Modeling Impacts of Viticultural and Environmental Factors on 3-Isobutyl-2-Methoxypyrazine in Cabernet franc Grapes

Discussion

Grapes and wine produced in cool regions and cool years are reported to have higher IBMP concentrations (Allen et al. 1991, Falcão et al. 2007, Lacey et al. 1991), but we did not observe this phenomenon. In 2008 and 2009, the average growing degree accumulation (°C) from 1 May to 50 DAA across sites was 894 and 799, respectively, and IBMP concentrations at 50 DAA were significantly higher (69%) at all sites in 2008. In concordance with other studies (Hashizume and Umeda 1996, Lacey et al. 1991), the decrease in IBMP from 50 DAA to harvest was lower in the cooler year (2009), but concentrations at harvest were still significantly lower in 2009 than 2008 at two sites and not significantly different at five sites as a result of the reduced accumulation. We constructed PLSR models, combining all sites and years to predict IBMP at 50 DAA and log-fold decrease in IBMP from 50 DAA to harvest (data not shown), and temperature was an important predictor in both models. Average temperature from anthesis to 50 DAA positively correlated with IBMP concentration at 50 DAA, and average temperature from 50 DAA to harvest positively correlated with the log-fold decrease in IBMP, suggesting that both pre- and postveraison temperatures are important determinants of final IBMP concentrations. Earlier studies cited above have focused on temperature during the ripening period and quantified IBMP after the onset of degradation, but did not address the impact of temperature on IBMP accumulation.

There was significant variation within and among sites in IBMP concentrations at both preveraison time points and harvest, but the site-to-site variation was greater (Table 3). The PLSR models indicated that IBMP accumulation was predicted by variables associated with vine vigor. In general, vines with higher vigor reflected in greater shoot length and diameter, cane weight, and other associated variables accumulated more IBMP. The most frequently used variable in 50 DAA models was the berry carbon isotope ratio. The carbon isotope ratio (δ¹³C) of grape leaves and berries correlates with predawn leaf water potential and is an indicator of vine water status (de Souza et al. 2003). In response to stress (e.g., drought), the stomatal aperture decreases, resulting in depletion of ¹²C and less discrimination against the heavier carbon isotope, ¹³C, resulting in an increase in δ¹³C. Berry δ¹³C was a better predictor of IBMP concentration at 50 DAA than leaf δ¹³C. Interestingly, leaf δ¹³C had an opposite regression coefficient of berry δ¹³C in several of the models. Because we only sampled sun-exposed leaves from nodes 15, 16, and 17, it is likely that berry δ¹³C was a better representation of the whole canopy over berry development. In all but two of the models for IBMP at 50 DAA, berry δ¹³C was negatively correlated with IBMP. Thus, vines with higher discrimination (i.e., less water stress) tended to be more vigorous and have fruit with higher IBMP concentrations. However, in the multisite models, δ¹³C loaded heavily on a different latent variable than the variables associated with vine vigor. This discrepancy could potentially be explained by other variables that influence vigor, including bud number, nutrient availability, rootstock, and crop level.

It is not entirely clear why MP accumulation should be linked to temperature or to water availability. Several metabolic processes in grapes, such as photosynthesis, are known to increase with increasing temperature to a point before decreasing (Keller 2010). However, only a single enzyme family associated with MP biosynthesis has been identified: two O-methyltransferases (VvOMT1/2) responsible for methylation of hydroxypyrazine (HP) intermediates (Dunlevy et al. 2010). To our knowledge, the temperature dependence of VvOMT1/2 activity has not been characterized. A recent report has characterized another V. vinifera OMT responsible for methylation of flavonoids (FAOMT), which showed four-fold greater activity at 37°C than at 25°C and maximum activity at 50°C (Lücker et al. 2010). While this could potentially explain greater MP accumulation at high temperatures, it is possible that the other uncharacterized steps in hydroxypyrazine synthesis are also temperature sensitive.

Although variables associated with vine vigor explained the majority of variance in IBMP concentration at 50 DAA across and within sites in each year, vigor did not appear to correlate well with explaining differences in IBMP between years. In 2009, vines were significantly more vigorous, p < 0.05, (average cane weight across sites, 65.7 ± 26 g) than the previous year (average cane weight across sites, = 46.1 ± 22 g). The most noticeable difference between the two years was that 2008 had higher preveraison temperatures (p < 0.05, Supplemental Table 2).

In field studies, preveraison cluster light exposure can reduce IBMP accumulation (Marais et al. 1999, Ryona et al. 2008). One exception, a reduction in IBMP accumulation in the absence of light, has been reported (Hashizume and Samuta 1999), but this experiment was performed on harvested unripe berries, and extrapolating these results to clusters still on the vine may not be appropriate. Cluster light exposure was not a predictor in any of the multisite models for IBMP concentration at 50 DAA. Because canopy management practices (e.g., leaf removal, shoot thinning, shoot positioning, hedging) were imposed by growers at different phenological stages and differing intensities across sites, it is possible that we did not capture the true dynamics of exposure during the preveraison period. Even with these caveats, the lack of predictive ability of cluster light exposure in the multisite study is not surprising; the differences in IBMP between fully shaded and fully exposed fruit within a vineyard do not usually exceed a factor of two (Marais et al. 1999, Ryona et al. 2008, Scheiner et al. 2010). However, the variation in IBMP among and within regions, including in our current study, is reported to exceed an order of magnitude (Allen et al. 1994, Ryona et al. 2008). Furthermore, in five of the single-site models for IBMP at 50 DAA, cluster light exposure, reported as cluster exposure flux availability (CEFA), was negatively correlated with IBMP but loaded to the same latent variables as the x variables associated with vigor. Thus, in all but one instance where CEFA contributed with a negative loading to a single-site model of IBMP accumulation, it was coincident with a positive loading from vigor metrics. By comparison, in 9 of the 15 single-site models where vigor metrics had positive loadings, CEFA had no contribution to the model. As previously observed, in contrast to vigor, CEFA was not useful in modeling IBMP accumulation in the multisite models. That may indicate that vine vigor independent of cluster exposure can have a greater influence than cluster exposure on IBMP accumulation, but better controlled experiments would be necessary to be conclusive.

In general, IBMP concentrations in Cabernet franc followed the expected pattern over the growing season of preveraison accumulation followed by postveraison degradation. However, in 2009, there was a 72% decrease in IBMP from 30 DAA to 50 DAA at one site (site 8), in contrast to several reports that IBMP peaks in concentration ~0 to 14 days before veraison (Roujou de Boubée et al. 2002, Ryona et al. 2008) as well as all other sites in our current studies. Because the results were highly anomalous, we confirmed the numbers by rerunning the samples, starting with berries, another two times. We also verified that peak shape and qualifier ion ratios were unaffected, indicating that interferences were unlikely. At site 8, the 50 DAA samples were collected ~10 days before veraison, indicating that IBMP did decrease earlier than previously reported. The vines at site 8 were highly vigorous and thinned to less than one cluster per shoot (average yield/pruning weight, 1.18, average clusters/shoot, 0.6) at ~20 DAA, but we cannot establish that this practice led to an early decrease in IBMP. At harvest, Brix and TA were 21.6 and 7.3 g/L, values not indicative of advanced maturity.

In contrast to a previous report (Ryona et al. 2008), no correlation was observed between preveraison and harvest IBMP concentrations. In this previous work, the study was performed in a single region (Finger Lakes, NY), and soluble solids accumulation and the time elapsed between veraison and harvest were comparable among sites. It may be that preveraison IBMP is not a useful predictor of harvest IBMP across regions which differ in their rate of maturation. In comparison to IBMP accumulation, our attempts to model IBMP decrease across sites were less successful, as several x variables important to modeling IBMP decrease within a given site did not behave consistently from site to site. For example, variables associated with vine vigor were almost equally likely to have negative or positive correlations in our single-site models (Figure 3). Vine vigor measurements were taken before veraison, thus we cannot establish if they were an accurate representation of vine growth after veraison. However, all growers in our study reported using similar practices postveraison: no leaf removal was performed after veraison, but shoot tips were hedged at all sites. Therefore, practices should not have resulted in any systematic bias. We did observe that vines at some sites were still actively growing at harvest, and vine vigor has been reported to delay fruit maturity (Carbonneau 1997). Thus, the opposing relationship between these measurements and decrease in IBMP from site-to-site may have resulted from differences in fruit maturation as affected by vigor. In accordance, another group (Roujou de Boubée et al. 2000) tentatively correlated a decreased rate in IBMP degradation in Cabernet Sauvignon to late-season vine growth induced by high rainfall. It is also well established that overcropping can slow fruit maturation (Jackson and Lombard 1993). In 11 of the single-site models, measurements of crop to vine size (crop load, yield per shoot, clusters per shoot) were important predictors of IBMP decrease, but both positive and negative correlations were observed depending on the site. At three of the four sites where measurements of crop to vine size (crop load, yield per shoot, clusters per shoot) negatively correlated with log-fold decrease in IBMP, fruit maturity was positively correlated with log-fold decrease in IBMP, indicating that vines with higher crop to vine size had less mature fruit. Several groups have reported correlations between IBMP concentrations and fruit maturity indices such as Brix and malic acid (Chapman et al. 2004, Hashizume and Umeda 1996, Roujou de Boubée et al. 2002). Although neither IBMP at harvest nor the IBMP decrease could be predicted by Brix, pH, and TA across multiple sites, similar to previous observations (Ryona et al. 2008), within a site the log-fold decrease in IBMP positively correlated with these simple maturity indices in six of the single-site models (Figure 3), a significant result (p < 0.05, chi-squared test).

In the wines vinified from the 5-vine panels at each site (20 in 2008 and 15 in 2009), IBMP concentrations were at or above the reported sensory detection threshold, 15 pg/mL (Roujou de Boubée et al. 2000) in only a single wine (Figure 4). We have seen similar results in other recent studies; for example, in 2007, >15 pg/mL IBMP in only two of 13 sites in the Finger Lakes (Ryona et al. 2008). The percentage of wines with IBMP exceeding 15 pg/mL is comparable to the percentage reported for warmer climates (Allen et al. 1994, Chapman et al. 2004), even though MP concentrations are reportedly higher in cool regions.

The concentration of IBMP in grapes was correlated with the IBMP concentration in wines in the individual vine panel vinifications (R² = 0.76, p < 0.05, one-way ANOVA). IBMP concentrations in some wines were higher than the grapes from their corresponding panels (Supplemental Table 3) because the wines represent weighted averages of multiple vines. That also likely resulted in a weaker correlation coefficient than is reported elsewhere (Ryona et al. 2009).

The intensity of herbaceous aroma of the Cabernet franc wines was not linearly correlated with IBMP (p > 0.05 in both years) (Figure 4). While a correlation between IBMP and herbaceousness has been reported in wines from New Zealand and Australia (Allen et al. 1991) and France (Roujou de Boubée et al. 2000), these studies included some wines with >30 pg/mL IBMP, at least two-fold over its reported sensory threshold of 15 pg/mL (Roujou de Boubée et al. 2000). In our work, IBMP ranged from well below to just above the sensory detection threshold in red wine, suggesting that IBMP is not a useful proxy for herbaceousness in N.Y. Cabernet franc in the sites under study. Similar observations have been made for California Cabernet Sauvignon with comparable IBMP concentrations (Preston et al. 2008). Fruity aromas can mask the perception of herbaceous aromas, and vice versa (Hein et al. 2009), and we observed a similar inverse correlation between fruity and vegetal aromas in our work (Supplemental Figure 2). Thus, the difference in vegetal aroma intensity may relate to an absence of fruity aromas or the presence of other herbaceous odorants (e.g., C₆ alcohols), as wines produced from grapes not known to produce significant levels of IBMP can also have herbaceous aromas (Guinard and Cliff 1987). As a caveat, the wines from the two studies were stored under different conditions following bottling (9 months at 12°C for the 2008 vintage vs. 5 months at 21°C for the 2009 vintage), which could affect the relative rates of formation or loss of other odorants such as esters across the two years.

The intensity of herbaceous aroma was modeled using viticultural and climatic measurements, and the single most important predictor was the δ¹³C of mature berries. Related variables (shoot length and average cane weight) also negatively correlated with herbaceous aroma. Wines made from grapes with a lower vine water status and vigor indicated by greater δ¹³C, shorter shoot length, and smaller cane weights were rated as having the highest intensity of herbaceous aromas (Figure 5). This observation is surprising, considering that lower water availability and reduced vine growth have been associated with less herbaceous aromas and with improved red wine quality in general (Jackson and Lombard 1993). However, many of these previous studies investigated systems with a wide range of water availabilities, either by natural causes or cultural practices (i.e., deficit irrigation). Here, the range of δ¹³C values within a year was <2‰, less than reported elsewhere (de Souza et al. 2003). Additionally, all sites had δ¹³C < −26‰, likely indicating similarly high water availability. The weaker predictive ability of water status for wine quality in years with higher water availability has been previously reported (Van Leeuwen 2010). In 2008, herbaceousness was also inversely correlated with classic maturity indices (pH, TSS*pH²). The latter metric was originally suggested for use in predicting maturity in hot climates (Coombe et al. 1980). However, at least in one year, it appears to be appropriate for predicting IBMP degradation in a cool climate.