Original research articles

Grapevine row orientation, vintage and grape ripeness effect on anthocyanins, flavan-3-ols, flavonols and phenolic acids: I. Vitis vinifera L. cv. Syrah grapes

Abstract

Phenolic concentrations are affected by environmental factors and grape cultivar, as well as viticultural practices, which should be considered when a specific phenolic profile is intended. The effect of microclimate induced by row orientation on phenolic compounds of Vitis vinifera L. cv. Syrah/101-14 Mgt grapes was investigated. Grapes were harvested from four vintages at 22 °Brix, 24 °Brix and 26 °Brix from N-S, E-W, NE-SW and NW-SE orientated grapevine rows. Phenolics were quantified in freeze-dried grape skins using an HPLC method. A vintage effect was only evident for specific individual phenolics for the four vintages. Grapes from NE-SW rows at 22 °Brix had the highest total flavonols and anthocyanins. Catechin was highest in grapes from N-S rows at 24 °Brix, whereas total phenolic acids, caffeic acid and total flavonols were highest from NW-SE rows at 24 °Brix and 26 °Brix. Isoquercetin was highest from NE-SW rows at 22 °Brix and 26 °Brix, whereas kaempferol and quercitrin were highest from E-W rows at 24 °Brix. The majority of phenolics was highest from NW-SE rows at 24 °Brix and 26 °Brix. The results indicate that row orientation affects phenolic development in Syrah grapes, but that vintage has a limited effect on phenolics. Light induced in the fruit zone positively affected the grape phenolic development of NE-SW rows at 22 °Brix and that of NW-SE rows at 24 °Brix and 26 °Brix. However, it is unlikely that a single index can be applied to all growing conditions and grape cultivars. Rows that allow moderate radiation intensity in the canopy (i.e., NE-SW and NW-SE during the day) seem more favourable for the development of phenolics than N-S and E-W rows. The light and berry temperature conditions in the fruit zone resulting from row orientation have the potential for widening the range of Syrah wine styles. In practice, a desirable row orientation may not be applicable to all environments. Management of the fruit zone remains an option for increasing or decreasing grape light exposure, irrespective of row orientation.

Introduction

Environmental factors, such as topography, precipitation and soil type, affect the growth of grapevines and grape composition (Downey et al., 2004), whereas viticultural practices, such as row orientation and canopy management, primarily affect the grapevine microclimate (Hunter et al., 2016), which in turn has an effect on the grape chemical composition.

Grape growers attempt to minimise the heterogeneity of grape material within a single vineyard parcel/block to improve wine quality (Downey et al., 2004; Hunter et al., 2010). The development of phenolics (secondary plant metabolites) is affected by environmental factors (Gómez-Alonso et al., 2007), viticultural practices (Pérez-Magariño and González-SanJosé, 2002) and plant genetics (Czemmel et al., 2009).

The most common phenolic compounds present in red wine grapes are non-flavonoids (i.e., derivatives of hydroxybenzoic and hydroxycinnamic acids (Ribéreau-Gayon et al., 2006)), and flavonoids (i.e., anthocyanins, flavones, flavanonols, flavan-3-ols and flavonols (Margalit, 2004; Monagas et al., 2005)). Anthocyanins, flavan-3-ols and flavonols are most abundant in grape skin, whereas phenolic acids are mainly present in the grape pulp.

Phenolic concentrations increase throughout berry development (Pérez-Magariño and González-SanJosé, 2006), with differing evolution depending on phenolic group (Mateus et al., 2002).

The relationship between phenolic composition and grape ripeness levels has been reported by numerous authors. Phenolic evolution as a consequence of a delay in sugar accumulation during the grape development of artificially shaded Cabernet-Sauvignon has been reported by Rojas-Lara and Morrison (1989), which was likely the result of decreased photosynthetic capacity of the vine. Fournand et al. (2006) found that free anthocyanins change to derivatised anthocyanins during the grape ripening period, and Guidoni and Hunter (2012) reported differences between the anthocyanin profiles of grapes at different ripeness levels. Guidoni and Hunter (2012) showed that malvidin 3-O-glucoside and malvidin 3-O-(6-p-coumaroyl)-glucoside were lower when at 22 °Brix compared to 24 °Brix and 26 °Brix ripeness levels, whereas acetylated anthocyanins were higher at a lower ripeness level. The high proportion of malvidin 3-O-glucoside found at 22 °Brix appeared to occur at the expense of the acetyl derivative, which is expected to be more affected by degradation reactions in grapes. Non-acetylated and acetylated glucosides are sensitive to degradation processes induced by high temperature, whereas coumaroyl-glucoside seems to be more stable under these conditions. Treatment did not have an effect on coumaroyl-glucosides. The “insensitivity” of coumaroyl-glucoside to treatment can be ascribed to their participation in intramolecular co-pigmentation (Brouilard and Dangles, 1994).

Giacosa et al. (2015) found that the modification of the chemical profile of Shiraz grapes was more due to changes during grape ripening than to viticultural practices. During the over ripeness stage of grapes (> 26 °Brix), individual anthocyanins can undergo some transformation independently of light and temperature conditions.

Roubelakis-Angelakis and Kliewer (1986) suggested that temperature and light play an independent regulating role in the flavonoid biosynthetic pathway to the point of the formation of dihydroflavonols, taking into consideration that anthocyanins and flavonols share the same pathway. Hunter et al. (2016, 2021) reported that radiation profiles in the canopy of Shiraz grapevines have implications for photosynthetic rates and grape berry temperature.

Hydroxylation levels of anthocyanins can be affected by not only UV radiation, but also temperature variation (Spayd et al., 2002). However, Alonso et al. (2016) and Mori et al. (2005) reported that anthocyanins may not always respond to radiation. Increased temperature in the fruit zone can result in grape chemical composition variability (Romboli et al., 2017), since the response of anthocyanins to UV radiation depends on the interaction of grape cultivar, grape ripeness and temperature factors (Jordan, 2017).

Gallic acid and most flavan-3-ols in grape skins do not respond to UV radiation, but p-coumaric, caffeic and ferulic acid, as well as gallocatechin increase in UV blocked grape bunches (Spayd et al., 2002). Yu et al. (2016) reported that increased temperature in the fruit zone was associated with an increase in epigallocatechin and a decrease in epicatechin in Merlot and Shiraz grapes respectively. Allegro et al. (2019) found that increased light interception in the canopy and increased berry temperature (after leaf removal) did not affect the total flavan-3-ols in the skins of Grechetto Gentile grapes; however, high levels of epicatechin were counterbalanced by low levels of epigallocatechin.

Sternad-Lemut et al. (2011) showed that hydroxycinnamic acids in Pinot noir grapes respond only slightly to leaf removal (exposure to light), indicating that solar radiation has a limited role in regulating phenolic acid biosynthesis. Light exclusion (by brown paper bagging) of Cabernet-Sauvignon grape bunches have been found to affect phenolic acids less than flavonoids; however, differences in hydroxycinnamic acid contents in grape berries during ripening have been found between light-excluded grape bunches and bunches exposed to light (Sun et al., 2019).

Rescic et al. (2016) reported increased concentrations of gallic acid in Istrian Malvasia grapes harvested at 24 °Brix, with increased light in the fruit zone brought about by leaf removal/shoot thinning of grapevines planted in N-S (north-south) rows. Work by Tessarin et al. (2014) on grapevines planted in east-west (E-W) directions showed that caffeic acid concentrations in Uva Longanesi grapes were higher after 50 % defoliation compared to no defoliation treatment.

Martínez-Lüscher et al. (2014) found lower concentrations of kaempferol and quercetin in Tempranillo grapes from N-S orientated vines receiving reduced light due to artificial shading compared to low vigour vines. Friedel et al. (2015) reported that boxed-in Riesling grape bunches from vines planted in an E-W direction, resulted in an increase in rutin, quercitrin and isoquercetin. In a study by Downey et al. (2004), artificially shaded Shiraz grapes at harvest were lower in flavan-3-ol content than exposed bunches; however, the decrease in total flavan-3-ols during berry ripening was greater in exposed bunches, to the extent that the levels were virtually the same in shaded and exposed berries at harvest. Scrafidi et al. (2013) reported higher concentrations of total flavan-3-ols in exposed Grillo grapes at 21 °Brix and planted in N-S rows than in boxed-in grapes, whereas Cortell et al. (2005) found that (-)-epigallocatechin concentrations in Pinot noir grapes at harvest, planted in an E-W direction, were positively correlated to increased light in the canopy and fruit zone.

Light intensity and light quality (UV-B and blue light) have been associated with the regulation of anthocyanin biosynthesis (Jaakola, 2013). Shiraz and Pinot noir grape bunches that develop in low vigour vines, as opposed to those that develop in high vigour vines, have increased concentrations of berry skin anthocyanins (Downey et al., 2004; Cortell et al., 2007). According to Joscelyne et al. (2007) and Ristic et al. (2007), artificially shaded Syrah grapes of vines planted in an E-W direction had lower concentrations of total anthocyanins than grape bunches from low vigour vines. Feng et al. (2015) reported that increased light brought about by 50 % leaf removal of Pinot noir N-S planted vines, resulted in an increase in total anthocyanins, compared to no leaf removal treatment. Martínez-Lüscher et al. (2014) found that reduced light (artificial shading) in the canopy of Tempranillo grapevines planted in a N-S direction, resulted in an increase in acylated malvidin-glucosides in grapes at 23 °Brix. Fernandes-De Oliveira and Nieddu (2016) reported higher concentrations of delphinidin- and petunidin 3-O-(6-O-p-coumaroyl) glucosides in artificially shaded (reduced UV-radiation) Bovale Grande and Cannonau grapes from N-S orientated vines than naturally exposed grapes.

In the cited literature, flavonols, flavan-3-ols and phenolic acids generally increased in grapes of vines exposed to light due to defoliation, whereas artificial shading or high vigour vines resulted in a decrease in these phenolics. Increased light due to defoliation also resulted in an increase in total anthocyanins, but a decrease in certain monomeric anthocyanin glucosides and anthocyanin glucoside coumaroylates.

Internationally, work on the effect of grapevine row orientation under realistic field conditions has not been published, and in South Africa, only limited investigations have been undertaken on the effect of row orienation per se on phenolics. It was demonstrated that, despite total anthocyanins and phenolics in Syrah grapes from rows with different orientations being relatively similar (with highest values found for the NW-SE (northwest-southeast) rows), wooded and unwooded wines produced from E-W orientated vines contained the lowest concentrations (Hunter and Volschenk, 2017).

The aim of this study was to investigate the effect of microclimate induced by different grapevine row orientations under realistic field conditions on the concentrations of selected individual anthocyanins, flavan-3-ols, flavonols, and phenolic acids in Syrah grapes, harvested at three grape ripeness levels over four consecutive seasons.

Materials and methods

1. Vineyard layout and management

An experimental vineyard was established in 2003 (Figure 1). The first full harvest was carried out five years after the establishment of the vineyard. The vineyard was already in full production when this experiment was undertaken. Syrah, clone SH 9C was grafted onto 101-14 Mgt rootstock. The vineyard was established on a flat terroir with clayey loam soil on the research farm of the Agricultural Research Council (ARC) in the Breede River Valley, Robertson (33.8021° S, 19.8875° E, 187 metres above sea level), South Africa (Hunter et al., 2016). Row and vine spacing were 2.7 m and 1.8 m respectively. The vines were trained to a vertical shoot positioning (VSP) trellis system. A cover crop of rye grass was sown after harvest (April) and treated with herbicide prior to bud burst (October). The grapevine canopies consisted of three to four leaf layers, side by side. The leaf layers were measured using the point quadrat method (Hunter et al., 2016). Grapevines were subject to supplementary irrigation every seven days.

Figure 1. Aerial view of different grapevine row orientations indicating experimental replicate blocks (1-5).

1 = East-west (low light penetration in the morning and afternoon in the canopy); 2 = North-south (high light penetration in the morning and afternoon in the canopy); 3 = Northwest-southeast (low light penetration in the afternoon and high light penetration in the morning in the canopy); 4 = Northeast-southwest (low light penetration in the morning and high light penetration in afternoon in the canopy).

The experimental design was a randomised complete block (Figure 1) with four different row orientations (i.e., N-S, E-W, NE-SW and NW-SE) replicated at random in each of five experimental blocks with a total surface area of 1860 m2. An experimental unit consisted of all the grapevines within a vineyard block of a specific row orientation within the experimental vineyard.

2. Temperature and radiation measurements

The methodology for the determination of ambient photosynthetic radiation profiles, berry pulp temperature profiles, seasonal canopy temperature and seasonal ambient/air temperature profiles is fully described in Hunter et al. (2016) and Hunter et al. (2021).

3. Grape samples

Sampling took place over four consecutive vintages (2008, 2009, 2010 and 2011) at three different ripeness levels; i.e., 22 °Brix, 24 °Brix and 26 °Brix. Syrah grapes in Robertson, South Africa, are normally harvested at 24 °Brix grape maturity for winemaking (Guidoni and Hunter, 2012). Part of the experimental design of this study was to include a grape ripeness level two degree below and two degrees above the harvest ripeness level, hence 22 °Brix and 26 °Brix.

Individual grape berries (100) were collected from the top, middle and bottom of the bunches before midday. Five subsamples of 100 berries each per experimental unit were selected to ensure sufficient material for further processing.

3.1 Grape berry preparation

The grape skins of each 100-berry sample were separated from the grape flesh and seeds by hand. The skins were rinsed with distilled water and blotted with laboratory absorptive paper. The skins were freeze-dried (Christ Beta 1-8 LSCbasic, Freeze-drier, 336 Osterode/Harz, Germany) and stored in airtight containers at 4 °C until required for analysis.

3.2. Extraction procedure

A method described by Castillo-Muñoz et al. (2007) was used for the extraction of phenolics. The freeze-dried grape skins were ground prior to extraction. Separate extractions and analyses were done for each experimental unit from the five block replicates. A volume of 30 mL methanol/formic acid/water (50:48.5:1.5) was added to 1.0 g of freeze-dried ground grape skins (n = 5). Extraction was performed in a dark room using a Variomag Poly15 Electronicrȕhrer (Avantor, Inc. Randor, Pennsylvania, USA) stirring for 20 min at 25 °C at a rate of 450 rpm. The samples were subsequently centrifuged using a Hettich® ROTANTA 460/460R centrifuge (Merck KgaA, Darmstadt, Germany) at 1000 x g for 15 min. The supernatant was filtered through a 0.45 µm nylon membrane syringe filter [Microsep, (Pty), Ltd, Johannesburg, South Africa] prior to high performance liquid chromatographic (HPLC) analysis.

4. Reagents

All solvents and acids used were of analytical grade (Merck, Johannesburg, South Africa). De-ionised water was supplied through a Modulab® water purification system (Separations Scientific, Johannesburg, South Africa).

5. Chromatography

A reversed-phase HPLC-diode array detection (RP-HPLC-DAD) method was used to separate, identify and quantify 23 target phenolics in the freeze-dried grape skin extracts. Quantification of anthocyanins, flavonols, flavan-3-ols and phenolic acids was performed on a SpectraSYSTEM HPLC instrument (Thermo Separations Products, Inc., New Jersey, USA) equipped with an auto-sampler (injection volume 20 μL), using a published method of Waterhouse et al. (1999). Detection was by means of photodiode array between 190 nm and 950 nm. ChromQuest software was utilised for data acquisition. Calibration curves for anthocyanins, flavan-3-ols, flavonols and phenolic acids were constructed by injection of standard reference sample solutions of different concentrations of individual phenolics (Table S1).

Calibration parameters and quantification of the target phenolics were performed using ChromQuest software. Separation was performed at 22 °C, using a polymer reversed-phase analytical column (PLRP-S 100 Å, 5 µm, 250 x 4.6 mm) with polystyrene divinylbenzene as stationary phase (Polymer Laboratories, Massachusetts, USA). Binary gradient elution was performed at 1 mL/min flow rate. The binary mobile phases comprised water/phosphoric acid [985:15 v/v (pH 1.35) - eluent A] and water/phosphoric acid/acetonitrile [185:15:800 v/v/v (pH 1.25) - eluent B]. The gradient programme is listed in Table 1. An analysis time of 90 min was preceded by a 20-min equilibration time. Anthocyanins were detected at 520 nm, flavan-3-ols at 280 nm, phenolic acids at 316 nm and flavonols at 360 nm. The identification of phenolics was confirmed by their relative retention times based on reference standards and UV-visible absorption characteristics. All reference standards and reagents used were of HPLC grade and supplied by Merck (Pty), Ltd and Sigma-Aldrich, Johannesburg, South Africa.

Table 1. Gradient programme used for the RP-HPLC-DAD separation of phenolics (flow rate 1 mL/min).


Time (min)

Eluent A composition (%)

Eluent B composition (%)

0

94.00

6.00

73

69.00

31.00

78

38.00

62.00

86

38.00

62.00

90

94.00

6.00

6. Statistics

Analysis of variance (ANOVA) per ripeness level was performed according to the experimental design, including the factors row orientation and vintage (repeated measures), on all variables assessed, using General Linear Models Procedure of SAS software (Version 9.4; SAS Institute Inc., Cary, USA). The Shapiro and Wilk test (Shapiro and Wilk, 1965) was performed to check for deviation from normality. Fisher’s least significant difference test was calculated at a 5 % level to compare means for significant effects (Ott, 1998). A probability level of 5 % was considered significant for all tests. Averages, variances and standard deviations for phenolic data were determined using XLSTAT 2016 (Version 18.06, Addinsoft, New York, USA). Principal component analysis was applied to phenolic compound data for each ripeness level, also using XLSTAT, to elucidate associations between row orientation and vintage and phenolic compounds.

Results

1. Seasonal temperature trends, berry temperature, air temperature and radiation in the canopy

Seasonal temperature trends, radiation profiles, berry pulp temperature profiles and air temperature relevant to this investigation have been fully discussed in Hunter et al. (2016) and Hunter et al. (2021). Average ambient temperatures (canopy) increased from approx. mid-December (around green pea-size) and maximum temperature frequently reached more than 30 °C onwards (Hunter et al., 2016). Micro-temperature profiles inside the canopy were not notably different among rows, except for generally slightly lower and slightly higher morning temperatures of E-W and N-S rows respectively (Hunter et al., 2016).

Berry pulp temperature increased during the late ripening stage from morning to afternoon and varied by approx. 3-8 °C (Table 2) (Hunter et al., 2016). The largest differences among row orientations were recorded in the morning and afternoon. At midday, average berry temperatures per orientation were, however, similar.

Table 2. Average berry pulp temperature (°C) for each canopy during grape ripening of Syrah grapes planted in four different row orientations (hand-held temperature probe measurements) (values represent averages of four consecutive seasons).


Table

Average berry pulp temperature (°C)

Average berry temp per day

Measured at 10:00 am

Measured at 13:00 pm

Measured at 16:00 pm

N-S

29.6a

31.9a

34.6b

32.0a

E-W

27.3b

32.3a

32.4c

30.6b

NE-SW

27.6b

32.3a

35.4a

31.7a

NW-SE

29.3a

32.3a

33.3bc

31.6a

p = 0.05

1.2

1.0

1.4

0.7

Average ambient (air) temperature at 10:00 am, 13:00 pm and 16:00 pm = 22.8 °C, 29.5 °C and 32.3 °C respectively.

The lowest average temperature is reported for E-W rows, being 1-1.4 °C lower than N-S, NE-SW and NW-SE rows, but still more than 2 °C higher than air temperature. In general, N-S rows resulted in grapes gaining heat in the morning, as well as the afternoon. This was also the scenario for NE-SW rows, but there was generally more heat gain during the afternoon. An E-W row mostly mitigated berry heat gain during the season. This also occurred for NW-SE rows, but with higher gains in the morning.

Depending on the month of the season, radiation outside the canopies ranged from 1400 to 1700 µmol m-2/s for all row orientations (Hunter et al., 2016). For all row orientations, the highest radiation inside the canopies was received during the first and second months of measurement at the beginning of the season (approx. berry set to pea berry size stage) when the canopies were not yet completely settled. Grapevines planted in E-W rows received a maximum photosynthetic active radiation (PAR) only at midday, peaking - depending on the month of the season - at ca. 45 - 1050 µmol m- 2/s inside the canopy (Hunter et al., 2016). During the last four months of measurement, the interior-canopy radiation reached 40 µmol m-2/s just after midday. The N-S rows received maximum PAR twice a day, peaking - depending on the month of the season - at ca. 600 µmol m- 2/s and 1050 µmol m- 2/s inside the canopy during late morning and late afternoon (highest values in the beginning of the season) (Hunter et al., 2016). These rows may be considered to cause a uniform light distribution in the canopy. The N-S rows maintained a higher interior canopy light interception in the morning and afternoon compared to E-W rows. During the last four months of measurement, the interior-canopy radiation consistently reached ca. 60 µmol m-2/s in the afternoon. The NE-SW rows received maximum PAR twice a day during mid-morning and afternoon (highest values in the beginning of the season), peaking - depending on the month of the season - at ca. 15 µmol m-2/s and 1050 µmol m-2/s radiation inside the canopy (Hunter et al., 2016). During the last four months of measurement, the interior-canopy radiation reached ca. 75 µmol m-2/s during the afternoon. Grapevines planted in a NW-SE direction also received maximum PAR twice a day during mid-morning and the afternoon (highest values at the beginning of the season), peaking - depending on the month of the season - at ca. 30 µmol m-2/s and 700 µmol m-2/s, measured inside the canopy (Hunter et al., 2016). During the last four months of measurement, the interior-canopy radiation reached ca. 60 µmol m-2/s during the morning.

2. Grape yield

Grape yield depends on factors such as climate, soil, grape cultivar/rootstock combination and cultivation conditions (Hunter et al., 2017). Light penetration within the canopy, which depends on the trellis system type, vine vigour, canopy density and management, PAR and water relations, has an effect on vine metabolism and biochemical processes and ultimately an effect on grape yield (Hunter et al., 2017). In this study, all the above factors were kept constant, except for PAR as it was altered by row orientation. Average grape yields for the four respective years of the experiment (2007/08–2010/11) at ripeness level 1 were 24.8, 16.4, 25.3 and 17.0 (N-S); 24.8, 13.7, 25.5 and 14.3 (E-W); 22.5, 15.3, 22.6 and 14.9 (NE-SW); and 23.7, 13.1, 26.5 and 15.0 (NW-SE) tonnes per hectare. At ripeness level 2, the yields were 23.0, 12.3, 25.3 and 16.3 (N-S); 21.9, 13.0, 23.0 and 15.1 (E-W); 21.9, 11.7 24.2 and 13.2 (NE-SW); and 21.8, 11.7, 20.8 and 14.6 (NW-SE) tonnes per hectare; whereas at ripeness level 3 the yields were 22.6, 12.2, 21.4 and 12.3 (N-S); 21.6, 10.5, 22.3 and 12.8 (E-W); 20.4, 10.1, 18.8 and 10.1 (NE-SW); and 20.9, 11.2, 21.3 and 10.8 (NW-SE) tonnes per hectare.

Average yields over the four years and three ripeness levels were 19.03, 18.23, 17.13 and 17.63 tonnes per hectare for the N-S, E-W, NE-SW and NW-SE orientations respectively. The E-W rows showed the lowest yield losses from low to high ripeness level (Hunter et al., 2017). A decreasing trend in grape yield during the last stages of ripening (> 25 °Brix) is commonly observed because of grape concentration decrease in berry mass and volume when grape ripening progresses after sugar accumulation peaks (Carlomagno et al., 2018). Hunter et al. (2017) showed that the N-S row orientation was more desirable in terms of yielding ability and stability. According to De Souza et al. (2019) row orientation of Syrah vines had a greater effect on grape composition than on grape yield. Considering yield differences between row orientations, Hunter and Volschenk (2017) confirmed that yield was a weak driver (if at all) of wine style/quality and that sensory profiles were rather driven by a combination of environmental (most likely mainly at microclimate level - radiation impacting on berry temperature and berry metabolism) and physiological factors; berry chemical/biochemical compounds involved in respiratory metabolism; and products of secondary metabolism that responded to radiation and temperature.

3. Analysis of variance p-values (2-factor ANOVA) for row orientation and vintage main effects and interaction to determine which effects are statistically significant

Row orientation by vintage interaction at 22 °Brix (Table 3) was not statistically significant for most compounds, except total phenolic acids, caffeic acid, ferulic acid, kaempferol, quercitrin, cyanidin 3-O-glucoside, petunidin 3-O-glucoside, petunidin 3-O-(6-O-acetyl) glucoside, delphinidin 3-O-(6-O-p-coumaroyl) glucoside and petunidin 3-O-(6-O-p-coumaroyl) glucoside. For row orientation by vintage interaction at 24 °Brix (Table 3), gallic acid, p-coumaric acid, isoquercetin, total anthocyanins, petunidin 3-O-glucoside, malvidin 3-O-glucoside, malvidin 3-O-(6-O-acetyl) glucoside and petunidin 3-O-(6-O-p-coumaroyl) glucoside were statistically significant. At 26 °Brix (Table 3), row orientation by vintage interaction was statistically significant for total flavan-3-ols, (+)-catechin, EGCG, petunidin 3-O-glucoside, peonidin 3-O-glucoside, delphinidin 3-O-glucoside, petunidin 3-O-(6-O-acetyl) glucoside, peonidin 3-O-(6-O-acetyl) glucoside, delphinidin 3-O-(6-O-p-coumaroyl) glucoside, petunidin 3-O-(6-O-p-coumaroyl) glucoside and malvidin 3-O-(6-O-p-coumaroyl) glucoside.

Table 3. Analysis of variance p-values (2-factor ANOVA) for row orientation and vintage main effects and interaction for Syrah grapes at 22 °Brix, 24 °Brix and 26 °Brix to determine which effects are statistically significant.


Phenolic compounds

22 °Brix

24 °Brix

26 °Brix

Main effects

Interaction

Main effects

Interaction

Main effects

Interaction

Row orientation

Vintage

Row orientation x vintage

Row orientation

Vintage

Row orientation x vintage

Row orientation

Vintage

Row orientation x vintage

Total flavan-3-ols

< 0.0001

0.0009

0.4099

0.0005

0.0143

0.5144

< 0.0001

0.2066

0.0284

(+)-Catechin

0.0015

0.2171

0.8429

< 0.0001

0.1517

0.9102

< 0.0001

0.0006

0.0137

(-)-Epicatechin

0.0037

0.0501

0.3766

0.1870

< 0.0001

0.1582

0.3470

0.2872

0.6771

EGCG1

< 0.0001

0.0027

0.1352

< 0.0001

0.4110

0.5803

0.0008

0.9208

0.0469

Total phenolic acids

< 0.0001

0.0064

0.0015

< 0.0001

0.7496

0.8200

< 0.0001

0.0636

0.4158

Gallic acid

0.0025

0.0039

0.4090

< 0.0001

0.0055

0.0078

< 0.0001

0.3873

0.1293

Caffeic acid

< 0.0001

< 0.0001

0.0005

< 0.0001

0.9951

0.9502

< 0.0001

0.0838

0.4907

p Coumaric acid

< 0.0001

0.5169

0.1445

< 0.0001

0.5285

0.0526

< .00001

0.0158

0.4288

Ferulic acid

< 0.0001

0.5144

0.0042

< 0.0001

0.5399

0.5249

0.0007

0.4721

0.8558

Rutin

0.3820

0.0341

0.3493

0.0228

0.0468

0.5053

0.0180

0.0116

0.3489

Total flavonols

< 0.0001

0.8483

0.0656

< 0.0001

0.2568

0.1704

< 0.0001

0.1640

0.4008

Isoquercetin

< 0.0001

0.4161

0.1723

< 0.0001

0.0140

0.0023

< 0.0001

0.0091

0.4195

Quercetin

< 0.0001

0.0605

0.2818

< 0.0001

0.2752

0.4879

0.0079

0.0503

0.0737

Kaempferol

< 0.0001

0.0895

0.0393

< 0.0001

0.1663

0.5133

< 0.0001

0.6634

0.4476

Quercitrin

< 0.0001

0.7451

0.0152

< 0.0001

0.3409

0.1366

< 0.0001

0.5543

0.4541

Total anthocyanins

0.0012

0.0010

0.6308

0.0023

0.0002

0.0374

0.9236

0.0006

0.1170

CyGluc2

< 0.0001

0.6716

< 0.0001

0.0020

0.0530

0.2107

0.0085

0.4473

0.4327

PetGluc2

< 0.0001

< 0.0001

0.0021

0.0003

0.0001

0.0002

< 0.0001

0.6105

< 0.0001

PeoGluc2

0.0017

0.0822

0.6622

0.2300

0.1910

0.5479

0.0070

0.0007

0.0351

MalGluc2

0.0051

< 0.0001

0.6973

0.0232

< 0.0001

0.0238

< 0.0001

0.0038

0.8793

DelGluc2

0.6010

0.0781

0.5539

0.1015

< 0.0001

0.1731

0.0111

0.0001

0.0304

PetGlucAc3

0.0005

0.2785

< 0.0001

0.2054

0.0423

0.9217

< 0.0001

0.2598

< 0.0001

PeoGlucAc3

0.0487

0.3406

0.5767

0.2725

< 0.0001

0.5022

0.0005

0.0167

< 0.0001

MalGlucAc3

0.0241

0.0887

0.2734

0.0249

0.0002

0.0175

0.0005

0.5906

0.8032

DelGlucCoum4

0.0017

0.0050

0.0216

0.0672

0.0333

0.9756

< 0.0001

0.0114

< 0.0001

PetGlucCoum4

< 0.0001

0.6020

< 0.0001

< 0.0001

0.0004

< 0.0001

< 0.00001

0.0042

< 0.0001

MalGlucCoum4

0.0623

0.2131

0.4000

0.0280

0.0828

0.2584

0.0008

< 0.0001

0.0216

1Epigallocatechin 3-O-gallate; 2Cyanidin-, petunidin-, peonidin-, malvidin- and delphinidin 3-O-glucoside; 3Petunidin-, peonidin- and malvidin 3-O-(6-O-acetyl) glucoside; 4Delphinidin-, petunidin- and malvidin 3-O-(6-O-p-coumaroyl) glucoside.

4. Phenolic compound concentration means for vintage main effects

The following results (means) are for compounds that were statistically significantly affected by the main effect only; i.e., vintage (Table 4). Certain compounds at 22 °Brix (Table 4) in 2008 and 2009 had generally lower concentrations of phenolics than in 2010 and 2011. The exception was gallic acid, for which the lowest levels were recorded in 2011 (low yield). Total flavan-3-ols and total anthocyanins were the highest in 2011 but were not different in 2010 (high yield), whereas malvidin 3-O-glucoside was the highest in 2011.

Table 4. Phenolic compound concentration (mg/L) means for vintage (2008, 2009, 2010 and 2011) main effects only at 22 °Brix, 24 °Brix and 26 °Brix.


Vintage

2008

2009

2010

2011

p-value

*Ambient min/max Temp

15.4-29.5 °C

15.0-29.7 °C

15.5-30.5 °C

16.7-31.0 °C

22 °Brix

Phenolics

Total flavan-3-ols

**11.820 ± 1.29c

12.137 ± 1.38bc

12.403 ± 1.34ab

12.713 ± 1.04a

0.0009

(-)-Epicatechin

4.958 ± 0.18b

5.022 ± 0.33ab

5.202 ± 0.39a

5.199 ± 0.39a

0.0501

EGCG1

1.596 ± 0.91b

1.666 ± 1.16b

1.890 ± 0.95a

1.941 ± 0.83a

0.0027

Gallic acid

1.510 ± 0.29a

1.680 ± 0.39a

1.515 ± 0.27a

1.313 ± 0.35b

0.0039

Rutin

0.652 ± 0.12a

0.585 ± 0.15ab

0.514 ± 0.18b

0.657 ± 0.13a

0.0341

Total anthocyanins

372.115 ± 59.87c

375.960 ± 60.70bc

411.303 ± 57.41ab

439.961 ± 69.47a

0.0010

Malvidin2

80.369 ± 18.65c

87.711 ± 26.64bc

95.139 ± 16.02b

120.428 ± 25.48a

< 0.0001

24 °Brix

Phenolics

Total flavan-3-ols

**12.387 ± 0.66b

12.811 ± 0.77a

13.157 ± 0.65a

12.897 ± 0.58a

0.0143

(-)-Epicatechin

5.005 ± 0.29b

5.072 ± 0.29b

5.670 ± 0.39a

5.119 ± 0.39b

< 0.0001

Rutin

0.685 ± 0.07ab

0.669 ± 0.09ab

0.723 ± 0.15a

0.635 ± 0.07b

0.0468

DelGluc3

1.765 ± 0.22c

2.156 ± 0.38ab

1.971 ± 0.31bc

2.351 ± 0.29a

< 0.0001

PetGlucAc4

2.884 ± 0.43b

3.185 ± 0.37ab

3.316 ± 0.67a

3.368 ± 0.34a

0.0423

PeoGlucAc4

10.201 ± 1.42b

12.493 ± 1.85a

11.270 ± 1.55b

13.179 ± 1.56a

< 0.0001

DelGlucCoum5

6.841 ± 1.38b

7.333 ± 1.25ab

8.014 ± 1.36a

7.685 ± 1.45ab

0.0333

26 °Brix

Phenolics

p Coumaric acid

**24.071 ± 3.96ab

24.059 ± 3.89ab

25.346 ± 4.92a

23.588 ± 3.75b

0.0158

Rutin

0.589 ± 0.06b

0.594 ± 0.06b

0.613 ± 0.07b

0.666 ± 0.05a

0.0116

Isoquercetin

2.406 ± 1.01bc

2.713 ± 1.01a

2.622 ± 1.01ab

2.198 ± 0.78c

0.0091

Ouercetin

7.291 ± 1.19a

6.642 ± 1.29ab

6.440 ± 1.12b

6.362 ± 1.16b

0.0503

Total anthocyanins

334.007 ± 34.54c

350.910 ± 39.76bc

360.768 ± 30.63ab

380.778 ± 40.48a

0.0006

Malvidin2

73.751 ± 17.24c

86.864 ± 19.58ab

82.221 ± 15.83bc

92.965 ± 18.46a

0.0038

*Average ambient temperatures for January, February and March (harvest time). **Means given for compounds with significant vintage main effects as indicated in Table 3. Different letters in the same row indicate significant differences in the content of the measured variables among the different row orientations according to Fischer's least significant difference test (p = 0.05). 1Epigallocatechin 3-O-gallate; 2Malvidin 3-O-glucoside; 3Delphinidin 3-O-glucoside; 4Petunidin- and Peonidin 3-O-(6-O-acetyl) glucoside; 5Delphinidin 3-O-(6-O-p-coumaroyl) glucoside.

A vintage effect on phenolic compounds at 24 °Brix (Table 4) was not found, except for total flavan-3-ols in 2008 (high yield), which were lower than in 2009, 2010 and 2011. Epicatechin (-) was highest in 2010 (high yield). Statistically significant differences among vintages for the remaining compounds were not found. At 26 °Brix, only rutin was highest in 2011 (low yield) (Table 4).

5. Phenolic compound concentration means for row orientation main effects

The following results (Table 5) are for compounds statistically significantly affected by main effects only; i.e., row orientation (Table 3).

Total flavan-3-ols, (+)-catechin, gallic acid, total flavonols, and peonidin 3-O-glucoside the were lowest from the N-S rows at 22 °Brix (Table 5), but the highest concentrations of total flavonols, isoquercetin and total anthocyanins were found in grapes from NE-SW rows with peonidin 3-O-glucoside the highest in the NW-SE rows. The lowest p-coumaric acid and malvidin 3-O-(6-O-acetyl) glucoside were recorded from the NW-SE rows. Generally, the lowest phenolic concentrations were from the N-S rows. The majority of phenolics quantified were lowest in grapes form N-S rows.

Ferulic acid, EGCG, total flavonols, quercetin and kaempferol were lowest from the N-S rows at 24 °Brix (Table 5) and (+)-catechin was highest from the N-S rows. The highest levels of total phenolic acids, caffeic acid and total flavonols were found in grapes at 24 °Brix from the NW-SE rows, whereas kaempferol and quercitrin were highest from the E-W rows (Table 5). Concentration differences were evident for total phenolic acids and caffeic acid among rows with lowest levels from the NE-SW rows. Cyanidin 3-O-glucoside was lowest in grapes at 24 °Brix (Table 5) from the E-W rows.

Table 5. Phenolic compound concentration (mg/L) means for row orientation main effects only at 22 °Brix, 24 °Brix and 26 °Brix.


Phenolics

Row orientation

p-value

E-W1

NE-SW1

N-S1

NW-SE1

22 °Brix

Total flavan-3-ols

*11.401 ± 0.84b

13.199 ± 0.52a

11.008 ± 0.79c

13.404 ± 0.72a

< 0.0001

(+)-Catechin

5.386 ± 0.45a

5.588 ± 0.35a

5.100 ± 0.50b

5.529 ± 0.48a

0.0015

(-)-Epicatechin

5.230 ± 0.40a

4.986 ± 0.35b

4.931 ± 0.14b

5.214 ± 0.32a

0.0037

EGCG2

0.784 ± 0.34b

2.625 ± 0.35a

0.976 ± 0.35b

2.660 ± 0.37a

< 0.0001

Gallic acid

1.504 ± 0.24a

1.669 ± 0.36a

1.173 ± 0.35b

1.621 ± 0.23a

0.0025

p Coumaric acid

31.723 ± 4.53a

25.671 ± 3.62b

29.117 ± 5.47ab

16.031 ± 2.11c

< 0.0001

Total flavonols

16.056 ± 1.44b

24.302 ± 2.10a

13.032 ± 1.38c

15.534 ± 1.28b

< 0.0001

Isoquercetin

1.711 ± 0.29c

3.095 ± 0.47a

1.569 ± 0.25c

2.693 ± 0.40b

< 0.0001

Quercetin

5.041 ± 0.79a

4.065 ± 1.95b

5.419 ± 0.90a

4.600 ± 0.64ab

< 0.0001

Total anthocyanins

407.968 ± 67.24b

452.845 ± 70.78a

387.571 ± 46.59bc

352.615 ± 41.01c

0.0012

PeoGluc3

14.802 ± 2.68ab

14.159 ± 2.07b

11.221 ± 2.18c

16.286 ± 4.64a

0.0017

MalGluc3

101.381 ± 29.37ab

113.582 ± 28.18a

86.223 ± 19.71bc

84.368 ± 19.97c

0.0051

PeoGlucAc4

11.203 ± 2.46a

10.909 ± 2.07a

10.429 ± 1.52ab

9.242 ± 1.47b

0.0487

MalGlucAc4

57.721 ± 11.69a

60.936 ± 16.43a

56.496 ± 13.93a

43.638 ± 12.06b

0.0240

24 °Brix

Total flavan-3-ols

*12.475 ± 0.57b

13.209 ± 0.74a

12.477 ± 0.61b

13.102 ± 0.59a

0.0005

(+)-Catechin

5.121 ± 0.26c

5.452 ± 0.33b

5.774 ± 0.49a

5.214 ± 0.28c

< 0.0001

EGCG2

1.993 ± 0.42b

2.663 ± 0.32a

1.460 ± 0.16c

2.772 ± 0.33a

< 0.0001

Total phenolic acids

85.961 ± 6.62b

57.244 ± 3.23d

63.865 ± 5.10c

117.728 ± 6.84a

< 0.0001

Caffeic acid

48.391 ± 6.25b

15.036 ± 1.20d

24.648 ± 3.24c

83.658 ± 6.31a

< 0.0001

p Coumaric acid

28.301 ± 3.51b

34.413 ± 3.03a

34.723 ± 4.89a

25.084 ± 1.82c

< 0.0001

Ferulic acid

6.996 ± 1.12a

5.541 ± 1.21b

2.905 ± 0.75c

6.915 ± 0.91a

< .00001

Total flavonols

16.711 ± 2.06b

16.440 ± 1.33b

13.836 ± 1.94c

34.406 ± 2.72a

< .00001

Rutin

0.643 ± 0.10b

0.632 ± 0.11b

0.713 ± 0.09a

0.715 ± 0.09a

0.0228

Quercetin

5.921 ± 1.48a

6.012 ± 1.01a

5.358 ± 1.09b

5.976 ± 1.95a

< 0.0001

Kaempferol

1.569 ± 0.21a

1.304 ± 0.07b

0.726 ± 0.28c

1.317 ± 0.074b

< 0.0001

Quercitrin

7.226 ± 1.02a

6.531 ± 0.92b

6.286 ± 0.99b

4.572 ± 1.56c

< 0.0001

CyGluc3

1.964 ± 0.26c

2.288 ± 0.48b

2.654 ± 0.57a

2.406 ± 0.54ab

0.0020

MalGlucCoum5

15.276 ± 2.71bc

14.547 ± 2.33c

16.403 ± 2.90ab

16.871 ± 2.69a

0.0280

26 °Brix

Total phenolic acids

*62.689 ± 4.05b

45.687 ± 5.90c

65.727 ± 4.22b

83.101 ± 5.83a

< .0001

Gallic acid

1.728 ± 0.24b

1.656 ± 0.32b

1.630 ± 0.24b

2.207 ± 0.34a

< .0001

Caffeic acid

29.942 ± 2.22c

18.491 ± 4.04d

31.803 ± 3.48b

48.986 ± 5.09a

< .0001

p Coumaric acid

27.207 ± 2.620a

18.567 ± 2.80b

25.866 ± 1.58a

26.007 ± 1.51a

< .0001

Ferulic acid

7.217 ± 1.01a

6.333 ± 1.02b

5.832 ± 0.57bc

5.266 ± 0.53c

0.0007

Total flavonols

16.921 ± 0.95c

19.041 ± 1.06b

16.530 ± 1.62c

27.448 ± 2.72a

< .0001

Rutin

0.593 ± 0.06b

0.637 ± 0.06a

0.594 ± 0.07b

0.633 ± 0.07a

0.0180

Isoquercetin

1.629 ± 0.26c

3.561 ± 0.57a

1.623 ± 0.26c

2.962 ± 0.56b

< .0001

Quercetin

6.102 ± 0.85b

7.330 ± 0.74a

6.658 ± 1.67b

6.558 ± 1.18b

0.0079

Kaempferol

1.682 ± 0.17b

1.403 ± 0.164c

1.219 ± 0.06d

1.993 ± 0.23a

< .0001

Quercitrin

7.510 ± 0.72b

6.745 ± 0.91c

7.029 ± 1.15bc

15.933 ± 1.60a

< .0001

CyGluc3

2.000 ± 2.00b

2.209 ± 2.20ab

2.324 ± 2.32a

2.426 ± 2.42a

0.0085

MalGluc3

78.425 ± 13.25b

74.599 ± 13.98b

103.821 ± 18.84a

78.957 ± 13.32b

< .0001

MalGlucAc4

65.654 ± 15.01b

60.996 ± 8.42b

63.239 ± 14.31b

86.945 ± 12.41a

0.0005

*Means given for compounds with significant row orientation main effects as indicated in Table 3. Different letters in the same row indicate significant differences in the content of the measured variables among the different row orientations according to Fischer's least significant difference test (p = 0.05). 1East-West; 1Northeast-Southwest; 1North-South; 1Northwest-Southeast. 2Epigallocatechin 3-O-gallate; 3Peonidin-, malvidin and cyanidin 3-O-glucoside; 4Peonidin- and malvidin 3-O-(6-O-acetyl) glucoside; 5Malvidin 3-O-(6-O-p-coumaroyl) glucoside.

Statistically significant differences in malvidin 3-O-(6-O-coumaroyl) glucoside were not found among any of the row orientations for grapes at 24 °Brix (Table 5). Similar to grapes at 22 °Brix, most phenolics quantified at 24 °Brix were lowest from the N-S rows.

Total phenolic acids, gallic acid, caffeic acid, total flavonols, kaempferol, quercitrin and malvidin 3-O-(6-O-acetyl) glucoside were highest from the NW-SE rows at 26 °Brix (Table 5). Statistically significant differences in caffeic acid and kaempferol among all row orientations were evident. Caffeic- and p-coumaric acid were lowest from the NE-SW rows, whereas ferulic acid was highest in grapes from E-W rows. Ferulic acid was not significantly different among the remaining rows.

Isoquercetin and quercetin were both highest from the NE-SW rows at 26 °Brix (Table 5). Malvidin 3-O-glucoside was highest from the N-S rows with no statistically significant differences among the E-W, NE-SW and NW-SE rows, whereas malvidin 3-O-(6-O-acetyl) glucoside was highest from the NW-SE rows. No statistical significant differences among the E-W, N-S and NE-SW rows for malvidin 3-O-(6-O-acetyl) glucoside were found. The majority of phenolics of this study quantified in grapes at 26 °Brix were highest from the NW-SE rows.

Generally, caffeic acid was lowest from the NE-SW rows at 24 °Brix and 26 °Brix, whereas total flavonols and kaempferol lowest from the N-S rows at 22 °Brix and 24 °Brix and 24 °Brix and 26 °Brix (Table 5). Highest total phenolics, caffeic acid and total flavonols were found in grapes from the NW-SE rows at 24 °Brix and 26 °Brix and isoquercetin was highest from the NE-SW rows at both 22 °Brix and 26 °Brix. NW-SE (low light exposure in the morning) was found to lead to increased concentrations of most phenolics measured at 24 °Brix and 26 °Brix ripeness.

6. Principal component analysis

The PCA biplot (Figure 2) of the first two principal components (PC1 and PC2) illustrates the association of phenolic compounds at 22 °Brix in 2008, 2009, 2010 and 2011 with the N-S, E-W, NE-SW and NW-SE rows, explaining 59.42 % of the variation in the data. The major source of variation is row orientation with PC1 separating the NE-SW row from the E-W, N-S and NW-SE rows, while PC2 separates the NW-SE row from the E-W and N-S rows. Variables with the highest squared cosine values (data not shown) on PC1 are (+)-catechin, caffeic acid, ferulic acid, total flavonols, isoquercetin, quercetin, kaempferol, total anthocyanins, petunidin 3-O-glucoside, malvidin 3-O-glucoside, delphinidin 3-O-(6-O-p-coumaroyl) glucoside and petunidin 3-O-(6-O-p-coumaroyl) glucoside. These variables were positively associated with the NE-SW rows.

Flavan-3-ols, epigallocatechin 3-O-gallate, total phenolic acids, p-coumaric acid, peonidin 3-O-(6-O-acetyl) glucoside and malvidin 3-O-(6-O-acetyl) glucoside had the highest squared cosine values on PC2. The NW-SE rows were positively associated with flavan-3-ols, but negatively associated with total phenolic acids.

Figure 2. Principal component analysis bi-plot illustrating the association of 23 individual phenolic compounds measured in grapes harvested in 2008, 2009, 2010 and 2011 at 22 °Brix from N-S, E-W, NE-SW and NW-SE rows. Gluc = glucoside; Coum = coumaroyl; EGCG = Epigallocatechin 3-O-gallate.

Principal component 3 explains only an additional 13.85 % of variation (data not shown) and does not improve interpretability; it was therefore not included in the biplot. Only quercitrin, (-)-epicatechin and peonidin 3-O-glucoside had the highest squared cosine value on PC3. Close groupings of the same rows from different vintages may be an indication that the largest variation in phenolic content are between rows, while variation between vintages are less pronounced.

At 24 °Brix, the PCA biplot (Figure 3) of the first two principal components (PC and PC2) illustrates the association of phenolic compounds in 2008, 2009, 2010 and 2011 with the N-S, E-W, NE-SW and NW-SE rows, explaining 57.61 % of the variation in the data. In general, the different vintages and rows separated and grouped reasonably well.

Figure 3. Principal component analysis bi-plot illustrating the association of 23 phenolic compounds measured in grapes harvested in 2008, 2009, 2010 and 2011 at 24 °Brix from N-S, E-W, NE-SW and NW-SE row orientations. Gluc = glucoside; Coum = coumaroyl; EGCG = Epigallocatechin-3-O-gallate.

The main source of variation is row orientation with PC1 separating NW-SE from E-W, NE-SW and N-S rows, while PC2 separates individual vintages within rows. Variables with the highest squared cosine values (≥ 0.5) on PC1 were EGCG, phenolic acids, caffeic acid, p-coumaric acid, ferulic acid, total favonols, quercetin, quecitrin and petunidin 3-O-glucoside. Phenolic acids and flavonols were mainly associated with the NW-SE rows.

Catechin (+), total anthocyanins, malvidin 3-O-glucoside, delphinidin 3-O-glucoside, peonidin 3-O-(6-O-acetyl) glucoside and malvidin 3-O-(6-O-acetyl) glucoside had the highest squared cosine values on PC2. Gallic acid, rutin and cyanidin 3-O-glucoside had the highest squared cosine values on PC3, but it explained only an additional 15.17 % of variation in the biplot. It does not improve interpretability and was therefore not included in the biplot.

At 26 °Brix, the PCA biplot (Figure 4) of the first two principal components (PC1 and PC2) illustrates the association of phenolic compounds in 2008, 2009, 2010 and 2011 with the N-S, E-W, NE-SW and NW-SE rows, explaining 54.75 % of the variation in the data. The different vintages and rows showed excellent separation and grouping. PC1 mainly separates NW-SE from the other rows, while PC2 separates NE-SW from N-S and E-W. The phenolic compounds with the highest squared cosine values (≥ 0.5) on PC1 were total flavan-3-ols, (+)-catechin, total phenolic acids, gallic acid, caffeic acid, ferulic acid, total flavonols, kaempferol, quercitrin and malvidin 3-O-(6-O-acetyl) glucoside. NW-SE rows were mainly associated with flavonols and phenolic acids. Epigallocatechin 3-O-gallate, p-coumaric acid, isoquercetin, quercetin and petunidin 3-O-(6-O-acetyl) glucoside have the highest squared cosine values on PC2 (data not shown).

Most anthocyanins were associated with E-W rows and flavan-3-ols with NE-SW. Peonidin 3-O-glucoside, malvidin 3-O-glucoside and malvidin 3-O-(6-O-coumaryol) glucoside have the highest squared cosine values on PC3, but they contributed only an additional 13.76 % to the variability in the PCA biplot; PC3 does not improve interpretability and was therefore not included in the biplot.

Figure 4. Principal component analysis bi-plot illustrating the association of 23 phenolic compounds measured in grapes harvested in 2008, 2009, 2010 and 2011 at 26 °Brix from the N-S, E-W, NE-SW and NW-SE rows. Gluc = glucoside; Coum = coumaroyl; EGCG = Epigallocatechin-3-O-gallate.

Discussion

The results show that row orientation and grape ripeness effects were detectable for individual phenolic compounds and phenolic classes. Generally, vintage effect was not evident among the phenolics quantified, except for gallic acid and malvidin 3-O-glucosides in 2011 (22 °Brix), epicatechin and total flavan-3-ols in 2008 and 2010 (24 °Brix) respectively, and rutin in 2011 (26 °Brix). Hunter et al. (2016) reported that climatic conditions among vintages were similar, reaching comparable maturity indices in all four seasons; therefore, the vintage factor was not taken into account and the grapes per ripeness level were considered as replications.

Ambient (air) temperature of the experimental vineyard during grape development never exceeded 35 °C (Hunter et al., 2016). Ambient temperature between 30 °C and 35 °C is the critical temperature range for flavonoid and non-flavonoid biosynthesis (Tarara et al., 2008; Bonada et al., 2015). Temperatures above 35 °C can be detrimental to, for example, anthocyanin and flavonol accumulation, since they share the same flavonoid biosynthetic pathway (Bonada et al., 2015).

Considering the relatively uniform vegetative characteristics of the canopy architecture (Figure 1), canopy interior temperature primarily reflected ambient (air) temperature at meso-level (Hunter et al., 2017) and therefore temperature profiles inside the canopy did not show marked differences among the different rows. Canopy temperature per se consequently played a minor role in the final berry temperature of VSP-trellised vines of the different rows. Despite this, berry temperature and light penetration were slightly affected by the canopy orientation. Berry temperature of grapes from the E-W and NE-SW rows were slightly lower than grapes from the N-S and NW-SE rows in mid-morning (Table 2). However, at around midday, significant differences in temperature among the rows were not found. In late afternoon, grapes from the NE-SW rows had the highest temperature. This may be a consequence of the high levels measured for total flavonols, isoquercetin and total malvidin 3-O-glucosides at 22 °Brix and isoquercetin and quercetin measured at 26 °Brix. The respective berry temperatures of the N-S and E-W rows in the late afternoon were significantly different, but those of the N-S and NW-SE rows were not different. Nevertheless, on average over the four consecutive seasons, only grapes from the E-W rows were significantly lower in temperature (Table 2).

Beggs and Wellman (1994) reported that increased light exposure of grape berries, and not temperature, enhances anthocyanin biosynthesis. Downey et al. (2006) reported that increased grape bunch temperature can result in a decrease in total anthocyanin content, and Cagnasso et al. (2011) reported that cooler climates seem to favour anthocyanin accumulation in Carema grapes. Anthocyanins can increase at higher grape ripeness levels as reported by Guidoni and Hunter (2012), who showed that increased anthocyanin levels in Shiraz grapes is related to cooler temperatures and moderate light penetration under the control of photoreceptors during the final stages of ripening, which may prolong anthocyanin synthesis. Despite this, these flavonoids can also undergo polymerisation during the last ripening phase before harvest, leading to a decrease in anthocyanin levels. Therefore, separating the effects of temperature and light on grape composition is difficult because many of the biochemical pathways are light and temperature (solar radiation) sensitive (Spayd et al., 2002).

Anthocyanins reached their highest levels at 22 °Brix from the N-S rows, whereas flavonols and flavan-3-ols seemed to be the highest at 24 °Brix from the same row orientation (Minnaar, 2018). Anthocyanin concentrations tended to decrease as grape ripeness levels increased. Hunter et al. (2021) reported that anthocyanins in Shiraz grapes from N-S rows decreased as the grapes ripend. Phenolic acids were not substantially affected by grape ripeness levels but were highest at 24 °Brix from N-S rows.

For E-W rows, flavan-3-ols and phenolic acids reached their highest levels at 22 °Brix, after which a decrease occurred as the grapes ripened further (Minnaar, 2018). Jordão et al. (2001) reported flavan-3-ols of Castelão Francês and Touriga Francesa grapes to decrease during the grape maturation process. Flavonols, on the other hand, appeared to be highest at a ripeness level of 24 °Brix. This is similar for grapes from N-S rows of this study. Anthocyanins tended to already be highest at 22 °Brix, after which a decrease occurred as the grapes ripened. This is similar to the findings for the grapes from N-S rows. The grapes from both the N-S and E-W rows tended to reach their highest levels of anthocyanins and flavonol at 22 °Brix and 26 °Brix respectively.

The NE-SW rows brought about only a slight variation in flavan-3-ol levels from 22 °Brix to 26 °Brix ripeness level. Flavonols and phenolic acids ostensibly reached their highest levels at 22 °Brix, after which a decrease occurred as the grapes ripened (Minnaar, 2018). Anthocyanins seemed to reach their highest levels at 22 °Brix, as was the case for the grapes from N-S and E-W rows.

Northwest-southeast rows were the cause of flavan-3-ols already reaching their highest levels at 22 °Brix, after which a decrease occurred as the grapes ripened. Flavonols, phenolic acids and anthocyanins tended to be highest at 24 °Brix. Grapes from NE-SW rows seemed to reach their highest anthocyanin, phenolic acid and flavonol levels before those from NW-SE rows, but they tended to reach their highest flavan-3-ol levels before those from the NE-SW rows (Minnaar, 2018). Certain treatments (rows) had no effect on phenolics (no main effects, p-values not significant) which means that row direction prevented sufficient UV-B radiation from entering the canopy, thereby delaying accumulation, but did not completely inhibit synthesis.

Monomeric anthocyanins not affected by treatment (not significant main effects) maybe a result of the shift in anthocyanin composition during grape maturation due to the hydroxylation of the B-ring (Ryan and Revilla, 2003); this is an indication of post maturity and the process in which cells irreversibly stop dividing and enter a state of permanent growth arrest without undergoing cell death, also known as senescence. In contrast to findings by Guidoni and Hunter (2012), Ryan and Revilla (2003), and Pérez-Magarino and González-SanJosé (2006) reported that maximum accumulation of anthocyanins occurred when grape sugar levels reached between 20 and 25 °Brix in Tempranillo and Cabernet-Sauvignon grapes planted in an E-W direction. This is in agreement with the results of this study, in which maximum anthocyanin accumulation already occurred at 22 °Brix, but in grapes from NE-SW rows. Furthermore, De la Hera Orts et al. (2005) reported that maximum anthocyanin levels were reached at between 20 and 24 °Brix in Monastrell grapes, but Fourmand et al. (2006) found that anthocyanins remained unchanged from 20 °Brix to 26 °Brix.

Chorti et al. (2010) reported increased anthocyanins in artificially (partially) shaded Nebbiolo grapes at harvest and planted in N-S rows (northern-hemisphere), compared to grape bunches without shading. Partially shaded grapes from N-S rows with limited light penetration in the canopy can be considered to be similar to the NW-SW rows of this study in terms of light penetration (low light penetration in the afternoon). Contrary to Chorti et al. (2010), Bubola et al. (2017) and Feng et al. (2017) have reported increased total anthocyanins in Teran grapes at 23 °Brix and Pinot noir grapes at 22 °Brix from N-S-orientated vines. Vines underwent 50 % defoliation thereby increasing the light penetration and temperature in the fruit zone, compared to no leaf removal.

Ultraviolet-B radiation can upregulate chalcone synthase; i.e., flavonoid biosynthesis and phenylalanine ammonia-lyase in the phenylpropanoid genes pathway (Jansen et al., 1998). Flavonols can therefore increase when grapes are exposed to increased light penetration in the canopy (Alonso et al., 2016; Pastore et al., 2017a) and expression of gene encoding flavonol synthase in grape skins (Pastore et al., 2013). Flavonols are sensitive to changes in environmental conditions and can respond as a protectant against UV-containing light. Temperatures above 35 °C can, however, have a detrimental effect on flavonol biosynthesis (Pastore et al., 2017b). In contrast to the above, flavonol levels in this study were lowest in grapes from N-S rows (high light penetration all day) at 22 °Brix, 24 °Brix and 26 °Brix; this is similar to work by Cohen et al. (2012), who found lower concentrations of flavonols in Merlot grapes at 22 °Brix of vines planted in N-S rows subjected to heating in the fruit zone (via heat blowers), compared to grapes at ambient temperature. Increased temperature in the fruit zone may have inhibit flavonol biosynthesis.

In this study, flavonols that decreased during grape ripening (Table 5) may be attributed to oxidation through coupled reactions and may act as co-pigments with anthocyanins in co-pigmentation processes (Bimpilas et al., 2015). The increase in flavonols (i.e., grapes from NW-SE rows at 24 °Brix and 26 °Brix) could be due to the high light penetration in the canopy in the morning, resulting in increased grape bunch temperature (Table 2), which stimulates flavonol biosynthesis; some UV radiation is required to stimulate gene transcription to produce flavonol synthase (Holton and Cornish, 1995). Flavonol synthase catalysis the conversion of dihydrokaemferol to kaempferol and dihydroquercetin to quercetin, among other flavonols. Friedel et al. (2015) have also reported that flavonols increased in Riesling grape bunches at harvest with increased light penetration in the canopy (E-W direction) due to leaf removal in the fruit zone. It seems that flavonol biosynthesis favours increased light penetration in the fruit zone in the morning; i.e., in NW-SE rows.

In the present study, the decrease in total phenolic acids in grapes from the E-W, NE-SW and NW-SE rows as grapes ripened (24 °Brix to 26 °Brix) may be a result of down-regulated flavonoid biosynthesis due to limited light penetration in these canopies. Grapes from N-S rows showed a slight increase in phenolic acids with ripeness. Additionally, the differences in grape phenolic acid levels among rows may be a consequence of these reactive compounds changing to galloylated derivatives when exposed to increased PAR, thereby decreasing free acids (Jordão et al., 2001). Trihydroxybenzoic acids in grapes do not usually respond to UV radiation; hydroxycinnamic acids, however, respond to variation in light penetration in the canopy (Spayd et al., 2002), which could have contributed to the observed differences in their concentrations in grapes depending on the row orientation in this study (Table 5). Rescic et al. (2016) reported that total phenolic acids were higher in Uva Longanesi and Istrian Malvasia grapes from E-W and N-S rows respectively at 22 °Brix after 50 % defoliation, compared to vines with no defoliation treatment. In this study, the phenolic acid levels were highest in grapes from NW-SE rows at 24 °Brix and 26 °Brix.

Flavan-3-ol levels in grapes from the NE-SW and NW-SE rows at both 22 °Brix and 24 °Brix were higher than those in grapes from to N-S and E-W rows. Similar results have been reported by Romboli et al. (2017), who found increased flavan-3-ol levels in low vigour Sangiovese grape planted in NE-SW rows at harvest, compared to high vigour vines. In the present study, total flavan-3-ols generally increased until grape ripeness reached 24 °Brix, after which it was not affected by the treatment as grapes ripened to 26 °Brix.

In a study by Downey et al. (2004), flavan-3-ol levels in artificially shaded Shiraz grapes during berry ripening were found to be lower than in exposed bunches; however, the decrease in total flavan-3-ols during berry ripening was greater in exposed bunches, to the extent that the levels were virtually the same in shaded and exposed berries at harvest. Pastore et al. (2017a) reported flavan-3-ols to have minimal response to environmental factors; however, PAR rather than UV radiation had a positive effect on flavan-3-ol synthesis. In addition, Scrafidi et al. (2013) reported higher concentrations of total flavan-3-ols in exposed Grillo grapes at 21 °Brix and planted to N-S rows than in boxed-in grapes. In contrast to work by Pastore et al. (2017a) and Scrafidi et al. (2013), the results of this study show that grapes from N-S rows (high light penetration all day) had the lowest levels of total flavan-3-ols. However, in agreement with the flavan-3-ol levels found in this study, Allegro et al. (2019) reported that increased light penetration in the canopy/fruit zone of Grechetto Gentile grapes did not increase the accumulation of flavan-3-ols. Higher levels of total flavan-3-ols, phenolic acids and flavonols are to be expected in N-S rows (high light penetration in the canopy), compared to E-W, NE-SW and NW-SE rows; however, this was not the case in this study. The low levels of total flavan-3-ols and total flavonols at 22 °Brix and total flavonols at 24 °Brix may be due to over-exposure of grape bunches to light (i.e., high initial PAR at 22 to 24 °Brix grape ripeness), which could have inhibited some metabolic processes (Downey et al., 2006).

The variation in phenolic concentrations reported in the cited literature was likely caused by defoliation and artificial shading, resulting in grape bunches being over- or under-exposed to light. Artificial shading of grape bunches (light manipulation/boxed-in) caused a decrease in certain phenolic compounds and over-exposure/artificial heating an increase. The results reported by international authors were therefore primarily due to the manipulation of the canopy and fruit zone in addition to “natural” row orientation creating a microclimate. The cited results may, however, be linked to the results and deductions of this study regarding the effect of different row orientations on the light penetration in the fruit zone. Hunter et al. (2016) have reported that the microclimate profiles (light) of canopies are affected by changes in row orientation.

This is the first investigation in South Africa to demonstrate the effect of grapevine row orientation on individual phenolic compounds in Syrah grapes, as well as the combined effect of vintage and grape ripeness level, although vintage did not play a major role. The results showed that phenolic compound biosynthesis involves the interaction of light and grape ripeness (and temperature). The effect of different light regimes brought about by the different row orientations and grape ripeness on phenolic compound development was shown.

Under realistic field conditions (i.e., different row orientations), planting grapevines in N-S and NE-SW rows can result in higher temperatures in the fruit zone (Hunter et al., 2016) than planting in E-W and NW-SE rows. An increase in temperature in the fruit zone resulting from grapevine row direction has been found to cause a decrease in certain phenolics, which is probably due to a combination of both degradation and biosynthesis inhibition (Mori et al., 2005; Tarara et al., 2008; Feng et al., 2017). In this study, NE-SW and NW-SE row orientation generally had a positive effect on phenolic development, which may be linked to shorter periods of light penetration/exposure in the fruit zone; i.e., low light exposure in the morning for NE-SW and low light exposure in the afternoon for NW-SE rows. However, the effect of light on the biosynthesis of phenolics can vary depending on the grape cultivar (Rustioni et al., 2011; Bonada and Sadras, 2014). Ambient temperatures above 35 °C may inhibit phenolic biosynthesis even under different light regimes. Nevertheless, light intensity differences in the fruit zone are an important factor for phenolic compound development/accumulation, irrespective of row orientation per se (Koyama et al., 2012; Zoratti et al., 2014). Accumulation is also dependent on a combination of moderate exposure of grape bunches to light and moderate temperature in the canopy.

Within the context of global warming, many grape growers are facing premature véraison due to early-season temperature increases, which could lead to sunburn of grape-berry skin tissue, as well as the inhibition of the biosynthesis of, for example, anthocyanins and flavan-3-ols (Hunter et al., 2010). Achieving phenolic maturity when grape sugar content and acidity are at optimum levels can sometimes be challenging in warm, semi-arid wine-growing regions, such as in Robertson in South Africa. As a result, growers have to either wait for phenolic maturity, at the cost of producing wines with high alcohol content and sometimes too low acidity, or harvest at desired sugar-acid levels and risk producing wines with poor colour and unripe tannins.

The desirable conditions for grapevines growing in a warm semi-arid environment are moderate to low light penetration of the canopy. However, the orientation of vineyard rows has proven to be one of the viticultural practices enabling successful canopy microclimate manipulation, leading to grape phenolic improvement. Since in practice, it may not be possible to achieve suitable grapevine row orientation in all environments, the management of the fruit zone by means of pruning, shoot positioning, removal of infertile shoots and leaf removal remains an option for increasing/decreasing grape exposure to light, irrespective of row orientation.

Conclusion

The results indicate that row orientation and ripeness level can be applied to control phenolic development. The effect of vintage, however, cannot be controlled. In the southern hemisphere, light conditions induced in the grape bunch zone primarily in the NE-SW (at 22 °Brix) and NW-SE (at 24 °Brix and 26 °Brix) rows positively affected most phenolics. Grapes from these row orientations could result in Syrah wines with an improved phenolic profile and have the potential to extend the range of Syrah wine styles. However, it is unlikely that a single factor can be applied to all growing conditions and grape cultivars. It is advised that viticulture-related factors, such as fertility, berry set, berry size and berry yield, be taken into account before making a decision on a specific row orientation. It is important to consider the characteristics of specific growing regions when determining the effect of row orientation on grape phenolic content. In the light of this, the results of this study can be applied to grapevines planted in specific row orientations in clayey loam soil and trained to a VSP trellis system. Commercial vineyard parcels/blocks, different terroirs and the hombre influencia can also have an effect on grape phenolic profiles. Therefore, further research is needed to understand the associations that exist among vine phenology, microclimate/light penetration and grape phenolic content.

Acknowledgements

The authors would like to thank the Agricultural Research Council and Winetech for providing funding and infrastructure. This research forms part of a PhD thesis obtained by P Minnaar at Stellenbosch University, South Africa.

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Authors


Phillip Minnaar

minnaarp@arc.agric.za

Affiliation : Post-Harvest & Agro-Processing Technologies, ARC Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch, 7600

Country : South Africa


Marieta Van Der Rijst

Affiliation : Agricultural Research Council, Biometry, Private Bag X5026, Stellenbosch 7599, Stellenbosch, 7600

Country : South Africa


JJ Hunter

Affiliation : Plant Protection and Viticulture Division, ARC Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch, 7600

Country : South Africa

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