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. REFERENCES Allen M 1960 The uptake of metallic ions by leaves of apple trees. 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