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Colour response of ‘Cripps’ Pink’ apples to postharvest irradiation is influenced by maturity and temperature
Scientia Horticulturae 90 (2001) 31±41
Colour response of `Cripps' Pink' apples to postharvest irradiation is in¯uenced by maturity and temperature Evelyn Marais, Gerard Jacobs, Deirdre M. Holcroft* Department of Horticultural Science, University of Stellenbosch, Private Bag X1, Matieland 7602, Stellenbosch, South Africa Accepted 21 October 2000
Abstract Preliminary experiments indicated that postharvest irradiation enhanced anthocyanin synthesis in `Cripps' Pink' apples. Consequently this system was used to understand the effects of maturity and temperature on anthocyanin synthesis. Fruits harvested 5, 4, 3, 2 and 1 weeks prior to predicted commercial harvest did not develop a red blush in response to postharvest irradiation (72 h with high-pressure sodium lights at 208C). Fruits harvested at maturity rapidly developed a red blush in response to irradiation. Mature `Cripps' Pink' apples harvested from two growing regions were subjected to 120 h of irradiation at 6 or 208C following 0, 2 or 5 days at 0.58C. Fruits harvested from the cooler area (Ceres) and irradiated immediately showed greater red colouration at 68C than at 208C. The difference between the temperatures was no longer signi®cant after fruits have been stored for 5 days at 0.58C prior to irradiation. Fruits harvested from the warmer area (Grabouw) were consistently redder when irradiated at 68C than 208C. In a separate experiment, fruits were stored for 20 days at 0.58C prior to irradiation at either 68C or 20/68C (day/night). The alternating temperature resulted in better colour of fruits from both areas. In a ®nal experiment, well-coloured `Cripps' Pink' apples were irradiated at 378C to simulate periods of high temperature that occur in summer. The hue angle of the fruits increased from 29.3 to 48.38 after 144 h of light indicating a loss of red colour. The anthocyanin concentration decreased over 50% during the same period. Fruits kept at 378C in the dark did not lose red colour. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Anthocyanin; Red blush; Light; Malus domestica Borkh.; Pink LadyTM
*
Corresponding author. Tel.: 27-21-8084105; fax: 27-21-8082121. E-mail address: [email protected] (D.M. Holcroft). 0304-4238/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 2 5 6 - 9
32
E. Marais et al. / Scientia Horticulturae 90 (2001) 31±41
1. Introduction The development of a red blush on bicoloured apples is required to satisfy export standards. The red colour is a result of anthocyanin pigments, particularly cyanidin 3-galactoside (idaein). However, perceived fruit colour is also in¯uenced by chlorophyll, carotenoids, and other ¯avonoids, (Lancaster, 1992) as well as the physico-chemical environment in the cell (Brouillard et al., 1997). Anthocyanin biosynthesis requires light. Fruits produced deep inside the canopy where light levels are low, or where light has been arti®cially excluded, synthesise little or no anthocyanin (Saure, 1990), whereas fruits exposed to 70% of full sunlight develop good colour (Heinecke, 1966). Arti®cial light sources have been used successfully to improve colour in red or bicoloured apples (Arakawa et al., 1985; Saks et al., 1990; Marais et al., 2001). However, both fruit maturity, and temperature, interact with light and affect anthocyanin synthesis in apples (Faragher, 1983). Even under conditions of high light in apples, anthocyanins are synthesised only at two distinct phenological stages in fruit development. The ®rst occurs in young fruit (<1 cm) during cell division, and is not economically important (Lancaster, 1992). The second takes place during maturation of the fruit, when anthocyanin concentrations increase 3±5-fold. This is usually coincident with a decrease in chlorophyll concentration and an increase in carotenoid concentration (Knee, 1972; Faragher and Brohier, 1984; Lancaster, 1992; Ju et al., 1995; Reay et al., 1998). Low night temperatures (<208C) stimulate anthocyanin production and high temperatures inhibit it (Saure, 1990). Night temperatures of 108C resulted in better colour development than night temperatures of 21±268C (Creasy, 1968; Blankenship, 1987). While low night temperatures cannot replace the light requirement, they can improve anthocyanin synthesis in poorly exposed tissue (Creasy, 1968). Creasy (1968) found that warm nights and hot days (>328C) suppressed biosynthesis of the pigment. When fruits were kept in controlled temperature chambers at cool nights and warm days, an environment conducive to good colour development, a single day at high temperatures could adversely affect colour development. Marais (1995) observed a loss of red colour in bicoloured pears prior to harvest when day temperatures exceeded 358C. A similar response has been observed on apples (W.J. Steyn, personal communication). These experiments were designed to understand the factors that affect colour development in `Cripps' Pink' apples (marketed as `Pink Lady' when they satisfy certain colour requirements). The objectives were to determine the effect of (1) maturity, (2) a short cold period prior to irradiation, and (3) temperature conditions during irradiation, on the ability of the fruit to form a red blush. We hypothesised that high temperatures not only prevent anthocyanin synthesis, but can cause anthocyanin degradation.
E. Marais et al. / Scientia Horticulturae 90 (2001) 31±41
33
2. Material and methods 2.1. Experiment 1 In 1999, 30 `Cripps' Pink' apples were harvested at weekly intervals from Lourensford farm, Somerset West, South Africa (latitude: 338100 S, longitude: 198200 E) until the optimum harvest date, resulting in six harvest dates. Fruits were randomly divided into six replicates of ®ve fruits per replicate and placed in growth chambers at 208C. Fruits were irradiated continuously with light from 400 W highpressure sodium lamps (HPS) (Philips SON-T, 253 mW W 1) which have an emission spectrum from 400 to 800 nm, with a peak at about 605 nm. In our previous research, HPS lights resulted in a better response than UV-B plus incandescent lights, possibly because the acrylic layer between the light source and fruits in the chamber absorbed UV (Marais et al., 2001). The irradiance, measured with a Decagon light meter (AccuPAR version 4.1, Decagon devices, Pullman, Washington, USA) approximately 1 m from the light source was 22±24 W m 2. The green sides of the fruits were exposed to the light source for 144 h. Colour measurements of the fruits were taken before irradiation, and again every 24 h. 2.2. Experiment 2 In 1999, fruits were harvested at commercial maturity from two areas, Ceres and Grabouw, with climatic differences that typically result in a variation in colour. Fruits grown in the Ceres region (De Eike farm, latitude: 338140 S; longitude: 198140 E; altitude: 890 m) usually develop better red colour and this is attributed to lower night temperatures than experienced in Grabouw (De Rust farm, latitude: 348100 S; longitude: 198070 E; altitude: 330 m). Fruits were either irradiated immediately or stored for 2 or 5 days at 0.58C before irradiation. Chambers were at either 68C or 208C and fruits were irradiated for 120 h. Six single-fruit replicates were used for each treatment from each area. Colour measurements were made after 120 h, and then exposed sides of fruits were peeled for anthocyanin analysis. Control fruits kept in the dark at room temperature (ca. 208C) were also peeled for anthocyanin analysis. 2.3. Experiment 3 Fruits harvested from Ceres and Grabouw at the same time as in experiment 2 were stored for 20 days at 0.58C, after which their colour was measured, and they were placed at either 68C or an alternating temperature of 6/208C (12 h each). Six single-fruit replicates from each area were irradiated for 120 h, after which colour measurements were made and the exposed sides of the fruits were peeled for anthocyanin measurement.
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E. Marais et al. / Scientia Horticulturae 90 (2001) 31±41
2.4. Experiment 4 Well coloured `Cripps' Pink' apples were harvested from De Eike farm in 1999. After measuring their colour, fruits were placed at 378C. Fruits were exposed to irradiation for 0, 24, 48, 72, 96, 120 or 144 h, while control fruits were covered with white polystyrene. Seven single-fruit replicates were used for each irradiation period. 2.5. Measurements Circles were marked on the fruits and external colour was measured in the marked area using a colorimeter (model NR-3000, Nippon Denshoku Kogyo, Tokyo, Japan). Although chroma, hue angle and lightness values were recorded, only the hue angle data are presented, since they best represent change in colour from green to red. Hue angle h arctangentb=a refers to the angle formed by a line from the origin to the intercept of the a (x-axis) and b (y-axis) co-ordinates, where 0 red, 90 yellow, 180 green and 270 blue (McGuire, 1992). When anthocyanin concentration was measured, the exposed sides of the fruits were peeled with a potato peeler, and replicates were pooled to ensure enough tissue for analysis. Fruit peel was frozen at 808C, lyophilised and ground to a ®ne powder. Anthocyanin extraction was based on the procedure of Siegelman and Hendricks (1958). Fifteen millilitres of 1% HCl in methanol solution was added to 0.5 g of sample and left for 18 h at 58C in the dark. Samples were centrifuged for 10 min at 12 000gn. Absorbance of the supernatant was measured at 530 nm on a spectrophotometer (DU1 Series 64; Beckman, CA). A standard curve was obtained with idaein chloride (cyanidin 3-galactoside) (Carl Roth, Karlsruhe, Germany). Anthocyanin concentration was expressed as mg g 1 dry peel. 2.6. Statistical analyses The data were analysed with the General Linear Models (GLM) procedure of SAS1 (SAS Institute, 1996. SAS1 release 6.12, Cary, NC). 3. Results 3.1. Experiment 1 Fruits harvested 5, 4, 3, 2 and 1 weeks prior to the optimum harvest date showed only slight decreases in hue angle (<12.28) in response to irradiation. At commercial harvest, red colour development was rapid and a decrease of 62.58 in hue angle was measured after 72 h of irradiation (data not shown).
E. Marais et al. / Scientia Horticulturae 90 (2001) 31±41
35
Fig. 1. Mean maximum and mean minimum temperatures from fruit set in September 1998 until harvest in April 1999 measured at Ceres and Grabouw. Values used for April were calculated up until the harvest date.
3.2. Experiment 2 Mean maximum and minimum temperatures recorded from both Ceres and Grabouw for the period from anthesis (September 1998) to harvest (mid-April 1999) indicated that lower maximum and minimum temperatures were experienced in Ceres, except in April (Fig. 1). The cold storage period prior to irradiation had no signi®cant effect on changes in hue angle for fruit from Grabouw, although 5 days at 0.58C appeared to result in an increase in anthocyanin concentration in all treatments, particularly when fruits were subsequently irradiated at 68C for 120 h (Table 1). The most noticeable effect on hue angle was that of temperature during irradiation. Fruits irradiated at 68C showed greater decreases in hue angle than fruits irradiated at 208C P 0:0001. These decreases in hue angles were mirrored by apparent increases in anthocyanin concentrations, which were greater at 68C, regardless of pre-irradiation storage.
36
Table 1 The effects of irradiation at different temperatures on hue angle and anthocyanin concentration of `Cripps' Pink' apples grown in Grabouw, and stored for 0, 2 or 5 days at 0.58C prior to irradiationa Duration of irradiation (h)
Temperature after storage (8C)
Final hue angle (8C)
0 120 120
20 20 6
104.7 106.4 78.4
1.6 4.6 29.2
91 126 212
2
0 120 120
20 20 6
106.4 99.2 70.5
1.3 4.7 31.6
85 125 212
5
0 120 120
20 20 6
102.6 92.0 68.9
2.1 14.2 33.1
139 160 250
Grabouw 0
Significance Storage LIN Storage QUAD Temperature(6 vs. 208C) LIN Storage LIN by temperature LIN Storage QUAD by temperature a
Control fruits were stored in the dark at room temperature (ca. 208C).
± ± ± ± ±
D hue angle (8)
0.0804 0.6739 0.0001 0.4175 0.5039
Anthocyanin concentration (mg g 1)
± ± ± ± ±
E. Marais et al. / Scientia Horticulturae 90 (2001) 31±41
Storage (days)
Table 2 The effects of irradiation at different temperatures on hue angle and anthocyanin concentration of `Cripps' Pink' apples grown in Ceres, and stored for 0, 2 or 5 days at 0.58C prior to irradiationa Duration of irradiation (h)
Temperature after storage (8C)
Final hue angle (8C)
0 120 120
20 20 6
107.0 86.1 67.3
1.2 21.9 38.9
115 203 235
2
0 120 120
20 20 6
108.7 82.6 64.9
1.2 25.1 45.0
81 193 285
5
0 120 120
20 20 6
107.6 64.5 66.3
1.1 38.9 38.7
128 240 255
Ceres 0
Significance Storage LIN Storage QUAD Temperature (6 vs. 208C) LIN Storage LIN by temperature LIN Storage QUAD by temperature a
± ± ± ± ±
D hue angle (8)
0.1806 0.8142 0.0180 0.1341 0.3583
Anthocyanin concentration (mg g 1)
± ± ± ± ±
E. Marais et al. / Scientia Horticulturae 90 (2001) 31±41
Storage (days)
Control fruits were stored in the dark at room temperature (ca. 208C).
37
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E. Marais et al. / Scientia Horticulturae 90 (2001) 31±41
Changes in hue angle in the fruits harvested from Ceres were not affected by the cold temperature storage period prior to irradiation. Without light, the hue angle of fruits stored at room temperature did not change. Fruits irradiated at 68C had greater decreases in hue angles and apparent increases in anthocyanin concentrations than fruits irradiated at 208C, regardless of storage prior to irradiation (Table 2). However, fruits from Ceres that were irradiated at 208C responded differently than fruits from Grabouw. Fruits from Ceres which were irradiated immediately, or stored for 2 days before irradiation, showed decreases of 21.9 and 25.18, respectively, compared to the approximately 4.58 decreases for fruits from Grabouw. The decrease in hue angle for fruits stored for 5 days was the same (398), regardless of whether they were irradiated at 6 or 208C (Table 2). The apparent anthocyanin concentration was higher when fruits were irradiated at the lower temperature (68C) compared to irradiation at 208C. 3.3. Experiment 3 The hue angle responses of fruits from Grabouw were signi®cantly different when irradiated at 68C or alternating 6/208C P 0:0001 (Table 3). Alternating temperatures resulted in the greatest decrease in hue angle (708), with a corresponding apparent anthocyanin concentration of 654 mg g 1. Irradiation at 68C resulted in a decrease in hue angle value of 22.48 and apparent anthocyanin concentration of 207 mg g 1 (Table 3). Similar results were measured for fruits Table 3 The effects of irradiation with HPS light at different temperature regimes on `Cripps' Pink' apples, grown in Grabouw and Ceres, and stored for 20 days at 0.58C prior to irradiationa Duration of irradiation (h)
Temperature during treatment (8C)
Final hue angle (8C)
Grabouw 0 120 120
20 6/20 6
106.5 39.8 76.0
Significance 6/208C vs. 68C Ceres 0 120 120 Significance 6/208C vs. 68C a
± 20 6/20 6
107.3 41.5 93.0 ±
D hue angle (8) 0.5 70.0 22.4 0.0001 1.8 65.3 18.3 0.0001
Control fruits were stored in the dark at room temperature (ca. 208C).
Anthocyanin concentration (mg g 1) 131 654 207 ± 166 677 202 ±
E. Marais et al. / Scientia Horticulturae 90 (2001) 31±41
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Table 4 The effects of the combination of a high temperature (378C), with or without irradiation, on the hue value and anthocyanin concentration of `Cripps' Pink' apples Treatment
Duration of treatment (h)
Final hue angle (8C)
D hue angle (8)
Anthocyanin concentration (mg g 1)
378C light
0 24 48 72 96 120 144
29.3 32.0 36.6 44.5 43.3 48.3
3.4 8.4 12.0 18.4 19.8 24.9
740 681 471 452 372 341 284
378C dark
0 24 48 72 96 120 144
25.6 26.0 26.3 25.9 23.1 25.5
0.8 2.5 2.9 2.2 2.5 0.7
743 757 738 809 684 774 742
Significance Within light Within dark Light vs. dark
± ± ±
0.0006 0.4881 0.0006
± ± ±
from Ceres, with irradiation at 6/208C resulting in signi®cantly better colour P 0:0001 and higher apparent anthocyanin concentration than fruits stored at 68C (Table 3). 3.4. Experiment 4 When fruits were irradiated with HPS light at 378C hue angle increased linearly with time, corresponding to a loss in red colour (Table 4). The apparent anthocyanin concentration decreased by 62% during 144 h of irradiation. Fruits at 378C, without any irradiation, showed no change in hue angle P 0:4881 or anthocyanin concentration after 144 h. 4. Discussion Chalmers et al. (1973) found that fruits developed colour 14±21 days before maturity, and Proctor and Lougheed (1976) found that the onset of rapid red colour formation in `McIntosh' apples was about 20 days before harvest.
40
E. Marais et al. / Scientia Horticulturae 90 (2001) 31±41
However, we found that `Cripps' Pink' apples must be close to commercial maturity before anthocyanin synthesis occurred, even under inductive conditions. Chalmers et al. (1973) proposed that in immature fruits anthocyanin degradation potential may exceed synthesis potential, whereas in mature fruits synthesis potential may exceed degradation potential. The change in synthesis potential correlates with phenylalanine ammonia lyase (PAL) activity since the enzyme is more active in ripe fruit than unripe fruit (Faragher, 1983). Faragher (1983) concluded that ripening has a greater effect on anthocyanin synthesis than temperature. Storage at low temperatures ( 0.58C) for as little as 5 days prior to irradiation improved colour and increased anthocyanin concentrations. This positive effect of low temperature on anthocyanin synthesis in apples has been noted previously (Creasy, 1968; Faragher, 1983). Little colour developed at an irradiation temperature of 208C in fruits from Grabouw, whereas the hue angle values of the fruits from Ceres showed signi®cant decreases at this temperature. Fruits from the Grabouw area may have a greater need for a period of cold prior to irradiation in order to enhance anthocyanin synthesis. In both cases hue angle values decreased with increasing storage duration, but the magnitude of these decreases was greater for fruits from Ceres, than from Grabouw. Our results showed that alternating temperatures of 6/208C were more effective in improving colour than a constant temperature of 608C, and that 68C was, in turn, more effective than a constant temperature of 208C. Anthocyanin concentration and PAL enzyme activity was greater in `Red Spy' apples that received alternating 6/188C than fruits kept at a constant 188C (Tan, 1979). Although experiments 2 and 3 cannot be compared statistically, it was interesting to observe that the response at 68C differed between the two experiments. The decreases in hue angle were greater in experiment 2 (318 for Grabouw, and 418 for Ceres) than in experiment 3 (228 for Grabouw, and 188 for Ceres). The only difference between these fruits was the extent of the storage period. It seems that the ability to produce colour at this temperature decreased with extended low temperature storage. Bishop and Klein (1975) found that the ability of apples to synthesise anthocyanin under light decreased sygmoidally with storage prior to irradiation, but apples may maintain considerable capacity for anthocyanin formation after 6 months of storage or even longer. Creasy (1968) observed that high day temperatures inhibit the synthesis of anthocyanin, while Blankenship (1987) found that red skin colour was adversely affected by high night temperatures. Our data have clearly shown that high temperatures alone do not cause this response, and that loss of colour is caused by a combination of high temperatures and exposure to light. The mechanism of the degradation of anthocyanins in intact apple cells is unknown. In conclusion, the response of `Cripps' Pink' apples to postharvest irradiation was affected by both fruit maturity and temperature conditions during irradiation.
E. Marais et al. / Scientia Horticulturae 90 (2001) 31±41
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Lower temperatures enhanced synthesis of anthocyanins, while high temperatures resulted in anthocyanin degradation. References Arakawa, O., Hori, Y., Ogata, R., 1985. Relative effectiveness and interaction of ultraviolet-B, red and blue light in anthocyanin synthesis of apple fruit. Physiol. Plant. 64, 323±327. Bishop, R.G., Klein, R.M., 1975. Photo-promotion of anthocyanin synthesis in harvested apples. HortScience 10, 26±127. Blankenship, S.M., 1987. Night-temperature effects on rate of apple fruit maturation and fruit quality. Sci. Hort. 33, 205±212. Brouillard, R., Figueiredo, P., Elhabiri, M., Dangles, O., 1997. Molecular interactions of phenolic compounds in relation to the colour of fruit and vegetables. In: Thomas-Barberan, F.A., Robins, R.J. (Eds.), Phytochemistry of Fruit and Vegetables. Oxford Science Publications, Oxford, pp. 29±49. Chalmers, D.J., Faragher, J.D., Raff, J.W., 1973. Changes in anthocyanin synthesis as an index of maturity in red apple varieties. J. Hort. Sci. 48, 387±392. Creasy, L.L., 1968. The role of low temperature on anthocyanin synthesis in `McIntosh' apples. Proc. Am. Soc. Hort. Sci. 93, 716±724. Faragher, J.D., 1983. Temperature regulation of anthocyanin accumulation in apple skin. J. Exp. Bot. 34, 1291±1298. Faragher, J.D., Brohier, R.L., 1984. Anthocyanin accumulation in apple skin during ripening: regulation by ethylene and phenylalanine ammonia-lyase. Sci. Hort. 22, 89±96. Heinecke, D.R., 1966. Characteristics of McIntosh and red delicious apples as in¯uenced by exposure to sunlight during the growing season. Proc. Am. Soc. Hort. Sci. 89, 10±13. Ju, Z., Liu, C., Yuan, Y., 1995. Activities of chalcone synthase and UDPGal:¯avonoid-3-oglycosyltransferase in relation to anthocyanin synthesis in apple. Sci. Hort. 63, 175±185. Knee, M., 1972. Anthocyanin, carotenoid, and chlorophyll changes in the peel of `Cox's Orange Pippin' apples during ripening on and off the tree. J. Exp. Bot. 23, 184±196. Lancaster, J.E., 1992. Regulation of skin color in apples. Grit. Rev. Plant Sci. 10, 487±502. Marais, E., Jacobs, G., Holcroft, D.M., 2001. Postharvest irradiation enhances anthocyanin synthesis in apples but not in pears. HortScience, in press. Marais, G.F., 1995. Optimisation of the economic biomass production of `Forelle' pears (Pyrus communis L.). M.Sc. Thesis (Agric). University of Stellenbosch, Stellenbosch, South Africa. McGuire, R.G., 1992. Reporting of objective color measurements. HortScience 27, 1254±1255. Proctor, J.T.A., Lougheed, E.C., 1976. The effect of covering apples during development. HortScience 11, 108±109. Reay, P.F., Fletcher, R.H., Thomas, V.J., 1998. Chlorophylls, carotenoids and anthocyanin concentrations in the skin of `Gala' apples during maturation and the in¯uence of foliar applications of nitrogen and magnesium. J. Sci. Food Agric. 76, 63±71. Saks, Y., Sonego, L., Ben-Arie, R., 1990. Arti®cial light enhances red pigmentation, but not ripening, of harvested `Anna' apples. HortScience 25, 547±549. Saure, M.C., 1990. External control of anthocyanin formation in apple. Sci. Hort. 42, 181±218. Siegelman, H.W., Hendricks, S.B., 1958. Photocontrol of anthocyanin synthesis in apple skin. Plant Physiol. 33, 185±190. Tan, S.C., 1979. Relationships and interactions between phenylalanine ammonia-lyase, phenylalanine ammonia-lyase inactivating system and anthocyanin in apples. J. Am. Soc. Hort. Sci. 104, 581±586.
Colour response of `Cripps' Pink' apples to postharvest irradiation is in¯uenced by maturity and temperature Evelyn Marais, Gerard Jacobs, Deirdre M. Holcroft* Department of Horticultural Science, University of Stellenbosch, Private Bag X1, Matieland 7602, Stellenbosch, South Africa Accepted 21 October 2000
Abstract Preliminary experiments indicated that postharvest irradiation enhanced anthocyanin synthesis in `Cripps' Pink' apples. Consequently this system was used to understand the effects of maturity and temperature on anthocyanin synthesis. Fruits harvested 5, 4, 3, 2 and 1 weeks prior to predicted commercial harvest did not develop a red blush in response to postharvest irradiation (72 h with high-pressure sodium lights at 208C). Fruits harvested at maturity rapidly developed a red blush in response to irradiation. Mature `Cripps' Pink' apples harvested from two growing regions were subjected to 120 h of irradiation at 6 or 208C following 0, 2 or 5 days at 0.58C. Fruits harvested from the cooler area (Ceres) and irradiated immediately showed greater red colouration at 68C than at 208C. The difference between the temperatures was no longer signi®cant after fruits have been stored for 5 days at 0.58C prior to irradiation. Fruits harvested from the warmer area (Grabouw) were consistently redder when irradiated at 68C than 208C. In a separate experiment, fruits were stored for 20 days at 0.58C prior to irradiation at either 68C or 20/68C (day/night). The alternating temperature resulted in better colour of fruits from both areas. In a ®nal experiment, well-coloured `Cripps' Pink' apples were irradiated at 378C to simulate periods of high temperature that occur in summer. The hue angle of the fruits increased from 29.3 to 48.38 after 144 h of light indicating a loss of red colour. The anthocyanin concentration decreased over 50% during the same period. Fruits kept at 378C in the dark did not lose red colour. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Anthocyanin; Red blush; Light; Malus domestica Borkh.; Pink LadyTM
*
Corresponding author. Tel.: 27-21-8084105; fax: 27-21-8082121. E-mail address: [email protected] (D.M. Holcroft). 0304-4238/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 2 5 6 - 9
32
E. Marais et al. / Scientia Horticulturae 90 (2001) 31±41
1. Introduction The development of a red blush on bicoloured apples is required to satisfy export standards. The red colour is a result of anthocyanin pigments, particularly cyanidin 3-galactoside (idaein). However, perceived fruit colour is also in¯uenced by chlorophyll, carotenoids, and other ¯avonoids, (Lancaster, 1992) as well as the physico-chemical environment in the cell (Brouillard et al., 1997). Anthocyanin biosynthesis requires light. Fruits produced deep inside the canopy where light levels are low, or where light has been arti®cially excluded, synthesise little or no anthocyanin (Saure, 1990), whereas fruits exposed to 70% of full sunlight develop good colour (Heinecke, 1966). Arti®cial light sources have been used successfully to improve colour in red or bicoloured apples (Arakawa et al., 1985; Saks et al., 1990; Marais et al., 2001). However, both fruit maturity, and temperature, interact with light and affect anthocyanin synthesis in apples (Faragher, 1983). Even under conditions of high light in apples, anthocyanins are synthesised only at two distinct phenological stages in fruit development. The ®rst occurs in young fruit (<1 cm) during cell division, and is not economically important (Lancaster, 1992). The second takes place during maturation of the fruit, when anthocyanin concentrations increase 3±5-fold. This is usually coincident with a decrease in chlorophyll concentration and an increase in carotenoid concentration (Knee, 1972; Faragher and Brohier, 1984; Lancaster, 1992; Ju et al., 1995; Reay et al., 1998). Low night temperatures (<208C) stimulate anthocyanin production and high temperatures inhibit it (Saure, 1990). Night temperatures of 108C resulted in better colour development than night temperatures of 21±268C (Creasy, 1968; Blankenship, 1987). While low night temperatures cannot replace the light requirement, they can improve anthocyanin synthesis in poorly exposed tissue (Creasy, 1968). Creasy (1968) found that warm nights and hot days (>328C) suppressed biosynthesis of the pigment. When fruits were kept in controlled temperature chambers at cool nights and warm days, an environment conducive to good colour development, a single day at high temperatures could adversely affect colour development. Marais (1995) observed a loss of red colour in bicoloured pears prior to harvest when day temperatures exceeded 358C. A similar response has been observed on apples (W.J. Steyn, personal communication). These experiments were designed to understand the factors that affect colour development in `Cripps' Pink' apples (marketed as `Pink Lady' when they satisfy certain colour requirements). The objectives were to determine the effect of (1) maturity, (2) a short cold period prior to irradiation, and (3) temperature conditions during irradiation, on the ability of the fruit to form a red blush. We hypothesised that high temperatures not only prevent anthocyanin synthesis, but can cause anthocyanin degradation.
E. Marais et al. / Scientia Horticulturae 90 (2001) 31±41
33
2. Material and methods 2.1. Experiment 1 In 1999, 30 `Cripps' Pink' apples were harvested at weekly intervals from Lourensford farm, Somerset West, South Africa (latitude: 338100 S, longitude: 198200 E) until the optimum harvest date, resulting in six harvest dates. Fruits were randomly divided into six replicates of ®ve fruits per replicate and placed in growth chambers at 208C. Fruits were irradiated continuously with light from 400 W highpressure sodium lamps (HPS) (Philips SON-T, 253 mW W 1) which have an emission spectrum from 400 to 800 nm, with a peak at about 605 nm. In our previous research, HPS lights resulted in a better response than UV-B plus incandescent lights, possibly because the acrylic layer between the light source and fruits in the chamber absorbed UV (Marais et al., 2001). The irradiance, measured with a Decagon light meter (AccuPAR version 4.1, Decagon devices, Pullman, Washington, USA) approximately 1 m from the light source was 22±24 W m 2. The green sides of the fruits were exposed to the light source for 144 h. Colour measurements of the fruits were taken before irradiation, and again every 24 h. 2.2. Experiment 2 In 1999, fruits were harvested at commercial maturity from two areas, Ceres and Grabouw, with climatic differences that typically result in a variation in colour. Fruits grown in the Ceres region (De Eike farm, latitude: 338140 S; longitude: 198140 E; altitude: 890 m) usually develop better red colour and this is attributed to lower night temperatures than experienced in Grabouw (De Rust farm, latitude: 348100 S; longitude: 198070 E; altitude: 330 m). Fruits were either irradiated immediately or stored for 2 or 5 days at 0.58C before irradiation. Chambers were at either 68C or 208C and fruits were irradiated for 120 h. Six single-fruit replicates were used for each treatment from each area. Colour measurements were made after 120 h, and then exposed sides of fruits were peeled for anthocyanin analysis. Control fruits kept in the dark at room temperature (ca. 208C) were also peeled for anthocyanin analysis. 2.3. Experiment 3 Fruits harvested from Ceres and Grabouw at the same time as in experiment 2 were stored for 20 days at 0.58C, after which their colour was measured, and they were placed at either 68C or an alternating temperature of 6/208C (12 h each). Six single-fruit replicates from each area were irradiated for 120 h, after which colour measurements were made and the exposed sides of the fruits were peeled for anthocyanin measurement.
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E. Marais et al. / Scientia Horticulturae 90 (2001) 31±41
2.4. Experiment 4 Well coloured `Cripps' Pink' apples were harvested from De Eike farm in 1999. After measuring their colour, fruits were placed at 378C. Fruits were exposed to irradiation for 0, 24, 48, 72, 96, 120 or 144 h, while control fruits were covered with white polystyrene. Seven single-fruit replicates were used for each irradiation period. 2.5. Measurements Circles were marked on the fruits and external colour was measured in the marked area using a colorimeter (model NR-3000, Nippon Denshoku Kogyo, Tokyo, Japan). Although chroma, hue angle and lightness values were recorded, only the hue angle data are presented, since they best represent change in colour from green to red. Hue angle h arctangentb=a refers to the angle formed by a line from the origin to the intercept of the a (x-axis) and b (y-axis) co-ordinates, where 0 red, 90 yellow, 180 green and 270 blue (McGuire, 1992). When anthocyanin concentration was measured, the exposed sides of the fruits were peeled with a potato peeler, and replicates were pooled to ensure enough tissue for analysis. Fruit peel was frozen at 808C, lyophilised and ground to a ®ne powder. Anthocyanin extraction was based on the procedure of Siegelman and Hendricks (1958). Fifteen millilitres of 1% HCl in methanol solution was added to 0.5 g of sample and left for 18 h at 58C in the dark. Samples were centrifuged for 10 min at 12 000gn. Absorbance of the supernatant was measured at 530 nm on a spectrophotometer (DU1 Series 64; Beckman, CA). A standard curve was obtained with idaein chloride (cyanidin 3-galactoside) (Carl Roth, Karlsruhe, Germany). Anthocyanin concentration was expressed as mg g 1 dry peel. 2.6. Statistical analyses The data were analysed with the General Linear Models (GLM) procedure of SAS1 (SAS Institute, 1996. SAS1 release 6.12, Cary, NC). 3. Results 3.1. Experiment 1 Fruits harvested 5, 4, 3, 2 and 1 weeks prior to the optimum harvest date showed only slight decreases in hue angle (<12.28) in response to irradiation. At commercial harvest, red colour development was rapid and a decrease of 62.58 in hue angle was measured after 72 h of irradiation (data not shown).
E. Marais et al. / Scientia Horticulturae 90 (2001) 31±41
35
Fig. 1. Mean maximum and mean minimum temperatures from fruit set in September 1998 until harvest in April 1999 measured at Ceres and Grabouw. Values used for April were calculated up until the harvest date.
3.2. Experiment 2 Mean maximum and minimum temperatures recorded from both Ceres and Grabouw for the period from anthesis (September 1998) to harvest (mid-April 1999) indicated that lower maximum and minimum temperatures were experienced in Ceres, except in April (Fig. 1). The cold storage period prior to irradiation had no signi®cant effect on changes in hue angle for fruit from Grabouw, although 5 days at 0.58C appeared to result in an increase in anthocyanin concentration in all treatments, particularly when fruits were subsequently irradiated at 68C for 120 h (Table 1). The most noticeable effect on hue angle was that of temperature during irradiation. Fruits irradiated at 68C showed greater decreases in hue angle than fruits irradiated at 208C P 0:0001. These decreases in hue angles were mirrored by apparent increases in anthocyanin concentrations, which were greater at 68C, regardless of pre-irradiation storage.
36
Table 1 The effects of irradiation at different temperatures on hue angle and anthocyanin concentration of `Cripps' Pink' apples grown in Grabouw, and stored for 0, 2 or 5 days at 0.58C prior to irradiationa Duration of irradiation (h)
Temperature after storage (8C)
Final hue angle (8C)
0 120 120
20 20 6
104.7 106.4 78.4
1.6 4.6 29.2
91 126 212
2
0 120 120
20 20 6
106.4 99.2 70.5
1.3 4.7 31.6
85 125 212
5
0 120 120
20 20 6
102.6 92.0 68.9
2.1 14.2 33.1
139 160 250
Grabouw 0
Significance Storage LIN Storage QUAD Temperature(6 vs. 208C) LIN Storage LIN by temperature LIN Storage QUAD by temperature a
Control fruits were stored in the dark at room temperature (ca. 208C).
± ± ± ± ±
D hue angle (8)
0.0804 0.6739 0.0001 0.4175 0.5039
Anthocyanin concentration (mg g 1)
± ± ± ± ±
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Storage (days)
Table 2 The effects of irradiation at different temperatures on hue angle and anthocyanin concentration of `Cripps' Pink' apples grown in Ceres, and stored for 0, 2 or 5 days at 0.58C prior to irradiationa Duration of irradiation (h)
Temperature after storage (8C)
Final hue angle (8C)
0 120 120
20 20 6
107.0 86.1 67.3
1.2 21.9 38.9
115 203 235
2
0 120 120
20 20 6
108.7 82.6 64.9
1.2 25.1 45.0
81 193 285
5
0 120 120
20 20 6
107.6 64.5 66.3
1.1 38.9 38.7
128 240 255
Ceres 0
Significance Storage LIN Storage QUAD Temperature (6 vs. 208C) LIN Storage LIN by temperature LIN Storage QUAD by temperature a
± ± ± ± ±
D hue angle (8)
0.1806 0.8142 0.0180 0.1341 0.3583
Anthocyanin concentration (mg g 1)
± ± ± ± ±
E. Marais et al. / Scientia Horticulturae 90 (2001) 31±41
Storage (days)
Control fruits were stored in the dark at room temperature (ca. 208C).
37
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E. Marais et al. / Scientia Horticulturae 90 (2001) 31±41
Changes in hue angle in the fruits harvested from Ceres were not affected by the cold temperature storage period prior to irradiation. Without light, the hue angle of fruits stored at room temperature did not change. Fruits irradiated at 68C had greater decreases in hue angles and apparent increases in anthocyanin concentrations than fruits irradiated at 208C, regardless of storage prior to irradiation (Table 2). However, fruits from Ceres that were irradiated at 208C responded differently than fruits from Grabouw. Fruits from Ceres which were irradiated immediately, or stored for 2 days before irradiation, showed decreases of 21.9 and 25.18, respectively, compared to the approximately 4.58 decreases for fruits from Grabouw. The decrease in hue angle for fruits stored for 5 days was the same (398), regardless of whether they were irradiated at 6 or 208C (Table 2). The apparent anthocyanin concentration was higher when fruits were irradiated at the lower temperature (68C) compared to irradiation at 208C. 3.3. Experiment 3 The hue angle responses of fruits from Grabouw were signi®cantly different when irradiated at 68C or alternating 6/208C P 0:0001 (Table 3). Alternating temperatures resulted in the greatest decrease in hue angle (708), with a corresponding apparent anthocyanin concentration of 654 mg g 1. Irradiation at 68C resulted in a decrease in hue angle value of 22.48 and apparent anthocyanin concentration of 207 mg g 1 (Table 3). Similar results were measured for fruits Table 3 The effects of irradiation with HPS light at different temperature regimes on `Cripps' Pink' apples, grown in Grabouw and Ceres, and stored for 20 days at 0.58C prior to irradiationa Duration of irradiation (h)
Temperature during treatment (8C)
Final hue angle (8C)
Grabouw 0 120 120
20 6/20 6
106.5 39.8 76.0
Significance 6/208C vs. 68C Ceres 0 120 120 Significance 6/208C vs. 68C a
± 20 6/20 6
107.3 41.5 93.0 ±
D hue angle (8) 0.5 70.0 22.4 0.0001 1.8 65.3 18.3 0.0001
Control fruits were stored in the dark at room temperature (ca. 208C).
Anthocyanin concentration (mg g 1) 131 654 207 ± 166 677 202 ±
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Table 4 The effects of the combination of a high temperature (378C), with or without irradiation, on the hue value and anthocyanin concentration of `Cripps' Pink' apples Treatment
Duration of treatment (h)
Final hue angle (8C)
D hue angle (8)
Anthocyanin concentration (mg g 1)
378C light
0 24 48 72 96 120 144
29.3 32.0 36.6 44.5 43.3 48.3
3.4 8.4 12.0 18.4 19.8 24.9
740 681 471 452 372 341 284
378C dark
0 24 48 72 96 120 144
25.6 26.0 26.3 25.9 23.1 25.5
0.8 2.5 2.9 2.2 2.5 0.7
743 757 738 809 684 774 742
Significance Within light Within dark Light vs. dark
± ± ±
0.0006 0.4881 0.0006
± ± ±
from Ceres, with irradiation at 6/208C resulting in signi®cantly better colour P 0:0001 and higher apparent anthocyanin concentration than fruits stored at 68C (Table 3). 3.4. Experiment 4 When fruits were irradiated with HPS light at 378C hue angle increased linearly with time, corresponding to a loss in red colour (Table 4). The apparent anthocyanin concentration decreased by 62% during 144 h of irradiation. Fruits at 378C, without any irradiation, showed no change in hue angle P 0:4881 or anthocyanin concentration after 144 h. 4. Discussion Chalmers et al. (1973) found that fruits developed colour 14±21 days before maturity, and Proctor and Lougheed (1976) found that the onset of rapid red colour formation in `McIntosh' apples was about 20 days before harvest.
40
E. Marais et al. / Scientia Horticulturae 90 (2001) 31±41
However, we found that `Cripps' Pink' apples must be close to commercial maturity before anthocyanin synthesis occurred, even under inductive conditions. Chalmers et al. (1973) proposed that in immature fruits anthocyanin degradation potential may exceed synthesis potential, whereas in mature fruits synthesis potential may exceed degradation potential. The change in synthesis potential correlates with phenylalanine ammonia lyase (PAL) activity since the enzyme is more active in ripe fruit than unripe fruit (Faragher, 1983). Faragher (1983) concluded that ripening has a greater effect on anthocyanin synthesis than temperature. Storage at low temperatures ( 0.58C) for as little as 5 days prior to irradiation improved colour and increased anthocyanin concentrations. This positive effect of low temperature on anthocyanin synthesis in apples has been noted previously (Creasy, 1968; Faragher, 1983). Little colour developed at an irradiation temperature of 208C in fruits from Grabouw, whereas the hue angle values of the fruits from Ceres showed signi®cant decreases at this temperature. Fruits from the Grabouw area may have a greater need for a period of cold prior to irradiation in order to enhance anthocyanin synthesis. In both cases hue angle values decreased with increasing storage duration, but the magnitude of these decreases was greater for fruits from Ceres, than from Grabouw. Our results showed that alternating temperatures of 6/208C were more effective in improving colour than a constant temperature of 608C, and that 68C was, in turn, more effective than a constant temperature of 208C. Anthocyanin concentration and PAL enzyme activity was greater in `Red Spy' apples that received alternating 6/188C than fruits kept at a constant 188C (Tan, 1979). Although experiments 2 and 3 cannot be compared statistically, it was interesting to observe that the response at 68C differed between the two experiments. The decreases in hue angle were greater in experiment 2 (318 for Grabouw, and 418 for Ceres) than in experiment 3 (228 for Grabouw, and 188 for Ceres). The only difference between these fruits was the extent of the storage period. It seems that the ability to produce colour at this temperature decreased with extended low temperature storage. Bishop and Klein (1975) found that the ability of apples to synthesise anthocyanin under light decreased sygmoidally with storage prior to irradiation, but apples may maintain considerable capacity for anthocyanin formation after 6 months of storage or even longer. Creasy (1968) observed that high day temperatures inhibit the synthesis of anthocyanin, while Blankenship (1987) found that red skin colour was adversely affected by high night temperatures. Our data have clearly shown that high temperatures alone do not cause this response, and that loss of colour is caused by a combination of high temperatures and exposure to light. The mechanism of the degradation of anthocyanins in intact apple cells is unknown. In conclusion, the response of `Cripps' Pink' apples to postharvest irradiation was affected by both fruit maturity and temperature conditions during irradiation.
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Lower temperatures enhanced synthesis of anthocyanins, while high temperatures resulted in anthocyanin degradation. References Arakawa, O., Hori, Y., Ogata, R., 1985. Relative effectiveness and interaction of ultraviolet-B, red and blue light in anthocyanin synthesis of apple fruit. Physiol. Plant. 64, 323±327. Bishop, R.G., Klein, R.M., 1975. Photo-promotion of anthocyanin synthesis in harvested apples. HortScience 10, 26±127. Blankenship, S.M., 1987. Night-temperature effects on rate of apple fruit maturation and fruit quality. Sci. Hort. 33, 205±212. Brouillard, R., Figueiredo, P., Elhabiri, M., Dangles, O., 1997. Molecular interactions of phenolic compounds in relation to the colour of fruit and vegetables. In: Thomas-Barberan, F.A., Robins, R.J. (Eds.), Phytochemistry of Fruit and Vegetables. Oxford Science Publications, Oxford, pp. 29±49. Chalmers, D.J., Faragher, J.D., Raff, J.W., 1973. Changes in anthocyanin synthesis as an index of maturity in red apple varieties. J. Hort. Sci. 48, 387±392. Creasy, L.L., 1968. The role of low temperature on anthocyanin synthesis in `McIntosh' apples. Proc. Am. Soc. Hort. Sci. 93, 716±724. Faragher, J.D., 1983. Temperature regulation of anthocyanin accumulation in apple skin. J. Exp. Bot. 34, 1291±1298. Faragher, J.D., Brohier, R.L., 1984. Anthocyanin accumulation in apple skin during ripening: regulation by ethylene and phenylalanine ammonia-lyase. Sci. Hort. 22, 89±96. Heinecke, D.R., 1966. Characteristics of McIntosh and red delicious apples as in¯uenced by exposure to sunlight during the growing season. Proc. Am. Soc. Hort. Sci. 89, 10±13. Ju, Z., Liu, C., Yuan, Y., 1995. Activities of chalcone synthase and UDPGal:¯avonoid-3-oglycosyltransferase in relation to anthocyanin synthesis in apple. Sci. Hort. 63, 175±185. Knee, M., 1972. Anthocyanin, carotenoid, and chlorophyll changes in the peel of `Cox's Orange Pippin' apples during ripening on and off the tree. J. Exp. Bot. 23, 184±196. Lancaster, J.E., 1992. Regulation of skin color in apples. Grit. Rev. Plant Sci. 10, 487±502. Marais, E., Jacobs, G., Holcroft, D.M., 2001. Postharvest irradiation enhances anthocyanin synthesis in apples but not in pears. HortScience, in press. Marais, G.F., 1995. Optimisation of the economic biomass production of `Forelle' pears (Pyrus communis L.). M.Sc. Thesis (Agric). University of Stellenbosch, Stellenbosch, South Africa. McGuire, R.G., 1992. Reporting of objective color measurements. HortScience 27, 1254±1255. Proctor, J.T.A., Lougheed, E.C., 1976. The effect of covering apples during development. HortScience 11, 108±109. Reay, P.F., Fletcher, R.H., Thomas, V.J., 1998. Chlorophylls, carotenoids and anthocyanin concentrations in the skin of `Gala' apples during maturation and the in¯uence of foliar applications of nitrogen and magnesium. J. Sci. Food Agric. 76, 63±71. Saks, Y., Sonego, L., Ben-Arie, R., 1990. Arti®cial light enhances red pigmentation, but not ripening, of harvested `Anna' apples. HortScience 25, 547±549. Saure, M.C., 1990. External control of anthocyanin formation in apple. Sci. Hort. 42, 181±218. Siegelman, H.W., Hendricks, S.B., 1958. Photocontrol of anthocyanin synthesis in apple skin. Plant Physiol. 33, 185±190. Tan, S.C., 1979. Relationships and interactions between phenylalanine ammonia-lyase, phenylalanine ammonia-lyase inactivating system and anthocyanin in apples. J. Am. Soc. Hort. Sci. 104, 581±586.