Haselgrove, Botting, van Heeswijck, Høj, Dry, Ford & Iland Canopy microclimate and berry composition 141

Canopy microclimate and berry composition:

The effect of bunch exposure on the phenolic composition
of Vitis vinifera L cv. Shiraz grape berries
L. HASELGROVE1, D. BOTTING1,2, R. van HEESWIJCK1,2,
P.B. HØJ1,2,3, P.R. DRY1,2, C. FORD1,2 and P.G. ILAND1,2,4
1
Department of Horticulture,Viticulture and Oenology,The University of Adelaide ,
Waite Campus, PMB1, Glen Osmond SA 5064
2
CRC for Viticulture, PO Box 154, Glen Osmond SA 5064
3
The Australian Wine Research Institute, PO Box 197, Glen Osmond SA 5064
4
Corresponding author: P.G. Iland, facsimile +61 8 8303 7116, e-mail patrick.iland@adelaide.edu.au

Abstract
Studies on the effect of light exposure on specific phenolic compounds of berries from Shiraz vines
grown in a hot climate are reported. Berries that had developed on bunches receiving high levels of
ambient light generally had the highest relative levels of quercetin-3-glucoside and a lower proportion of
their malvidin anthocyanins as the coumarate derivative, compared to berries that had developed on
bunches in shaded canopy conditions. The response of total anthocyanin levels to treatment conditions
was variable and depended on the degree of bunch shading and the resultant berry temperature. It
appears that a high degree of bunch exposure in hot climates is not conducive to optimal anthocyanin
accumulation in berries. The interactive effects of light and temperature on berry phenolic content and
concentration are discussed.

Keywords: grape berry, bunch exposure, light, temperature, phenolics, anthocyanins, quercetin-3-glucoside

Introduction this paper we examine the relationship between bunch

The importance of the effect of canopy microclimate on
berry composition was initially raised by Shaulis et al.
(1966) in their investigations with Concord grapevines
trained to the Geneva Double Curtain (GDC) training
system. This work stimulated others, including Smart
(1985a, 1985b), and Carbonneau (1987a, 1987b) to
study light interception within grapevine canopies, using
a range of trellis and training systems. Smart (1985a)
adopted the term ‘microclimate’ to define the environ-
mental conditions within the immediate vicinity of the
leaves and fruit.
In grapevine canopies, depending on canopy archi-
tecture, leaves and bunches can develop in conditions
varying from heavily shaded (shaded canopies) through
to fully exposed (open canopies). Generally, berries that
develop in open canopy conditions, as opposed to those
that develop in shaded canopy conditions, have higher
juice sugar concentration (measured as total soluble
solids), improved acid balance (lower juice pH and high-
er titratable acidity), less incidence of unripe herbaceous
fruit characters and often increased concentration of
berry phenolics, including anthocyanins (Gladstones
1992). More recently, there has been interest in the effect
of canopy microclimate on individual phenolic com-
pounds of black grapes (for example, Price et al. 1995). In
exposure and the levels of both quercetin-3-glucoside
and various forms of anthocyanins, respectively in Shiraz
berries sampled from bunches with varying degrees of
bunch exposure on vines grown in a hot climate.
Materials and methods
Description of trial site
The vineyard site was the Cooperative Research Centre
for Viticulture (CRCV) trellis trial site at Qualco, located
in the Riverland district of South Australia, approximate-
ly 200 kilometres NE of Adelaide. The climate is described
as being hot and arid with a mean January temperature
of approximately 22.8°C, low rainfall, averaging 130 mm
per growing season (Smart and Dry 1980) and with 1642
biologically effective day degrees (Gladstones 1992). Soils
in the treatment blocks were relatively uniform, typified
by loamy, coarse sand. The vines used in the experiment
were cv. Shiraz, clone BVRC32 grown on Ramsey root-
stock and planted in 1991. Row and vine spacing were
3.3 metres and 2.5 metres respectively, with the rows
orientated in an east-west direction.
A trial investigating the effects of various trellis sys-
tems on fruit composition had been established at this
site. The trellis systems, incorporated in a randomised
142 Canopy microclimate and berry composition
complete block design, were High Single Cordon (HSC),
Minimally Pruned (MP), Smart-Dyson (SD), Scott Henry
(SH) and Vertical Shoot Positioned (VSP) (Smart and
Robinson 1992). Sections of this trellis trial were used in
the experiments described below.
Experiment 1 – comparison of the composition of berries
sampled from either fully exposed or fully shaded bunches,
under artificially created conditions
Vines were trained to the SH configuration (spur
pruned), and pruned to approximately 20 nodes per
metre of cordon. Bunches on the lower cordon were used
for all treatments. Prior to application of the treatments,
leaves were removed in the vicinity of the bunches along
the upper and lower cordons to achieve fully exposed
bunches along the lower cordon. Additionally, bunches
were selectively removed from shoots on the lower cor-
don, leaving one bunch per shoot, this being the lower
bunch. Treatments were applied when berries were at
the peppercorn stage (Stage 29; Modified E-L system,
Coombe 1995). Two levels of bunch exposure were
imposed, fully shaded and fully exposed. Bunches on
shoots where leaves had been removed represented the
fully exposed treatment. The fully shaded treatment was
achieved by completely enclosing the bunches within a
cylindrical wire cage covered with aluminium foil. The
bottom was left open to provide ventilation for the
bunches. The inner surface was spray-painted black to
absorb any stray light entering through the bottom of the
cage. The cages were fastened to the cordon wire by a
short string, and the top of the foil was completely sealed.
Treatments were applied in a completely randomised
experimental design. Two replicates of each treatment
were applied to each of 16 vines, giving a total of 32
replicates per treatment. Each vine was randomly
allocated to be sampled at a given berry maturity.
Bunches were harvested at four different sampling occa-
sions between veraison and late maturity, four replicates
of each treatment being sampled at each time. Prior to
the removal of the bunches at sampling, each bunch was
marked to identify the north-facing side.
Fifteen berries were randomly removed from each
sample bunch separately from the following three posi-
tions: (a) north-facing berries, (b) berries positioned in
the interior of the bunch and (c) south facing berries.
Each sample of 15 berries was further subdivided into
one lot of five berries and another of ten berries. Each lot
of five berries was separately pressed and total soluble
solids determined on the expressed juice. The other lots
of ten berries were stored frozen (–80°C) for later extrac-
tion and analysis of phenolic compounds. Prior to extrac-
tion and analysis, six berries were randomly sampled
from each pool of ten berries and their skins removed
while the berries were partly frozen. Each sample of skins
was separately weighed, shredded and a known weight
taken for extraction and analysis of phenolic composi-
tion by HPLC. Only data for the north-facing side of fully
exposed and fully shaded bunches is reported here, so as
to evaluate the extremes of bunch exposure.
The temperature within the bunches of both treat-
Australian Journal of Grape and Wine Research 6, 141–149, 2000
ments was monitored using thermistor probes positioned
within the interior of the bunch. Data was collected using
a 16-channel macro 32 Starlog Data Logger. All replicates
from both treatments on two of the trial vines were
monitored continuously for six days in mid January, with
measurements taken at five-minute intervals.
Experiment 2 – comparison of the composition of berries
sampled from bunches with different degrees of exposure to light
under natural conditions
A composite random 50-berry sample was taken
separately from bunches positioned either in the centre
(termed ‘centre’) or the north-exposed side (termed
‘north’) of four separate HSC trellised vines at different
maturity stages during the period of berry ripening. After
dividing each sample (four replicates of each treatment,
at each sampling date) into two 25-berry lots, one lot
was processed to determine juice total soluble solids and
the other lot was frozen prior to extraction and analysis
of phenolic compounds.
The temperature within four ‘centre’ and four ‘north’
bunches was monitored as described in Experiment 1,
but in this case monitoring occurred over the period
between berry set and maturity. The temperature values
for ‘centre’ and ‘north’ bunches were separately aver-
aged and the difference in temperature calculated at each
recording.
An estimate of bunch exposure was obtained by mea-
suring photosynthetically active radiation (PAR) with a
Sunfleck Ceptometer (Decagon Devices Inc., Cambridge,
England) positioned (at least five times) in the bunch
zone either in the centre of the canopy or on the north
facing side of the canopy. Each reading was expressed as a
percentage of ambient light intensity. Readings for each
set of measures were averaged to calculate an estimate of
bunch exposure.
Experiment 3 – comparison of the composition of berries
sampled from bunches on different trellis systems
Random samples of 100 berries were taken separately
from bunches on four vines of each trellis system (HSC,
MP, SD, SH and VSP) at approximately a berry maturity
of 23 to 24°Brix. After dividing each sample into two 50-
berry lots, one 50-berry lot was processed to determine
juice total soluble solids, while the other lot was stored as
frozen berries for extraction and analysis of phenolic
compounds.
An estimate of bunch exposure for the bunches on
vines of each trellis system was obtained by placing the
ceptometer in the bunch zone of vines in each trellis sys-
tem and taking readings at various angles to the sun.
Each reading was expressed as a percentage of ambient
light intensity. Readings for each set of measures were
averaged to calculate an estimate of bunch exposure.
Extraction of phenolic compounds from skins and berries
Extraction of phenolic compounds was carried out with
either 95% v/v aqueous acidified ethanol (pH 1) at 70°C
for five minutes (for skin sections from experiment 1) or
with 50% v/v aqueous acidified ethanol (pH 2) at 25°C
Haselgrove, Botting, van Heeswijck, Høj, Dry, Ford & Iland
for one hour (for portions of whole berry extracts from
Experiments 2 and 3). The extraction conditions used for
Experiment 1 were essentially that of Price et al. (1995)
and for Experiments 2 and 3, that of Iland et al. (1996).
HPLC analysis
A Beckman System Gold HPLC, comprising a Beckman
126 NM solvent module coupled to a Beckman 168
photo-diode array detector was used for all analyses. A
C18 reverse phase column was used (250 × 2.5 mm i.d.;
201TP54), maintained at 30°C and protected by a guard
column of the same material. Gradient elution was
employed, with Solvent A being 1% v/v phosphoric acid
in water, and Solvent B being 1% phosphoric acid in
80% v/v acetonitrile, with a flow rate of 0.6 mL/min.
Gradient conditions were: 0 min, 100% A, 0% B; 30 min,
65% A. 35% B; 40 min 0% A, 100% B; 45 min, 100% A,
0% B; 55 min, 100% A, 0% B. The photo-diode array
recorded spectral scans at a frequency of 2 Hz, over a 4
nm interval, between 200–600 nm.
Anthocyanins were monitored at 520 nm. To confirm
the identity of peaks of the anthocyanin profile of skin
and berry extracts, peak fractions eluting during the
HPLC were analysed by liquid chromatography-mass
spectrometry (LC-MS). Distinct mass data for compounds
showing maximal absorbance in the λ = 520 nm range,
with HPLC retention times of t = 28.3, t = 32.8 and t =
36.2 min respectively, were observed (data not shown).
The data identified the three major HPLC peaks in the
berry skin extracts as malvidin-3-glucoside, malvidin-
3-(acetyl)-glucoside and malvidin-3-(p-coumaryl)-
glucoside, respectively. The LC-MS results and other
HPLC chromatograms indicated that the three malvidin
derivatives accounted for the majority of the antho-
cyanins present (generally > 75 %). The elution order of
the anthocyanin compounds appeared to be identical to
previously published studies (Baldi et al. 1995, Roggero et
al. 1986, Wulf and Nagel 1978). Concentrations of
malvidin-3-glucoside and its acylated derivatives in skin
and berry extracts were calculated from the absorbance
value corresponding to maximum peak height using a
reported extinction coefficient of malvidin-3-glucoside of
500 (100 mL/g/cm) (Somers and Evans 1974, Somers
and Evans 1977).
Quercetin-3-glucoside was monitored at 353 nm
(Price 1994). The composition of the peak observed in
chromatograms of skin and berry extracts, which eluted
at the same retention time (31.5 min) as pure quercetin-
3-glucoside was obtained by electrospray mass spectro-
metry. This technique also showed that quercetin-3-
glucuronide co-eluted with quercetin-3-glucoside under
these HPLC conditions. To determine the proportion of
quercetin-3-glucoside in the peak, this fraction was
collected and treated with Helix promatia glucosidase, a
glucosidase enzyme of narrow specificity. This enzyme
hydrolyses quercetin-3-glucoside and therefore in
preparations treated with this enzyme the glucuronide
alone now elutes at the retention time of 31.5 min. The
hydrolysate and an untreated control were analysed via
HPLC using similar conditions as previously. Comparison
Canopy microclimate and berry composition 143
of the peak areas before and after hydrolysis indicated
that quercetin-3-glucoside represented 60% of the peak
area; a figure which is consistent with the ratio deter-
mined in leaves of Moroccan Vitis vinifera cultivars by
Hmamouchi et al. (1996), and which was adopted for all
samples. The concentration of quercetin-3-glucoside in
skin and berry extracts was quantified by comparing the
area of the peak corresponding to quercetin-3-glucoside
with a standard curve obtained from pure quercetin-
3-glucoside and accounting for the factor of 60% as
determined above.
Resveratrol was monitored at 307 nm, based on the
chromatogram of a solution of pure resveratrol. The
concentration of resveratrol in skin and berry extracts
was quantified by comparing the area of the peak cor-
responding to resveratrol with a standard curve obtained
from pure resveratrol.
Expression of results
Although a number of anthocyanins were separated by
HPLC analysis, only the data from the analysis of malvi-
din-3-glucoside and its acylated derivatives is reported
here; collectively they are referred to as anthocyanins.
Results were expressed in equivalents of malvidin-3-
glucose on either the basis of content (amount per berry)
or concentration (amount per gram berry weight). In the
developmental studies (Experiments 1 and 2) changes in
composition over time were evaluated on a content basis
as this removes the influence of changes in berry weight
and provides a valid interpretation of variations in
absolute amounts of components during berry ripening.
When comparisons were made between treatments
where berry weight differed, data were assessed on a con-
centration basis rather than content basis, since this
approach relates values obtained to a similar area of skin,
and thus more readily reflects component response to
light interception. In all cases, comparisons were made
between berries harvested from different treatments at a
similar stage of berry maturity (measured as °Brix) to
avoid the interference of degree of maturity on com-
ponent content and concentration.
Statistical analysis
In Experiments 1 and 2 differences between the measures
of berry weight and berry composition due to the dif-
ferent treatments were assessed by one-way analysis of
variance, while in Experiment 3, relationships between
the estimate of bunch exposure and berry composition
were evaluated by linear regression analyses.
Results
Experiment 1 – comparison of the composition of berries
sampled from either fully exposed or fully shaded bunches
under artificially created conditions
There was no significant difference in the juice total
soluble solids or average berry weight between the two
treatments at each sampling date, except for the third
sampling date when the weight of shaded berries was
significantly higher than that of the exposed berries
(Table 1).
144 Canopy microclimate and berry composition Australian Journal of Grape and Wine Research 6, 141–149, 2000

Table 1. Juice total soluble solids and average berry

weights for berries sampled from artificially shaded and
exposed treatments at four sampling occasions during

Quercetin-3-glucoside
ripening of Shiraz grapes (Experiment 1). Each value is

per berry (mg)

the mean of four replicates of each treatment.

Sampling Juice TSS2 Stat. Average berry Stat.

occasion1 (°Brix) Signif.3 weight (g) Signif.3
Shaded Exposed Shaded Exposed

0 7.8 8.1 ns 0.91 0.87 ns

15 13.9 14.8 ns 1.33 1.29 ns
Days post veraison
35 21.6 21.3 ns 1.58 1.36 *
46 25.3 25.8 ns 1.04 1.15 ns Figure 2. The change in quercetin-3-glucoside (mg per berry) for
berries sampled from the shaded (■) and exposed (■ ■ ) treatments at
1. Days after veraison
2. Total soluble solids.
various sampling occasions during ripening of Shiraz grapes
3. * indicates treatment means are significantly different at the P < 0.05 level; (Experiment 1). Each value represents the mean of four replicates
ns indicates no significant difference. and vertical bars indicate the standard error of the mean for each
value.

Exposed 25 average berry weights differed, the concentration of

anthocyanins was significantly higher in berries from the
Relative absorbance

Shaded 20
exposed bunches (data not shown). Generally, at all
(per berry)

15 sampling stages, the percentage of the anthocyanins that

10
5 the anthocyanins that existed as malvidin-3-(p-
0 coumaryl)-glucoside was higher in the shaded berries
0 15 35 46
Days post veraison
Figure 1. The change in anthocyanins per berry (relative absorbance
units per berry) for berries sampled from the shaded (■ ) and
exposed (■■ ) treatments at various sampling dates during ripening of
Shiraz grapes (Experiment 1). Each value represents the mean of
four replicates and vertical bars indicate the standard error of the
mean for each value.
For both treatments, anthocyanin levels per berry
increased until the third sampling date (approximately
22°Brix) and then decreased. For the second and third
sampling occasions, anthocyanin levels per berry were
significantly higher in the exposed treatment, but at the
last sampling date (approximately 26°Brix ) there was no
significant difference between the values for the two
treatments (Figure 1). At the third sampling date, where
existed as malvidin-3-glucoside was higher in the
exposed berries (Table 2). In contrast the percentage of
(Table 2). The percentage that existed as the acetyl deriv-
ative was only significantly higher in berries of the shad-
ed treatment at the second sampling occasion; on all
other sampling occasions there was no significant differ-
ence between treatments (Table 2).
At all sampling dates, the level of quercetin-3-gluco-
side per berry was significantly higher in berries from
exposed bunches than in berries from shaded bunches
(Figure 2). A similar trend was evident when these were
expressed on a concentration basis (data not shown).
At all sampling dates, the concentration of resvera-
trol in berry extracts was undetectable.
Measures of the temperature of exposed and shaded
bunches indicated that in the daylight hours the temper-
ature of the exposed berries was on average 0.6°C higher
than that of the shaded berries, with the maximum
difference being plus 2.6°C during the period 12 noon to

Table 2. The percentage of each form of malvidin-3-glucoside of the total malvidin-3-glucosides of berries from artifi-
cially fully exposed and fully shaded bunches at four sampling occasions (Experiment 1).

Percentage of the various forms of Malvidin-3-glucoside

Sampling Malvidin-3-glucoside Malvidin-3-(acetyl)-glucoside Malvidin-3-(p-coumaryl)-glucoside
date1 Shaded Exposed Significance2 Shaded Exposed Significance2 Shaded Exposed Significance2

0 43 46 ns 25 23 ns 32 31 ns
15 41 54 * 24 21 ** 35 25 *
35 43 51 * 23 23 ns 34 26 *
46 38 49 * 24 25 ns 38 26 *

1. Days after veraison.

2. Level of significance: * and* * indicate treatment means are significantly different at the P < 0.05 and P < 0.01 level, respectively; ns indicates no
significant difference.
Haselgrove, Botting, van Heeswijck, Høj, Dry, Ford & Iland Canopy microclimate and berry composition 145

50 Figure 3. Variation over time

in temperature of ripening
berries in bunches situated in
40 the centre of the canopy is
shown as A. The difference in
temperature of berries in
Bunch temperature (°C)

30 bunches located in the

‘north’ side compared with
those in the ‘centre’ is shown
20 as B (Experiment 2).

10

–10
5 Jan 12 Jan 19 Jan 26 Jan 2 Feb 9 Feb 16 Feb 23 Feb 2 Mar 9 Mar

Table 3. Comparison of °Brix, average berry weight, and the coumarate form was significantly higher in berries
phenolic composition of berries from bunches in either from ‘centre’ bunches (Table 3).
the ‘centre’ or ‘north’ positions of the canopy, at a similar The estimate of bunch exposure, derived from the
berry maturity (Experiment 2) ceptometer measures, was in the order of 40% and 80%
for the ‘centre’ and ‘north’ positioned bunches, respec-
Centre North Significance1 tively. Ambient irradiance varied between 1850 and 1950
(shaded) (exposed) µmole quanta/m2s PPFD.
During berry development, the temperature of the
°Brix 24 24 ns ‘north’ bunches was generally higher than that of the
Berry weight 0.86 0.71 ns ‘centre’ bunches. At various stages, this difference was as
Total anthocyanins 0.95 0.50 *** high as 10°C (Figure 3).
(mg per berry)
Total anthocyanins 1.11 0.70 *** Experiment 3 – comparison of composition of berries from
(mg per g berry weight) bunches on different trellis systems
% malvidin-3-glucoside 44 51 * The estimate of bunch exposure was positively correlated
% malvidin-3-(acetyl)-glucoside 26 26 ns (r2 = 0.83, P = 0.03) with the concentration of quercetin-
% malvidin-3-(p-coumaryl)-glucoside 30 23 * 3-glucoside and negatively correlated (r2= 0.73, P = 0.06)
with the percentage of anthocyanins that existed as the
Quercetin-3-glucoside 0.056 0.127 ***
(mg/berry) coumarate derivative in these berries, respectively (Figure
Quercetin-3-glucoside 0.065 0.178 *** 4). There was no significant relationship between the esti-
(mg per g berry weight) mate of bunch exposure and the concentration of antho-
cyanins (r2 = 0.57, P = 0.14 ) of berries sampled from the
1. * and *** indicate that treatment means are significantly different at different trellis systems (Figure 4).
the P < 0.05 and the P < 0.001 level, respectively; ns indicates no
significant difference.
Discussion

4 pm (data not shown). The maximum temperature of Anthocyanins

the exposed berries was in the order of 30 to 35°C (data
not shown).
Experiment 2 – comparison of the composition of berries
sampled from bunches with different degrees of exposure to
light under natural conditions
When compared at a similar maturity (based on °Brix),
berries sampled from the bunches situated in the ‘centre’
of the canopy had significantly higher content and
concentration of anthocyanins and lower content and
concentration of quercetin-3-glucoside compared to
berries sampled from exposed bunches (Table 3). The
percentage of the total malvidin-3-glucosides in the
malvidin-3-glucoside form was significantly higher in
berries from the ‘north’ bunches, while the percentage in
From the outcomes of our three experiments, it appears
that anthocyanin metabolism responds to changes in both
light and temperature conditions. In conditions where
bunches are heavily shaded, as in Experiment 1, it is likely
that light is a limiting factor in the accumulation of
anthocyanins during the early stages of ripening. In
Experiment 2, it seems that while berries on both the
‘centre’ and ‘north’ bunches are receiving adequate light
for anthocyanin production, the temperature conditions
of the ‘north’ bunches may be a limiting factor (Figure 3).
In this site it is likely that the berry temperature is suffi-
ciently high in the ‘north’ bunches (generally being over
35°C during the afternoon period) to inhibit anthocyanin
synthesis and/or to increase anthocyanin degradation for
a considerable time period during the day.
146 Canopy microclimate and berry composition Australian Journal of Grape and Wine Research 6, 141–149, 2000

1.5 that Pinot Noir berries showed maximum colour accu-

per g berry weight (mg) (a)
1.4 mulation at low light intensities, of less than 18% ambi-
ent photosynthetic photon flux density (PPFD). Similarly,
Anthocyanins

1.3
anthocyanin concentrations in Merlot berries were high-
1.2 est when the percentage of incident radiation at the
1.1 bunch zone was in the order of 10% and decreased as
the percentage of incident radiation increased to 18%
1.0
(Mabrouk and Sinoquet 1998). Furthermore, Keller and
0.9 Hrazdina (1998) showed that for Cabernet Sauvignon,
the concentration of total anthocyanins in berries was
65 70 75 80 85 90 95
almost as high at 20% sunlight interception as it was at
Estimate of bunch exposure 100%. It would seem then, that if light conditions within
a canopy are such that the bunches receive sufficient light
45 of moderate intensity, then light is not necessarily a lim-
(b )
iting factor for anthocyanin synthesis. However, these
% coumarate form

40 effects may also be temperature-related (Mabrouk and

Sinoquet 1998). Enzymes involved in the anthocyanin
35 biosynthetic pathway exhibit an optimum temperature
range for activity. Studies by Pirie (1977) suggest that this
30 range is between 17 and 26°C. The high berry tempera-
tures experienced by the exposed bunches in Experiment
25 1 and 2 (where temperature conditions often exceeded
35°C) are therefore likely to inhibit anthocyanin syn-
65 70 75 80 85 90 95
thesis.
Estimate of bunch exposure Management practices that create a canopy architec-
ture where bunches receive sufficient light for antho-
0.200 cyanin synthesis, but where berries are protected from
(c)
excessive berry heating, would seem appropriate for the
per g berry weight (mg)
Quercetin-3-glucoside

0.175 production of fruit with optimal levels of anthocyanins

0.150
0.125
0.100
65 70 75 80 85 90
Estimate of bunch exposure
Figure 4. Relationships between berry anthocyanin concentration
(a), percentage of the coumarate derivative of the total malvidin-3-
glucosides (b) and berry quercetin-3-glucoside (c) with the estimate
of bunch exposure of the different trellis systems (Experiment 3).
The literature supports the above hypothesis. Previous
reports indicate that anthocyanin synthesis is directly reg-
ulated by both the light exposure and the temperature
conditions to which a grape bunch is subjected (Pirie and
Mullins 1980, Hrazdina et al. 1984, Smart et al. 1985a,
Crippen and Morrison 1986, Smart et al. 1988).
Furthermore, studies conducted by Kliewer (1970) in
which night and day temperatures were controlled at two
light intensities indicated that both temperature and light
had a significant effect on anthocyanin production and
that these effects were relatively greater on anthocyanin
production than on sugar accumulation. In general, high-
er light intensities are considered to promote greater
anthocyanin accumulation (Macheix et al. 1990), how-
ever this response appears to depend on the range of light
intensity under investigation. Dokoozlian (1990) found
for vines grown in hot climates. The actual degree of
canopy openness and bunch exposure required for
optimal anthocyanin accumulation is difficult to define.
As a guide to the degree of openness, scientific studies
and opinion would suggest that a desirable canopy for
vines grown in hot climatic conditions is one where
bunches are moderately exposed; conditions which are
95 often described by the phrase ‘dappled light within the
canopy’. In Experiment 3, there was not a strong rela-
tionship between berry anthocyanin concentration and
the estimate of bunch exposure for berries sampled from
the different trellis systems (Figure 4). This is not un-
expected, as here, even though there was variation in
bunch exposure, it was not large; the estimate of bunch
exposure varied from 71% for VSP to 88% for MP.
Bunches were probably sufficiently well exposed in all
trellis systems to ensure that light was not a limiting fac-
tor for anthocyanin production. Also, other factors such
as crop load and vine balance may have affected the
development of anthocyanins in fruit from the different
trellis systems.
It must be appreciated that the effects of light and
temperature on anthocyanin production may also be
different to their effects on metabolism of flavour com-
pounds within the berry. Although anthocyanin produc-
tion can apparently be optimal in moderately open
canopies, the impact of the degree of shading on berry
flavour profiles is less predictable. As shading is increased
there is a greater possibility of the development of unripe
herbaceous characters. Since the flavour profiles of
berries from shaded and exposed conditions were not
Haselgrove, Botting, van Heeswijck, Høj, Dry, Ford & Iland
assessed here, it is not possible to link these with antho-
cyanin responses; however this aspect should be
addressed in future studies. Interestingly, Hunter et al.
(1991) in a defoliation experiment with Cabernet
Sauvignon (where leaves were removed in the vicinity of
bunches), found only small differences in berry antho-
cyanin production, but nevertheless there were differ-
ences in wine character and quality, indicating that other
components, including varietal character, were modified
under altered light conditions.
A difficulty in stating an optimal level of bunch
exposure is that the measurement of exposure by a
ceptometer is taken at only one or a few stages in the
development of the vine and may not truly reflect the
continuous and cumulative interception of light by
bunches over the growing season. Measurement of light
interception by a ceptometer does, however, provide
some form of comparison between the light conditions
that exist in different canopy systems.
Another feature of this work relevant to practical
viticulture is the observed decline in total anthocyanin
content of berries during the later stages of ripening
(Figure 1). This trend was also apparent in the develop-
ment of anthocyanins in berries from ‘centre’ and ‘north’
bunches of Experiment 2 (data not shown). This
phenomenon of decline in absolute amounts of antho-
cyanins in the later stages of ripening has been reported
previously (Somers 1976, Roggera et al. 1986). Exami-
nation of the data from Experiment 1 and 2, suggests
that this decline represents a loss of all major forms of
malvidin-3-glucoside. The significance of this feature of
anthocyanin metabolism is that if fruit is left on the vine
in anticipation of the formation of riper flavour charac-
ters, it needs to be appreciated that this can be at the
expense of a loss of absolute amounts of total antho-
cyanins. Whether the anthocyanins are degraded or
incorporated into other molecules is not known. Keller
and Hrazdina (1998) suggest that breakdown of antho-
cyanins may be caused by glycosidase and peroxidase
activity in the grape skin vacuoles.
The accumulation of the coumarate derivative of mal-
vidin-3-glucoside was enhanced when berries developed
under relatively more shaded conditions (Table 2 and 3,
Figure 4). It has been reported (Leone et al. 1984) that
the coumarate form of malvidin-3-glucoside is preferen-
tially lost during winemaking. It is also less extractable
than the other forms when the anthocyanins are extract-
ed from berries with 10% v/v ethanol (Iland unpub-
lished). These observations may help to explain why, in
some situations, berries with similar concentrations of
total anthocyanins may produce wines with different
intensity of colour. Wines produced from berries with a
higher proportion of their anthocyanins in the coumarate
form may suffer a relatively larger loss of anthocyanins
during processing. However, the practical significance of
the differences in concentration of the coumarate deriva-
tive observed in these studies, in the order of 10%,
remains to be proven.
Although this relationship between bunch shading
and enhanced coumarate production appears not to have
Canopy microclimate and berry composition 147
been previously reported, Castia et al. (1992) in their
investigations did report a difference in the degree of
esterification of anthocyanins in berries sampled over a
number of years, a feature they attributed to differences
in macroclimate between years. Other authors (Iacono
et al. 1994, Dry et al. 1999 and Keller and Hrazdina
1998) have shown that a shift in the metabolism of
the different anthocyanins occurs under altered light con-
ditions. In the studies of Keller and Hrazdina (1998),
cyanidin-3-glucoside was most sensitive to light condi-
tions, decreasing with increasing shade, while malvidin-
3-glucoside was least affected. Similarly, Iacono et al.
(1994) found that shading lowered the percentages of
delphinidin, cyanidin and petunidin monoglucosides of
the anthocyanin pool of berries.
Quercetin-3-glucoside
Quercetin is a flavonol compound synthesised in the
epidermal cells of berry skin and sequestered as various
glycosides in the central vacuoles (Price 1994). Flavonols
are synthesised in the upper epidermis, while antho-
cyanins and other phenolic compounds accumulate in
the lower epidermis (Stafford 1990), and it appears that
regulation of the pathways of flavonols and anthocyanins
occurs independently, both spatially and temporally
(Price 1994). Quercetin is predominantly present in the
glycosylated form in grape berries (Price et al. 1995,
Spanos and Wrolstad 1990). It has been suggested that
these glycosides act as UV screening compounds, helping
to protect the plant tissue from damage (Flint et al. 1985).
Levels of quercetin-glucoside in Pinot Noir berries
have been shown to be strongly correlated with the
degree of fruit exposure (Price et al. 1995). In this current
study with Shiraz berries, in all three experiments, the
accumulation of quercetin-3-glucoside was enhanced in
berries from bunches with greater light exposure, sup-
porting the findings of Price et al. (1995). Enhanced
levels of quercetin-3-glucoside may play a role in co-
pigmentation, helping to stabilise the colour of wine
produced from those berries (Price et al. 1995). However,
for the Shiraz grapes investigated in this report, the con-
centration of quercetin-3-glucoside relative to the con-
centration of anthocyanins is low (approximately 10%)
and whether these levels of quercetin-3-glucoside are
sufficient to have a major impact on colour stability in
practice requires further investigation.
In Experiment 1, in the fully exposed berries, con-
siderable synthesis of quercetin-3-glucoside had already
occurred in the exposed samples during the period lead-
ing up to veraison (Figure 2). Similar results were shown
by Price et al. (1995) and Keller and Hrazdina (1998).
These results are of considerable interest as they suggest
that there are highly active glycosyl-transferase enzymes
present in berries well before veraison.
Resveratrol
Resveratrol is produced in the epidermal cells of grape
berries, and to a lesser degree in the seeds, with the con-
centration in the pulp being almost negligible (Creasy
and Coffee 1988, Jeandet et al. 1991, Jeandet et al.
148 Canopy microclimate and berry composition
1995). Resveratrol is produced in the berries of certain
cultivars in varying quantities, in response to fungal
infection (Langcake and Pryce 1977b, Langcake and
McCarthy 1979, Langcake 1981) or tissue damage result-
ing from irradiation with short-wave (< 260 nm) UV light
(Langcake and Pryce 1977a, Langcake and McCarthy
1979, Pool et al. 1981, Jeandet et al. 1992a). While there
is no record in the literature of resveratrol being isolated
from either leaves or berries without prior induction by
pathogens, wounding or UV light, it is conceivable that
increased light penetration may be able to induce some
degree of resveratrol synthesis.
In Experiment 1, no resveratrol was found in any of
the samples of shaded or exposed berries. This result is
not unexpected as there was very little disease incidence
on the vines at this site. The inability to detect resveratrol
in these berry samples sourced from a range of light
conditions, does however indicate that the metabolism of
resveratrol is not as sensitive to light conditions as antho-
cyanins or quercetin-3-glucoside. It should be noted that
no analysis was undertaken for the glucosylated resvera-
trol metabolite, piceid (Waterhouse and Lamuela-
Raventos 1994).
Conclusion
A high degree of bunch exposure in hot climates may
not be desirable for the production of anthocyanins in
berries. In these situations, while some exposure to light
may be appropriate, high berry temperatures resulting
from full exposure of berries are likely to inhibit antho-
cyanin metabolism.
Generally, shaded bunch conditions favour the metab-
olism of the coumarate derivative of malvidin-3-gluco-
side, which may have implications for colour expression
and stability in wines made from those berries.
Exposed bunch conditions enhance the formation of
quercetin-3-glucoside in berries. It is important to appre-
ciate that this may be coupled with either higher or lower
levels of anthocyanins as the metabolism of these com-
pounds can respond differently to altered light and tem-
perature regimes. It appears that the level of quercetin-3-
glucoside in berries may be an indicator of the degree of
fruit exposure.
In climates where temperatures regularly exceed
35°C, moderately open canopies appear to create the best
conditions for berry development. Berries developing in
moderately open canopies are likely to have optimal
levels of anthocyanins with minimal enhancement of
the coumarate derivative, and relatively high levels of
quercetin-3-glucoside, although not necessarily at the
maximum levels possible for this compound.
Acknowledgements
Financial support was provided by the CRC Viticulture
and the Grape and Wine Research and Development
Corporation. We thank Southcorp Wines for providing
the experimental sites and Ms Renata Ristic for technical
support.
Australian Journal of Grape and Wine Research 6, 141–149, 2000
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Manuscript received: 21 December 1999
Revised manuscript received: 27 April 2000