This study aimed to investigate flavan-3-ol and anthocyanin evolution and composition under altered microclimatic conditions in Cabernet Sauvignon grown in the Stellenbosch Wine of Origin District.
Flavonols are colourless compounds which are synthesised in the skins and accumulate after flowering and during ripening (Price et al., 1995; Spayd et al., 2002; Downey et al., 2003a). They contribute to wine colour by forming co-pigments with anthocyanins (Asen et al., 1972; Scheffeldt & Hrazdina et al., 1978; Boulton, 2001). Moreover, flavonols are UV protectants and act as free radical scavengers (Flint et al., 1985; Smith & Markham, 1998). Anthocyanins are the pigmented compounds responsible for the colour in red grapes and wine (Ribereau-Gayon & Glories, 1986). Anthocyanins are synthesised and accumulate from véraison in the berry skins of most grape cultivars and contributes to the red and purple colour of the fruit (Adams, 2006). However, some Vitis vinifera cultivars, i.e. Alicante Bouschet, and non Vitis vinifera, i.e. hybrid cultivars, show anthocyanins also in the pulp and are known as teinturier cultivars (Guan et al., 2012). These two groups of flavonoids are influenced by environmental factors like the proanthocyanidins in Part 1 and 2 of this series.
Materials and methods
The site and methods have been discussed in Part 1 of this series. Flavonol evolution, anthocyanin evolution and the composition were determined with a RP-HPLC method at the same sampling dates as in Part 1 and 2.
Results and discussion
Flavonol accumulation commenced after fruit set until harvest in both seasons (Figure 1). Throughout both seasons, quercetin-3-O-glucoside and quercetin-3-O-glucuronide were the most abundant flavonol glycosides, while quercetin-3-O-rutinoside and quercetin-3-O-galactoside were present in smaller quantities (data not shown). This contradicts the findings of Mattivi et al. (2006) who reported that myricetin is the major flavonol in Cabernet Sauvignon. In both seasons, the patterns of accumulation were characterised by an increase after fruit set reaching a maximum four and five weeks post-véraison in 2010/2011 and 2011/2012, respectively, followed by small fluctuations in 2010/2011 or a decrease in 2011/2012 (Figure 1). Downey et al. (2006) also found a decrease in flavonols per berry two to four weeks after véraison in both exposed and shaded fruit within one season, while the flavonol content fluctuated from véraison until harvest in the other seasons.
Flavonol concentration and content were higher in the LRW treatment when compared with the other treatments (Figure 1). Similar patterns of accumulation were seen in the STD and LRW treatments in 2011/2012. The treatments with the UV-B exclusion sheets had the lowest flavonol concentration and content throughout ripening for both seasons (Figure 1). The results also indicate a clear seasonal impact on flavonol evolution during ripening and are due to the significant impact of the season on the light quality and quantity. This is in agreement with the findings of several other authors (Price et al., 1995; Haselgrove et al., 2000; Spayd et al., 2002; Downey et al., 2004) who reported that shaded fruit had lower flavonol glucosides at harvest or during berry development in, respectively, Cabernet Sauvignon, Shiraz and Merlot noir. Martinez-Lüscher et al. (2019) suggested that flavonol profile is a reliable indicator to assess canopy architecture and exposure of red wines to solar radiation. Flavonol concentration and content clustered together over the two seasons (Figure 1). This indicates that flavonol synthesis is independent within the grape berry skin.
The data suggest that UV-B radiation plays an important role in the photo-protection of the berry against light exposure. Increased sunlight radiation resulted in high levels of UV exposure, leading to increased flavonol levels. Consequently, the flavonol concentration and content are dependent on the light quality. This is in agreement with other studies describing that fruit exposed to different light qualities had higher flavonol glucosides (Crippen & Morrison, 1986; Downey et al., 2003). The latter phenomenon was also confirmed by Flint et al. (1985) and Berli et al. (2011). These authors suggested that flavonols act as UV screening compounds, protecting the plant tissue from the light damage during berry ripening. In this way, the accumulation of phenols takes place in the epidermal cell vacuoles of leaf tissue and grape berries, thereby protecting the photosynthetic mesophyll tissue (Flint et al., 1985; Macheix et al., 2005).
FIGURE 1. Developmental changes in the flavonol concentration expressed as mg/g fresh skin weight and content (mg/berry) during berry development under different light conditions: (a) 2010/2011 flavonol concentration, (b) 2011/2012 flavonol concentration, (c) 2010/2011 flavonol content, and (d) 2011/2012 flavonol content. Each value represents the mean of five replicates ± standard error.
Anthocyanin evolution and composition
The trend of anthocyanin accumulation differed between the two seasons (Figure 2). The 2010/2011 season was characterised by an increase in anthocyanin concentration and content from véraison and a decrease from 90 – 116 DAA (Figure 2a). The 2011/2012 season was characterised by an increase after véraison between 68 and 82 DAA, a decrease between 83 and 96 DAA, followed by another increase from 96 – 110 DAA, and a decrease from 110 – 130 DAA (Figure 2b). The STD and LRW treatments (treatments that were consistent over the two seasons) showed similar concentrations and contents at 48 DAA with a maximum at 76 DAA and similar levels at 116 DAA in the 2010/2011 season (Figure 2). Overall, there was no significant difference in the anthocyanin concentration and content in both the STD and LRW treatments for both seasons. The treatments with the UV-B exclusion sheets did not vary significantly from the STD and LRW treatments in 2010/2011. The mean anthocyanin concentration and content of the STD-UV-B and LRW-UV-B treatments in 2010/2011 were also similar. However, in the 2011/2012 season, the shaded LR (-UV-B, 2xOp50) had the highest overall concentration and content when compared to the other treatments. The LR (-UV-B, 2xUHI) treatment had the lowest concentration and content (data not shown), while it had the highest light exposure in addition to UV-B exclusion. Conflicting treatment results indicate that the season had a significant impact. Overall, there was no significant difference in the anthocyanin concentration and content in both the STD and LRW treatments for both seasons (Table 1). The mean anthocyanin concentration and content of the STD-UV-B and LRW-UV-B treatments in 2010/2011 were also similar (Table 1).
FIGURE 2. Developmental changes in the skin anthocyanin concentration expressed as mg/g fresh skin weight and content (mg/berry) during berry development under different light conditions: (a) 2010/2011 anthocyanin concentration, (b) 2010/2011 anthocyanin content, (c) 2011/2012 anthocyanin concentration, and (d) 2011/2012 anthocyanin content. Each value represents the mean of five replicates ± standard error.
Mono-glucosides, acetyl-glucoside and coumaroyl-glucoside derivatives of delphinidin, petunidin, peonidin and malvidin were determined in both seasons (data not shown). The accumulation of the individual anthocyanins commenced at véraison, at 48 DAA and 68 DAA, in 2010/2011 and 2011/2012, respectively, which is in agreement with the findings of other researchers (Ryan & Revilla, 2003; Downey et al., 2006; Mori et al., 2007). In the total anthocyanin pool, mono-glucoside was the predominant form, while acetyl-glucoside and coumaroyl-glucoside forms were present in lower proportions (data not shown). Malvidin-3-O-glucoside was the dominant anthocyanin and malvidin-3-O-acetyl glucoside was the major acylated anthocyanin in all treatments in both seasons (data not shown).
The individual anthocyanin composition in 2010/2011 was not significantly different for most of the anthocyanins among treatments. However, in 2011/2012 significant differences were observed in all the derivatives except for petunidin coumaroyl-glucoside. This indicates that the higher temperatures experienced in 2010/2011 had a larger impact rather than PAR. This also resulted in similar concentrations and contents of anthocyanins in the STD and LRW treatments in 2010/2011 and 2011/2012, respectively. The second season (2011/2012) was cooler, resulting in shifts in the anthocyanin profiles, confirming the findings of other authors who found vintage effects to play an important role in anthocyanin composition (Crippen & Morrison, 1986; Gao & Cahoon, 1994). From these results there was not a clear trend in anthocyanin accumulation in grape related to a different light exposure. Inconsistent treatment effects indicated that seasonal (climatic) impact was greater than any impact due to treatment.
This study highlighted the complexity of working under vineyard conditions to investigate the complex interaction of abiotic factors on berry metabolites. The novelty of our study involves the work being conducted under actual vineyard conditions in South Africa, which experiences high levels of UV radiation, while other studies were based on experimental setups. Flavonol and anthocyanin evolution is dependent on the prevailing light quality/quantity and temperatures during berry development in a particular season. Flavonol accumulation was significantly impacted in treatments that restricted UV-B light in the bunch zone, resulting in significant decreases in flavonol biosynthesis in both seasons studied. Therefore, it can be concluded that the light quality is the main abiotic driver of skin flavonol biosynthesis regulation. Anthocyanin concentration and content were largely influenced by the season and not the treatments applied, suggesting a synergistic influence of both light quantity and temperature with limited impact due to UV-B exclusion.
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