on the specific anthocyanin profile.

Anthocyanin biosynthesis is regulated by internal and external physiological and environmental conditions and metabolomic profiles can give some insight into these regulations and biosynthetic pathways (Downey et al. 2006, Mazzuca et al. 2005). The metabolic profile in terms of quantity and pattern of polyphenolics and lipids of non-vinifera grapes including two hybrid cultivars and five American native Vitis species was explored by Ruocco et al. Total

33 anthocyanin content varied significantly by genotype and some, but not all, wild genotypes were characterized by the presence of diglucosides (Ruocco et al. 2017). In some hybrid and native varieties, delphinidin derivatives were found to be the most abundant components of the anthocyanin profiles as compared to the V. vinifera cultivars, in which malvidin derivatives were the most abundant (Ruocco et al. 2017). Although Mazzuca et al., among many others, proposed differentiating V. vinifera and hybrid wines using their respective anthocyanin profile because 3,5-diglucosides are specific to hybrids, the absence of diglucosides cannot be considered sufficient evidence to support this identification (Balik et al. 2013, Mazzuca et al.

2005). The diglycosylated trait is sometimes absent when hybrids are backcrossed with

European grapevine varieties, resulting in an absence of diglycosidic anthocyanins within hybrid cultivars (Balik et al. 2013).

One “unusual” characteristic of hybrid wines is the difference in color evolution over aging compared to V. vinifera wines. Sims et al. describes color stability in terms of accumulation of polymeric anthocyanins; a wine that browns to a greater extent and loses more color under aging conditions would be considered “less stable.” It’s been found that diglucosidic anthocyanins are more susceptible to browning than monoglucosidic anthocyanins

(Sims and Morris 1985). A study by Burtch et al. analyzed the aging characteristics of mono- and diglucosidic forms of the five primary wine anthocyanidins in model wine solution with additions of catechin and acetaldehyde. In this study, individual diglucosides converted to polymeric pigment more slowly than monoglucosides. In a model solution with both monoglucosides and diglucosides combined, the reaction rate of monoglucosides was slower than that of monoglucosides alone (Burtch et al. 2017). Interspecific hybrid wines containing high concentrations of diglucosides will form less polymeric pigment than wines containing

34 primarily monoglucosides because they are less reactive than monoglucosides and have a greater tendency to brown (Burtch et al. 2017, Robinson et al. 1966, Sims and Morris 1985).

This instability of monoglucosides allow for them to readily react and polymerize, therefore stabilizing the chromophore. Diglucosides convert more slowly, resulting in slower polymer formation and lower concentration of polymeric pigment. This therefore results in less chromophore protection and less stable color over aging (Burtch et al. 2017). The color evolution from purple-red to brick-red is often not observed in hybrid wines because pyranoanthocyanins cannot be formed from diglucosides (Burtch et al. 2017). This is because the second glucose located at the C5 position blocks the reaction (Burtch et al. 2017, He et al.

2012b, Sims and Morris 1985) Hybrid wines generally also have lower concentrations of condensed tannins which are key components of polymeric pigment formation (Burtch et al.

2017, Manns et al. 2013). Overall the color evolution of hybrid wines is affected by the slower rates of polymeric pigment formation of diglucosides, their inability to form stable red-orange pyranoanthocyanins, and the competition from diglucosides when both monoglucosides and diglucosides exist that slows the formation of polymeric pigment by monoglucosides (Burtch et al. 2017).

V. Viticultural and Winemaking Practices: Influence on Anthocyanins

Environmental conditions, viticultural practices, and winemaking techniques can affect grape phenolic composition and concentration as well as the profile of the final wine (Kennedy et al. 2006, Mazza et al. 1999). Each cultivar of wine grape has a general color “fingerprint,” or profile of anthocyanins. Anthocyanin profile describes the relative distribution of anthocyanins within a cultivar. With the application of different techniques or conditions, the

35 total concentration of anthocyanins can be altered, but the anthocyanin profile itself often stays the same (Arozarena et al. 2002, Cacho et al. 1992, Mazza et al. 1999, Ryan and Revilla 2003).

Climate, soil conditions, season, and cultivar can affect anthocyanin profiles (Mazza et al.

1999).

Vineyard techniques and environmental conditions can potentially alter the phenolic composition in grapes prior to winemaking. In general, weaker sun and lower precipitation levels result in reduced anthocyanins and other flavonoids (Dokoozlian and Kliewer 1996,

Papoušková et al. 2011, Wicks and Kliewer 1983). However, some studies have shown no change in total anthocyanins with shading or decreased anthocyanins with high light levels.

Shading of foliage in the vineyard can decrease the flavonoid content of the grapes and alter the temperature and humidity of the canopies (Buttrose et al. 1971, Crippen and Morrison 1986,

Haselgrove et al. 2000, Kliewer 1977, Rojas-Lara and Morrison 1989, Smart et al. 1988).

Increased temperature in the plant can increase the rate of metabolic processes, but at high temperatures (roughly 30 deg C in grapevines) many metabolic processes stop or are significantly reduced (Dokoozlian and Kliewer 1996, Jones 2014, Kobayashi et al. 1965,

Reynolds et al. 1986). Spayd et al. artificially cooled more exposed fruit and heated less exposed fruit, and found that the cooled and exposed fruit increased in anthocyanin content, while the heated, less-exposed fruit evinced a reduction in anthocyanin levels (Spayd et al.

2002). In Shiraz grapes, Downey et al. found that when maintaining microclimate parameters such as temperature and humidity, there was no difference in anthocyanin accumulation between shaded fruit and exposed fruit (Downey et al. 2004). Based on these and other studies, it is generally understood that anthocyanin biosynthesis is affected more by temperature than light where increased berry temperature can limit anthocyanin accumulation (Bergqvist et

36 al. 2001, Downey et al. 2004, Haselgrove et al. 2000, Kliewer and Lider 1968, Spayd et al.

2002). Spayd et al. and Bergqvist et al. noted anywhere from a 3°C to an 11°C difference in berry temperature between exposed and shaded berries, while Kliewer et al. noted a range of

0°C to 20°C difference (Bergqvist et al. 2001, Kliewer and Lider 1968, Spayd et al. 2002).

Diurnal differences in temperature can further complicate the analysis of the effects of temperature and light on anthocyanin accumulation; Mori et al. found that lower night temperature (15°C) resulted in greater anthocyanin accumulation than a constant temperature of 30°C (Mori et al. 2005). Shifts in anthocyanin composition can also occur based on temperature and light; studies have shown that shaded fruit have a higher proportion of trihydroxylated anthocyanins based on delphinidin, petunidin, and malvidin (Downey et al.

2004, Spayd et al. 2002). A proportional increase in coumaroyl glucosides has also been seen with higher temperatures (Downey et al. 2004, Spayd et al. 2002). These shifts in composition in response to temperature suggest that warm-climate fruit might tend to have a higher proportion of malvidin, petunidin, and delphinidin coumaroyl derivatives, while cool-climate, shaded fruit might have more peonidin and cyanidin nonacylated glucosides and acetylglucosides (Downey et al. 2006, 2004, Spayd et al. 2002). However, these anthocyanin compositions are largely based on cultivar, and it has not yet been established how each of these components contribute to final wine color (Downey et al. 2006). Canopy management for bunch exposure, row orientation, and trellising, in addition to irrigation and yield regulation, likely can be used to influence the temperature in viticultural systems (Downey et al. 2006). However, differences among region and grape cultivars makes understanding how to optimize wine color in the vineyard challenging.

37 Many of these studies have analyzed V. vinifera cultivars, and research on the effects of viticultural practices on phenolic content in hybrid grape cultivars is somewhat limited. A study by Sun et al. evaluated the effects of shoot thinning and two harvest times on Maréchal Foch, an interspecific French hybrid grape grown commonly in the Finger Lakes and in eastern parts of the United States and Canada (Smiley 2016). This study indicated that shoot thinning results in decreased yield components and increased berry anthocyanins and soluble solids (Sun et al.

2011). However, a corresponding anthocyanin increase was not seen in the final wine. Later harvest also increased soluble solids and anthocyanin content, and the final wine showed increased anthocyanin content (Sun et al. 2011). Corot Noir responded variably to cluster and shoot thinning with some changes in wine anthocyanin, berry skin tannin, berry seed tannin, and wine tannin concentrations; overall, anthocyanin in the wine (but not the berry) increased in the second year with the shoot thinning treatment (Sun et al. 2012). However, it is uncertain if the results of these treatments balance out the cost. Due to the high vigor of some hybrids, implementing these techniques would increase labor costs (Sun et al. 2012). The influence of a single pre-veraison leaf and lateral shoot removal treatment on the juice of cold climate interspecific hybrid grape cultivars Brianna, La Crescent, Frontenac, Marquette, and Petite

Pearl over three growing seasons was analyzed by Scharfetter et al. In the final year of the study, the wines made from these hybrids were also analyzed. For the red cultivars Frontenac,

Marquette, and Petite Pearl the treatment increased the total phenolic content and monomeric anthocyanin concentration in both the juice and the wine. This increase was associated with increased berry temperature and higher photosynthetically active radiation in the fruit zone.

Hybrids that have lower tannin extractability and contain a large proportion of diglucoside anthocyanins could benefit from exposed fruit zone treatments (Scharfetter et al. 2019). In a

38 study by Morris et al., French-American hybrid grapes Chancellor and Villard noir both showed greater red color after shoot thinning compared to the control (Morris et al. 2004). The effects of regulated deficit irrigation treatments on hybrids have also been analyzed. Baco Noir, a

French-American hybrid, accumulated the most anthocyanins when they were under slight water stress (Balint and Reynolds 2017). Generally, this change in anthocyanin content does occur aside from the effect deficit irrigation has on berry size, which would increase the concentration of the compounds relative to total berry weight. However, the total anthocyanin change is likely due to the changes in structure and development of the skins rather than a change to the biosynthesis pathway (Downey et al. 2006). Vineyard additions such as abscisic acid, which has shown to increase anthocyanin accumulation, and gibberellins or the growth regulators such as forchlorfenuron, which have shown to decrease levels of anthocyanins, need to be explored in interspecific hybrids to analyze the possibility of using these vineyard techniques to modulate color (Downey et al. 2006).

Many different winemaking techniques can be implemented to alter the final profile of the wine. Total concentrations of phenolics are affected by practices including extended maceration, processing temperature, thermovinification, and enzyme and tannin additions (He et al. 2012a, Sacchi et al. 2005). A study by Manns et al. demonstrates that traditional vinification treatments, including pectolytic enzyme addition, exogenous tannin addition, and cold soak, are ineffective at enhancing phenolic compounds in hybrid wines (Manns et al.

2013). The hot press treatment in this study did result in an increase of certain phenolic compounds, but also in an increase of diglucosides anthocyanins that may produce hues perceived as undesirable in wine (Manns et al. 2013). However, none of these increases carried over to the final wines (Manns et al. 2013). Other methods that have shown variable results in

39 increasing the phenolic content of V. vinifera wines, such as carbonic maceration or yeast selection, could be tested to further investigate their efficacy on improving phenolic content in hybrid wines (Sacchi et al. 2005). A study by Nicolle et al. showed that co-fermentation of red grapes with white pomace can be used to modulate hybrid wine color composition. As some hybrid wines can have color much darker than traditional wines, this method can lighten wine color to make the wines more acceptable to consumers (Nicolle et al. 2018). Sims and Morris found that with the hybrid red grape Chancellor, sensory panelists rated the darker red color of juice treated with heat extraction higher than the lighter red color of juice with 24-hour skin contact or the bluish juice that was immediately pressed (Sims and Morris 1987). This study also found that 24-hour skin contact juice and immediately pressed juice browned to an unacceptable level after 5 months of aging, while the heat treated juice did not brown at all

(Sims and Morris 1987). With Brazilian hybrids Isabel, Rúbea, and Cora grapes, pre-drying was ineffective due to the destruction of anthocyanins during thermal treatment, but submerged cap techniques resulted in wines with higher anthocyanin content and color intensity that resembled traditional V. vinifera wines (de Castilhos et al. 2017, 2015). Another study by de

Castilhos et al. showed that submerged cap treatments in Isabel and BRS Violeta wines had a higher acceptance for appearance and aroma driven by parameters for total phenolic content, anthocyanins, and CIEL*a*b* (de Castilhos et al. 2016). In Isabel grapes, UV-C light exposure increased monomeric anthocyanin content, but the anthocyanin profile remained unchanged

(Maurer et al. 2017).

Different aging practices can also affect the color evolution of wine. Mild oxygenation during storage can improve wine quality by stabilizing wine color because oxygen is involved directly or indirectly in the formation of polymeric anthocyanin pigments (He et al. 2012b).

40 Micro-oxygenation affects wine color in a method comparable to oak barrel maturation because it results in a lower concentration of monomeric anthocyanins and a higher concentration of polymeric anthocyanins and pyranoanthocyanins (He et al. 2012b). However, red wines with lower phenolic content, such as interspecific hybrid wines, might not be affected the same way by micro-oxygenation (He et al. 2012b). Anthocyanins can interact with other compounds extracted from oak during aging, including gallic, ferulic, vanillic, syringic, and ellagic acids, ellagitannins, and tannins. During aging on lees, yeast cell walls could interact with or adsorb anthocyanins (He et al. 2012b).

It is necessary to further explore effects of viticultural and oenological treatments on hybrid grapes and wines in order to understand the mechanisms in which anthocyanins unique to hybrids interact throughout the growing, fermentation, and aging processes.

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60

CHAPTER 2

Effect of Sun Exposure on the Evolution and Distribution of Anthocyanins in Interspecific Red Hybrid Winegrapes

Introduction

The development of interspecific hybrid cultivars designed to combat environmental challenges and overcome the weaknesses of traditional wine grape vines has made commercial wine grape growing possible in regions of the United States and the world that previously were considered poorly suited to wine grape production (Atucha et al. 2018, Burgess 2017, Sabbatini and Howell 2014). Corot noir, Maréchal Foch, and Marquette are three economically significant cultivars of interest that represent the three major categories of interspecific hybrids: neo-

American, French-American, and riparia-based cold hardy, respectively. These recent hybrid grapes are capable of producing high quality wines that are increasingly more “vinifera-like,” meaning they make wines of excellent quality that are less distinct from V. vinifera wines than early hybrids (Pellechia 2018, Stafne 2019, Yin et al. 2019).

Anthocyanins are water-soluble molecules that contribute the red, purple, and blue colors to red grapes and wine (Mazza and Francis 1995, Ribéreau-Gayon et al. 2006). Color is a critical parameter of wine quality determination because whether consciously or unconsciously, the consumer first analyzes wine according to its color (Aleixandre-Tudó et al.

2015, Cheynier et al. 2006b, Lawless and Heymann 2010, Moreno-Arribas and Polo 2009,

Somers and Evans 1974). Non-vinifera grapes, including interspecific hybrids and native varieties, can include mono- and diglucoside anthocyanins (He et al. 2012a, Waterhouse et al.

2016). Diglucosides are more stable than monoglucosides but are less pigmented at wine pH and are more susceptible to browning because they do not form polymeric pigment as readily

61 (Boulton 2001, Burtch 2017, He et al. 2012a, Hrazdina et al. 1970, Manns et al. 2013, Mazza and Brouillard 1987, Robinson et al. 1966). In addition, cool-climate areas like New York that utilize disease-resistant and cold-hardy hybrid grapes generally also don’t have the climate conditions conducive to producing dark-colored red wines (Jackson 2017a). Hybrid wines can also sometimes have a purple or blue hue, rather than the traditional red or brick hue. This is likely due to the high proportion of anthocyanins other than malvidin (Manns et al. 2013,

Romero et al. 2008). A further understanding of the color composition of, and reactions between, components in interspecific hybrid wines is necessary to optimize their production.

Anthocyanin biosynthesis and degradation are affected by physiological and environmental conditions (Downey et al. 2006, Mazzuca et al. 2005). The application of different viticultural techniques can alter the concentration of anthocyanins and other phenolics in grapes, juice, and wine (Downey et al. 2006, Kennedy et al. 2006, Mazza et al. 1999,

Mazzuca et al. 2005). In general, weaker sun levels result in reduced anthocyanins and other flavonoids (Dokoozlian and Kliewer 1996, Papoušková et al. 2011, Wicks and Kliewer 1983).

Increased temperature in the plant can increase the rate of metabolic processes, but at high temperatures (roughly 30°C in grapevines) many metabolic processes stop or are significantly reduced (Dokoozlian and Kliewer 1996, Jones 2014, Kobayashi et al. 1965, Reynolds et al.

1986). When separating the impacts of light and temperature on anthocyanin accumulation, it’s been found that generally anthocyanin biosynthesis is more affected by temperature than by light, where increased berry temperature can limit anthocyanin accumulation (Bergqvist et al.

2001, Kliewer and Lider 1968, Spayd et al. 2002). Differences anywhere from a 0°C to a 20°C in berry temperature between exposed and shaded berries have been observed, with larger changes often in warmer climate regions (Bergqvist et al. 2001, Kliewer and Lider 1968, Spayd

62 et al. 2002). Diurnal differences in temperature can further complicate the attempt to separate effects of temperature and light on anthocyanin accumulation (Mori et al. 2005). Shifts in anthocyanin composition can also occur based on temperature and light, with a higher proportion of trihydroxylated anthocyanins (delphinidin, petunidin, and malvidin) in shaded samples as compared to dihydroxylated anthocyanins (cyanidin and peonidin) (Downey et al.

2004, Spayd et al. 2002, Guidoni 2002).

However, much of the research on the effect of viticultural practices on anthocyanin accumulation has been on V. vinifera cultivars. Some viticultural treatments have been tested on hybrid grapes, but results have been variable (Balint and Reynolds 2017, Morris et al. 2004,

Scharfetter et al. 2019, Sun et al. 2011, 2012). With Maréchal Foch, shoot thinning increased anthocyanin content in the berry but not the final wine while later harvest timing increased anthocyanin content through to the final wine (Sun et al. 2011). With Corot noir, cluster and shooting thinning resulted in an increase in anthocyanins in the wine but not in the berry, and due to the high vigor of some hybrids, implementing these techniques can be costly (Sun et al.

2012). Scharfetter et al. found that a pre-veraison leaf and lateral shoot removal treatment increased the total phenolic and monomeric anthocyanin concentration in both the juice and the wine of the red cultivars Frontenac, Marquette, and Petite Pearl (Scharfetter et al. 2019). This increase was associated with increased berry temperature and higher photosynthetically active radiation in the fruit zone (Scharfetter et al. 2019). Similarly, two French-American hybrid grapes, Chancellor and Villard noir, both showed greater red color after cluster and shoot thinning compared to the control (Morris et al. 2004). Baco noir, another French-American hybrid, accumulated the most anthocyanins when it was under slight water stress from regulated deficit irrigation treatments (Balint and Reynolds 2017). Overall, these studies have shown

63 evidence that anthocyanin accumulation in interspecific hybrids can be affected by viticultural practices, but further research is needed to separate the impacts of light and temperature and to optimize the effects of these practices.

This research aims to define the distribution of anthocyanins within three economically significant interspecific cool-climate hybrid grape cultivars (Vitis spp): Corot noir, Maréchal

Foch, and Marquette. Monomeric anthocyanins were extracted from shaded and unshaded fruit from each cultivar at multiple points between veraison and harvest and analyzed via HPLC to explore the evolution of anthocyanins over the course of ripening and to clarify the effects of vine microclimate on red hybrid color. Light exposure and berry and air temperature were monitored in Corot noir in both years and berry temperature was monitored in Maréchal Foch in 2019 to represent generalized vine microclimate.

Materials and Methods

Sample Collection

Beginning two weeks after veraison, fruit samples were collected from three hybrid grapevine cultivars located at three vineyards in the Finger Lakes region of New York: Corot

Noir from the Finger Lakes Teaching and Demonstration Vineyard (Penn Yan), Maréchal Foch from Prejean Vineyard (Dundee), and Marquette from Stever Hill Vineyard (Branchport). Each vineyard had three replication blocks of treatment (leaf-pulled) vines and three replication blocks of control (shaded) vines. Leaf-pulling was performed at fruit set. One 100-berry sample was collected from each of the six blocks at each of the three vineyards at multiple timepoints from veraison to harvest. Samples were collected at two, three, or four timepoints (T1, T2, T3,

T4) each season as dictated by season length (Table 2.1). Berries were picked by hand blindly

64 within the designated block and placed into labeled clear plastic bags. All samples were frozen at 0C until sample collection was completed.

Table 2.1. Dates of veraison (50%), sampling, and harvest for each grape cultivar.

2018 Veraison T1 T2 T3 T4 Harvest

Corot noir 13 Aug 22 Aug 7 Sept 21 Sept 9 Oct 10 Oct Maréchal 1 Aug 23 Aug 7 Sept 21 Sept - 4 Oct Foch Marquette 26 July 22 Aug 7 Sept 11 Sept - 22 Sept

2019 Veraison T1 T2 T3 T4 Harvest

Corot noir 24 Aug 29 Aug 15 Sept 27 Sept 12 Oct 14 Oct Maréchal 16 Aug 30 Aug 15 Sept 27 Sept - 29 Sept Foch Marquette 8 Aug 30 Aug 15 Sept - - 16 Sept

Solvents and Instrumentation

All solvents, including hydrochloric acid, methanol, type 1 water, phosphoric acid, ethyl acetate, and acetonitrile (Fisher Chemical, Fair Lawn, NJ or VWR International, LLC,

Mississauga, ON, Canada), were high-performance liquid chromatography (HPLC) grade. An

Agilent Model 1260 Infinity series HPLC consisting of an in-line vacuum degasser, autosampler, binary pump, diode array detector, and thermostated column compartment controlled via Chemstation software (Rev. B.04.03-SP2 [105] with spectral pack) was used.

The HPLC was equipped with a Kinetex pentafluorophenyl (PFP) column (100 mm  2.1 mm,

2.6 μm particle size) fitted with a KrudKatcher guard filter (Phenomenex, Torrance, Calif.,

U.S.A.) controlled via a column-switching valve.

65 Counting and Weighing Samples

Samples were removed from the freezer (-20°C) and 100 berries were counted and weighed in a tared plastic cup on a balance to record berry weight. Samples were then returned to the freezer until processing.

Separating Skin from Pulp

Samples were placed in the refrigerator to gradually thaw, then berries were individually squeezed between thumb and forefinger to eject the pulp. Skins were stored in labeled 50 mL foil-wrapped Falcon tubes and returned to the freezer. Pulp was put into a stomacher bag and placed in a Seward Stomacher 400 Circulator (Seward, West Sussex, England) for 2 minutes at

300 rpm. Pulp soluble solids (as Brix) was tested with a Misco Palm Abbe #PA201 Digital

Refractometer (Misco, Solon, OH) and then transferred to labeled clear plastic bags, which were stored in an aluminum envelope in the freezer.

Freeze-Drying Skins

Aluminum boats were folded and labeled to fit 6 boats within 45 cm x 18.5 cm freeze- dryer trays. Skins were taken directly from freezer and spread out in a single layer on an aluminum boat. Samples were then run in a 25-hour freeze-dryer cycle in a Medium Home

Freeze Dryer (Harvest Right, North Salt Lake, UT) in 2018 and a Thermocouple Vacuum

Gauge Freeze Dryer (The Virtis Company, Gardiner, NY) in 2019. Once dry, skins were put into labeled clear plastic bags. Each sample was weighed and then stored in an aluminum envelope in the freezer. Samples were protected from light exposure with low ambient lighting and aluminum foil covering throughout processing.

66 Grinding Skins

Freeze-dried skins were ground in a Retsch MM200 MixerMill Ball Mill (Verder

Scientific, Inc., Newton, PA). Two grinder capsules with two balls in each capsule were cleaned with ethanol and thoroughly dried using Kimwipes. Grape skins were removed from the plastic bag and both grinder capsules were filled. The samples were shaken in the ball mill for 110 s at

25 rev/s. Using a funnel and a small brush, ground skins were transferred into labeled 15 mL plastic Falcon tubes. Grinder capsules and balls were cleaned with ethanol and thoroughly dried before continuing to the next sample.

Methanol Extraction

This extraction method was based off of protocols designed by Jeffrey et al. and Manns et al. (Jeffery et al. 2008, Manns and Mansfield 2012). Individual samples were removed from the freezer and 3 g were immediately weighed on a balance using a weigh boat. The 3 g of sample was added to a foil-wrapped 250 mL Erlenmeyer flask with 50 mL of 80% methanol solution, some of which was used to rinse out the weigh boat. The sample was mixed with a spatula and stoppered with a neoprene stopper and covered in parafilm, then placed in an ice bath in a Branson 2800 sonicator (Branson, Danbury, CT) and secured with a weight ring. Each sample was sonicated for 20 minutes and swirled at five-minute intervals for the duration to prevent cavitation and standing waves. After sonication, the sample was filtered through a

Buchner funnel with a #1 filter into a foil-wrapped collection flask. The filter cake was washed with a 25 mL aliquot of 80% methanol. The filter cake was then returned to the Erlenmeyer flask with 50 mL 80% methanol and the sonication, filtering, and rinsing was repeated. All filtrate in collection flask was transferred to a round-bottom flask, using 25 mL 80% methanol to rinse out the previous collection flask. Filtrate was reduced to 10-30 mL using a Büchi

67 Rotavapor R-200 rotary evaporator (Büchi, New Castle, DE) at 25 kPa pressure and moderately fast speed in a 32C water bath. The filtrate was then diluted back to 50 mL with Type I water and kept frozen in a foil-wrapped Falcon tube until further analysis.

Solid-Phase Extraction

Up to 8 samples that had completed methanol extractions were removed from the freezer and defrosted in the fridge overnight. Samples were shaken well by hand and sonicated for 5 minutes. Two solid-phase extraction manifolds, J.T. Baker, Inc (Avantor, Radnor, PA) and

Burdick & Jackson (Honeywell, Charlotte, NC) were set up with tube racks and eight sets of

Varian Bond Elut solvent reservoirs, funnel adaptors, Oasis HLB 3cc 60 mg cartridges (Waters,

Taunton, MA), and stopcocks. To precondition each cartridge, 3 mL of 100% methanol and subsequently 3 mL 0.01 N HCl were run through under gravity and discarded. Then, 2 mL of the sample was applied to the cartridge and allowed to drain under gravity to maintain a flow rate below 1-2 mL/min. This elution was discarded and 2 mL of 0.01 N HCl was similarly run through under gravity and discarded to elute sugars and organic acids. The SPE manifolds were attached to vacuum and the cartridges were dried under light vacuum pressure for 5 minutes at

20 PSI. Monomeric compounds were then eluted with 40 mL of acidified acetonitrile solution

(95:5 acetonitrile:0.01 N HCl) using the solvent reservoirs. This elution was run under gravity and collected in large culture tubes.

Samples were placed in a test tube rack in a 30C water bath under a continuous stream of nitrogen (15-30 PSI) and dried. Once completely dry, 3 mL ethyl acetate was added to each test tube and gently swirled and discarded to remove non-anthocyanin monomeric compounds.

This ethyl acetate rinse was repeated 3 times and samples were dried again under nitrogen.

68 When dry, 1 mL 0.01 N HCl was added to dissolve the solid anthocyanin. This was then passed through a 0.2 m polyethersulfone (PES) filter into an amber HPLC vial and immediately analyzed.

HPLC Analysis

HPLC conditions and method were followed from Manns et al. (2012) for

Anthocyanins, full range (PFP) (Manns and Mansfield 2012).

Prepared samples were analyzed on an Agilent 1260 Infinity Series HPLC using a

Kinetex Core-Shell 100 mm x 2.1 mm pentafluorophenyl (PFP) column packed with 2.6 m diameter particles with a 100 Å pore size fitted with an inline Krudkatcher guard filter

(Phenomenex).

Standards of oenin chloride (malvidin-3-glucoside) and malvin chloride (malvidin-3,5- glucoside) were purchased from Extrasynthese (Genay Cedex, France). Dilutions of 5, 10, 50,

100, 500, and 1000 mg/L were prepared using 0.01 N HCl. Standards were filtered through a

0.2 m PES filter and analyzed using the same HPLC protocol as the samples.

Statistical Analyses

Analysis of variance (ANOVA) was performed using RStudio software Version

1.2.5042 (RStudio Team 2020) to determine significant differences (p<0.05, p<0.01, and p<0.001) in the quantification of anthocyanins between timepoints, treatment, and the interaction of timepoint and treatment. More specifically, a linear mixed effect model using the

Kenward-Rogers approach to estimate denominator degrees of freedom was used with fixed effects of time, treatments, and the interaction and a random effect of panel to control for the repeated measures. The mixed model was fit to the log-transformed quantification values in

69 order to make sure that the model assumptions of normality and homogenous variance were met.

Results

Across all cultivars, the samples that underwent the leaf-pulling treatment (exposed samples, E) did not have significantly different concentrations of total anthocyanins compared to the control (shaded samples, S). However, the concentration of certain individual compounds increased or decreased within each cultivar between timepoints. In all spectra, the group of peaks following the elution of malvidin-3-glucoside around 20 minutes were identified as

“modified anthocyanins,” consisting of various acetylated and coumarylated anthocyanin mono- and di-glucosides (Manns and Mansfield 2012). These acylated anthocyanins will need to be further analyzed for identification.

A total of ten to eleven peaks were consistently identified in the HPLC spectra for each cultivar in both Y1 and Y2. These peaks were labeled Peak 1 – Peak 10 or Peak 1 – Peak 11 until identification can be verified using mass spectrometry. Tentative identifications were assigned based on calculated retention factor (k = retention time/void volume), which were compared to calculated k values from Manns et al. with a 2.5% accepted variability (Manns and

Mansfield 2012). Tentative identities are listed within each cultivar (Tables 2.4, 2.6, and 2.7).

Corot noir

In Y1, two short humps appeared in the chromatogram after Peak 7 and after Peak 9 in some of the samples and were labeled Peak 7a and Peak 9a respectively; these peaks appeared gradually within each of the 6 total samples at each timepoint during that year (Figure 2.2,

Table 2.2).

70 Figure 2.1. Representative HPLC chromatogram for Corot noir (Year 1, Timepoint 1, Treatment (Exposed), Panel 2).

Figure 2.2. Representative HPLC chromatogram for Corot noir (Year 1, Timepoint 4, Control (Shaded), Panel 2).

Table 2.2. Number of appearances of extra peaks in Corot noir at each sampling (Year 1).

Peak T1 T2 T3 T4 7a 0 3 5 6 9a 0 0 5 6

In Y1, E samples showed a significantly higher concentration of each compound in

Peaks 1, 2, and 3 (Table 2.3). E samples had a significantly lower concentration of the compounds in Peaks 8 and 10.

71 Table 2.3. Anthocyanins with accumulation significance between E and S samples for Corot noir (Year 1) (* p<0.05; ** p<0.01; *** p<0.001).

Peak p-value Significance 1 0.0009 *** 2 0.0020 ** 3 0.0125 * 8 0.0168 * 10 0.0019 **

In E samples in Y2, none of the compounds were significantly changed except peak 10, tentatively identified as malvidin-3-glucoside (p=0.0430) (Table 2.4). This compound showed a significantly lower accumulation in E samples compared to S samples, though there was no significant effect on total anthocyanins.

72 Table 2.4. Tentative identifications based on retention factor and their relative distributions in Corot noir.

Percent of Year Peak Anthocyanin Total Profile 1 Cyanidin-3,5-diglucoside 8.4 2 Petunidin-3,5-diglucoside Delphinidin-3-glucosidea 2.2 3 Peonidin-3,5-diglucoside 11.0 4 Cyanidin-3-glucoside 31.4 5 Pelargonidin-3-glucoside 2.5 Year 6 Petunidin-3-glucoside 7.9 1 7 Malvidin-3,5-diglucoside 13.7 7a Malvidin-3,5-diglucoside 2.5 8 Peonidin-3-glucoside 12.2 9 Unknown 1.6 9a Unknown 1.3 10 Malvidin-3-glucoside 5.3 1 Unknown 4.9 2 Unknown 2.5 3 Unknown 9.5 4 Peonidin-3,5-diglucoside 31.2 Year 5 Unknown 2.9 2 6 Pelargonidin-3-glucoside 8.1 7 Malvidin-3,5-diglucoside 14.3 8 Peonidin-3-glucoside 16.2 9 Unknown 2.4 10 Malvidin-3-glucoside 8.0 a Two possible identities based on proximity to calculated k value.

73 Figure 2.3. Individual peak means over the course of ripening for Corot noir (Year 1) (* p<0.05; ** p<0.01; *** p<0.001).a

A Peak 1 B Peak 2

Shaded Exposed Shaded Exposed *** 250 60 *** 50 200 *** 40 ** 150 *** 30 ** 100 20 * 50 * 10

0 0 Concentration (mg/L) Concentration (mg/L) Concentration 1 2 3 4 1 2 3 4 Timepoint Timepoint

C Peak 3 D Peak 8

Shaded Exposed Shaded Exposed 300 * 300 * 250 250 * 200 200 150 * 150 100 100 50 50

0 0 Concentration (mg/L) Concentration 1 2 3 4 (mg/L) Concentration 1 2 3 4 Timepoint Timepoint

E Peak 10 Shaded Exposed

140 ** 120 ** * 100 * 80 * 60 40 20 0 1 2 3 4 (mg/L) Concentratioin Timepoint

a Error bars show standard error. Only peaks with significance shown. Concentration (mg/L) in malvidin-3- glucoside equivalents.

74 Figure 2.4. Total anthocyanin content means over the course of ripening for Corot noir (Year 1).a

Shaded Exposed

2000

1800 1600

1400 1200

1000

800 Concentration (mg/L) Concentration 600

400 200

0 1 2 3 4

Timepoint

a Error bars show standard error. Concentration (mg/L) in malvidin-3-glucoside equivalents.

75 Figure 2.5. Anthocyanin composition during ripening for Corot noir (Year 1).a

A Shaded (S) 600 Peak 1

550 Peak 2

500 Peak 3 450 Peak 4 400 Peak 5 350 300 Peak 6 250 Peak 7

(mg/L) Concentration 200 Peak 7a 150 Peak 8 100 Peak 9 50 0 Peak 9a 1 2 3 4 Peak 10 Timepoint

B Exposed (E) Peak 1 600 Peak 2 550 500 Peak 3

450 Peak 4

400 Peak 5 350 Peak 6 300 Peak 7 250

(mg/L) Concentration 200 Peak 7a 150 Peak 8

100 Peak 9 50 Peak 9a 0 1 2 3 4 Peak 10 Timepoint

a Error bars show standard error. Concentration (mg/L) in malvidin-3-glucoside equivalents.

76 Figure 2.6. Individual peak means over the course of ripening for Corot noir (Year 2) (* p<0.05; ** p<0.01; *** p<0.001).a

Peak 10

Shaded Exposed 180 ** 160 140 120 100 80

60

40 (mg/L) Concentration 20 0 1 2 3 4 Timepoint a Error bars show standard error. Only peaks with significance shown. Concentration (mg/L) in malvidin-3- glucoside equivalents.

Figure 2.7. Total anthocyanin content means over the course of ripening for Corot noir (Year 2).a

Shaded Exposed

1800 1600 1400

1200 1000

800

Concentration (mg/L) 600 400

200 0 1 2 3 4 Timepoint a Error bars show standard error. Concentration (mg/L) in malvidin-3-glucoside equivalents.

77 Figure 2.8. Anthocyanin composition during ripening for Corot noir (Year 2).a

A Shaded (S) Peak 1 550 500 Peak 2 450 Peak 3 400 Peak 4 350

Peak 5 300 250 Peak 6 200 Concentration Concentration (mg/L) Peak 7 150

100 Peak 8

50 Peak 9 0 Peak 10 1 2 3 4 Timepoint

B Exposed (E) 550 Peak 1

500 Peak 2 450 Peak 3 400 Peak 4 350 300 Peak 5

250 Peak 6 200 (mg/L) Concentration Peak 7 150 Peak 8 100 Peak 9 50

0 Peak 10

1 2 3 4

Timepoint

a Error bars show standard error. Concentration (mg/L) in malvidin-3-glucoside equivalents.

78 Maréchal Foch

In Y1, a peak appeared in the chromatogram after Peak 8 in a few samples and was labeled 8a. In Y2, a peak appeared after Peak 3 in only a few samples and was labeled 3a (Table

2.5). In addition, the Maréchal Foch samples did not consistently show the same number of peaks; for example, in the second and third timepoint during the second year, two early peaks that appeared in the first sample were not evident in later samples. During the first year, all of the samples from the first timepoint had 6-7 peaks while the second and third timepoints later in the ripening process had 11-12 peaks.

Table 2.5. Number of appearances of extra peaks in Maréchal Foch at each sampling.

Peak T1 T2 T3 3 (Y2 only) 0 6 0 8 (Y1 only) 0 0 2

In Y1, E samples had a significantly lower concentration of the compound corresponding to Peak 9 (p=0.0211). No other peaks were significantly affected, and there was no significant effect on total anthocyanins.

For Y2, Peak 2 in the E samples showed a significantly higher anthocyanin content compared to the S samples (p=0.0160). Peak 8 (p=0.0499) in the E samples showed a significant interaction between timepoint and treatment, but not a significant effect from treatment alone

(p=0.0499). Total anthocyanins were not significantly affected.

79 Figure 2.9. Representative HPLC chromatogram for Maréchal Foch (Year 2, Timepoint 1, Control (Shaded), Panel 2).

Figure 2.10. Representative HPLC chromatogram for Maréchal Foch (Year 2, Timepoint 3, Control (Shaded), Panel 3).

80 Table 2.6. Tentative identifications based on retention factor and their relative distributions in Maréchal Foch.

Year Peak Anthocyanin Percent of Total Profile 1 Cyanidin-3,5-diglucoside 0.4 2 Delphinidin-3-glucoside Petunidin-3,5-glucosidea 0.4 3 Peonidin-3,5-diglucoside 2.4 4 Cyanidin-3-glucoside 29.1 5 Unknown 0.7 Year 6 Petunidin-3-glucoside 5.8 1 7 Malvidin-3,5-diglucoside 6.2 8 Peonidin-3-glucoside 21.1 8a Unknown 0.4 9 Unknown 0.5 10 Unknown 2.0 11 Malvidin-3-glucoside 31.0 1 Unknown 0.8 2 Unknown 1.3 3 Peonidin-3,5-diglucoside 23.3 3a Unknown 4.3 4 Unknown 1.2 Year 5 Pelargonidin-3-glucoside 5.4 2 6 Malvidin-3,5-diglucoside 4.9 7 Peonidin-3-glucoside 22.1 8 Unknown 0.8 9 Unknown 2.6 10 Malvidin-3-glucoside 33.2 a Two possible identities based on proximity to calculated k value.

81 Figure 2.11. Individual peak means over the course of ripening for Maréchal Foch (Year 1) (* p<0.05; ** p<0.01; *** p<0.001).a

A Peak 1 B Peak 9

Shaded Exposed Shaded Exposed 8 10 * 7 9 8 * 6 * 7 5 6 4 5

3 4 3 2

2

Concentration (mg/L) Concentration (mg/L) Concentration 1 1 0 0 1 2 3 1 2 3 Timepoint Timepoint

a Error bars show standard error. Only peaks with significance shown. Concentration (mg/L) in malvidin-3- glucoside equivalents.

Figure 2.12. Total anthocyanin content means over the course of ripening for Maréchal Foch (Year 1).a

Shaded Exposed

2500

2000

1500

1000

Concentration (mg/L) Concentration 500

0 1 2 3 Timepoint

a Error bars show standard error. Concentration (mg/L) in malvidin-3-glucoside equivalents.

82 Figure 2.13. Anthocyanin composition during ripening for Maréchal Foch (Year 1).a

A Shaded (S) Peak 1 800 Peak 2 700 Peak 3 600 Peak 4 500 Peak 5 400 Peak 6

300 Peak 7 Peak 8

Concentration (mg/L) Concentration 200

Peak 9 100 Peak 10 0 1 2 3 Peak 11

Timepoint

B Exposed (E) 800 Peak 1 Peak 2 700 Peak 3 600 Peak 4 500 Peak 5

400 Peak 6

300 Peak 7 (mg/L) Concentration 200 Peak 8 Peak 9 100 Peak 10 0 1 2 3 Peak 11 Timepoint

a Error bars show standard error. Concentration (mg/L) in malvidin-3-glucoside equivalents.

83 Figure 2.14. Individual peak means over the course of ripening for Maréchal Foch (Year 2) (* p<0.05; ** p<0.01; *** p<0.001).a

A Peak 2 B Peak 4

Shaded Exposed

30 30 *** ** 25 25 20 20

15 15 10 10

5 Concentration (mg/L) Concentration (mg/L) Concentration 5

0 0 1 2 3 1 2 3 Timepoint Timepoint

C D Peak 6 Peak 8 Shaded Exposed Shaded Exposed

200 14 180 * * 12 160 140 10 120 8 100 6 80 60 4

40 Concentration (mg/L) Concentration (mg/L) Concentration 2 20 0 0 1 2 3 1 2 3 Timepoint Timepoint a Error bars show standard error. Only peaks with significance shown. Concentration (mg/L) in malvidin-3- glucoside equivalents.

84 Figure 2.15. Total anthocyanin content means over the course of ripening for Maréchal Foch (Year 2).a

Shaded Exposed 3000

2500

2000

1500

(mg/L) Concentration 1000

500

0 1 2 3 Timepoint

a Error bars show standard error. Concentration (mg/L) in malvidin-3-glucoside equivalents.

85 Figure 2.16. Anthocyanin composition during ripening for Maréchal Foch (Year 2).a

A Shaded (S) Peak 2 800 Peak 3 700 Peak 4 600 Peak 5 500

Peak 6 400

Concentration Concentration (mg/L) 300 Peak 7

200 Peak 8

100 Peak 9 0 Peak 10 1 2 3 Timepoint

B Exposed (E) Peak 2 800 Peak 3 700 Peak 4 600

Peak 5 500

Peak 6 400

(mg/L) Concentration 300 Peak 7

200 Peak 8 100 Peak 9 0 Peak 10 1 2 3 Timepoint

a Error bars show standard error. Concentration (mg/L) in malvidin-3-glucoside equivalents.

86 Marquette

In Y1, a number of extra peaks appeared around Peak 1, 2, 3, 5, and 7. Because of their sporadic appearance, they were omitted from analysis. Only Peak 1a in Y1, which only appeared at T1, showed a significant difference in accumulation (p=0.0488). Total anthocyanins were not significantly affected in Y1. For Y2, none of the compounds showed a significant difference between E and S. Total anthocyanins in E samples were significantly different at T1 (p=0.0438), but not significantly affected at T2.

Figure 2.17. Representative HPLC chromatogram for Marquette (Year 1, Timepoint 2, Treatment (Exposed), Panel 2).

Figure 2.18. Representative HPLC chromatogram for Marquette (Year 2, Timepoint 2, Control (Shaded), Panel 1).

87 Table 2.7. Tentative identifications based on retention factor and their relative distributions in Marquette.

Year Peak Anthocyanin Percent of Total Profile 1 Cyanidin-3,5-diglucoside 5.4 2 Delphinidin-3-glucoside 2.4 3 Peonidin-3,5-diglucoside 8.4 4 Cyanidin-3-glucoside 27.9 5 Pelargonidin-3-glucoside Petunidin-3-glucosidea 3.0 Year 1 6 Malvidin-3,5-diglucoside Petunidin-3-glucosidea 11.3 7 Unknown 12.7 8 Unknown 14.7 9 Unknown 0.8 10 Unknown 2.4 11 Malvidin-3-glucoside 11.1 1 Unknown 2.1 2 Unknown 1.8 3 Unknown 4.3 4 Peonidin-3,5-diglucoside 23.8 5 Unknown 3.3 Year 2 6 Pelargonidin-3-glucoside 9.2 7 Malvidin-3,5-diglucoside 8.8 8 Peonidin-3-glucoside 20.3 9 Unknown 1.2 10 Unknown 4.4 11 Malvidin-3-glucoside 20.7 a Two possible identities based on proximity to calculated k value. Concentration (mg/L) in malvidin-3-glucoside equivalents.

88 Figure 2.19. Individual peak means over the course of ripening for Marquette (Year 1) (* p<0.05; ** p<0.01; *** p<0.001).a

Peak 2

Shaded Exposed 30

* 25

20

15

10

(mg/L) Concentration 5

0 1 2 3 Timepoint

a Error bars show standard error. Only peaks with significance shown. Concentration (mg/L) in malvidin-3- glucoside equivalents.

Figure 2.20. Total anthocyanin content means over the course of ripening for Marquette (Year 1).a

Shaded Exposed

900 800 700

600 500 400

300

(mg/L) Concentration 200 100

0 1 2 3 Timepoint

a Error bars show standard error. Concentration (mg/L) in malvidin-3-glucoside equivalents.

89 Figure 2.21. Anthocyanin composition during ripening for Marquette (Year 1).a

A Shaded (S) 250 Peak 1

Peak 2 200 Peak 3

Peak 4 150 Peak 5 Peak 6 100

(mg/L) Concentration Peak 7 Peak 8 50 Peak 9

Peak 10 0 1 2 3 Peak 11 Timepoint

B Exposed (E) 250 Peak 1 Peak 2

200 Peak 3 Peak 4 150 Peak 5 Peak 6

Peak 7 100 (mg/L) Concentration Peak 7a Peak 8 50 Peak 9

Peak 10 0 1 2 3 Peak 11 Timepoint

a Error bars show standard error. Concentration (mg/L) in malvidin-3-glucoside equivalents.

90 Figure 2.22. Individual peak means over the course of ripening for Marquette (Year 2) (* p<0.05; ** p<0.01; *** p<0.001).a

A B Peak 8 Peak 10 Shaded Exposed Shaded Exposed

250 40 * * 35 200 30 25 150 20 100 15 10

50

Concentration (mg/L) Concentration (mg/L) Concentration 5

0 0 1 2 1 2

Timepoint Timepoint

C Peak 11

Shaded Exposed 250

** 200

150

100

50 (mg/L) Concentration

0 1 2 Timepoint

a Error bars show standard error. Only peaks with significance shown. Concentration (mg/L) in malvidin-3- glucoside equivalents.

91 Figure 2.23. Total anthocyanin content means over the course of ripening for Marquette (Year 2) (* p<0.05; ** p<0.01; *** p<0.001).a

Shaded Exposed 1200 *

1000

800

600

400

Concentration (mgL) Concentration 200

0 1 2 Timepoint

a Error bars show standard error. Concentration (mg/L) in malvidin-3-glucoside equivalents.

92 Figure 2.24. Anthocyanin composition during ripening for Marquette (Year 2).a

A Shaded (S) 300 Peak 1

Peak 2

250 Peak 3

Peak 4 200 Peak 5 150 Peak 6

Peak 7 Concentration (mg/L) 100 Peak 8

50 Peak 9 Peak 10 0 1 2 Peak 11

Timepoint

B Exposed (E) 300 Peak 1 Peak 2 250 Peak 3 Peak 4 200

Peak 5

150 Peak 6 Peak 7 (mg/L) Concentration 100 Peak 8 50 Peak 9

Peak 10 0 1 2 Peak 11 Timepoint

a Error bars show standard error. Concentration (mg/L) in malvidin-3-glucoside equivalents.

93 Viticulture

Figure 2.25. Light exposure (PAR) in exposed (E) and shaded (S) fruit zones of Corot noir.a

A 28 Sept - 9 Oct 2018 1400.0

1200.0

)

1 1000.0 -

s

2 - 800.0

mol mol m

µ 600.0

( PAR 400.0

200.0 0.0 28/9/18 30/9/18 2/10/18 4/10/18 6/10/18 8/10/18 10/10/18

Exposed Shaded

B 28 Sept - 9 Oct 2019 µ 1400.0

1200.0

)

1 1000.0

-

s 2

- 800.0

mol m µ 600.0

PAR ( PAR 400.0

200.0

0.0 28/9/19 30/9/19 2/10/19 4/10/19 6/10/19 8/10/19 10/10/19 Exposed Shaded a PAR = photosynthetically active radiation

94 Table 2.8. Average berry temperature difference (°C) between exposed and shaded samples.

Cultivar Year All Hours Daytime Only (7 AM – 6 PM) 2018 0.29 0.84 Corot noir 2019 0.30 0.84 Marquette 2019 0.12 0.44

Table 2.9. Average berry temperature (°C).

Daytime Only Cultivar Year Treatment All Hours (7 AM – 6 PM) Exposed 15.68 18.19 2018 Shaded 15.52 17.72 Corot noir Exposed 17.31 19.82 2019 Shaded 15.70 17.49 Exposed 20.35 23.69 Marquette 2019 Shaded 20.47 24.13

Discussion

In all cultivars, whether the exposed fruit or the shaded fruit had a higher anthocyanin concentration was extremely variable between compounds. This is representative of the statistical outcome that although certain anthocyanins significantly increased or decreased due to the leaf-pulling treatment, total anthocyanin content was not significantly affected in the exposed (E) samples of any of the cultivars (Figures 2.4, 2.7, 2.12, 2.15, 2.20, 2.23). However, the group of modified anthocyanins that eluted after 20 minutes will need to be analyzed prior to drawing conclusions on the effects of this viticultural treatment on total anthocyanins.

Studies by Sun et al. demonstrated similar results; viticultural treatments including shoot and cluster thinning sometimes increased berry anthocyanins in Maréchal Foch and Corot noir, but overall results were variable in the final anthocyanin content and the viticultural techniques could not be reliably used to change the anthocyanin content of the final wine (Sun

95 et al. 2011, 2012). In contrast, Scharfetter et al. found that total phenolic content and monomeric anthocyanin concentration in both the juice and the wine of three cold climate cultivars

(Frontenac, Marquette, and Petite Pearl) increased with a single pre-veraison leaf and lateral shoot removal treatment (Scharfetter et al. 2019). This difference may be due to the timing of the viticultural treatment, differences between cultivars, or higher berry temperatures and photosynthetically active radiation in the fruit zone.

Certain unique peaks appeared sporadically in addition to the typical 10-11 peaks analyzed for each cultivar; the reason for their appearance varies. Some appeared without much pattern, as demonstrated in Maréchal Foch and Marquette. These could be a result of carryover in the column or compound degradation over the course of analysis. However, for Corot noir, these peaks appeared and seemed to be progressively larger over the course of ripening. This could suggest that the final anthocyanin composition of Corot noir is influenced by the appearance of new compounds later in ripening.

Corot noir

Generally, for both Y1 and Y2, the concentration of anthocyanins was successively higher as the fruit ripened. Many of the individual anthocyanins seemed to increase over the course of ripening or increase and then level out later in ripening (Figures 2.5, 2.8). The total anthocyanin content seemed to gradually increase over ripening as well (Figures 2.4, 2.7).

For Y1 and Y2 in both S and E samples, Peak 4 had the highest concentration of all of the anthocyanins, tentatively identified as cyanidin-3-glucoside or peonidin-3,5-diglucoside

(Figures 2.5, 2.8; Table 2.4).

In Y1, Peaks 1 and 2 had a higher concentration in E samples throughout ripening and

Peak 3 had a higher concentration in E samples at the second and fourth timepoint (Figures

96 2.3A, 2.3B, 2.3C). Peaks 8 and 10 had a higher concentration in S samples later in ripening

(Figures 2.3D, 2.3E). These peaks were tentatively identified as cyanidin-3,5-diglucoside

(Peak 1), petunidin-3,5-diglucoside or delphinidin-3-glucoside (Peak 2), peonidin-3,5- diglucoside (Peak 3), peonidin-3-glucoside (Peak 8), and malvidin-3-glucoside (Peak 10)

(Table 2.4). In Y2, Peak 10 had a higher concentration in S samples at the end of ripening, tentatively identified as malvidin-3-glucoside (Figure 2.6, Table 2.4). These changes in individual anthocyanins could affect the overall total hue of the wine (He et al. 2012a, Jackson

2017, Manns et al. 2013, Romero et al. 2008).

Maréchal Foch

In Maréchal Foch, not all of the individual anthocyanins increased linearly between timepoints over ripening. In Y1, some peaks increased and then decreased, or decreased and then remained level (Figure 2.13). However, some peaks did increase and remain level over ripening (Figure 2.13). In Y2, many of the peaks showed a decrease and then an increase or seemed to remain level before increasing (Figure 2.16). These trends could indicate that certain anthocyanin structures were converting into others, or that these specific anthocyanins were degrading due to oxidation (Boss et al. 1996, VanderWeide et al. 2018). Overall, the total anthocyanin content seemed to gradually increase (Figures 2.12, 2.15).

In Y1, Peaks 4, 8, and 11 had the highest concentration at T1 and T2, but by harvest,

Peak 8 decreased to a level similar to the other anthocyanins (Figure 2.13). These compounds were tentatively identified as cyanidin-3-glucoside (Peak 4), peonidin-3-glucoside (Peak 8), and malvidin-3-glucoside (Peak 11) (Table 2.6). In Y2, Peaks 3, 7, and 10 had the highest concentration over the course of ripening (Figure 2.16). These compounds were tentatively

97 identified as peonidin-3,5-diglucoside (Peak 3), peonidin-3-glucoside (Peak 7), and malvidin-

3-glucoside (Peak 10) (Table 2.6).

In Y1, Peak 1 showed a significantly higher amount in E samples at T1, tentatively identified as cyanidin-3,5-diglucoside (Figure 2.11A). However, this difference at the beginning of ripening did not continue through to harvest. In addition, Peak 9 showed a significantly higher concentration in S samples through the T2 and T3 (Figure 2.11B). In Y2,

Peaks 2, 4, 6, and 8 showed a significantly higher concentration in E samples just prior to harvest, indicating that sun exposure had a significant effect on the final accumulation of these individual anthocyanins (Figure 2.14).

Marquette

As in Maréchal Foch, individual anthocyanins did not seem to progressively accumulate in Marquette. In Y1, many of the compounds increased and then decreased (Figure 2.21). This pattern is largely seen in E samples; many of the S samples increased progressively or decreased and then increased (Figure 2.21). In Y2, each peak within S samples seemed to consistently decrease, while roughly half of the E samples decreased, and half of the E samples increased

(Figure 2.24). Only two timepoints, rather than three, were collected in Y2 due to the sudden harvest of the Marquette grapes following the collection of the second sample set.

In Y1, total anthocyanins increased then decreased in E samples, and decreased then increased in S samples (Figure 2.20). In Y2, total anthocyanins increased in E samples, while they decreased in S samples (Figure 2.23). Certain changes in climate surrounding the dates of sampling could have affected the anthocyanin composition of the grapes at that specific timepoint. According to the National Centers for Environmental Information, at the first sampling (T1) in Y1 there was some rain before and no extreme temperature changes. At T2 in

98 Y1, there had been no rain for multiple days and the region had been experiencing a heat spike for some days. At T3 in Y1, there was rain before and the day of and a large drop in temperature.

In Y2, there was some rain before and a slight temperature increase at T1 and little rain and a relative decrease in temperature at T2 (NCEI 2020). These changes in climate could account for the trends shown by Marquette, notably that the exposed samples had higher anthocyanin concentrations during the heat spike. The seemingly opposite trends between shaded and exposed likely could have been clarified if more samples had been collected over the course of ripening.

In Y1, Peak 4 had the highest concentration, tentatively identified as cyanidin-3- glucoside (Figure 2.21, Table 2.7). In Y2, Peaks 4, 8, and 11 had the highest concentration

(Figure 2.24). These were tentatively identified as peonidin-3,5-diglucoside (Peak 4), peonidin-3-glucoside (Peak 8), and malvidin-3-glucoside (Peak 11), respectively (Table 2.7).

Viticulture

Although a large difference was observed in PAR (photosynthetically active radiation) in both 2018 and 2019 in Corot noir between E and S samples, a corresponding difference in berry temperature change was not observed in Corot noir (Figure 2.25, Table 2.8). It is generally understood that temperature has a greater influence on anthocyanin biosynthesis than light (Bergqvist et al. 2001, Downey et al. 2004, Haselgrove et al. 2000, Kliewer and Lider

1968, Spayd et al. 2002). In V. vinifera, it has been shown that increased light exposure can positively affect anthocyanin accumulation while higher temperatures can negatively affect anthocyanin accumulation (Bergqvist et al. 2001, Haselgrove et al. 2000, Spayd et al. 2002).

These higher temperatures often correspond to the increased direct heating, incident radiation, or increased air temperature (Downey et al. 2006). It is likely that the observed temperature

99 differences in the berries were less pronounced than those stated in the literature due to the cool, cloudy climate of the Finger Lakes (Bergqvist et al. 2001). There is also a factor of wind velocity to be considered which was not recorded, which can cool the grapes despite incident radiation (Millar 1972, Smart and Sinclair 1976, Spayd et al. 2002). It is possible that although light exposure was much higher, the lack of temperature change within the berries resulted in the variable changes observed in anthocyanins within each cultivar. However, some temperature increase in the plant can increase metabolic processes, and it seems that in grapes the optimal color accumulation range is usually 20-30°C. On the higher end of this range is where anthocyanin accumulation can become inhibited. Many of the studies that have shown that decreased temperature increased anthocyanin biosynthesis recorded peak temperatures well above 30°C, but the average berry temperatures in Corot noir never reached above 20°C, while the average daytime berry temperatures in Maréchal Foch remained below 25°C (Table 2.9).

Therefore, it is possible that the grapes in vineyards in the Finger Lakes are still below this

“optimal temperature range” to increase anthocyanin accumulation. In addition, it is well- known that the effects of both light and temperature on anthocyanin accumulation are critically cultivar dependent (Downey et al. 2006). Overall, the cost and labor of leaf removal in these interspecific hybrid cultivars will not conclusively alter anthocyanin accumulation based on this study alone; further research is necessary to identify the individual effects of light and temperature on hybrid color composition.

Conclusion

This study analyzed how the anthocyanin profile of three different interspecific hybrid grape cultivars evolved over the course of ripening, and how these profiles were affected by a

100 leaf-pulling treatment. Results suggest that the leaf-pulling treatment does not significantly affect the total anthocyanin concentration within any of these three grape cultivars. However, individual anthocyanin concentrations within each cultivar changed in response to excess sun exposure, demonstrating that the anthocyanin profile itself can be significantly altered by this vineyard treatment. Changes in concentrations of individual anthocyanins can potentially alter the hue of the wine. Although photosynthetically active radiation increased greatly with the leaf-pulling treatment, a negligible change in berry temperature was observed. With the results of this study, recommendations cannot yet be made on the efficacy of this vineyard treatment to adjust final wine color. However, this study has set the groundwork to understand the anthocyanin evolution of interspecific hybrid cultivars and to further optimize hybrid wine color.

101

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CHAPTER 3

RESEARCH CHALLENGES AND FUTURE WORK

Research Challenges

Using HPLC as the sole qualitative measurement for this experiment presented challenges, as HPLC retention times can only be used to report tentative identities due the closeness and occasional overlap within the identity ranges. In addition, the nature of the HPLC can result in slight shifts between runs or between different columns. To verify the identities of the monomeric anthocyanins analyzed, another identification method, such as mass spectrometry, would need to be used. There is also a lack of published data to compare to on individual anthocyanins within these interspecific hybrid cultivars, making an identification verification method necessary.

The nature of doing a vineyard trial comes with inherent challenges. First, the impact of annual variations made it challenging to identify patterns within cultivars. Although this study was not comparing vintages, repeated measurements can be helpful in identifying the efficacy of the vineyard treatment or patterns within anthocyanin accumulation. Because there are often large variations between vintages due to climate, the measurements repeated over two years have to be analyzed as singular entities and not as repeated data. Second, there are other factors that could affect anthocyanin content of the berries tested, including recent rainfall and climate on the day of sampling. However, these factors cannot be controlled for in a vineyard environment, and so sampling took place in potentially different conditions. It is unclear if this could significantly change the results of berries harvested. Third, sampling protocol had to work around the businesses that offered the samples. For example, sampling could only occur on

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certain days around spray scheduling; this prevented keeping a strict sampling schedule and potentially affected the study. In addition, Marquette berries needed sudden harvesting, and a third sample set for Year 2 could not be acquired. These are all examples of challenges associated with a vineyard trial.

The project design to sample 3-4 times over the course of ripening ideally would demonstrate the way each individual anthocyanin evolved between veraison and harvest.

However, following data analysis, it has become clear that perhaps more data points were needed to get a full picture of how the anthocyanins evolved over time.

The nature of anthocyanins and the rapid reactions they undergo to degrade or stabilize was a large challenge in this project. Many precautions were taken to try to diminish the effects of these reactions, such as elimination of light during processing and freezing samples prior to and following extraction. Despite these precautions, it is still unclear if the anthocyanin profile results are an exact snapshot of the composition of the grapes in the vineyard. The appearance of random peaks sporadically in the HPLC spectra could be evidence that the extraction method did not successfully extract only monomeric compounds; however, these peaks could also represent new compounds formed or carryover within the column.

Future Work

There is still much work left to be done in understanding and optimizing interspecific hybrid grapes for wine production. More research needs to be done to understand the reactions of diglucosidic anthocyanins to understand their effect on the total wine matrix. Experiments to compare the degradation products of diglucosidic anthocyanins with those of monoglucosidic anthocyanins can elucidate the reactions these compounds undergo and how these reactions

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affect the wine matrix. Because extraction and retention of phenolic compounds, especially tannins, is a large challenge in hybrid wine production, a better understanding of these reactions within the wine matrix is necessary.

For this project in particular, identifications must be verified using a second method of analysis in addition to HPLC. These identifications will also include the group of modified anthocyanins that eluted after 20 minutes. By verifying the composition of acylated anthocyanins, the anthocyanin profiles of these cultivars can be fully defined and the effects of this viticultural treatment on total anthocyanins can be re-evaluated. Following the verification of identities, the effects on groups of anthocyanins can be compared such as monoglucosides and diglucosides or dihydroxylated anthocyanins vs trihydroxylated anthocyanins.

Further research can clarify how changes in concentrations of individual anthocyanins affect hue angle or intensity of the resulting juice, and if these changes carry over into the resulting wine. It is necessary to understand how much of a change in the concentration of each individual anthocyanin is needed to have an effect on visible wine color, and how these changes are perceived by consumers or trained wine critics. Depending on the cultivar, the color challenges with hybrid winemaking vary; some are too vibrant, while others are not vibrant enough, and some have too much of a blue, pink, or purple hue. All of these examples are anecdotal, and it is important to verify empirically what color challenge hybrid winemaking faces in order to find solutions.

Other existing challenges faced in hybrid winemaking, such as acidity control, phenolic extraction, and occasional varietal off-aromas are other areas of research that need to be studied.

In addition, it is critical to optimize the market for interspecific hybrid wines. Hybrid cultivars are critical for their disease resistance and cold hardiness but maintain a lower-quality

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reputation in the market. By adjusting the marketing of these wines and optimizing the production quality, interspecific hybrid cultivars can enhance the wine industry in regions less suitable to V. vinifera production.

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