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J Sci Food Agric 1998, 76, 63È71
Chlorophylls, Carotenoids and Anthocyanin
Concentrations in the Skin of ‘Gala’ Apples during
Maturation and the Influence of Foliar
Applications of Nitrogen and Magnesium
Peter F Reay1* Robert H Fletcher2 and V J (Gary) Thomas2
1The Horticulture and Food Research Institute of New Zealand, Private Bag 11030, Palmerston North,
New Zealand
2The Agricultural Research Institute of New Zealand, Private Bag 11008, Palmerston North, New Zealand
(Received 11 July 1995 ; revised version received 25 November 1996 ; accepted 6 May 1997)
Abstract : “GalaÏ apple fruit were sampled over two growing seasons, to determine the e†ects
of maturation, and of foliar applications of nitrogen and magnesium on pigment concentrations and colour development in maturing fruit. Treatments of urea and/or magnesium sulphate sprays were applied to the trees in a commercial orchard. The chlorophyll, carotenoid
and anthocyanin concentrations in the fruit skin were determined for both the blush and
back sides over 15 harvests in 1991È1992. In 1990È1991, the skin chlorophyll concentrations
were determined only for the back side of the fruit. The fruit nitrogen, magnesium and
sulphur concentrations were determined near maturity. The concentrations of chlorophyll
and carotenoids in apple skin decreased from the Ðrst harvest in 1991È1992 as the fresh
weight increased from 12 g, 40 days from Ñowering to 140 g, 138 days from Ñowering. Chlorophyll continued to decrease whereas carotenoid concentrations increased after 138 days
and until at least 153 days. The average skin chlorophyll and carotenoid concentrations were
higher on the blush side of the fruit than on the back. The total chlorophyll per fruit
increased from the Ðrst harvest to a peak about 70 days after Ñowering in 1990È1991 and
then decreased over the remainder of the period studied. The time of this peak was about 40
days later in the 1991È1992 season compared with the 1990È1991 season. The total carotenoid increased up to about 50 days after Ñowering in 1991È1992, Ñuctuated and then
decreased up to 138 days when the amount increased again. The ratio of carotenoids to
chlorophylls was highest for the Ðrst three harvests, Ñuctuated somewhat up to c 138 days
after Ñowering after which the ratio increased rapidly. The anthocyanin concentrations were
low and Ñuctuated up to 130 days from Ñowering and then increased markedly only on the
blush side at 130 days, about 14 days before the carotenoids started to increase. The treatment of the tree canopy with urea increased the chlorophyll and carotenoid concentrations in
the fruit skin, 14È20 days after the Ðrst application until after maturity, more so on the blush
side than on the back side. These increased concentrations were maintained up to the harvest
after which carotenoid concentrations increased. The urea treatment lessened the increase of
anthocyanin concentrations in the blush side skin of the fruit, at maturity. At maturity, the
nitrogen concentration of fruit from urea treated trees was higher than those from untreated
trees. The magnesium sulphate treatment of the canopy increased the chlorophyll and carotenoid concentration on the back of the fruit within 20È30 days from commencing spraying.
The “ground colourÏ scores for the back sides of the fruit (used for fruit picking and grading)
were greener for the urea treated fruit and reached an acceptable score about two weeks later
than the untreated fruit.
J Sci Food Agric 76, 63È71 (1998)
Key words : apple ; fruit ; colour ; maturity ; chlorophyll ; carotenoids ; anthocyanin ; urea ;
nitrogen ; magnesium
* To whom correspondence should be addressed.
63
( 1998 SCI.
J Sci Food Agric 0022-5142/98/$17.50.
Printed in Great Britain
P F Reay, R H Fletcher, V J T homas
64
INTRODUCTION
With all apple cultivars, quality in the marketplace is
determined in the Ðrst instance by a striking visual
appearance. For red apple cultivars, the achievement of
a red blush over much of the surface is of particular
importance, but this blush development often occurs at
or past harvest maturity. Red blush is produced by high
anthocyanin concentrations together with low chlorophyll concentrations in the skin. Blush development
should be complete before the fruit are mature to ensure
the highest quality. The measurement of maturity can
be achieved with a number of fruit characteristics, but
from a practical standpoint the skin background colour
(or backcolour) underlying the red blush is amongst the
most useful. Red skinned fruit reach optimum maturity
with a light green back colour in the continuum
between dark green and golden yellow. The green
can be most easily perceived towards the back of
the fruit (shaded side) but while not perceived as such
under the red blush, the green reduces the intensity
of red, which is enhanced by the decline in green with
maturity.
The colour of apple fruit is determined primarily by
the relative amounts of the pigments in the fruit skin :
the anthocyanins, chlorophylls and carotenoids. For
“Golden DeliciousÏ apples the change from green to
yellow in the whole skin is associated with a decline in
chlorophyll concentration and a small increase in that
of the carotenoids (Workman 1963). During the ripening of “CoxÏs Orange PippinÏ apples, the chlorophyll
concentrations in the whole skin falls throughout ripening along with the carotenoid concentrations but the
latter begins to increase just before the climacteric
(Knee 1972). In “Golden DeliciousÏ apples (Workman
1963), chlorophyll concentrations in 1960 fell from July
to the end of September, as in “CoxÏs Orange PippinÏ
apples, whereas in 1961 the concentrations increased
from July to August and then decreased from August to
October. In “Golden DeliciousÏ carotenoid concentrations fell after the beginning of September and then
increased from October, well before the climacteric. In
green “Granny SmithÏ fruit the chlorophyll and carotenoid concentrations falls from December to May with
a slowing of the decrease near maturity (Mussini et al
1985), but without the increase of carotenoid observed
for “CoxÏs OrangeÏ and “Golden DeliciousÏ. These
studies were carried out on the whole fruit skin, bulked
from a number of apples. Workman (1963) observed
that the blush side of “Golden DeliciousÏ had a 50%
higher chlorophyll concentration than the back,
whereas the carotenoid concentrations were similar.
The yellowing of the back or “green cheekÏ ground
colour of “GalaÏ is correlated with increased ethylene
evolution that occurs with increasing maturity (Walsh
and Volz 1990). The changes in green and yellow pigments associated with these colours may be di†erent on
the back when compared with the blush side of the fruit.
For “McIntoshÏ trees sprayed with urea in spring,
chlorophyll concentrations were higher in the leaves
from mid-summer to autumn (Fisher and Cook 1950).
At harvest the percentage of fancy fruit (receiving the
highest return) was reduced from 74 to 58% by three
sprays and to 52% by four urea sprays. Foliar applications of urea during summer on “CoxÏs Orange PippinÏ
increased the chlorophyll and carotenoid concentrations and decreased the anthocyanin concentration in
the fruit skin, at harvest (Kaether 1965). In contrast,
foliar urea applied to “Golden DeliciousÏ, on a dwarÐng
rootstock, increased the yellow score of the fruit at
harvest (Ferree and Cahoon 1987). The e†ects of urea
were measured on fruit only at harvest and the measurements do not show the timing of sprays in relation
to their e†ects.
Foliar applications of magnesium salts have also been
reported to overcome the yellowing and browning of
leaves associated with magnesium deÐciency (Walker
and Fisher 1957) and may therefore also a†ect fruit
colour. In the orchard investigated, magnesium deÐciency symptoms were visible in leaves of “Granny
SmithÏ and its potential e†ect on fruit colour were investigated.
In this study, the e†ects of maturation and the foliar
application of nitrogen and magnesium on the pigmentation and colour development of “GalaÏ apples were
determined. The chlorophyll, carotenoid and anthocyanin concentrations and colour in the skin from the
blush and back sides of “GalaÏ apples were measured
during the greater part of fruit growth up to the closing
of commercial harvest.
EXPERIMENTAL
The multi-leader 15-year-old “GalaÏ apple trees used
were spaced 5 m along the rows and 8 m between rows
(220 trees ha~1) in a commercial orchard (Donella) on a
fertile loam, at Hastings, Hawkes Bay, New Zealand.
The rows of trees were oriented east-west, and the trees
had been interplanted with “GalaÏ Ðve years previously
(which acted as guard trees) and had not received fertiliser for the previous ten years. The individual trees in
three rows were assigned at random to the treatments,
in blocks of four. Four blocks of trees (16 trees, four
replicates) were used in 1990È1991 and Ðve blocks,
including the previous four, in 1991È1992. The time of
Ñowering was taken when half of the Ñowers had
opened : 15 October in 1990 and 10 October in 1991.
The treatments of urea (1%, w/v plus 0É01% “CitiwetteÏ)
and magnesium sulphate hydrate (1%, w/v plus “CitiwetteÏ), and both combined, were applied with a motorised portable lance sprayer with the spray directed
upwards under the canopy until the leaves dripped. The
Ðrst sprays were applied on the 8 Dec 1990 and on the
12 Dec 1991, when annual shoot growth had terminat-
Chlorophylls, carotenoids and anthocyanin concentrations in the skin of Gala apples
ed. Eight sprays were applied in total at weekly intervals, at least one day prior to forecast rain, to ensure
uptake of most of the applied nitrogen and magnesium
(Cook and Boynton 1952 ; Allen 1960). Fruit were
sampled weekly ; the Ðrst sample was taken 1 week prior
to the Ðrst treatment in 1990 and 4 weeks in 1991. Fruit
were picked from the lower outer zone of the tree
canopy, two each from the north and south sides of
each tree. The Ðnal sample was taken in the last week of
the 3 week commercial harvest period. In 1991È1992,
sufficient fruit to complete the subsequent samplings
were tagged just prior to the commercial harvest. These
were left on the tree by the fruit pickers so that the same
population of fruit was sampled throughout the commercial harvest.
The fruit were weighed and sampled within 1 day of
harvesting. The fresh weight and also in 1991È1992 the
weight submerged in water was determined. The surface
area was estimated from this volume, by assuming the
fruit was a sphere. For 1990È1991 the density was taken
as 0É89 for this calculation. In 1990È1991, the back of
each fruit was sampled for the determination of colour
and chlorophyll concentrations. In the 1991È1992
season the back and the blush sides of each fruit were
sampled for the determination of colour, chlorophyll,
carotenoid and anthocyanin concentrations. The
ground colour score (1È10) was determined on the back
side of the fruit with New Zealand Apple and Pear
Marketing Board Gala/Royal Gala ground colour
charts. The reddest blush area of fruit skin (ie the
blushed side), adjacent to the equator and at least 3 cm2
in area was sampled for the analysis of the blush side.
An area of skin on the back, opposite to the blushed
area and of similar size was sampled for the analyses on
the back of the fruit. On each side, three 8 mm diameter
cores were punched with a sharpened stainless-steel
tube : one for anthocyanin, one for chlorophyll and one
for carotenoid determination. On the blush side the
colour of the skin disc punched for anthocyanin was
determined and on the back the colour of the skin
determined from the disc punched for chlorophyll determination. Colour was measured with a Minolta CR-221
colour meter, with a 3 mm diameter measuring head
which did not include the gloss component of the reÑected light. The colour was described in the L *a*b* (1976
CIE colour system). The plugs were then removed and
the skin sliced o† at 3 mm from the skin for chlorophyll
and carotenoid and at 1 mm for the anthocyanin determinations.
Anthocyanins were extracted from skin discs with
isopropanol/water/lactic acid (3 ml, 95 : 3 : 2, v/v) in
vacuo for 10 min and then stood overnight at 4¡C. An
additional 1 ml water was added to each sample which
was mixed, stood for 2 h and centrifuged at
2000 ] RCF. Absorbances were measured with a Pye
Unicam SP6 spectrophotometer (6 nm bandpass) in a
1 cm Ñow cell. The absorbance for anthocyanin was
65
measured at 530 nm and the anthocyanin concentration
calculated from the extinction coefficient of an ideainÈ
HCl solution in this solvent compared with that in
methanolÈHCl (Siegelman and Hendricks, 1959). Ideain
hydrochloride was obtained from PlantTech (UK), The
University, Reading, UK. The chlorophylls were
extracted with 2 ml dimethylformamide(DMF)-triethanolamine carbonate (prepared by saturating a stock
solution of triethanolamine in dimethylformamide
(50 : 50, v/v) with carbon dioxide and diluting 100 fold)
in vacuo for 20 min and then overnight at 4¡C. Acetone/
dimethylformamide/water (3 ml, 80 : 19 : 1, v/v) was
added, the solution mixed, stood for 1 h at room temperature, mixed and centrifuged at 2000 ] RCF. The
absorbance was measured at 662É5 and 645 nm
on separate aliquots in a 4 cm Ñowcell. The concentrations of chlorophyll a and b together were calculated
from the absorbances. The extinction coefficients were
calculated for chlorophyll a and b in the dimethylformamide/acetone/water extractant, by comparison
of the absorbances measured in this solvent with their
absorbances in dimethylformamide for which extinction coefficients were available (Inskeep and Bloom
1985). PuriÐedchlorophyll a and b were prepared
from a leaf extract by separation on a starch column,
transferred into these solvents and the absorbances
immediately measured. The calibration measurements
were made on both the SP6 spectrophotometer and
a Cary 1E scanning spectrophotometer (0É2 nm
bandpass). The decrease of chlorophyll absorbance
in the DMF-triethanolaminecarbonate extracting
solvent was found to be less than 5% after 3 days
at 4¡C. Reagents were Analar grade, unless otherwise
stated.
The sample discs for carotenoid determinations were
frozen, stored at [10¡C until extracted with 1 ml
DMF-triethanolaminecarbonate and 0É3 g anhydrous
sodium sulphate in vacuo for 0É5 h, then stood at
[10¡C overnight and then warmed to room temperature. A solution of hydroxylamine (GPR grade,
0É1 ml of a 50%, w/v, solution in water and neutralised
with sodium hydroxide) was added (Ogawa and Shibata
1965) and the samples then stood in the dark for 1 h (to
convert the chlorophyll b to a derivative with shorter
wavelength at the blue end of the spectrum and remove
interference with the carotenoids at 475 nm). Then 1 ml
acetone, 0É3 g anhydrous sodium sulphate, 1 ml isopropanol and 3 ml petroleum spirit (“AnalarÏ, 120È160¡C
bp) were added and the tubes vortexed and shaken for
0É5 h in the dark. Following this, potassium chloride
solution (5% w/v, 3É5 ml) was added and the tubes vortexed and centrifuged at 1000 ] RCF for 20 min. The
upper phase was transferred to another glass tube, 1 ml
isopropanol was added and mixed. The absorbance was
read at 475 nm and the concentration of total carotenoids calculated from a speciÐc absorption coefficient
of 2050 (Litchtenthaler, 1987) corrected for the recovery
66
of a b-carotene (Sigma Chemical Co) standard carried
through the procedure in parallel.
Fruit for the determination of nitrogen was harvested
on the 12th and 14th harvests at 125 and 138 days after
Ñowering and fruit for magnesium was harvested on the
thirteenth harvest at 131 days after Ñowering. Fruit was
harvested from the North aspect of the trees and
sampled. Fruit was cut in half across the blush-back
axis and cores (8 mm diameter) were punched through
at the equator on the blush and the back sides of each
fruit, then tapered to a point at the centre, parallel
to the fruit axis, and two cores from the opposing sides bulked for each fruit, weighed and dried at
120¡C. The fresh cores for organic nitrogen determination were extracted in isopropanol/oxalic acid. The
dried cores for magnesium and sulphur analysis were
digested with nitric acid as for Reay and Waugh
(1981). Magnesium and sulphur were determined
in 1 M hydrochloric acid solution of the residue by
Plasma Emission Spectrophotometry (ICP) with an
ARL 3200.
For the analysis of organic nitrogen, the cores were
extracted with isopropanol/oxalic acid solution
(2É2% w/v, 8 ml per g fresh weight), stored at [ 10¡C
for 2 days and then lithium carbonate solution added
(1% LiOH monohydrate, w/v, in water, saturated with
carbon dioxide, 0É8 ml per ml of isopropanol/oxalic).
The samples were stored at 4¡C for a further two
days. The extraction solution was decanted and the volume of water in the sample was added to that of the
extract to give the total volume of extract. The residue
was reextracted with isopropanol/acetic acid/water
(50/1/49, v/v, 5 ml per g FW) for 2 days at 4¡C,
to remove residual soluble nitrogen. An aliquot
of the extract and the whole residue were dried
at 120¡C in glass test tubes after the addition of
1È4 drops of 0É2 M sulphuric acid. The samples were
digested with concentrated sulphuric acid at 200¡C
for 2 days. Then the glass tubes were sealed and
heated at 400¡C for 10 h, and ammonia determined
(Reay 1985).
The area of the skin punched out was estimated from
the paper discs. Concentrations were calculated per unit
area and the amounts per fruit calculated from this
together with the total area of the skin. Statistical
analysis was carried out with an ANOVA. Harvests
prior to the treatment applications were analysed
separately from those sampled after applications commenced. Urea, magnesium sulphate and the blocks of
trees were treated as main e†ects split for tree aspect
and harvests. Where apple side (blush and back) was
considered in the analysis, the harvest e†ect was further
split for the blush and back sides of the apples.
The subsamples (fruit) were averaged before analysis
and the Error was derived from the sums of the
block interactions and the third and higher order
interactions.
P F Reay, R H Fletcher, V J T homas
RESULTS AND DISCUSSION
Chlorophyll, carotenoid and anthocyanin concentrations
in fruit skin
The chlorophyll concentration in the skin (blush side
and back averaged) decreased more than Ðve-fold in the
fruit from the control (unsprayed) trees from 42 to 153
days after Ñowering in 1991È1992 (Fig 1(a)). This
decrease in the chlorophyll concentration with time is
similar to but more linear than that found for “Granny
SmithÏ (Mussini et al 1985) and similar to the 1961 data
of Workman (1963) for “Golden DeliciousÏ. The average
total chlorophyll content per fruit for the Ðrst harvest at
42 days after Ñowering was already 60% of the
maximum content which was reached 62 days later (Fig
1(b)). The total content of chlorophyll then decreased
more than three fold from 104 to 153 days after Ñowering. On the blush side, the total chlorophyll content
from 42 to 104 days after Ñowering increased more
rapidly than that on the back and reached a higher
maximum (Fig 1(b)), to then decrease in similar proportions and the amount remained higher than that for the
back side. The average carotenoid concentrations in the
fruit skin remained constant for the Ðrst three harvests
and then decreased more rapidly than the chlorophyll
concentrations over the next three. After this, the concentrations then declined steadily until 138 days, during
the commercial harvest period, after which the carot-
Fig 1. Chlorophyll (Chl) and carotenoid (Car) in the skin of
“GalaÏ fruit from the control trees in 1992 : (a) the concentrations for blush and back sides averaged, and (b) the total contents of pigment per fruit for blush and back sides separately.
Chlorophylls, carotenoids and anthocyanin concentrations in the skin of Gala apples
67
Fig 2. The average carotenoid/chlorophyll ratios in the skin
of “GalaÏ fruit from all treatments combined on the blush side
and back side.
enoid concentrations then increased (Fig 1(a)). The total
carotenoid amount reached a maximum at 48È55 days
after Ñowering, then fell slightly but with a slight
increase after 100 days to another maximum on the
blush side near the time of maximum chlorophyll before
falling to a minimum at 138 days (Fig 1(b)). The carotenoid increase near ripening is similar to that found for
“CoxÏs Orange PippinÏ (Knee 1972) but occurred later
than that shown for the yellow “Golden DeliciousÏ
(Workman 1963 ; Gorski and Creasy 1977). The carotenoid to chlorophyll ratio (found for the treatment
averages) was highest for the Ðrst three harvests, fell and
rose slightly until after 138 days when it increased
markedly (Fig 2). The ratios were slightly higher on the
blush side of the fruit (P \ 0É01), especially at 153 days
from Ñowering.
The average chlorophyll concentration on the back
side of the fruit for the last four harvests in 1992 was
signiÐcantly higher (P \ 0É05) than that found in 1991
(Table 1). The average fresh weights were also slightly
higher in 1992 than in 1991 so that the total skin chlorophyll in 1992 was nearly twice that in 1991. This difference arose from a later decrease of total chlorophyll
in 1992, commencing after 111 days from Ñowering (see
Fig 1(b)), whereas in 1991 total chlorophyll fell after 74
days, with a similar rate of decline to that in 1992.
The anthocyanin concentrations Ñuctuated at low
values during much of the growth period and then near
TABLE 1
The average chlorophyll content of fruit skin from the back
side and the average fresh weights for the last four harvests
from control trees in 1991 and 1992.
Y ear
1991
1992
Chlorophyll
nm cm~2
nm per fruit
1É09 ^ 0É09
1É74 ^ 0É17
144 ^ 12
242 ^ 27
Fresh weight (g)
127É7 ^ 3É3
142É7 ^ 4É1
Fig 3. The average anthocyanin values of the skin for the
blush side and back of “GalaÏ fruit from the minus urea treatments : (a) the concentration of anthocyanin, and (b) the total
content of anthocyanin in the skin from the blush and back
sides of the fruit.
maturation (125 days from Ñowering) increased markedly on the blush side (Fig 3). The concentration on the
back Ñuctuated less up to 125 days, subsequently
increased and then declined in contrast to that on the
blush side (Fig 3). The increases in anthocyanin concentrations for both sides of the fruit were about 14 days
ahead of the increase in carotenoid concentrations.
Similar changes were observed for the whole skin of
“CoxÏs Orange PippinÏ (Knee 1972) where the increase of
anthocyanin was three-fold over the period studied,
which began ten days prior to the rise in carotenoid
concentrations, compared with the greater than Ðve fold
increase determined here with the blush side skin of
“GalaÏ. The rate of increase of the total content of
anthocyanin per “GalaÏ fruit was slightly higher than
that of the anthocyanin concentration and was related
to the increase of apple size (Fig 3).
The a†ect of three aspect and fruit side on chlorophyll,
carotenoid and anthocyanin concentrations
Fruit from the southern aspect of the tree had signiÐcantly higher (P \ 0É05) chlorophyll and carotenoid
concentrations than the northern aspect. The back of
the fruit had signiÐcantly lower concentrations of chlorophyll and carotenoids in the skin (P \ 0É001) than the
blush side (Table 2). Workman (1963) reported on data
with a greater di†erence than that here, although it was
concluded that the two fruit sides are similar in their
chlorophyll and carotenoid concentrations. While both
the southern tree aspect and the back side of the “GalaÏ
fruit could be expected to have lower light intensities,
the pigment responses did not consistently follow this
trend, in that the chlorophyll concentrations were
higher on the southern aspect. There was no signiÐcant
e†ect of tree aspect on fruit weight (P [ 0É05) and chlo-
P F Reay, R H Fletcher, V J T homas
68
TABLE 2
The average chlorophyll (chl), carotenoid (car) and anthocyanin concentrations in the skin of “GalaÏ
apples for all treatments over the period following the start of urea applications (harvests 5 to 16)a
Fruit side
SigniÐcance
Tree aspect
Blush
Back
North
South
SigniÐcance
Chlorophyll
(nmol cm~2)
Carotenoid
(nmol cm~2)
Car/Chl
ratio
Anthocyanin
(nmol cm~2)
5É35
4É71
***
4É90
5É16
*
1É91
1É45
***
1É64
1É73
*
0É54
0É47
***
0É51
0É49
ns
14É0
7É9
***
11É7
10É3
**
a The signiÐcance of the di†erences is shown as : * P \ 0É05, ** P \ 0É01, *** P \ 0É001.
rophyll and carotenoid concentrations were not signiÐcantly inÑuenced by tree aspect ] fruit side interaction
(P [ 0É05). The carotenoid/chlorophyll ratio was signiÐcantly higher on the blush side compared with the back
(P \ 0É01). There was a small but signiÐcant harvest] fruit side interaction for both chlorophyll and carotenoid concentrations (P \ 0É01), where the blush side
had higher concentrations of these pigments especially
in the earlier harvests which is shown in the data for the
total amounts (see Fig 1(b)).
The anthocyanin concentration on the blush side of
the fruit was signiÐcantly greater than that on the back
side and greater for fruit on the north aspect of the tree
(P \ 0É001). There was no signiÐcant (P [ 0É05) tree
aspect ] fruit side interaction.
back of the fruit where the increase was apparent 28
days after the Ðrst spray. The di†erence between the
average chlorophyll for the urea treated and untreated
fruit decreased as the chlorophyll concentrations
declined, but fruit skin on the blush side from the urea
sprayed trees remained higher in chlorophyll up to and
including the Ðnal harvest (Fig 4). The e†ect of urea
sprays on the chlorophyll concentration of the back of
the fruit was similar for 1990È1991 and 1991È1992
(Table 4), even though the concentration of chlorophyll
was less in 1990È1991. Over the last two harvests, the
carotenoid concentrations on the blush side increased
more after the urea treatment than without urea
(P \ 0É05) (Fig 5 and Table 3). The urea treated fruit
had similar carotenoid to chlorophyll ratios to the
untreated fruit from 42 to 138 days after which the urea
The inÑuence of urea and magnesium sulphate sprays on
fruit skin colour and chlorophyll, carotenoid and
anthocyanin concentrations
Spraying urea onto the canopy of the tree signiÐcantly
increased the chlorophyll and carotenoid concentrations of the skin on both the blush and back sides of the
fruit (P \ 0É01, Fig 4 and Table 3). The urea increased
the average chlorophyll and carotenoid concentration
on the blush side after two sprays and the increases
occurred within 14 days of the Ðrst spray. Subsequent
sprays did not further increase the concentrations relative to the untreated fruit. The e†ect was less for the
TABLE 3
The e†ect of urea on the average carotene and anthocyanin
concentrations in the “GalaÏ fruit skin for the last four harvests
in 1992
Urea
Minus
Plus
Carotenoid (nm cm~2)
Anthocyanin (nm cm~2)
Blushside
Backside
Blushside
Backside
1É06 ^ 0É04
1É45 ^ 0É07
0É78 ^ 0É03
1É01 ^ 0É04
27É8 ^ 1É2
24É2 ^ 1É3
12É0 ^ 0É6
12É4 ^ 0É6
Fig 4. The e†ect of eight weekly urea sprays on the skin chlorophyll concentrations of “GalaÏ fruit. The Ðrst spray was
applied a day after the fourth sampling (day 62).
Chlorophylls, carotenoids and anthocyanin concentrations in the skin of Gala apples
69
Fig 5. The average carotenoid/chlorophyll ratios in the blush
side skin of “GalaÏ fruit from the minus urea and the plus urea
treatments.
treated fruit had relatively more carotenoids. Anthocyanin concentrations on the blush side of the fruit were
signiÐcantly reduced over the last four harvests by the
urea sprays (P \ 0É001) (Table 3). The di†erences
became apparent for the last harvests when the anthocyanin concentrations had increasedÈthis was some 8
weeks after the urea sprays had begun.
The magnesium sulphate treatment signiÐcantly
increased (P \ 0É05) the average chlorophyll and carotenoid concentrations over the last four harvests for the
back side of the fruit (Table 4) but not the blush side.
The di†erences were relatively small, and were greater
in 1991. An increase in chlorophyll and carotenoid concentrations after magnesium application has not been
previously reported. There was no signiÐcant e†ect of
magnesium sulphate sprays on the average of the colour
parameters a* and b* (P [ 0É05), which indicated that
the increase of yellow carotenoid balanced the e†ect of
the increase of green chlorophyll on the hue of the skin.
The a* colour values (colour space parameter for red
and green) for the fruit skin on the blush and back sides
were lowered (greener) and the b* values were higher
(were more yellow) with urea sprays, more on the blush
side (Fig 6) than the back side. On the blush side the a*
values increased to the tenth harvest (day 111), fell for
the eleventh harvest (day 117) and then rose steadily up
to the Ðnal harvest (Fig 6(a)). The b* values mirrored
those of the a* values (Fig 6(b)). The decrease at the
TABLE 4
The e†ect of urea on the average chlorophyll content of the
backside fruit skin for the last four harvests in 1991 and 1992
Y ear
Chlorophyll (nm cm~2)
Urea
1991
1992
Magnesium
Minus
Plus
Minus
Plus
1É27 ^ 0É09
1É76 ^ 0É10
1É68 ^ 0É1
2É16 ^ 0É10
1É36 ^ 0É09
1É89 ^ 0É11
1É59 ^ 0É10
2É03 ^ 0É10
Fig 6. (a) Average a* values, and (b) b* values (L *a*b* colour
system) for the blush side of “GalaÏ apples for the minus and
plus urea treatments. The Ðrst urea spray was applied a day
after the fourth sampling (day 62).
10th harvest was reÑected by a decrease of anthocyanin
and an increase of chlorophyll and carotenoid concentrations. It is possible that greener fruit were inadvertently sampled from the trees for the 11th and 12th
harvests. For the back of the fruit, the a* values for the
last four harvests, which were in the commercial harvest
period, were less with urea sprays ([6É7 ^ 0É5 compared with [2É6 ^ 0É9) and b values were higher
(27É9 ^ 0É6 compared with 23É2 ^ 0É6). This may be a
reÑection of the higher chlorophyll and carotenoid concentrations found over this time.
The e†ect of urea and magnesium sulphate sprays on the
nitrogen, magnesium and sulphur content of the fruit
The organic nitrogen concentration in fruit from urea
treated trees was signiÐcantly greater than that from the
minus urea trees (27É5 lmol N g~1 FW compared with
18É7 lmol N g~1 FW, P \ 0É001, sampled at harvests
12 and 14, 1990È1991). The nitrogen concentration
increased slightly and signiÐcantly (P \ 0É05) from
harvest 12(21É8 lmol N g~1 FW) to harvest
14(24É4 lmol N g~1 FW). These increases were primarily in the soluble nitrogen fraction, whereas the
insoluble (protein) fraction was little a†ected. The magnesium and sulphur contents of fruit from magnesium
sulphate treated trees were higher but not signiÐcantly
so than those from untreated trees (eg 1É70 lmol Mg
and 1É72 lmol S g~1 FW compared with 1É66 lmol Mg
70
and 1É64 lmol S g~1 FW, P [ 0É05).
The response of “GalaÏ fruit colour, chlorophyll,
carotenoids and anthocyanin concentrations to sprays
of urea and/or magnesium sulphate on the whole tree
occurred with no apparent modiÐcation of the growth
pattern, in contrast with early season applications of
urea to “McIntoshÏ (Fisher et al 1948). The ureanitrogen has therefore increased the chlorophyll and
carotenoid concentrations of the skin either by direct
uptake or perhaps indirectly through the leaves or
through other metabolites such as carbohydrates. A
general relationship has often been observed between
chlorophyll and nitrogen concentrations in leaves. This
ratio is decreased at higher illuminances (Evans 1993)
by a decrease in the chlorophyll amount, whereas in the
fruit here the blush side had higher chlorophyll concentrations. However “GalaÏ fruit have much less chlorophyll in the skin than is present in leaves. The loss of
chlorophyll in detached tobacco leaves was reduced
during senescence by ammonium salts and urea, similar
to the e†ect of some cytokinins (Singh and Latham
1992). For N deÐcient seedlings of tobacco in tissue
culture exogenous sucrose reduced both protein and
chlorophyll concentrations in the shoot (Paul and Stitt
1993). The increase in chlorophyll (and carotenoids)
observed here may relate to a decrease in the senescence
of the skin or to a change in the partioning of carbohydrate to the fruit. In “Golden DeliciousÏ apples, the
nitrogen supply, the nitrogen timing and the fruit/leaf
ratios within groups of nitrogen treatments, inÑuenced
the colour of the fruit in late October (Hansen 1980).
The increase of chlorophyll and carotenoid observed in
“GalaÏ apples with urea sprays is consistent with both a
greater amount per apple and the maintenance of the
maximum chlorophyll and carotenoid content for a
longer period. This di†erence persisted up to the last
harvest, when the e†ect on anthocyanin was also the
most apparent. In a comparison of the 2 years investigated, the di†erence in chlorophyll concentrations at
harvest relate to the timing of the decline in the total
amount which in 1990È1991 commenced after 70 days
from Ñowering, compared with 100 days in 1991È1992.
These pigment changes were also apparent in the
ground colour score at harvest (NZAPMB, see
Experimental) which is used to grade the maturity of
fruit for export packout. The range from 1 to 10 for
“GalaÏ is from light green to pale yellow. The percentages of fruit with a score of 4 to 7 (required for export)
from the minus and plus urea treatments were signiÐcantly di†erent at 65% and 25%, respectively,
(P \ 0É05) in the Ðrst week of the commercial harvest.
The minus urea fruit all met export grade standards by
the second week but the plus urea fruit did not reach
the grade standard until the third and last week of the
harvest. Such a di†erence could result in a lesser
packout for export.
The pigment concentrations and the colour values at
P F Reay, R H Fletcher, V J T homas
maturity and harvest were signiÐcantly a†ected by
treatments earlier in the growing season, prior to the
decline of total chlorophyll and carotenoids, which
accompanies maturation. The subsequent changes with
maturation maintained the relative pigment concentrations almost to the Ðnal harvest. The e†ect of urea in
reducing red fruit colour was twofold in that the
decrease of red colour, from lower anthocyanin concentrations was enhanced by an increase of chlorophyll
which also reduced the red colour of the skin and therefore fruit attractiveness. These results indicate that the
nitrogen supply or status of orchard trees early in the
growing season has a signiÐcant impact on the Ðnal
fruit appearance.
ACKNOWLEDGEMENTS
The authors wish to thank M Lewis, M Broadhurst, B
Wilson and M Ballintyne for skilled technical assistance, Dr J Lee for ICP analyses, Dr D S Tustin and J
Lancaster for helpful discussions, D Hirst, Donella
Orchards, Hastings, for providing the “GalaÏ apple trees
and fruit and the New Zealand Foundation for Science
and Technology for Ðnancial assistance.
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