This study aimed to investigate flavan-3-ol biosynthesis under altered microclimatic conditions in Cabernet Sauvignon grown in the Stellenbosch Wine of Origin District. This was part of a larger study in which flavonoid evolution and composition by manipulating the light in the fruit zone were investigated.
Cabernet Sauvignon is one of the most planted red grape cultivars globally, which is also true for the Stellenbosch Wine of Origin District in South Africa. Despite the importance of this variety for winemaking, there are still important research questions that should be addressed. One such question is about the impact of light quality and quantity in interaction with temperature on berry flavonoid evolution/biosynthesis at the microclimatic/bunch level. Flavonoids perform major roles in plants, such as pollen fertilisation, auxin transport regulation, pigmentation, defence against pathogens and pests, and protection from ultraviolet (UV) radiation (Blancquaert, 2019). The three main groups of flavonoids identified in red grape berries are flavan-3-ols (tannin), anthocyanin and flavonols.
Flavan-3-ols include a range of polyphenolic compounds that include flavan-3-ol monomers, dimers and various oligomers and polymers that are connected by interflavan linkages (C4-C8 or C4-C6) called condensed tannins or proanthocyanidins (Adams, 2006). Proanthocyanidins are the most abundant class of grape phenols in the grape berry and are present in the seeds, skins, pulp and stems (Adams, 2006). The accumulation of flavonoids and their genes is up-regulated with exposure to light, while shading down-regulates the gene expression (Ryan and Revilla, 2003; Mori et al., 2007). A number of factors have been identified that can influence flavonoid accumulation and composition in grapes. This includes abiotic factors, such as light, temperature and water status, as well as cultivar, crop level, nutritional status, soil type and plant growth regulators (Blancquaert et al., 2019).
Materials and methods
A Cabernet Sauvignon/101-14 Mgt in a Stellenbosch University vineyard was studied over two years (2010/2011 and 2011/2012). The vineyard has a north-west/south-east row orientation and are trained on a six-wire vertical trellis system under drip irrigation (supplemental). Four treatments were applied in both seasons:
- No lateral shoot or leaf removal in the bunch zone (STD, shaded bunches).
- Leaf removal on the western side of the bunch zone (leaf removal west (LRW), exposed bunches in the afternoon sun). To study the effect of UV light on fruit growth and composition, a UV sheet (Perspex® Opal 050, Perspex South Africa (Pty) Ltd, Umbogintwini), reducing the UV-B radiation, was added to the control/STD.
- (STD-UV-B) and leaf removal west.
- (LRW-UV-B) treatment in 2010/2011.
UV-B-suppression sheets were installed on both sides of the canopy after the leaves were removed during the 2011/2012 season. In addition to the Perspex® Opal 050 sheets (used in 2010/2011), a clear acrylic UV sheet (extruded high impact (UHI)) was used during the 2011/2012 season. The latter resulted in the following treatments in 2011/2012: (iii) LR (-UV-B, -PAR) shaded without leaves and laterals, and (iv) LR (-UV-B, 2xUHI) (Figure 1). These sheets were installed just after fruit set at ±35 cm above the cordon and suspended on 1.2 m custom-made poles with hinges to open for sampling and the spraying program. Microclimate was monitored within the canopy and bunch zone with a Tinytag and light incidence was measured in (PAR) with a ceptometer. Sampling occurred at regular intervals from fruit set until harvest in both seasons. Monomeric and polymeric procyanidins (seed) and proanthocyanidins (skin) tannins were quantified using RP-HPLC.
FIGURE 1. The experimental layout created by leaf removal and UVB attenuation and optical properties of ‘Perspex’® and acrylic UV sheets.
Results and discussion
The accumulation of fruit thermal degree-days (DD, microclimate) was affected by the season and by the treatments (Table 1). The DD among the treatments in the 2011/2012 season was lower than that in the 2010/2011 season. The pattern of growing degree accumulation varied among the two seasons, as the macroclimate in the 2010/2011 season was characterised by continuous drought and heat throughout the summer (Vinpro, 2011). On the other hand, the 2011/2012 season was, however, considered as an ideal growing season with a cool, and lengthened, harvesting period without rain or prolonged heat (Vinpro, 2012).
Evolution of grape seed procyanidins and proanthocyanidins during ripening
The main seed flavan-3-ol monomers identified were (+)-catechin, (−)-epicatechin and (−)-epicatechin-3-O-gallate (Blancquaert, 2015). Procyanidin B2 (EC-(4β-8)-Ec) was the most abundant dimer in the seeds of the two measured: B1 (EC-(4β-8)-Cat) and B2 (Blancquaert, 2015). The concentration and content of individual monomers and dimers followed a similar pattern increasing from fruit set (13 – 22 DAA in 2010/2011 and 36 – 40 DAA in 2011/2012) (Figure 2a and 2b). A maximum was reached close to véraison (48 DAA in 2010/2011 and 54 – 68 DAA in 2011/2012) followed by a decrease until harvest (116 DAA in 2010/2011 and 130 DAA in 2011/2012) in both seasons (Figure 2a and 2b). When evaluating STD and LRW, the two treatments that were consistent between the two seasons, the mean monomer and dimer concentrations and contents of STD treatment, were similar between the seasons (Figure 2a and 2b). These results indicate minimal light and temperature effects, which are consistent with the findings of Dokoozlian and Kliewer (1996), and Downey et al. (2004), who suggested that shading resulted in minimal variation in seed chemistry. The pattern of seed tannin concentration (mg/g seed) and content (mg/berry) differed, according to RP-HPLC determination, between the investigated seasons (Figure 3). The 2010/2011 season was characterised by an increase in the seed tannin concentration and content until véraison (48 DAA), followed by a decrease and another increase from 76 DAA in all the treatments until harvest (Figure 3a and 3b). In the 2011/2012, tannin accumulation increased until véraison or two weeks prior to véraison and then fluctuated, except for treatment UHI (Figure 3b). Our results are in agreement with the findings of other authors, who reported that the genes responsible for seed tannin biosynthesis are switched on after fertilisation (Dixon et al., 2005).
FIGURE 2. Developmental changes in the seed monomeric and dimeric concentration (mg/g seed weight) and content (mg/berry) during berry development under different light conditions: (a) 2010/2011 Grape seed flavan-3-ol monomer and dimer concentration, (b) 2010/2011 grape seed flavan-3-ol monomer and dimer content, (c) 2011/2012 grape seed flavan-3-ol monomer and dimer concentration, and (d) 2011/2012 grape seed monomer flavan-3-ol content. Each value represents the mean of five replicates ± standard error.
FIGURE 3. Pattern of seed tannin concentration expressed as mg/g fresh seed weight and content expressed as mg/berry during berry development under different light conditions: (a) 2010/2011 Grape seed tannin concentration, (b) 2011/2012 grape seed tannin content, (c) 2011/2012 grape seeds tannin concentration, and (d) 2011/2012 grape seed tannin content. Each value represents the mean of five replicates ± standard error.
Evolution of grape skin procyanidins and proanthocyanidins during ripening
The main flavan-3-ol monomers identified in the skins were (+)-catechin, (−)-epicatechin and (−)-epicatechin-gallate; the dimers were EC-(4β-8)-Cat (B1) and EC-(4β-8)-EC (B2) in both seasons (Blancquaert, 2015). The accumulation pattern of skin monomers and dimers differed among the two seasons (Figure 4a and 4b). In general, the shaded STD treatment had the highest flavan-3-ol monomer and dimer concentrations and contents in 2010/2011 (Figure 2a). However, the other shaded treatment (STD-UV-B) had the lowest concentration and content. In the latter, UV-B radiation was additionally influenced, indicating the potential impact of UV-B radiation on flavan-3-ol synthesis. Differences in the concentration and content of total skin tannins were observed between the two seasons among all the treatments (Figure 5). Overall, the skin tannin content was higher in the 2010/2011 season when compared with the 2011/2012 season. Our results show that skin tannin reaches a maximum at véraison followed by a decrease. This study indicates that light quantity and quality have a potential impact on flavan-3-ol and tannin accumulation in the skin.
FIGURE 4. Developmental changes in the skin monomer and dimer concentration (mg/g skin) and content (mg/berry) during berry development under different light conditions: (a) 2010/2011 Grape skin monomer and dimer concentration, (b) 2010/2011 grape skin monomer and dimer content, (c) 2011/2012 grape skin monomer and dimer concentration, and (d) 2011/2012 grape skin monomer and dimer content. Each value represents the mean of five replicates ± standard error.
FIGURE 5. Developmental changes in the skin total tannin (mg/g skin) and content (mg/berry) during berry development under different light conditions: (a) 2010/2011 Grape skin total concentration, (b) 2010/2011 grape skin total content, (c) 2011/2012 grape skin total tannin concentration 2011/2012, and (d) grape skin total content in 2011/2012. Each value represents the mean of five replicates ± standard error.
The results suggested that tannin evolution is dependent on the prevailing light quality/quantity during berry development in a particular season, while leaves and laterals at the bunch zone seemed not to impact flavan-3-ol metabolism under the seasonal conditions studied. The bulk of both seed and skin monomers, dimers and tannin was synthesised just after fruit set and reached a maximum at véraison, after which it decreased in both seasons. The post-véraison decrease of the seed and skins monomers, dimers and tannin concentration and content is ascribed to a reduction in the extractability of the tannin post-véraison. The skin tannin increases/decreases observed during berry growth could be ascribed to the pattern of expression of flavonoid pathway genes reported by Boss et al. (1996).
I hypothesise that the light quality and quantity are a potential factor affecting the final skin total tannin concentration and content. Skin tannins, therefore, play a photo-protective role within the berry. This study highlights the importance of including seed number data and dry mass data to enhance interpretation. The applied treatments in this study did not introduce significant temperature differences. Treatments did result in differences in light quantity and quality, which had only a marginal impact on skin flavan-3-ol synthesis and no effect on seed tannin. In the case of skin tannin, there was a hint of increased skin tannin with light exposure, but this was only visible in the 2010/2011 seasons, indicating that seasonal variability had a larger impact than the individual treatments applied to alter the light quantity and quality.
– For more information, contact Erna Blancquaert at firstname.lastname@example.org.