A. Seed color is significantly browned by treatment conditions
A color index (CI) was calculated to link all the three colorimeter parameters and better visualize the change in seed color during natural ripening and in response to treatments. Natural ripening led to significant seed browning (decrease in the index) in both PN and CS, however, the change in CI was over 7-times greater in CS than PN (Table 1). This might be linked to higher concentrations of phenolic compounds in PN seeds compared to other vinifera cultivars (Revilla, Alonso, & Kovac, 1997). This result may also reflect the larger size of PN seeds compared to other vinifera cultivars, and therefore, a difference between PN and CS in the extractability of phenolic compounds and their susceptibility to oxidation (Mattivi et al., 2009). Finally, PN berries were slightly less ripe (-1.5 °Brix) than CS despite harvesting fruit based on identical maturity in the field, which could influence the impact of natural ripening (Supplemental Table 1).
The incubation period impacted both cultivars significantly (Figure 1, Supplementary Figure 1). However, for each cultivar and sampling time, the point at which treatments changed from ‘time 0’ was different (Supplemental Figure 1). Seeds from all treatments in both cultivars browned significantly after 3 h at veraison. Meanwhile at harvest, seeds required 24 h and 6 h for PN and CS, respectively. For this reason, all three time points (3 h, 6 h, 24 h) were included in Table 1, which reveals a significant effect of the incubation time on seed color in both cultivars. Similar browning between cultivars during seed incubation (without the presence of pulp and skin tissues) suggests that the differences observed during natural ripening between PN and CS were attributed to physical components of the seeds.
In both PN and CS, freezing treatments induced an immediate browning of the seed color that lasted until 6 h (Figure 1, Supplemental Figure 1). While some effects were observed at veraison, freezing had a more significant impact on seeds at harvest. This confirms our previous work which reported a significant CI change after 3 h post-freezing (Rustioni et al., 2018; VanderWeide et al., 2020). The cause for this greater effect on lignified seeds is intriguing, as lignification reduces the susceptibility of plant cells to freezing damage (Sano et al., 2016).
The effect of heating was impactful at veraison, with T20I40 having the darkest seeds in both cultivars after 24 h (Table 1, Figure 1). In contrast, harvest samples were unaffected by heating, contributing to the non-significant effect of heating on color when both timings are considered (Table 1). As seeds ripen, the inner-most cell layers of the outer integument lignify, making the seed less penetrable (Ristic & Iland, 2005). This process diminishes the conductance of heat through these cell layers, as is important during seed dormancy (Sano et al., 2016).
B. Natural ripening and artificial ripening oppositely influence the seed flavan-3-ol profile by the gallyolation pattern of compounds
Among variables in this experiment, cultivar accounted for the largest variability in the seed extract flavan-3-ol profile (Figure 2). The majority of compounds influenced by cultivar were non-galloylated, while galloylated flavan-3-ols explained little variance (Figure 2, Figure 3A, B). This may be because non-galloylated flavan-3-ols, such as (+)-catechin and (-)-epicatechin, are represented in the highest concentrations in grape seeds and vary greatly between cultivars (Mattivi et al., 2009). Likewise, Núñez et al., (2006) reported that non-galloylated flavan-3-ols are negatively correlated with mDP (differ greatly by berry maturity). This suggests that the substantial oxidation and polymerization experienced by these compounds during fruit ripening may also influence their cultivar-dependent concentrations (Kennedy et al., 2000). Meanwhile, alterations in mono-galloylated flavan-3-ols are thought to be more season-dependent (Núñez et al., 2006).
In contrast, natural ripening and freezing treatment induced a larger change to galloylated compounds compared to non-galloylated ones (Figure 3, Supplementary Tables 3, 4). With respect to natural ripening, the compound with the greatest relative decrease was epicatechin-3-O-gallate, a result observed previously (Ferrer-Gallego, García-Marino, Miguel Hernández-Hierro, Rivas-Gonzalo, & Escribano-Bailón, 2010; Kennedy et al., 2000). Additionally, there was a less visible effect on non-galloylated compounds. This led to both having a significant impact on the ratio of galloylated to non-galloylated flavan-3-ols (Table 2). Galloylated flavan-3-ols are formed by a condensation reaction using gallic acid (Watrelot & Norton, 2020). This structure provides three additional hydroxyl groups capable of donating electrons. Galloylated flavan-3-ols have a higher free radical quenching capacity (DPPH) compared to non-galloylated ones, such as catechin and epicatechin (Colon & Nerín, 2016; Roy et al., 2010). However, the oxygen radical absorbance capacity (ORAC) assay revealed the opposite trend (Ishimoto et al., 2012; Roy et al., 2010). This difference in antioxidant results may lie in the enhanced ability of galloylated flavan-3-ols to penetrate lipid bilayers due to their more lipophilic nature (Karas, Ulrichová, & Valentová, 2017; Narukawa et al., 2010). Our previous work showed that freezing disrupts cell membranes and walls (VanderWeide et al., 2020), suggesting that this may allow galloylated compounds to become exposed to oxygen. Additionally, flavan-3-ols with galloyl groups were less stable at neutral pH (Narukawa et al., 2010). More recently, Kim et al., (2020) revealed that phenolics with a pyrogallol group were highly oxidizable under a pH=10. This suggests that seeds out of wine-like (low pH) solution may promote oxidation of galloylated phenolics.
Heating treatment had an inconsistent effect on individual flavan-3-ol compounds. B-type and C-type procyanidins and total procyanidins were significantly increased and decreased in PN and CS, respectively. This led to no significant effect on the ratio of galloylated to non-galloylated flavan-3-ols extracted from seeds in either cultivar (Table 2).
C. Relationships between seed color and flavan-3-ols composition.
The Pearson’s correlation coefficients between the seed color index and seed flavan-3-ol groups (galloylated, non-galloylated, and their ratio) are displayed in Table 3. When the data from both cultivars are considered together, all flavan-3-ol groups are correlated strongly with the color index (CI), reflective of the relationship between seed phenolic concentration and color (Rustioni & Failla, 2016). However, when examined on a cultivar-dependent basis, these correlations were only completely upheld in CS (Table 3). In PN, the relationship between the CI and total flavan-3-ols, galloylated flavan-3-ols, and the ratio of galloylated flavan-3-ols to non-galloylated compounds were significant. But there was no relationship between CI and non-galloylated flavan-3-ols. This suggests that despite the large decrease in non-galloylated flavan-3-ols during natural ripening, that galloylated flavan-3-ols may play a large role in seed color determination in proportion to their concentration. Given the location of compounds largely comprising the non-galloylated fraction in vacuoles of cells, it may be that differences in extractability of compounds may be influencing this difference between cultivars (Mattivi et al., 2009). Future works will seek to establish whether the structural characteristics of seeds influence seed coloration during ripening and in response to artificial ripening treatments.
D. Implications of artificial ripening on grape and wine quality
Grape growers often utilize seed color and organoleptic properties as a basis for harvest maturity of fruits. This study reveals that seed color may not a be a sufficient marker alone to determine grape ripeness due to the large variability among cultivars in the concentration of compounds related to both of these parameters (Rustioni & Failla, 2016). It is well established that galloylated flavan-3-ols are a source of astringency in red grapes wines (Ćurko et al., 2014; Gombau et al., 2020; Narukawa et al., 2010). Flavan-3-ol monomers are also well known to impact bitterness in wine (Sáenz-Navajas et al., 2015). The results from this study provide some implications for wine quality based on these facts. Wines made with seeds subjected to freezing treatment should be less bitter and astringent due to the significant decrease in galloylated phenolics, which are also understood to more strongly interact with bitter taste receptors due to their lipophilic nature (Narukawa et al., 2010). However, freezing may in turn promote oxidative polymerization of procyanidins; a process known to enhance wine astringency (Vidal et al., 2003). The degree of procyanidin polymerization and galloylation were not analyzed in this study but will be a focus of future works. Likewise, given the potential differences in treatment effectiveness in must or wine solution (Oszmianski, Romeyer, Sapis, & Macheix, 1986), future works will consider this aspect to truly understand whether this technique could positively influence red wine quality.