PROGRAMAS DE VIGILANCIA Y CONTROL DE ZOONOSIS EN LA PRODUCCIÓN PRIMARIA

More detailed analyses of the total flavonoids were carried out in potted berries from four CE experiments. These compounds were separated and analysed by HPLC (section 3.3.3). The main flavonols were also identified by LC-MS analysis as described previously (section 3.3.4).

Potted vines at 10 weeks post bud burst

Figure 5.3 shows the flavonoid composition in berries at 10 weeks post bud burst (2-3 weeks pre- veraison) during the experimental period. At 10 weeks post bud burst, the major flavonols in berries were quercetin-3-O-glucuronide (RT 15.7), quercetin-diglucoside (RT 9.2) and quercetin-3-O-glucoside (RT 14.8). Quercetin-3-O-glucuronide had the highest concentration in berries. The average concentration of quercetin-3-O-glucuronide maintained at 9.7 μg/g, which was over 100% and 500%, higher than quercetin-diglucoside and quercetin-3-O-glucoside, respectively. Additionally, a decreasing pattern was observed in quercetin-3-O-glucuronide in the control berries, with the concentration decreasing from 11.6 to 8.5 μg/g during the experimental period (p<0.05). Similarly, quercetin-diglucoside, the second highest flavonol in potted berries at 10 weeks post bud burst, significantly decreased from 5.8 to 3.4 μg/g in both the Control and UV-B treatments throughout time points measured (p<0.05).

When compared with the Control treatment, a significant UV-B induction was observed in major flavonols in the UV-B treatment (Figure 5.3b). This UV-B induction in flavonols came up after 3 days of UV-B exposure and reflected in the increases of quercetin-3-O-glucuronide, quercetin-3-O-glucoside and quercetin-3-O-rutinoside (RT 13.8). For example, quercetin-3-O-glucuronide was maintained at about 11.6 μg/g in berries from the beginning to 3 days of UV-B exposure. From the third day, the concentration showed a significant increase and reached 17.6 μg/g at the end of the 7-day UV-B exposure. Consistent UV-B induction was observed in quercetin-3-O-glucoside and quercetin-3-O- rutinoside in the UV-B treatment. The concentration of quercetin-3-O-glucoside significantly increased from 1.4 to 2.8 μg/g, while quercetin-3-O-rutinoside increased from 0.75 to 1.96 μg/g throughout the experimental period. However, quercetin-diglucoside, the second highest flavonol in berries at 10 weeks post bud burst, did not significantly affected by UV-B radiation.

Figure 5.3 The effects of UV-B radiation on flavonoid composition in Sauvignon blanc berries at 10 weeks post bud burst

Data shown are the average mean ± standard error of three replicates (n=3). *Significant difference comparing the Control treatment according to Two-way ANOVA test with time points and treatments as specifications (*P<0.05, **P<0.01). The treatments are: Control light, berries collected from the Control treatment with PAR exposure; UV-B radiation, berries collected from the UV-B treatment with PAR+UV-B exposure. Flavonols are shown as retention time as separated by HPLC analysis. Major flavonols identified by LC-MS are quercetin- diglucoside (RT 9.2), quercetin-3-O-rutinoside (RT 13.8), quercetin-3-O-glucoside (RT 14.8), quercetin-3-O- glucuronide (RT 15.7), kaempferol-3-O-glucoside (RT 16.6), and kaempferol-3-O-glucuronide (RT 17.2).

Potted vines at 12 weeks post bud burst

When compared with berries at 10 weeks post bud burst (Figure 5.3a), berries at 12 weeks post bud burst (around veraison) contained lower concentrations of flavonols (Figure 5.4a). Quercetin-3-O- glucuronide (RT 15.7) continued to be the major flavonol in berries at 12 weeks post bud burst, but the concentration was significantly lower than that in berries at 10 weeks post bud burst (4 μg/g compares to 9.7 μg/g). A quantitative decrease was also found in quercetin-diglucoside (RT 9.2). The average concentration of quercetin-diglucoside was 5.4 μg/g in berries at 10 weeks post bud burst, which significantly declined to 1.3 μg/g in berries at 12 weeks post bud burst (p<0.01). However, there was no significant changes found in these flavonols throughout the experimental period in the Control treatment. The concentration of quercetin-3-O-glucuronide maintained relatively static at round 4 μg/g from 4 hours to 5 days in the control berries. Similarly, quercetin-3-O-glucoside (RT 14.8) and quercetin-diglucoside also showed relatively static concentrations throughout the whole experimental period in the Control treatment.

Consistent with berries at 10 weeks post bud burst, a significant UV-B response in flavonols was also found in berries at 12 weeks post bud burst. As shown in Figure 5.4b, the concentrations of flavonols in berries in the UV-B treatment began to increase after 3 days of UV-B exposure. Quercetin-3-O- glucuronide (RT 15.7) was the major component of flavonols that significantly increased in response to UV-B exposure. Quercetin-3-O-glucuronide remained at 4.0 μg/g at the beginning of the experimental period (from 4 hours to 2 days). After 3 days of UV-B exposure, quercetin-3-O- glucuronide showed a significant increase and the concentration reached a final value of 10.2 μg/g at the end of the 5-day UV-B exposure. Significant increases were also detected for quercetin-3-O- rutinoside (RT 13.8) and quercetin-3-O-glucoside (RT 14.8) in the UV-B treatment. Quercetin-3-O- rutinoside and quercetin-3-O-glucoside in berries at 12 weeks post bud burst showed similar concentrations (around 1.1 μg/g) at the beginning of the experimental period. After 5 days of UV-B exposure, the concentration of quercetin-3-O-rutinoside significantly increased to 1.8 μg/g while quercetin-3-O-glucoside increased to 1.5 μg/g in the UV-B treatment.

Figure 5.4 The effects of UV-B radiation on flavonoid composition in Sauvignon blanc berries at 12 weeks post bud burst

Data shown are the average mean ± standard error of three replicates (n=3). *Significant difference comparing the Control treatment according to Two-way ANOVA test with time points and treatments as specifications (*P<0.05, **P<0.01). The treatments are: Control light, berries collected from the Control treatment with PAR exposure; UV-B radiation, berries collected from the UV-B treatment with PAR+UV-B exposure. Flavonols are shown as retention time as separated by HPLC analysis. Major flavonols identified by LC-MS are quercetin- diglucoside (RT 9.2), quercetin-3-O-rutinoside (RT 13.8), kaempferol-3-O-rutinoside (RT 14.6), quercetin-3-O- glucoside (RT 14.8), quercetin-3-O-glucuronide (RT 15.7), and kaempferol-3-O-glucoside (RT 16.6).

Potted vines at 14 weeks post bud burst

Figure 5.5 shows flavonoid composition in berries at 14 weeks post bud burst (about 1-2 weeks post- veraison). Compared to berries at earlier developmental stages (10 and 12 weeks post bud burst), the main changes in flavonoid composition in response to berry development were reflected in quercetin- 3-O-glucuronide (RT 15.7), quercetin-diglucoside (RT 9.2) and quercetin-3-O-glucoside (RT 14.8). Quercetin-3-O-glucuronide continued to be the major component of flavonols in berries at 14 weeks post bud burst. However, the concentration significantly decreased from 9.7 to 3.2 μg/g in berries from 10 to 14 weeks post bud burst (P<0.05). Quercetin-diglucoside showed a similar decreasing pattern during berry development. The average concentration of quercetin-diglucoside in berries at 10 weeks post bud burst was 5.4 μg/g, which declined to 1.3 μg/g in berries at 12 weeks post bud burst and finally dropped to an undetectable level in berries at 14 weeks post bud burst. In contrast, quercetin- 3-O-glucoside showed a significant increase during the post-veraison period. The concentration of quercetin-3-O-glucoside increased significantly from 1 to 1.6 μg/g in berries from 12 to 14 weeks post bud burst (P<0.01). Additionally, an unknown peak (RT 10.4) was detected in berries at 14 weeks post bud burst. This peak was not detectable in berries at earlier developmental stages measured.

A significant UV-B response was observed in flavonols in berries at 14 weeks post bud burst (Figure 5.5b). After 3 days of PAR+UV-B and PAR exposure respectively, berries in the UV-B treatment contained significantly higher concentrations of flavonols than berries in the Control treatment. Significant increases induced by UV-B radiation were detected in most of individual flavonols separated from HPLC analysis, especially in quercetin-3-O-glucuronide (RT 15.7) and quercetin-3-O-glucoside (RT 14.8). The concentration of quercetin-3-O-glucuronide was maintained at around 3.0 μg/g at the beginning of the experimental period (from 4 hours to 2 days). After 3 days of UV-B radiation, the concentration showed a significant increase and the profile finally increased to 4.8 μg/g after 7 days. Quercetin-3-O-glucoside also showed a significant increase in response to UV-B exposure. After 7 days of UV-B exposure, the concentration of quercetin-3-O-glucoside increased to 5.3 μg/g in the UV-B treatment, which was 231% higher than the Control treatment.

Figure 5.5 The effects of UV-B radiation on flavonoid composition in Sauvignon blanc berries at 14 weeks post bud burst

Data shown are the average mean ± standard error of three replicates (n=3). *Significant difference comparing the Control treatment according to Two-way ANOVA test with time points and treatments as specifications (*P<0.05, **P<0.01). The treatments are: Control light, berries collected from the Control treatment with PAR exposure; UV-B radiation, berries collected from the UV-B treatment with PAR+UV-B exposure. Flavonols are shown as retention time as separated by HPLC analysis. Major flavonols identified by LC-MS are quercetin-3-O- rutinoside (RT 13.8), kaempferol-3-O-rutinoside (RT 14.6), quercetin-3-O-glucoside (RT 14.8), quercetin-3-O- glucuronide (RT 15.7), and kaempferol-3-O-glucoside (RT 16.6).

Potted vines at 17 weeks post bud burst

As shown in Figure 5.6a, the major flavonols in berries at 17 weeks post bud burst (4-5 weeks post- veraison) were shown as quercetin-3-O-glucuronide (RT 15.7), quercetin-3-O-glucoside (RT 14.8) and kaempferol-3-O-glucoside (RT 16.6). Quercetin-3-O-glucoside continued increasing in response to berry development. The concentration of quercetin-3-O-glucoside in berries increased from 1.6 μg/g at 14 weeks post bud burst to 2.5 μg/g at 17 weeks post bud burst (p<0.01). However, quercetin-3-O- glucuronide and kaempferol-3-O-glucoside did not show significant changes during berry development. Quercetin-3-O-glucuronide was maintained relatively static at 3.2 μg/g in berries from 14 to 17 weeks post bud burst. The concentration of kaempferol-3-O-glucoside was around 1.2 μg/g in berries at 17 weeks post bud burst, similar as in berries at 14 weeks post bud burst.

Consistent with earlier developmental stages, significant increases in flavonols were detected after UV-B exposure in berries at 17 weeks post bud burst (Figure 5.6b). When compared to berries with PAR exposure, flavonols significantly increased in berries with PAR+UV-B exposure. These increases occurred after 3 days of UV-B exposure, and reflected in three major flavonols. These flavonols were quercetin-3-O-glucoside (RT 14.8), quercetin-3-O-glucuronide (RT 15.7) and kaempferol-3-O-glucoside (RT 16.6). Quercetin-3-O-glucoside showed most significantly UV-B response compared with other flavonols, which increased from 3.8 to 25.8 μg/g (579%) after 7 days of UV-B exposure. Quercetin-3- O-glucuronide also showed a significant UV-B response. The concentration of quercetin-3-O- glucuronide increased from 3.2 to 7.2 μg/g (125%) after 7 days of UV-B exposure. A UV-B response was detected in kaempferol-3-O-glucoside in berries at 17 weeks post bud burst as well. The concentration of kaempferol-3-O-glucoside significantly increased from 1.2 to 7.1 μg/g (491%) during the experimental period.

Figure 5.6 The effects of UV-B radiation on flavonoid composition in Sauvignon blanc berries at 17 weeks post bud burst

Data shown are the average mean ± standard error of three replicates (n=3). *Significant difference comparing the Control treatment according to Two-way ANOVA test with time points and treatments as specifications (*P<0.05, **P<0.01). The treatments are: Control light, berries collected from the Control treatment with PAR exposure; UV-B radiation, berries collected from the UV-B treatment with PAR+UV-B exposure. Flavonols are shown as retention time as separated by HPLC analysis. Major flavonols identified by LC-MS are quercetin-3-O- rutinoside (RT 13.8), quercetin-3-O-glucoside (RT 14.8), quercetin-3-O-glucuronide (RT 15.7), kaempferol-3-O- glucoside (RT 16.6), and kaempferol-3-O-glucuronide (RT 17.2).

5.3

Discussion