Introduction

Tannins and anthocyanins are phenolic compounds that are essential to maintain wine quality, because they contribute astringency and colour to the finished wine. Condensed tannins are oligomers (3 to 9 units) and polymers (≥ 10 units) of flavan-3-ols, including (–)-epicatechin (EC), (+)-catechin (C), (–)-epicatechin-3-O-gallate (ECG), and (–)-epigallocatechin (EGC) (Bordiga et al., 2011), which are biosynthesised in skin and seed tissues (Kennedy, 2008). Those flavan-3-ols are linked through C4-C8 or C4-C6 interflavan bonds, and the number of subunits corresponds to the mean Degree of Polymerisation (mDP) of tannins. Tannins are responsible for texture and mouthfeel properties in wine, such as astringency, (Casassa & Harbertson, 2014), due to their ability to interact, form complexes, and aggregate with salivary proteins, thus decreasing lubrication and resulting in a mouth-drying or puckering sensation (Watrelot et al., 2019). Anthocyanins are found in the skins, and sometimes flesh, of fruits and petals of flowers (Kennedy, 2008; Lattanzio et al., 2012). Anthocyanins are present in several forms including malvidin-3-O-glucoside and malvidin-3,5-O-diglucoside. During the process of red winemaking, anthocyanins are extracted from grape skins and flesh, react with oxygen and other phenolic compounds, and are responsible for the colour intensity, stability, and hue of red wines.

Red wines made from interspecific cold-hardy (i.e., non-Vitis vinifera) grape cultivars have lower tannin concentration than red wines made from V. vinifera cultivars (Springer & Sacks, 2014; Watrelot & Norton, 2020). Examples of these cultivars include Marquette, Frontenac, Crimson Pearl, and Marechal Foch, which are grown in regions with winter temperatures too cold for V. vinifera to survive, such as the U.S. midwest and southeastern Canada. In particular, Marquette is one of the most widely planted cold-hardy cultivars. Additionally, interspecific grape cultivars are being considered worldwide as a potential mitigation for climate-related stressors on wine grape production (Santos et al., 2020). Low tannin concentration leads to low red wine quality indicators, such as high prevalence of oxidative aromas, low astringency, and poor colour stability (Watrelot & Norton, 2020). However, the final phenolics concentration in red wines depends on many factors; these include their initial content in grapes, which is influenced by viticultural practices, and their extractability during winemaking (Manns et al., 2013; Springer & Sacks, 2014). Various viticultural practices have been used to manage the phenolic content in grapes and thereby impact wine quality. Two practices that have shown promise on V. vinifera are nitrogen fertilisation (Delgado et al., 2004; Portu, et al., 2015a; Portu, et al., 2015b; Portu, et al., 2017a; Portu, et al., 2017b) and the application of plant growth regulators (LoGiudice et al., 2004; Mhetre et al., 2021; Portu, et al., 2017a; Thomidis et al., 2018; Vaquero-Fernandez et al., 2009).

Nitrogen fertilisation is widely used in agriculture, because nitrogen is an essential nutrient for plant growth (Metay et al., 2015). More specifically, vine nitrogen status is important for wine quality as it affects the amount of yeast assimilable nitrogen (YAN) in grapes, and adequate YAN (> 140 mg N/L) is necessary for yeasts during alcoholic fermentation to avoid the formation of reductive aromas (Bell & Henschke, 2005). Typically, nitrogen supplementation is applied to the soil, and various forms of nitrogen have been used (e.g., ammonium nitrate, urea) as a fertiliser on grapevines to modify the content of phenolic compounds with mixed results (Delgado et al., 2004; Hui et al., 2021). For example, Delgado et al. (2004) reported higher anthocyanin concentrations and increased tannin degree of polymerisation after fertilisation with nitrogen and potassium; however, astringency was not affected despite larger tannins. Hui et al. (2021) found an increase in the content of total anthocyanins, total flavanols, and total flavonols after spray treatment with urea. Alternately, nitrogen can be applied as a foliar spray with advantages over soil application including improved usage by the vine (up to 70 %), faster incorporation into plant tissues (one to two days vs three to four days), and reduced fertiliser waste and environmental risk (Cheng et al., 2021). Phenylalanine (Phe) is another source of nitrogen that has been used to target phenolics content, because Phe is a precursor for the biosynthesis of flavonoids via the phenylpropanoid (shikimate) pathway (Broeckling et al., 2012). In one study, an increase in total anthocyanins in V. vinifera cv. Tempranillo grape extracts were reported after Phe was applied (1.5 kg N/ha) to the vine canopy at véraison and one week after véraison (Portu, et al., 2015b). On the other hand, Phe applied at a lower rate (0.9 kg N/ha) resulted in no significant effect on phenolic content of whole grape extracts from Tempranillo vines (Portu, et al., 2017b); however, the two studies were conducted under different climatic conditions, and in different soils and vintages, which may have limited the effectiveness of the lower dose. Despite conflicting results of Phe effectiveness on phenolic content in grape extracts, the same group found Phe treatment increased tannin content by 30 % in Tempranillo wines over the control wines (Portu, et al., 2015a). Therefore, the use of Phe has potential to change the phenolic content in grapes and wines, which could benefit red wine quality when using cold-hardy grape cultivars initially low in tannin content.

Plant growth regulators, which are chemicals used to affect metabolic or developmental processes of plants (Rademacher, 2016; Ministry of Agriculture, 2022), have also been used to improve crop performance. Prohexadione calcium (ProCa, 3-oxido-4-propionyl-5-oxo-3-cyclohexene-carboxylate) is an industrially synthesised plant growth regulator with low toxicity, which has previously been shown to modify the sugar-acid balance and enhance aroma, thus improving the quality of fruit (Li et al., 2024). ProCa has led to an increase in phenolic compounds concentrations in V. vinifera cv. Xinomavro (Thomidis et al., 2018), which was attributed to a reduction in berry size due to the effects of ProCa as a late-stage gibberellin biosynthesis inhibitor that restrains cell expansion by limiting biosynthesis of the active forms of gibberellic acid (Rademacher, 2016). In the study conducted using Xinomavro, application of ProCa (250 mg/L, 1000 L/ha) resulted in higher concentrations of anthocyanins in grape skins and total phenolics in skins and seeds (Thomidis et al., 2018). Wines made from those ProCa treated vines also showed a higher tannin concentration, total phenolics, and colour intensity than control wines (Thomidis et al., 2018). Other studies have reported similar increases in colour intensity, total anthocyanins, and total phenolic compounds in juice and wine following ProCa treatment on various V. vinifera cultivars (Avizcuri-Inac et al., 2013; Lal et al., 2022; LoGiudice et al., 2004; Mhetre et al., 2021; Reinehr et al., 2021; Vaquero-Fernandez et al., 2009). Although treatment with Phe and ProCa did not show an effect on phenolic compounds in the skins and seeds of interspecific cold-hardy cultivar Marquette (Gapinski et al., 2024), extraction of phenolics was conducted with a non-wine matrix in a short time. For the current study, we wanted to evaluate the treatments during winemaking to understand if the changing fermentation matrix (i.e., change in ethanol concentration) disrupted binding/association between phenolics and cell wall material, and thus extractability of phenolics, as previously published by Bindon et al. (2014). Therefore, it was hypothesised that application of ProCa on interspecific cold-hardy cultivar Marquette grapevines would lead to similar outcomes, notably increased tannin concentrations in wines. The goal of this study was to evaluate the tannin and iron-reactive phenolics contents in grape juice and wine at bottling and after ageing after the application of Phe and ProCa on Marquette grapevines in the vineyard.

Materials and methods

1. Chemicals

Ammonium dihydrogen phosphate, potassium metabisulfite (KMBS), sodium dodecyl sulphate (SDS), acetone, acetonitrile (HPLC grade), D-fructose, ethyl alcohol (200 proof), ferric chloride, hydrochloric acid (37.5 %), methanol (HPLC grade), and ortho-phosphoric acid (extra pure, 85 % solution in water) were manufactured by Fisher Scientific (Fair Lawn, NJ). Triethanolamine (TEA) was purchased from Aqua Solutions (Deer Park, TX). Sodium hydroxide (0.1 N) was purchased from Lab Chem (Zelienople, PA). (–)-Epicatechin (purity ≥ 90 %), (+)-catechin hydrate (purity ≥ 98 %), glucose, L-tartaric acid, ammonium sulphate, and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (St. Louis, MO). Malvidin-3-O-glucoside (M3G) and malvidin-3,5-O-diglucoside (M35DG) were manufactured by Extrasynthese (Genay Cedex, France). Apogee® (27.5 % (w/w) prohexadione calcium) was purchased from BASF (Ontario, Canada). Induce®was purchased from Helena Agri-Enterprises (Collierville, TN, USA).

2. Vineyard treatments

The details of the vineyard treatments were as previously published (Gapinski et al., 2024). Briefly, cold-hardy interspecific (hybrid) Vitis spp. cv. Marquette grapevines were planted in 2011 at the Iowa State University (ISU) Horticulture Research Station in Ames, IA, USA (42° 06' N 93° 35' W, 300 m elevation, plant hardiness zone 5a, Clarion loam soil). The vines were trained on a single high-wire trellis system with north-south row orientation with 2.44 m between vines and 3.05 m between rows. Treatments were assigned to three-vine panels following a split-plot experimental design (Figure S1). The first and last panels in each row (i.e., panels 1 and 12) and all of row 3 were not used to assign treatments. Twenty-four of the remaining thirty panels were selected based on homogenous pruning weights and lack of dead vines and then were randomly assigned to four treatments with six replicates.

The treatments were as follows: phenylalanine (Phe), prohexadione calcium (ProCa), both prohexadione calcium and phenylalanine (ProCa+Phe), and a water-treated control (CTL), as previously published (Gapinski et al., 2024). Both Phe and ProCa were mixed with Induce® (0.6 mL/L) and ammonium sulphate (1.0 g/L) for foliar application on both sides of the vine canopy until the point of runoff using a 15 L backpack sprayer with a calibrated rate equivalent to 935 L per hectare (Solo Inc., Newport News, VA). Phe was applied at a rate of 100 mg/L at véraison and two weeks after the start of véraison. ProCa (50 mg/L) was applied every two weeks starting one week post bloom until two weeks after the start of véraison for a total of five treatments. ProCa was sprayed first on days when both treatments were applied. Cumulative rainfall between 1 April and harvest was 543 mm in 2022 and 472 mm in 2023. For the same time period, the average low temperature was 12 °C for both years, while the average high temperature was 25 °C in 2022 and 26 °C in 2023 (Gapinski et al., 2024).

3. Preparation of juices samples

Grape clusters were sampled at five phenological time points (one week post fruit set (PFS), three weeks PFS, véraison, mid-ripening, and harvest) in 2022 and in 2023 (Table S1), and transported to the laboratory in a cooler with ice packs within one hour of collection. Nine clusters per treatment (one cluster per vine) were collected randomly from different parts of the vines and pooled for analysis. At harvest, each three-vine panel was harvested separately. The yield for each panel was recorded and six clusters per panel were randomly sampled for basic chemical analysis. One hundred berries were randomly sampled from the nine clusters per treatment gathered at each time point throughout development and ripening (once per treatment), and from the six clusters per panel sampled at harvest (once for each panel, three per treatment). The juice obtained from the 100-berry samples was crushed by hand then chemically analysed.

4. Winemaking

Grapes were harvested on 26 August 2022 and 31 August 2023, sprayed with a 30 mg/L sulphur dioxide (SO₂) as a KMBS solution, and transported from the ISU Horticulture Research Station to the ISU winery for processing the same day. Each panel was processed separately. However, in 2022, one panel (M-ProCa-MW) yielded only bare stems and two other panels (M-Phe-NMW and M-CTL-NMW) did not yield enough for winemaking; therefore the clusters were added to the other panels of the same condition. Twenty-one wines were made in 2022, and 24 wines were made in 2023 according to the following procedure. Crushed and destemmed berries were placed into two-gallon (7.6 L) buckets. The median must weight was 14.1 kg in 2022 and 14.7 kg in 2023. The must was inoculated with Lalvin ICV D254 yeast (Scott Laboratories, Petaluma, CA, USA) with GoFerm™ yeast rehydration nutrient (Scott Laboratories, Petaluma, CA, USA) according to supplier instructions. Each fermentation was punched down twice per day after inoculation and throughout alcoholic fermentation. After 48 hours, the musts were co-inoculated with Lalvin VP41 bacteria (Scott Laboratories, Petaluma, CA, USA). Temperature, total soluble solids, and density (portable density meter DMA 35, Anton Paar, Ashland, VA, USA) were measured after the second punch down every day to monitor fermentation progress.

The wines were pressed at the completion of fermentation into one-gallon (3.8 L) jugs with an airlock and kept at room temperature (20 °C). Once malolactic fermentation was complete and checked by HPLC-DAD/RID (Gapinski et al., 2023), the wines were racked into half-gallon (1.9 L) jugs and 0.5 mg/L molecular SO₂ was added (as a 6.6 % (w/v) solution KMBS) according to each wine’s pH. Then, after flushing with argon, the wines were cold stabilised at 4.0 °C for four weeks. A volume of 6.6 % (w/v) KMBS solution was added at bottling to a target of 0.8 mg/L molecular SO₂. The wines were bottled in 750 mL green bottles, flushed with argon, and closed with cork #9. Samples of wines were collected for chemical analysis at bottling, and after five months of ageing.

5. Basic chemical parameters of juice and wine

Soluble solids (digital refractometer RF153, FLIR Commercial Systems Inc., Nashua, NH, USA), pH (Orion Star™ A211 Benchtop pH Meter, Thermo Fisher Scientific, Waltham, MA, USA), and titratable acidity (TA) were measured in juices and wines at all time points throughout winemaking and after ageing. Titratable acidity was determined by titrating 5 mL of sample with 0.1 N sodium hydroxide to an endpoint of pH 8.20 and expressed in tartaric acid equivalents. Two-mL aliquots were centrifuged (5 min at 11000 × g) prior to colour intensity and hue measurement in a 1 mm quartz cuvette with a Genesys 150 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) using VISIONlite Wine Analysis software (ascanis OHG, Ueberlingen, Germany). The software automatically scaled the results to a 1 cm path length after scanning the sample 380 to 780 nm at 5 nm intervals. The colour intensity and hue were evaluated in analytical triplicate for all juices during development and ripening, and in biological triplicate in juice at harvest and in all wines. An Agilent Hi-Plex H (7.7 × 300 mm, 8 µm) column protected with an H-plex guard column at 60 °C (Agilent Technologies, Santa Clara, CA, USA) was used on a reversed-phase high-performance liquid chromatograph (RP-HPLC, 1260 series, Agilent Technologies, Santa Clara, CA, USA) with a diode array detector (DAD) and refractive index detector (RID) to check the levels of malic acid and lactic acid during malolactic fermentation and ethanol. The isocratic mobile phase was 5 mM sulfuric acid at a flow rate of 0.7 mL/min. A 20 µL sample previously filtered at 0.45 µm using a PTFE filter was injected for 30 min. Ethanol was detected with the RID (cell temperature of 55 °C) and the concentration was calculated using an external calibration curve (Gapinski et al., 2023).

6. Total iron-reactive phenolics concentration

The total iron-reactive phenolics (IRP) concentration was measured in juices and wines following the Harbertson-Adams Assay (Harbertson et al., 2002). Briefly, background absorbance at 510 nm of 75 µL of sample in 800 µL sodium dodecylsulphate/triethanolamine buffer was recorded after a 10 min incubation at room temperature. Then, 125 µL ferric chloride reagent was added, the solution was vortexed, left at room temperature to incubate for 10 min, and the final absorbance at 510 nm was recorded. A blank of the same buffer (875 µL) and ferric chloride (125 µL) solutions without sample were used to evaluate the absorbance values. A calibration curve of (+)-catechin was used to express total iron-reactive phenolics as (+)-catechin equivalents.

7. Tannin concentration

Tannin concentration was quantified in juices and wines following a previously published method using RP-HPLC-DAD (1260 Infinity II, Agilent Technologies, Santa Clara, CA, USA) (Gapinski et al., 2024). The polystyrene divinylbenzene column (PLRP-S, 2.1 × 50 mm, 100 Å, 3 µm, Agilent Technologies, Santa Clara, CA, USA) was protected with a guard column of the same material (PRP-1, 3 × 8 mm, Hamilton Co., Reno, NV, USA) and held at 30 °C. A linear gradient at a flow rate of 0.30 mL/min was used as follows: time in min (% mobile phase B), 0 (14), 12.6 (34), 12.6−13.3 (34), 15.1 (70), 15.1−16.8 (70), 19.6 (14), and 19.6−28.0 (14). Mobile phases were 1.5 % (w/w), 85 % ortho-phosphoric acid (mobile phase A), and 20 % (v/v) mobile phase A in acetonitrile (mobile phase B). Five µL of previously filtered (0.45 µm PTFE filter) sample were injected and tannin quantification was carried out by first drawing a baseline at 0 mAU across the whole chromatogram, then integrating the peak between 16.8 to 19.8 min. An external calibration curve of (–)-epicatechin was used to express tannin concentration as (–)-epicatechin equivalents.

8. Anthocyanin content

Anthocyanin content was determined in wine at bottling and after ageing using RP-HPLC-DAD (1260 Infinity II, Agilent Technologies, Santa Clara, CA, USA) with a LiChrospher column (100-5 RP18 250 mm × 4.0 mm, 5 μm, Agilent Technologies) at 40 °C. A previously published mobile phase gradient at a flow rate of 0.5 mL/min was used (Ritchey & Waterhouse, 1999). Mobile phases were A: 50 mM ammonium dihydrogen phosphate adjusted to pH 2.5 with ortho-phosphoric acid, B: 20 % A with 80 % acetonitrile, and C: 0.2 M ortho-phosphoric acid adjusted to pH 1.5 with sodium hydroxide. Twenty µL of previously filtered (0.45 µm PTFE filter) sample were injected and anthocyanins were monitored at 520 nm with M3G and M35DG as external calibration standards for mono- and di-glucoside, respectively.

9. Statistical analysis

Data collected on juices were analysed using a three-way analysis of variance (ANOVA) for an additive effects linear regression model with random intercepts, for which the lme function from the nlme package (Pinheiro & Bates, 2023) in R statistical software (RStudio version 4.4.0) was used. Treatments were assigned using a split-plot design including row as a blocking factor, half rows assigned to mowed (MW) or not mowed (NMW) as the whole plot, and individual panels assigned to treatments as the subplots (Figure S1). MW and NMW panels for the same treatment were averaged for analysis. Values for time points one week PFS to mid-ripening are means of MW and NMW analytical triplicates ± standard deviation, and values at harvest are means of MW and NMW biological triplicates ± standard deviation. Mowing treatment was included in the model as a physical application in the vineyard but was not part of the study. The effect heredity principle (Hamada & Wu, 1992) was employed to aid interpretation of significant effects. See supplementary data for the R code (Code S1).

Data collected on wines were analysed with JMP Pro 17.0 software (SAS Institute Inc., Cary, SC, USA) using two-way (ethanol) and three-way (pH, titratable acidity, hue, colour intensity, iron-reactive phenolics, tannin, and anthocyanins) analysis of variance (ANOVA) followed by Tukey’s HSD test with α = 0.05 for the effects of year, time point, and treatment.

Results and discussion

1. Basic chemical parameters of juices

During berry development and ripening, the soluble solids and pH decreased from one week PFS to three weeks PFS and then increased to a maximum at harvest in both years, as expected (Table 1). Year had a significant effect on soluble solids and pH at each time point except harvest (Table 2). Additionally, the effects of ProCa, Year2023:ProCa, Year2023:Phe, and Year2023:ProCa:Phe on soluble solids were significant at three weeks PFS while Year2023:ProCa and Year2023:ProCa:Phe were also significant at one week PFS and véraison, respectively. The pH of juice was affected by Year2023:ProCa:Phe at véraison, and Year2023:ProCa and Year2023:Phe at mid-ripening, which indicates that year had more of an effect on juice pH than treatment with ProCa or Phe. Titratable acidity (TA) increased from one week PFS to véraison, when it reached a maximum for all four treatments in both years, then decreased to a minimum at harvest (Table 1). Statistical analysis showed TA was affected by year at every time point throughout development and ripening with lower TA in 2023 than in 2022, which could be related to warmer pre-véraison temperatures in 2023 lessening organic acid content (Blank et al., 2019). The effect of Year2023:ProCa on TA was significant at three weeks PFS, and the effect of Year2023:Phe was significant at véraison and mid-ripening. The three-way interaction between year, ProCa, and Phe was significant at three weeks PFS and véraison (Table 2). The effect of year on hue was significant at mid-ripening but on colour intensity at mid-ripening and harvest. Hue and colour intensity of juice were minimally affected by treatment. However, the effect of Phe treatment on hue was significant at harvest with higher hue when Phe was applied, which indicates a higher degree of yellowness than redness. This is contrary to the hypothesis that adding Phe would increase the biosynthesis rate of flavonoids, and potentially leading to higher anthocyanin content, thus deeper-coloured (i.e., redder) juice.

Overall, application of Phe and ProCa exhibited limited effect on basic chemical properties of juices including hue and colour intensity as previously reported on Tempranillo juices after application of Phe on vines (Portu, et al., 2015a; Portu, et al., 2015b; Portu, et al., 2017b) and on Beauty Seedless juices after application of ProCa on vines (Mhetre et al., 2021). However, the TA of Beauty Seedless juices was higher when ProCa was applied at 200 mg/L but lower when applied at 400 mg/L, compared to the control. Additionally, a study conducted with cultivars Grenache and Tempranillo reported no change in soluble solids, pH, or TA after application of ProCa at pre-bloom (Avizcuri-Inac et al., 2013), while another study across two years, also with Tempranillo, observed higher juice TA concentrations after ProCa application than control (Vaquero-Fernandez et al., 2009). Another study applied ProCa to Cabernet-Sauvignon and Cabernet franc observed increased colour intensity, which the authors attributed to a reduction in berry weight (LoGiudice et al., 2004). Therefore, ProCa seems to have variable effect on basic chemical properties, especially TA, and may be dependent on dose and cultivar or the climate and soil in which it is applied.

2. Iron-reactive phenolics and tannin concentrations in juices

The concentration of iron-reactive phenolics (IRP) in juice was highest at one week PFS (Figure 1), which was affected by year with higher IRP concentration in 2023 than in 2022 (Table 3). This could also be caused by the differences in temperature and precipitation, since warmer temperatures and less rainfall occurred in 2023, similar to the findings of Blank et al. (2019). From one week PFS, IRP concentration decreased through harvest, and IRP concentration was lower in 2023 for each time point. Although the interaction effects of Year2023:ProCa, Year2023:Phe, and Year2023:ProCa:Phe were significant at various time points, individual ProCa and Phe treatments did not have any significant effects on juice IRP concentrations at any time point (Table 3), which means year had more of an effect than treatment at the current application rates.

Similar to juice IRP concentration, juice tannin concentration was highest at one week PFS (Figure 2). At three weeks PFS, the tannin concentration decreased by 10- to 20-fold compared to one week PFS. At véraison and later, the juice tannin concentration was below the limit of quantification (< 35 mg/L) for all the treatments (Figure 2). Year had a significant negative effect at every time point, except véraison (Table 3), with lower juice tannin concentration in 2023 than in 2022. Also similar to juice IRP concentration, the effects of Year2023:ProCa and Year2023:Phe were significant at various time points; however, individual treatments showed no effect on juice tannin concentration (Table 3).

The low doses of Phe and ProCa used in this study may explain why limited effects on IRP and tannin concentrations were observed in juices. However, short contact time between juice, skins, and seeds during juice sample preparation may also be a factor, as other studies reported limited changes to total polyphenol index, total anthocyanins, and total flavanols in juice from Tempranillo and Grenache cultivars at harvest after application of Phe (Portu, et al., 2015b; Portu, et al., 2017a). Contrary to the present study, ProCa application on vines has been shown to increase total phenols and anthocyanins concentrations in Cabernet-Sauvignon and Cabernet franc juices (LoGiudice et al., 2004), in Beauty Seedless juices (Mhetre et al., 2021), and in Grenache and Tempranillo juices (Avizcuri-Inac et al., 2013), with no impact on total tannin concentrations. These results could indicate that higher doses than five applications of 50 mg/L per year of ProCa are needed to improve extraction of phenolic compounds in Marquette juices. Additionally, research on apples and pears (Halbwirth et al., 2002; Roemmelt et al., 2003), hops (Humulus lupulus, Kavalier et al. (2014)), and tomato (Ghiasi & Razavi, 2013) have demonstrated changes in activities of enzymes involved in phenolics biosynthesis. Specifically, Halbwirth et al. (2002) reported ProCa blocking formation of common flavonoids by inhibiting flavanone-3-hydroxylase (FHT), which means that production of leucocyanidins, and thus flavan-3-ols, is decreased, despite enhanced dihydroflavonol reductase (DFR) activity. Roemmelt et al. (2003) found a similar inhibition of FHT leading to higher hydroxycinnamic acids and lower falvan-3-ols and flavonols. Complementarily, when ProCa was applied to hops, researchers found an increase in metabolic precursors upstream from flavanon-3-hydroxylase (F3H) and decreases in flavonoid products (catechin, epicatechin, and quercetin) downstream from F3H due to F3H inhibition by ProCa. However, those differences were only present 7 and 15 days after treatment, and concentrations of the same metabolites returned to that of the control 22 days following treatment (Kavalier et al., 2014). Additionally, Ghiasi and Razavi (2013) reported increased phenylalanine ammonia-lyase (PAL) activity but no difference in total phenols content in tomato treated post-harvest with ProCa.

3. Basic chemical parameters of wines

As expected, pH in wines increased from harvest to bottling, and pH did not change after ageing (Table 4). The effect of year was significant with higher pH at bottling in 2022 (3.34 to 3.47) than in 2023 (3.23 to 3.29). A decrease in TA was observed between harvest and bottling, and TA was significantly affected by time point, year*time point, and year*time point*treatment. This indicates the winemaking process (e.g., cold stabilisation) changed TA more than treatment with ProCa or Phe, at least at the current doses. Ethanol concentration was significantly higher in 2023; however, as mentioned previously, soluble solids were not affected by year at harvest. It is likely that the difference in ethanol concentration was due to a one-day-longer fermentation in 2023 than 2022. Hue and colour intensity were affected by time point and year*time point, with higher hue and lower colour intensity after ageing than at bottling (Table 4). However, treatment had no effect, similar to previous reports considering Phe applications (Portu, et al., 2015a).

4. Iron-reactive phenolics, tannins, and anthocyanins concentrations in wines

Year had a significant effect on wine IRP and tannin concentrations (Table 5), which were higher in 2022 than in 2023 (Figure 3). The effect of time point was also significant with the highest IRP and tannin concentrations after ageing. The wines made from ProCa treated vines had the highest IRP and tannin concentrations, while the ProCa+Phe wines had the lowest concentrations across both years. Interestingly, the IRP concentration – averaged over year and time point – in the CTL wines was not different from either the Phe or ProCa treatments, but it was significantly higher than the ProCa+Phe treatment. Additionally, ProCa wines (72 mg/L) had significantly higher tannin concentration than ProCa+Phe wines (63 mg/L), but no treatment (including Phe, 68 mg/L) was significantly different from the CTL (67 mg/L).

Total anthocyanins were higher at bottling (347 mg/L) than after ageing (230 mg/L), with overall higher concentration in 2023 (312 mg/L) than 2022 (265 mg/L, Figure 4). Coupled with the changes observed for hue and colour intensity, the decrease in anthocyanins with ageing indicates formation of polymeric pigments over time (Picariello et al., 2017). Interestingly, the effect of treatment on total anthocyanins was significant (Table 6) with the highest concentration present in the CTL wines (308 mg/L), followed by Phe, ProCa, and ProCa+Phe (292, 287, and 267 mg/L, respectively, Figure 4). However, only the CTL and ProCa+Phe treatments were significantly different from each other. It is possible that there is an antagonistic effect between ProCa and Phe when both compounds are applied, similar to what has been previously proposed between Phe and methyl jasmonate (Portu, et al., 2017a). However, questions remain about exactly what that interaction may be. It is possible that the effects ProCa has on the phenylpropanoid pathway enzymes inhibited the ability for the added Phe to be converted to phenylpropanoid end products.

Previous work has shown limited or negative influence of foliar-applied nitrogen on wine phenolics concentrations when vine nitrogen status is within normal ranges and YAN is above threshold values (> 140 mg N/L), as they were in this study (Gapinski et al., 2024). For example, Cheng et al. (2021) concluded that foliar nitrogen supplementation is only beneficial in nitrogen-deficient vineyards, while Gutiérrez-Gamboa et al. (2017) found that flavonoid concentration in Cabernet-Sauvignon wines decreased, because YAN was sufficient. The short half-life of ProCa in plants (a few weeks) may have limited effectiveness at influencing phenolics concentration in grape berries by harvest, and thus the finished wines, since the final treatment of ProCa occurred about one month before harvest (Kavalier et al., 2014). Further, ProCa is a gibberellin inhibitor, which limits cell expansion, and may have affected cell wall porosity by thickening cell walls, thus limiting phenolics extraction. A previous study by Bindon et al. (2014) described changes in tannin content in grape skin extracts attributed to the degradation of the middle lamella (thus increasing surface area for tannin binding) as berries matured. Although the current dose of ProCa may not have been high enough to show an effect, the cell wall material in cold-hardy, hybrid grape cultivars may already be thicker than V. vinifera cultivars, resulting in lower tannin extractability, and the changes observed here could be due to different weather patterns between years leading to changes in cell wall material composition. Alternately or in combination, disruptions in enzyme activities for the normal phenylpropanoid pathway ending in flavan-3-ols by ProCa (as described above for juice) may have led to the formation of 3-deoxyflavans instead (Kavalier et al., 2014; Roemmelt et al., 2003).

Conclusion

Year and time point (both phenological and winemaking) affected basic chemical parameters of juices and wines more than treatments with Phe and/or ProCa. The same was observed for IRP and tannin concentrations. Treatment effectiveness may have been limited by low application rate (dose) or by inhibitory effects of ProCa on phenylpropanoid pathway enzymes. Although ProCa was hypothesised to act on the biosynthesis pathway, no effects were observed; therefore, interspecific cultivars such as Marquette may have some other way of regulating the pathway due to a genome that includes V. riparia heritage that differs from V. vinifera. More work is needed to better understand the effect of ProCa on those enzymes in interspecific cold-hardy grape cultivars, which would lead to insights on how to best use ProCa to modify phenolics content in those cultivars and the resulting wines. Future work will also include the use of abscisic acid as this plant hormone plays a significant role in plant stress response, which could impact biosynthesis of stress-mitigating phenolic compounds.

Acknowledgements

The authors acknowledge the contribution of Dr. Suzanne Slack, Olivia Meyer, Brooke Dietsch, and Jerimiah Johnson from the Department of Horticulture for their assistance with treatment application and mowing. Thank you to Reid-Vincent (Vinny) Paris from the Iowa State University Department of Statistics for assistance with checking statistical inferences and R code for analysis and graphics generation. We thank Nick Howell and Brandon Carpenter from the Iowa State University Horticulture Research Station and Yiliang Cheng, David Carter, Carmen Vavra, and Julain Sinkler from the Department of Food Science and Human Nutrition for their help with harvesting and processing the grapes. This work is a product of Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, USA, project N^o IOW05690, which is sponsored State of Iowa funds.