In the proposal for the 2012, we outlined three objectives: 1. Compare the rate of anthocyanin degradation in warm climate grapes vs cool climate grapes 2. Determine if other phenolic compounds also experience degradation in warm climates 3. Identify by-products of degradation and potential mechanisms for their formation We have continued to make progress on developing the methods needed to make the desired measurements and those results are outlined in the attached proposal. We have clarified the accumulation of anthocyanins in Cabernet Sauvignon over two vintages and believe we have discovered a hidden pool of metabolites in the phenolic synthesis pathway in Vitis vinifera.
Towards objectives 1 and 2, we have collected all the grape samples from two vineyards, one at the UC Davis Oakville vineyard and the other at the UC Kearney Agricultural Center between July and October 2012. We have cluster temperature data from each site, collected hourly. Six berries were collected from 3 vines treated with tracer and 2 control vines at each site approximately 8 times during the growing season. The berries will be extracted during February, and student assistants are currently being trained to undertake this exercise. We extract the skin of each berry separately to improve the statistical significance of our data. We have the instrumentation ready to analyze the extracts once they are prepared. Towards objective 3, we have collected data from berries incubated in the lab, at 25?C versus 45?C, with high levels of the 13C6 phenylalanine (Phe) with an M+6 mass. This provided anthocyanin metabolites with approximately 35%containing the M+6 label. This allows for facile detection of the anthocyanins and other metabolites. Berry skins from the 8 biological replicate pairs of samples were all extracted and the extracts analyzed by Chip-LC-MS, using a QToF detector. This detector has a very high resolution and mass accuracy, so that detected substances can be identified with more certainty.
The large and complex dataset was transferred to Austrian collaborators we identified at the Grape Research Coordination Network meeting. Using this technique, lists of potentially labeled molecular features were found in positive and negative mode analyses. Of these candidate ions, 42 positive and 11 negative molecular features were found to be statistically different between 25°C and 45°C treatments. Only a select few molecular features were found to be significantly higher in the 45°C treatment; these compounds present our best candidates for the degradation products of anthocyanins. From the list of molecular features believed to be derived from Phe13 metabolism, a pair-comparison t-test was performed over the 8 biological replicates to determine significant differences in the concentrations of labeled features. As expected, many phenolic compounds are susceptible to degradation under high temperatures. At least one of each anthocyanidin moiety appeared to be degraded under 45?C temperatures, although every single anthocyanin did not vary significantly between treatments.
Most interestingly, 3 features were found to be in greater quantity in the 45?C than in 25?C grape treatments. These features had m/z values of 433.113, 347.076, and 585.170. The molecular feature of mass 433.113 was tentatively identified as a benzyl alcohol dihexose. A similar molecular feature was found when researchers probed the degradation of anthocyanins in flower petals (Bar-Akiva et al. 2010). The feature with m/z 347.076 was tentatively identified as a syringetin aglycone. Syringetin, while present in grapes at low levels, is most likely an artifact of malvidin-3-glycoside created during ESI, since their molecular structures only vary in the oxidation states of the flavonoid ?C? ring and their retention times are nearly identical. This could easily be caused in conditions in which the pH is above 2, when anthocyanins undergo structural transformations. The final molecular feature identified 585.170 has not yet been identified, although if the molecular feature represents a product of anthocyanin degradation, it would have to be an adduct of some kind since the m/z is higher than many of the degrading anthocyanins. We were expecting more compounds that would increase at higher temps, but have found background noise is limiting our sensitivity, most likely due to the type of LC separation used (Chip-LC). At the present time, we are trying various methods to reduce that noise, through complicated data analyses, in hopes that the signals of the temperature sensitive and rising metabolites will be more evident.