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Carbohydrates as related to the heat-induced chilling tolerance and respiratory rate of ‘Fortune’ mandarin fruit harvested at different maturity stages
Postharvest Biology and Technology 25 (2002) 181– 191 www.elsevier.com/locate/postharvbio
Carbohydrates as related to the heat-induced chilling tolerance and respiratory rate of ‘Fortune’ mandarin fruit harvested at different maturity stages Nely Holland b, Hilary C. Menezes b, Marı´a T. Lafuente a,* a
Instituto de Agroquı´mica y Tecnologı´a de Alimentos, Consejo Superior de In6est. Cientif. C.S.I.C., Apartado de Correos 73, 46100 Burjassot, Valencia, Spain b Departamento de Tecnologia de Alimentos, FEA-Uni6ersidade Estadual de Campinas (UNICAMP), Caixa Postal: 6121, CEP 13083 -970, Campinas, Brazil Received 30 March 2001; accepted 18 October 2001
Abstract We have evaluated the effect of a heat-conditioning treatment (3 days at 37 °C) on respiratory rate, soluble carbohydrate and starch content of ‘Fortune’ mandarin fruit harvested at different maturity stages and stored at a chilling (2 °C) and a non-chilling (12 °C) temperature. The treatment was highly effective, increasing the tolerance of detached ‘Fortune’ fruit to chilling injury (CI). Changes in carbohydrate levels in response to the high temperature treatment, and during exposure of the non-conditioned and heat-conditioned fruit to 2 and 12 °C, appear to be mainly related to the consumption of carbohydrate reserves for respiration. The highest respiratory rate was found in fruit exposed to 37 °C. The respiration of non-conditioned fruit stored at 2 °C was lower than that of control fruit stored at 12 °C. Therefore, anomalous high respiratory activity did not occur during chilling in ‘Fortune’ mandarins. Sucrose appeared to be the most accessible sugar as a respiratory substrate in non-conditioned fruit. No relationship between the susceptibility to CI and changes in carbohydrates during storage of non-conditioned fruit at 2 °C was found. The heat treatment prevented the decline in sucrose content during chilling. After 30 days at 2 °C, the sucrose content of non-conditioned fruit harvested in December, January and March was about 79, 57 and 68% that of their respective heat-conditioned fruit. In contrast, heating the fruit favoured the loss of glucose, fructose and starch in fruit kept at 2 °C. These data suggest that sucrose but not glucose, fructose or starch could be involved in the heat-induced chilling tolerance of citrus fruit detached from the tree. The effect of the heat treatment preventing sucrose decline during storage of fruit at 2 °C was less relevant in fruit from December than in those harvested later in the season, although it was very effective in protecting fruit harvested at all maturity stages against chilling. Therefore, sucrose appears not to be a limiting factor for the heat-induced chilling tolerance in citrus fruit. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Citrus; Cold stress; Fructose; Glucose; Heat conditioning; Maturation; Respiration; Starch; Sucrose
* Corresponding author. Tel.: + 34-963900022; fax: + 34-963636301. E-mail address: [email protected] (M.T. Lafuente). 0925-5214/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 5 2 1 4 ( 0 1 ) 0 0 1 8 2 - X
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1. Introduction Carbohydrate levels and composition may influence the sensitivity of plant tissues to low temperature (Levitt, 1980; Purvis and Grierson, 1982). Chilling injury (CI) may occur in many citrus cultivars when exposed to low, non-freezing temperatures. Fruit of the hybrid ‘Fortune’ mandarin (Citrus clementina Hort. Ex Tanaka x Citrus reticulata, Blanca) show high sensitivity to CI, which is manifest as small brown pit-like depressions along the equatorial zones of the fruit. Previous investigations studying the role of carbohydrates on chilling tolerance of citrus fruit have focused mainly on the relationship between changes in carbohydrates in the flavedo (the outer pigmented portion of the peel) of fruit attached to the tree during maturity (Purvis and Grierson, 1982; Holland et al., 1999). Conflicting results have been found. Purvis and Grierson (1982) suggested that the increase in reducing sugar levels in the peel of grapefruit during winter could be a defence mechanism of citrus fruit attached to the trees to resist cold stress. Conversely, sugar accumulation in mandarins during winter did not precede their resistance to CI (Holland et al., 1999). The importance of soluble carbohydrates, especially sucrose, in freezing tolerance induced by cold acclimation in red raspberry plants has been recently shown (Palonen et al., 2000). Sugars are also involved in the acclimation response to chilling induced by mechanical stress in tomato leaves (Keller and Steffen, 1995). A comparison between the changes in chilling susceptibility of tomato seedlings and their starch content at different times during the light/dark cycle indicates that starch may also play a role in defending plant tissues against chilling (King et al., 1988). Through different experiments, we have shown that heating detached ‘Fortune’ mandarin fruit for 3 days at 37 °C and 90 – 95% RH considerably reduces cold-induced peel damage during the exposure of the fruit to 2 °C and that the efficacy of this high temperature conditioning treatment is not influenced by the maturity stage of the fruit (Lafuente et al., 1997; Sala and Lafuente, 1999; Gonzalez-Aguilar et al., 2000; Sanchez-Ballesta et al., 2000). Heat treatments have been shown to
induce cold tolerance in a wide range of horticultural crops (Lurie, 1998), but few studies have focused on the postharvest carbohydrate changes brought on by the imposition of heat treatments. Aung et al. (1988) showed that the heat treatment of lemons at 55 °C for 5 min significantly increased the sucrose content of the flavedo, while the raffinose content was unaffected and the glucose and fructose contents decreased slightly. It has been also reported that heat shock may lead to a reduction in sucrose cleavage and starch accumulation (Wang et al., 1993; Lafta and Lorenzen, 1995). How the high temperature affected the chilling response of these plant tissues or their carbohydrate metabolism, when transferred to low temperatures, was not examined. The influence of high temperatures on the respiratory rate in apples and other climacteric fruit such as tomatoes has been studied (Lurie, 1998). However, the relationship between sugar metabolism and respiratory rise in response to heat stress is little known. The effect of cold stress on carbohydrate changes in detached fruit as related to their respiratory rate and how those processes may be affected by the maturity stage of the fruit or by a heat conditioning treatment is also poorly understood. In response to other physiological processes, such as ripening, this relationship may vary among fruit. Thus, at a nonstress temperature, the rise in respiration in banana during ripening was temporally related to the net conversion of starch to soluble sugars, whereas in other fruit such as apples, accumulation of sugars was not associated with the respiratory rise (Mertens et al., 1987), and in kiwifruit, the rise in respiration did not occur until after the conversion of starch to sugars was effectively completed (MacRae et al., 1992). The aim of the present work was to study whether carbohydrate changes induced by high temperature conditioning may play a role in increasing tolerance of detached citrus fruit to chilling and how those changes may be related to the respiratory rate of fruit. To this end, we evaluated the effect of a high temperature conditioning treatment (3 days at 37 °C), which greatly increased chilling tolerance of the fruit, on the starch and soluble carbohydrate contents of the
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flavedo of ‘Fortune’ mandarins. The relationship between changes in the respiratory rate of the fruit and in the levels of carbohydrates was also examined. Fruit harvested at different maturity stages, differing in their susceptibility to CI, have been used to evaluate the pattern of carbohydrate accumulation during the storage of non-conditioned and heat-conditioned fruit at a chilling (2 °C) and a non-chilling (12 °C) temperature to better understand the role of carbohydrates in the protection of detached citrus fruit against CI.
2. Material and methods
2.1. Plant material and chilling and high temperature conditioning treatments Fruit of ‘Fortune’ mandarin (C. clementina Hort. Ex Tanaka x C. reticulata, Blanca) were harvested at three different stages of maturity during the season (December 9, January 27, and March 3). Fruit were harvested at random from adult trees growing in a commercial orchard at Sagunto (Valencia, Spain, latitude: 39° 28% 48¦ N; longitude: 00° 22% 52¦ W), immediately delivered to the laboratory and randomly divided into two lots. The first lot was subdivided into two groups, which were stored at 80– 90% relative humidity (RH) and 2 °C (chilled control) or 12 °C (nonchilled control) for up to 30 days. The second lot was used to evaluate the effect of heat conditioning on chilling-tolerance, respiratory rates and carbohydrate changes of the fruit. Fruit from this lot were subjected to a 3 days heat conditioning treatment at 90–95% RH and 37 °C and then subdivided into two groups which were stored at the same temperatures and RH as the freshly harvested (non-conditioned) fruit. For each different maturity stage and temperature regime, three replicates containing four fruit were used to periodically determine CO2 production, three replicates of 20 fruit each for CI evaluation and three of 10 fruit to analyze carbohydrate content. Flavedo tissue was collected from the total surface of fruit and frozen in liquid nitrogen for subsequent sugar and starch analysis.
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2.2. CI index The CI index was estimated weekly in heat-conditioned and non-conditioned fruit held at 2 and 12 °C. Brown pit-like depressions along the fruit are the main CI symptoms in ‘Fortune’ mandarins. CI was rated visually on a scale from 0 (no injury) to 3 (severe injury) according to the necrotic surface and the intensity of browning. A CI index was determined by summing the product of the number of fruit in each category multiplied by the score of each category, and then dividing this sum by the number of fruit evaluated (Lafuente et al., 1997). The results are the means of three replicate samples containing 20 fruit9 SE.
2.3. Sugar analysis Sugars were extracted from flavedo tissue by the method previously described by Purvis et al. (1979). One gram of flavedo was extracted three times with 10 ml of 80% boiling ethanol using a Polytron homogenizer. The combined ethanol extracts were filtered through a glass fibre filter pad and 2 ml of 2.1% raffinose added as an internal standard. The alcoholic solution was centrifuged, the supernatant vacuum-evaporated and the resulting residue dissolved in 5 ml water. One milliliter of this solution was purified by passing through a C-18 ‘Elud Bond’ cartridge from Varian (Harbor City, CA) and then filtering through a Millipore HV-4 filter (pore size 0.45 mm) (Ibe´ rica, S.A., Barcelona, Spain) for HPLC analysis as previously described (Holland et al., 1999). Sugars were eluted in 20 min at 85 °C using water as the mobile phase at a flow rate of 0.6 ml min − 1 and a 300× 7.8 mm Phenomenex column packed with a Rezex sulfonated polystyrene resin. A Waters 410 refractive index detector (Waters, Franklin, MA) was used and the sugars were quantified by peak area comparison using standard curves for sucrose, glucose and fructose, and raffinose as the internal standard. The results are the mean of three replicate samples of 10 fruit each9 SE.
2.4. Starch analysis Starch was determined from the insoluble
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residue obtained after ethanol extraction according to the method of Lafta and Lorenzen (1995). For starch content determination, 20 mg of the dried residue were rehydrated in 1 ml of water and heated for 1 h at 90 °C. The gelatinized starch sample was incubated with 1 ml of amyloglucosidase solution (10 units ml − 1, 20 mM NaF, 100 mM acetate buffer, pH 4.5) for 48 h at 40 °C, and the glucose released determined calorimetrically at 490 nm in a glucose oxidase-coupled reaction. The reagent contained 7 units ml − 1 glucose oxidase, 0.5 units ml − 1 peroxidase and 44 mM p-hydroxybenzoic acid in 100 mM phosphate buffer (pH 7.0). The starch content was expressed as mg of glucose released from starch per gram of dry flavedo tissue. About 60 mg of dry residue were obtained from 1 g of the freshly weighed flavedo. All the reagents were purchased from Sigma Chemical Co. (St. Louis, MO) except amyloglucosidase, which was obtained from Boehringer Mannheim Co. (Indianapolis, IN).
3. Results
3.1. Effect of high temperature conditioning on CI of ‘Fortune’ mandarin showing different susceptibility to CI Symptoms of CI were detected as pitting and rind staining distributed along the fruit in nonconditioned mandarins after 14 days storage at 2 °C. The CI index increased over 28 days at this chilling temperature, although the rate of increase varied from one stage of maturity to another. After 28 days at 2 °C, the CI index of the less mature fruit harvested in December was 1.4 (Fig. 1), whereas the CI indices of fruit harvested in January and March were 0.7 and 0.8, respectively (data not shown). No pitting or rind staining was observed in the flavedo of fruit kept at 12 °C or in fruit conditioned at 37 °C for 3 days and thereafter stored at 2 °C (Fig. 1) for any stage of maturity (data not shown).
2.5. Respiration At each time interval, CO2 production was measured by placing fruit from each treatment into individual 1.5 l glass containers and closing them for 3 h. Three replicates of four fruit were used for each determination. The concentration of CO2 was determined by injecting 1 ml gas samples, withdrawn using a syringe through a septum from the container headspace, into a Perkin– Elmer gas chromatograph (Valencia, Spain) equipped with a 1.5 m×2.0 mm Chromosorb 102 column from Supelco (Barcelona, Spain) and a thermal conductivity detector. CO2 levels were quantified by peak area comparison using a standard curve for CO2 and respiratory rates expressed in mmol CO2 kg − 1 min − 1. The CO2 standard was obtained from Abello-Oxı´genoLinde, S.A. (Valencia, Spain).
2.6. Statistics Values on the graphs represent means of three replicate samples. The corresponding SE values are indicated. Data were tested using ANOVA (F-test, P50.05).
Fig. 1. Changes in the chilling injury index of ‘Fortune’ mandarin fruit stored immediately after harvest (non-conditioned fruit) or after 3 days of conditioning at 37 °C and 90 – 95% RH (conditioned fruit), at a chilling (2 °C) or a non-chilling (12 °C) temperature. Fruit were exposed to the following treatments: (1) non-conditioned fruit stored at 2 °C ( ); (2) conditioned fruit stored at 2 °C (); (3) non-conditioned fruit stored at 12 °C ( ); (4) conditioned fruit stored at 12 °C ( ). Data shown correspond to fruit harvested in December. Values are the mean of three replicate samples containing 20 fruit 9SE.
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3.2. Changes in carbohydrates induced by high temperature conditioning in cold-stored fruit har6ested at different maturity stages The pattern of changes in soluble carbohydrates of freshly harvested (non-conditioned) or 3-day-heated (conditioned) fruit during holding at 2 or 12 °C are shown in Figs. 2 and 3. In general, sucrose levels declined in non-conditioned fruit kept at 2 or 12 °C (Fig. 2). The content of this sugar in the flavedo of the nonconditioned fruit harvested in December and March remained nearly constant during the first 10 days of holding at the chilling temperature (2 °C), but decreased afterwards. After 30 days, the sucrose content was about 59% that of freshly harvested fruit. Sucrose content decreased considerably after 10 days at 2 °C in fruit harvested in January (65% that of freshly harvested fruit) and kept decreasing for up to 30 days until about 36% of the initial levels. The content of sucrose decreased faster at the non-chilling temperature (12 °C) in fruit harvested at the three stages of maturity. During the whole period of storage, sucrose levels were significantly lower (P 5 0.05) in fruit kept at 12 than at 2 °C (Fig. 2). The levels of this soluble carbohydrate in the flavedo tissue were significantly reduced by heating fruit harvested in January at 37 °C for 3 days. However, no significant decrease occurred in fruit from December and March (Fig. 2). After 10 days storage at 12 °C similar sucrose levels were found in conditioned and non -conditioned fruit harvested at any maturity stage. By contrast, heat conditioning significantly influenced the pattern of changes in sucrose during holding of the fruit at the chilling temperature. Heating fruit harvested at the three maturity stages for 3 days at 37 °C avoided the decline in sucrose when fruit were exposed to 2 °C. Therefore, fruit exposed to these temperature conditions (37+2 °C) presented, in general, the highest levels of sucrose after prolonged storage. This effect was less relevant in the less mature fruit. The reducing sugars glucose and fructose showed similar patterns of changes in response
Fig. 2. Sucrose content in the flavedo of ‘Fortune’ mandarin fruit harvested at different maturity stages and stored immediately after harvest (non-conditioned fruit) or after 3 days of conditioning at 37 °C and 90 – 95% RH (conditioned fruit), at a chilling (2 °C) or a non-chilling (12 °C) temperature. Fruit were exposed to the following treatments: (1) non-conditioned fruit stored at 2 °C ( ); (2) conditioned fruit stored at 2 °C (); (3) non-conditioned fruit stored at 12 °C ( ); (4) conditioned fruit stored at 12 °C ( ). Values are the mean of three replicate samples containing 10 fruit 9SE.
to heating 12 °C (Fig. changes in holding of
or exposure of the fruit to 2 or 3). No clear trend in the pattern of these hexoses was observed during the non-conditioned fruit at 2 or
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12 °C when the effect of chilling temperature was compared in fruit harvested at different maturity stages. Both, glucose and fructose markedly decreased at 2 or 12 °C in fruit from January but not in fruit harvested early or later in the season (Fig. 3). Except in the less mature fruit, levels of reducing sugars were lower in fruit treated at 37 °C for 3 days (Fig. 3). In contrast to sucrose, heat-conditioned fruit stored at 2 °C had
the lowest levels of glucose and fructose. The hexose levels of fruit stored at the chilling temperature (2 °C) were considerably lower in conditioned than in non-conditioned fruit harvested at the three maturity stages. The levels of these sugars in the heat-conditioned fruit were, in general, lower in fruit kept at 2 than at 12 °C. The starch content was immediately reduced by holding the fruit at either 2 or 12 °C and by heating the fruit at 37 °C at all maturity stages
Fig. 3. Glucose (left panel) and fructose (right panel) content in the flavedo of ‘Fortune’ mandarin fruit stored immediately after harvest (non-conditioned fruit) or after 3 days of conditioning at 37 °C and 90 – 95% RH (conditioned fruit), at a chilling (2 °C) or a non-chilling (12 °C) temperature. Fruit were exposed to the following treatments: (1) non-conditioned fruit stored at 2 °C ( ); (2) conditioned fruit stored at 2 °C (); (3) non-conditioned fruit stored at 12 °C ( ); (4) conditioned fruit stored at 12 °C ( ). The harvest dates were: December 9 (A and B), January 27 (C and D), and March 3 (E and F). Values are the mean of three replicate samples containing 10 fruit 9 SE.
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decrease in starch during conditioning. In general, after 10 days storage, no significant differences in this carbohydrate were found between conditioned and non-conditioned fruit stored at 2 or 12 °C.
3.3. Effect of chilling and high temperature conditioning on the respiratory rate of fruit
Fig. 4. Starch content in the flavedo of ‘Fortune’ mandarin fruit harvested at different maturity stages and stored immediately after harvest (non-conditioned fruit) or after 3 days of conditioning at 37 °C and 90 –95% RH (conditioned fruit), at a chilling (2 °C) or a non-chilling (12 °C) temperature. Fruit were exposed to the following treatments: (1) non-conditioned fruit stored at 2 °C ( ); (2) conditioned fruit stored at 2 °C (); (3) non-conditioned fruit stored at 12 °C ( ); (4) conditioned fruit stored at 12 °C ( ). Values are the mean of three replicate samples containing 10 fruit 9 SE.
(Fig. 4). The levels of starch in non-conditioned fruit kept for 10 days at 2 or 12 °C were similar to those of fruit exposed for 3 days at 37 °C. Starch levels of the conditioned fruit did not decrease after transfer to low temperatures, except in the more mature fruit, which had a relative
The respiratory rate of the non-conditioned fruit always decreased during the 1st week of holding at 2 and 12 °C but thereafter showed only slight changes (Fig. 5). The CO2 production of freshly harvested fruit at the later stage of maturity was higher than that of fruit harvested early in the season. However, after storage at both temperatures, it was similar for fruit harvested at different maturity stages, and significantly higher levels of CO2 were consistently found in fruit kept at 12 °C than in those kept at 2 °C (P5 0.05) (Fig. 5, inset panels). Heating ‘Fortune’ mandarins at 37 °C and 90–95% RH considerably increased the respiratory rate of the fruit (Table 1). After 1 day, the CO2 production increased 3.7-fold in fruit harvested in December, three-fold in those from January, and 2.3-fold in the more mature fruit harvested in March. Fruit from all maturity stages produced similar levels of CO2 after 1 day of conditioning. These increases in the respiratory rises were followed by a decline during the 2nd and 3rd day of heating. The transfer of the 3 day heat-conditioned mandarins to 2 and 12 °C resulted in an immediate and noticeable decline in the respiratory rate, reaching levels similar to those of the non-conditioned mandarins (Fig. 5). This response was uniform for the fruit harvested at the three different maturity stages. As in non-heated fruit, the respiration rate remained nearly constant during storage of the fruit and was significantly lower in fruit kept at the chilling temperature (2 °C).
4. Discussion Sugars may have several beneficial effects in protecting plants against stresses (Ingram and Bartels, 1996). In the present work we have shown
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N. Holland et al. / Posthar6est Biology and Technology 25 (2002) 181–191 Table 1 Respiration of ‘Fortune’ mandarin fruit harvested at different maturity stages and exposed for up to 3 days at 37 °C and 90–95% RH Harvest date
Days at 37 °C
mmol CO2 kg−1 min−1
December 9
0 1 2 3
9.7 9 0.3 35.3 9 1.9 32.2 94.2 25.7 94.7
January 27
0 1 2 3
10.9 90.4 33.1 9 2.1 31.1 9 1.0 22.0 91.0
March 3
0 1 2 3
14.9 9 1.0 34.1 93.3 27.8 9 2.9 20.2 9 1.9
Values are mean of three replicate samples containing four fruit9 SE.
Fig. 5. Respiration of ‘Fortune’ mandarin fruit harvested at different maturity stages and stored at a chilling (2 °C) or non-chilling temperature (12 °C) immediately after harvest (non-conditioned fruit) or after 3 days of conditioning at 37°C and 90 – 95% RH (conditioned fruit). Fruit were exposed to the following treatments: (1) non-conditioned fruit stored at 2 °C ( ); (2) conditioned fruit stored at 2 °C (); (3) non-conditioned fruit stored at 12 °C ( ); (4) conditioned fruit stored at 12 °C ( ). Inset panels show values in a wider scale to better illustrate the differences between the respiratory rates of fruit held at 2 and 12 °C. Values are the mean of three replicate samples containing four fruit 9SE.
that sucrose levels declined in the flavedo of non-conditioned (non-heated) ‘Fortune’ fruit, harvested at any maturity stage, during storage at the chilling temperature (2 °C) (Fig. 2). However, levels of reducing sugars suffered little change in fruit harvested in December and March and decreased in fruit from January (Fig. 3). Interestingly, we found in previous work that the decrease in sucrose does not occur in the flavedo of fruit attached to the tree during their exposure to the more extreme minimum field temperatures, while the hexoses glucose and fructose increased (Holland et al., 1999). Purvis and Yelenosky (1983) reported that sucrose does not interchange between different tissues of the fruit at low temperature but is translocated into the fruit from other parts of the plant. Therefore, the loss of ability to maintain sucrose levels and to increase reducing sugars in detached mandarins held at 2 °C could be explained by the consumption of carbohydrate reserves for respiration in conjunction with the lack of sucrose transport from the plant, which in turn would be necessary to continuously increase reducing sugars in the fruit exposed to cold stress. Reducing sugars may be accumulated, however, in the flavedo tissue of
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detached grapefruit exposed to cold stress (Purvis and Rice, 1983). Although, anomalous high respiratory activity may occur during chilling in chilling-sensitive species (Lyons, 1973), we found that the respiratory rate of non-conditioned mandarins stored at 2 °C, and showing cold-induced peel damage, was lower than that of the fruit stored at 12 °C (Fig. 5). A significant positive correlation between respiratory rates and the sugar content has been shown in some plant tissues (Saglio and Pradet, 1980). Interestingly, the rate of decrease in sucrose content during the storage of the non-conditioned ‘Fortune’ fruit at 2 °C was always considerably lower than in fruit stored at 12 °C but less noticeable differences in the levels of reducing sugars or starch were found, in general, between fruit stored at both temperatures. This may suggest that sucrose appears to be the more accessible sugar as a respiratory substrate in the non-conditioned fruit. Our data also suggest that cold stress may not stimulate the increase in reducing sugars in nonconditioned detached fruit and that carbohydrate changes occurring in the flavedo of fruit harvested at different maturity stages in response to cold stress appear not to be related to variations in their susceptibility to CI. This result appears to reinforce our previous idea that the increase in reducing sugars in citrus fruit attached to the tree was more likely related to osmotic adjustment occurring during development and maturation, to assist the fruit in cell expansion, than to acclimation at low temperature to cope with chilling stress during the winter (Holland et al., 1999). High temperatures may result in a decrease (Savin and Nicolas, 1996) but also in an increase in carbohydrate levels in plants, which may be related to a shift in partitioning from one part of the plant to another (Lorenzen and Lafta, 1996). The beneficial effect of heat conditioning on increasing chilling resistance of plants is well known (Lurie, 1998), but the potential role of carbohydrates is still unclear. Heating detached ‘Fortune’ mandarin fruit for 3 days at 37 °C always avoided the development of CI symptoms in fruit stored at 2 °C. Although, fruit maturity affected changes in soluble sugars during heating, this treatment always avoided the decline of sucrose
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during storage of the fruit at the chilling temperature (2 °C) (Fig. 2). Heating muskmelons at 45 °C for 3 h prior to storage at 4 °C also inhibited the decline in sucrose content at the end of the cold storage period (Lingle and Dunlap, 1987). As these authors suggested, some of the processes responsible for sucrose degradation in the peel may have been heat-sensitive, but heat conditioning did not avoid the decline in sucrose in ‘Fortune’ fruit stored at the non-chilling temperature (12 °C). In contrast to sucrose, heated fruit kept at the chilling temperature presented the lowest levels of reducing sugars (Fig. 3). The increase in the respiratory rate occurring in ‘Fortune’ fruit during the high temperature conditioning treatment was, in general, accompanied by a decline in soluble sugars. Heat treatment of lemons at 55 °C for 5 min, however, significantly increased the sucrose content of the flavedo, while the glucose and fructose contents decreased slightly (Aung et al., 1988). The different patterns of change observed in response to heat treatment between these citrus species might be related in part to the severity of the stress imposed on them and therefore to the higher consumption of sucrose as a consequence of the higher heat-induced increase in the respiratory rate of ‘Fortune’ mandarins (3 days at 37 °C). Additionally, changes observed in the soluble sugars in the flavedo of ‘Fortune’ mandarins may be related to the effect of the different temperature regimes imposed on the fruit on the balance between sucrose synthesis and degradation. Thus, sucrose decreased while the reducing sugars increased in conditioned fruit from January and March stored at 12 °C. This metabolic change appears to require the conjunction of both thermal conditions (37+ 12 °C), since it did not occur either at 37+ 2 °C or in the unheated fruit stored at 12 °C (Figs. 3 and 4), and is also dependent on fruit maturity. This is in concordance with previous results from Lorenzen and Lafta (1996) showing that high temperature induced a significant increase in activities of enzymes involved in sucrose degradation in young potato leaves but not in mature leaves. Starch appears to be also used as a respiratory metabolite during heating of ‘Fortune’ fruit. This carbo-
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hydrate dramatically decreased at 37 °C and suffered smaller changes during storage of the fruit at 2 or 12 °C, which presented a lower respiratory rate than fruit held at 37 °C. Although, soluble sugars and starch may play a physiological role in defending plant tissues against chilling (Levitt, 1980; King et al., 1988), no relationship between the susceptibility of ‘Fortune’ mandarins to CI and changes in carbohydrates during cold storage was apparently found in non-conditioned fruit. The heat-conditioning treatment avoided the decrease in sucrose, favoured the loss of glucose, fructose and starch during holding of the fruit at the chilling temperature and greatly increased the tolerance of ‘Fortune’ mandarins to chilling. Therefore, sucrose but not glucose, fructose or starch could have a role in the heat-induced chilling tolerance of citrus fruit detached from the tree. Considering that the effect of the heat treatment on sucrose changes during storage of the fruit at 2 °C was scarcely relevant in the less mature fruit and that the heat treatment was at least as effective as in the more mature fruit increasing their chilling tolerance, it seems that the heat-induced changes in sucrose would not be a limiting factor in heat-induced chilling tolerance in citrus fruit.
Acknowledgements This work was supported by research grants (Project ALI 93-0117 and project ALI 96-0506C03-C1) of the Comisio´ n Interministerial de Ciencia y Tecnologı´a (CICYT), Spain. Holland was the recipient of a fellowship from the CNPq, Brazil and this study is part of her doctoral thesis. We gratefully acknowledge the technical assistance of D. Arocas.
References Aung, L.H., Obenland, D.M., Houck, L.G., 1988. Conditioning and heat treatments influence flavedo soluble sugars of lemon. J. Hort. Sci. Biotech. 73, 399 –402. Gonzalez-Aguilar, G.A., Zacarias, L., Perez-Amador, M.A., Carbonell, J., Lafuente, M.T., 2000. Polyamine content and chilling susceptibility are affected by seasonal
changes in temperature and by conditioning temperature in cold-stored ‘Fortune’ mandarin fruit. Physiol. Plant 108, 140 – 146. Holland, N., Sala, J.M., Menezes, H.C., Lafuente, M.T., 1999. Carbohydrate content and metabolism as related to maturity and chilling sensitivity of cv. ‘Fortune’ mandarins. J. Agric. Food Chem. 47, 2513 – 2518. Ingram, J., Bartels, D., 1996. The molecular basis of dehydration tolerance in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 377 – 403. Keller, E., Steffen, K.L., 1995. Increased chilling tolerance and altered carbon metabolism in tomato leaves following application of mechanical stress. Physiol. Plant 93, 519 – 525. King, A.I., Joyce, D.C., Reid, M.S., 1988. Role of carbohydrates in diurnal chilling sensitivity of tomato seedlings. Plant Physiol. 86, 764 – 768. Lafta, A.M., Lorenzen, J.H., 1995. Effect of high temperature on plant growth and carbohydrate metabolism in potato. Plant Physiol. 109, 637 – 643. Lafuente, M.T., Martı´nez-Te´ llez, M.A., Zacarias, L., 1997. Abscisic acid in the response of ‘Fortune’ mandarins to chilling. Effect of maturity and high-temperature conditioning. J. Sci. Food Agric. 73, 494 – 502. Levitt, J., 1980. Responses of plants to environmental stresses. In: Chilling, Freezing and High Temperature Stresses, vol. 1. Academic Press, New York, NY, p. 497. Lingle, S.E., Dunlap, J.R., 1987. Sucrose metabolism in netted muskmelon fruit during development. Plant Physiol. 84, 386 – 389. Lorenzen, J.H., Lafta, A.M., 1996. Effect of heat stress on enzymes that affect sucrose levels in potato shoots. J. Am. Soc. Hort. Sci. 121, 1152 – 1156. Lurie, S., 1998. Postharvest heat treatments. Postharvest Biol. Technol. 14, 257 – 269. Lyons, J.M., 1973. Chilling injury in plants. Annu. Rev. Plant Physiol. 24, 445 – 466. MacRae, E., Quick, W.P., Benker, C., Stitt, M., 1992. Carbohydrate metabolism during postharvest ripening in kiwifruit. Planta 188, 314 – 323. Mertens, E., Marcellin, P., Van Schaftingen, E., Hers, H.G., 1987. Effect of ethylene treatment on the concentration of fructose-2,6-bis-phosphate and on the activity of phosphofructokinase 2/fructose-2,6-biphosphatase in banana. Eur. J. Biochem. 167, 579 – 583. Palonen, P., Buszard, D., Donnelly, D., 2000. Changes in carbohydrates and freezing tolerance during cold acclimation of raspberry cultivars grown in vitro and in vivo. Physiol. Plant 110, 393 – 401. Purvis, A.C., Grierson, W., 1982. Accumulation of reducing sugar and resistance of grapefruit peel to chilling injury as related to winter temperatures. J. Am. Soc. Hortic. Sci. 107, 139 – 142. Purvis, A.C., Kawada, K., Grierson, W., 1979. Relationship between mid-season resistance to chilling injury and reducing sugar level in grapefruit peel. HortScience 14, 227 – 229.
N. Holland et al. / Posthar6est Biology and Technology 25 (2002) 181–191 Purvis, A.C., Rice, J.D., 1983. Low-temperature induction of invertase activity in grapefruit flavedo tissue. Phytochemistry 22, 831 – 834. Purvis, A.C., Yelenosky, G., 1983. Translocation of carbohydrates and proline in young grapefruit trees at low temperatures. Plant Physiol. 73, 877 – 880. Saglio, P.H., Pradet, A., 1980. Soluble sugars, respiration, and energy charge during aging of excised maize root tips. Plant Physiol. 6, 516 –519. Sala, J.M., Lafuente, M.T., 1999. Catalase in the heat-induced chilling tolerance of cold-stored hybrid ‘Fortune’ mandarin fruits. J. Agric. Food Chem. 47, 2410 –2414.
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Sanchez-Ballesta, M.T., Zacarias, L., Granell, A., Lafuente, M.T., 2000. Accumulation of PAL Transcript and PAL activity as affected by heat-conditioning and low-temperature storage and its relation to chilling sensitivity in mandarin fruits. J. Agric. Food Chem. 48, 2726 – 2731. Savin, R., Nicolas, M.E., 1996. Effects of short periods of drought and high temperature on grain growth and starch accumulation of two malting barley cultivars. Aust. J. Plant Physiol. 23, 201 – 210. Wang, F., Sanz, A., Brenner, M.L., Smith, A., 1993. Sucrose synthase, starch accumulation, and tomato fruit sink strength. Plant Physiol. 101, 321 – 327.
Carbohydrates as related to the heat-induced chilling tolerance and respiratory rate of ‘Fortune’ mandarin fruit harvested at different maturity stages Nely Holland b, Hilary C. Menezes b, Marı´a T. Lafuente a,* a
Instituto de Agroquı´mica y Tecnologı´a de Alimentos, Consejo Superior de In6est. Cientif. C.S.I.C., Apartado de Correos 73, 46100 Burjassot, Valencia, Spain b Departamento de Tecnologia de Alimentos, FEA-Uni6ersidade Estadual de Campinas (UNICAMP), Caixa Postal: 6121, CEP 13083 -970, Campinas, Brazil Received 30 March 2001; accepted 18 October 2001
Abstract We have evaluated the effect of a heat-conditioning treatment (3 days at 37 °C) on respiratory rate, soluble carbohydrate and starch content of ‘Fortune’ mandarin fruit harvested at different maturity stages and stored at a chilling (2 °C) and a non-chilling (12 °C) temperature. The treatment was highly effective, increasing the tolerance of detached ‘Fortune’ fruit to chilling injury (CI). Changes in carbohydrate levels in response to the high temperature treatment, and during exposure of the non-conditioned and heat-conditioned fruit to 2 and 12 °C, appear to be mainly related to the consumption of carbohydrate reserves for respiration. The highest respiratory rate was found in fruit exposed to 37 °C. The respiration of non-conditioned fruit stored at 2 °C was lower than that of control fruit stored at 12 °C. Therefore, anomalous high respiratory activity did not occur during chilling in ‘Fortune’ mandarins. Sucrose appeared to be the most accessible sugar as a respiratory substrate in non-conditioned fruit. No relationship between the susceptibility to CI and changes in carbohydrates during storage of non-conditioned fruit at 2 °C was found. The heat treatment prevented the decline in sucrose content during chilling. After 30 days at 2 °C, the sucrose content of non-conditioned fruit harvested in December, January and March was about 79, 57 and 68% that of their respective heat-conditioned fruit. In contrast, heating the fruit favoured the loss of glucose, fructose and starch in fruit kept at 2 °C. These data suggest that sucrose but not glucose, fructose or starch could be involved in the heat-induced chilling tolerance of citrus fruit detached from the tree. The effect of the heat treatment preventing sucrose decline during storage of fruit at 2 °C was less relevant in fruit from December than in those harvested later in the season, although it was very effective in protecting fruit harvested at all maturity stages against chilling. Therefore, sucrose appears not to be a limiting factor for the heat-induced chilling tolerance in citrus fruit. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Citrus; Cold stress; Fructose; Glucose; Heat conditioning; Maturation; Respiration; Starch; Sucrose
* Corresponding author. Tel.: + 34-963900022; fax: + 34-963636301. E-mail address: [email protected] (M.T. Lafuente). 0925-5214/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 5 2 1 4 ( 0 1 ) 0 0 1 8 2 - X
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1. Introduction Carbohydrate levels and composition may influence the sensitivity of plant tissues to low temperature (Levitt, 1980; Purvis and Grierson, 1982). Chilling injury (CI) may occur in many citrus cultivars when exposed to low, non-freezing temperatures. Fruit of the hybrid ‘Fortune’ mandarin (Citrus clementina Hort. Ex Tanaka x Citrus reticulata, Blanca) show high sensitivity to CI, which is manifest as small brown pit-like depressions along the equatorial zones of the fruit. Previous investigations studying the role of carbohydrates on chilling tolerance of citrus fruit have focused mainly on the relationship between changes in carbohydrates in the flavedo (the outer pigmented portion of the peel) of fruit attached to the tree during maturity (Purvis and Grierson, 1982; Holland et al., 1999). Conflicting results have been found. Purvis and Grierson (1982) suggested that the increase in reducing sugar levels in the peel of grapefruit during winter could be a defence mechanism of citrus fruit attached to the trees to resist cold stress. Conversely, sugar accumulation in mandarins during winter did not precede their resistance to CI (Holland et al., 1999). The importance of soluble carbohydrates, especially sucrose, in freezing tolerance induced by cold acclimation in red raspberry plants has been recently shown (Palonen et al., 2000). Sugars are also involved in the acclimation response to chilling induced by mechanical stress in tomato leaves (Keller and Steffen, 1995). A comparison between the changes in chilling susceptibility of tomato seedlings and their starch content at different times during the light/dark cycle indicates that starch may also play a role in defending plant tissues against chilling (King et al., 1988). Through different experiments, we have shown that heating detached ‘Fortune’ mandarin fruit for 3 days at 37 °C and 90 – 95% RH considerably reduces cold-induced peel damage during the exposure of the fruit to 2 °C and that the efficacy of this high temperature conditioning treatment is not influenced by the maturity stage of the fruit (Lafuente et al., 1997; Sala and Lafuente, 1999; Gonzalez-Aguilar et al., 2000; Sanchez-Ballesta et al., 2000). Heat treatments have been shown to
induce cold tolerance in a wide range of horticultural crops (Lurie, 1998), but few studies have focused on the postharvest carbohydrate changes brought on by the imposition of heat treatments. Aung et al. (1988) showed that the heat treatment of lemons at 55 °C for 5 min significantly increased the sucrose content of the flavedo, while the raffinose content was unaffected and the glucose and fructose contents decreased slightly. It has been also reported that heat shock may lead to a reduction in sucrose cleavage and starch accumulation (Wang et al., 1993; Lafta and Lorenzen, 1995). How the high temperature affected the chilling response of these plant tissues or their carbohydrate metabolism, when transferred to low temperatures, was not examined. The influence of high temperatures on the respiratory rate in apples and other climacteric fruit such as tomatoes has been studied (Lurie, 1998). However, the relationship between sugar metabolism and respiratory rise in response to heat stress is little known. The effect of cold stress on carbohydrate changes in detached fruit as related to their respiratory rate and how those processes may be affected by the maturity stage of the fruit or by a heat conditioning treatment is also poorly understood. In response to other physiological processes, such as ripening, this relationship may vary among fruit. Thus, at a nonstress temperature, the rise in respiration in banana during ripening was temporally related to the net conversion of starch to soluble sugars, whereas in other fruit such as apples, accumulation of sugars was not associated with the respiratory rise (Mertens et al., 1987), and in kiwifruit, the rise in respiration did not occur until after the conversion of starch to sugars was effectively completed (MacRae et al., 1992). The aim of the present work was to study whether carbohydrate changes induced by high temperature conditioning may play a role in increasing tolerance of detached citrus fruit to chilling and how those changes may be related to the respiratory rate of fruit. To this end, we evaluated the effect of a high temperature conditioning treatment (3 days at 37 °C), which greatly increased chilling tolerance of the fruit, on the starch and soluble carbohydrate contents of the
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flavedo of ‘Fortune’ mandarins. The relationship between changes in the respiratory rate of the fruit and in the levels of carbohydrates was also examined. Fruit harvested at different maturity stages, differing in their susceptibility to CI, have been used to evaluate the pattern of carbohydrate accumulation during the storage of non-conditioned and heat-conditioned fruit at a chilling (2 °C) and a non-chilling (12 °C) temperature to better understand the role of carbohydrates in the protection of detached citrus fruit against CI.
2. Material and methods
2.1. Plant material and chilling and high temperature conditioning treatments Fruit of ‘Fortune’ mandarin (C. clementina Hort. Ex Tanaka x C. reticulata, Blanca) were harvested at three different stages of maturity during the season (December 9, January 27, and March 3). Fruit were harvested at random from adult trees growing in a commercial orchard at Sagunto (Valencia, Spain, latitude: 39° 28% 48¦ N; longitude: 00° 22% 52¦ W), immediately delivered to the laboratory and randomly divided into two lots. The first lot was subdivided into two groups, which were stored at 80– 90% relative humidity (RH) and 2 °C (chilled control) or 12 °C (nonchilled control) for up to 30 days. The second lot was used to evaluate the effect of heat conditioning on chilling-tolerance, respiratory rates and carbohydrate changes of the fruit. Fruit from this lot were subjected to a 3 days heat conditioning treatment at 90–95% RH and 37 °C and then subdivided into two groups which were stored at the same temperatures and RH as the freshly harvested (non-conditioned) fruit. For each different maturity stage and temperature regime, three replicates containing four fruit were used to periodically determine CO2 production, three replicates of 20 fruit each for CI evaluation and three of 10 fruit to analyze carbohydrate content. Flavedo tissue was collected from the total surface of fruit and frozen in liquid nitrogen for subsequent sugar and starch analysis.
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2.2. CI index The CI index was estimated weekly in heat-conditioned and non-conditioned fruit held at 2 and 12 °C. Brown pit-like depressions along the fruit are the main CI symptoms in ‘Fortune’ mandarins. CI was rated visually on a scale from 0 (no injury) to 3 (severe injury) according to the necrotic surface and the intensity of browning. A CI index was determined by summing the product of the number of fruit in each category multiplied by the score of each category, and then dividing this sum by the number of fruit evaluated (Lafuente et al., 1997). The results are the means of three replicate samples containing 20 fruit9 SE.
2.3. Sugar analysis Sugars were extracted from flavedo tissue by the method previously described by Purvis et al. (1979). One gram of flavedo was extracted three times with 10 ml of 80% boiling ethanol using a Polytron homogenizer. The combined ethanol extracts were filtered through a glass fibre filter pad and 2 ml of 2.1% raffinose added as an internal standard. The alcoholic solution was centrifuged, the supernatant vacuum-evaporated and the resulting residue dissolved in 5 ml water. One milliliter of this solution was purified by passing through a C-18 ‘Elud Bond’ cartridge from Varian (Harbor City, CA) and then filtering through a Millipore HV-4 filter (pore size 0.45 mm) (Ibe´ rica, S.A., Barcelona, Spain) for HPLC analysis as previously described (Holland et al., 1999). Sugars were eluted in 20 min at 85 °C using water as the mobile phase at a flow rate of 0.6 ml min − 1 and a 300× 7.8 mm Phenomenex column packed with a Rezex sulfonated polystyrene resin. A Waters 410 refractive index detector (Waters, Franklin, MA) was used and the sugars were quantified by peak area comparison using standard curves for sucrose, glucose and fructose, and raffinose as the internal standard. The results are the mean of three replicate samples of 10 fruit each9 SE.
2.4. Starch analysis Starch was determined from the insoluble
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residue obtained after ethanol extraction according to the method of Lafta and Lorenzen (1995). For starch content determination, 20 mg of the dried residue were rehydrated in 1 ml of water and heated for 1 h at 90 °C. The gelatinized starch sample was incubated with 1 ml of amyloglucosidase solution (10 units ml − 1, 20 mM NaF, 100 mM acetate buffer, pH 4.5) for 48 h at 40 °C, and the glucose released determined calorimetrically at 490 nm in a glucose oxidase-coupled reaction. The reagent contained 7 units ml − 1 glucose oxidase, 0.5 units ml − 1 peroxidase and 44 mM p-hydroxybenzoic acid in 100 mM phosphate buffer (pH 7.0). The starch content was expressed as mg of glucose released from starch per gram of dry flavedo tissue. About 60 mg of dry residue were obtained from 1 g of the freshly weighed flavedo. All the reagents were purchased from Sigma Chemical Co. (St. Louis, MO) except amyloglucosidase, which was obtained from Boehringer Mannheim Co. (Indianapolis, IN).
3. Results
3.1. Effect of high temperature conditioning on CI of ‘Fortune’ mandarin showing different susceptibility to CI Symptoms of CI were detected as pitting and rind staining distributed along the fruit in nonconditioned mandarins after 14 days storage at 2 °C. The CI index increased over 28 days at this chilling temperature, although the rate of increase varied from one stage of maturity to another. After 28 days at 2 °C, the CI index of the less mature fruit harvested in December was 1.4 (Fig. 1), whereas the CI indices of fruit harvested in January and March were 0.7 and 0.8, respectively (data not shown). No pitting or rind staining was observed in the flavedo of fruit kept at 12 °C or in fruit conditioned at 37 °C for 3 days and thereafter stored at 2 °C (Fig. 1) for any stage of maturity (data not shown).
2.5. Respiration At each time interval, CO2 production was measured by placing fruit from each treatment into individual 1.5 l glass containers and closing them for 3 h. Three replicates of four fruit were used for each determination. The concentration of CO2 was determined by injecting 1 ml gas samples, withdrawn using a syringe through a septum from the container headspace, into a Perkin– Elmer gas chromatograph (Valencia, Spain) equipped with a 1.5 m×2.0 mm Chromosorb 102 column from Supelco (Barcelona, Spain) and a thermal conductivity detector. CO2 levels were quantified by peak area comparison using a standard curve for CO2 and respiratory rates expressed in mmol CO2 kg − 1 min − 1. The CO2 standard was obtained from Abello-Oxı´genoLinde, S.A. (Valencia, Spain).
2.6. Statistics Values on the graphs represent means of three replicate samples. The corresponding SE values are indicated. Data were tested using ANOVA (F-test, P50.05).
Fig. 1. Changes in the chilling injury index of ‘Fortune’ mandarin fruit stored immediately after harvest (non-conditioned fruit) or after 3 days of conditioning at 37 °C and 90 – 95% RH (conditioned fruit), at a chilling (2 °C) or a non-chilling (12 °C) temperature. Fruit were exposed to the following treatments: (1) non-conditioned fruit stored at 2 °C ( ); (2) conditioned fruit stored at 2 °C (); (3) non-conditioned fruit stored at 12 °C ( ); (4) conditioned fruit stored at 12 °C ( ). Data shown correspond to fruit harvested in December. Values are the mean of three replicate samples containing 20 fruit 9SE.
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3.2. Changes in carbohydrates induced by high temperature conditioning in cold-stored fruit har6ested at different maturity stages The pattern of changes in soluble carbohydrates of freshly harvested (non-conditioned) or 3-day-heated (conditioned) fruit during holding at 2 or 12 °C are shown in Figs. 2 and 3. In general, sucrose levels declined in non-conditioned fruit kept at 2 or 12 °C (Fig. 2). The content of this sugar in the flavedo of the nonconditioned fruit harvested in December and March remained nearly constant during the first 10 days of holding at the chilling temperature (2 °C), but decreased afterwards. After 30 days, the sucrose content was about 59% that of freshly harvested fruit. Sucrose content decreased considerably after 10 days at 2 °C in fruit harvested in January (65% that of freshly harvested fruit) and kept decreasing for up to 30 days until about 36% of the initial levels. The content of sucrose decreased faster at the non-chilling temperature (12 °C) in fruit harvested at the three stages of maturity. During the whole period of storage, sucrose levels were significantly lower (P 5 0.05) in fruit kept at 12 than at 2 °C (Fig. 2). The levels of this soluble carbohydrate in the flavedo tissue were significantly reduced by heating fruit harvested in January at 37 °C for 3 days. However, no significant decrease occurred in fruit from December and March (Fig. 2). After 10 days storage at 12 °C similar sucrose levels were found in conditioned and non -conditioned fruit harvested at any maturity stage. By contrast, heat conditioning significantly influenced the pattern of changes in sucrose during holding of the fruit at the chilling temperature. Heating fruit harvested at the three maturity stages for 3 days at 37 °C avoided the decline in sucrose when fruit were exposed to 2 °C. Therefore, fruit exposed to these temperature conditions (37+2 °C) presented, in general, the highest levels of sucrose after prolonged storage. This effect was less relevant in the less mature fruit. The reducing sugars glucose and fructose showed similar patterns of changes in response
Fig. 2. Sucrose content in the flavedo of ‘Fortune’ mandarin fruit harvested at different maturity stages and stored immediately after harvest (non-conditioned fruit) or after 3 days of conditioning at 37 °C and 90 – 95% RH (conditioned fruit), at a chilling (2 °C) or a non-chilling (12 °C) temperature. Fruit were exposed to the following treatments: (1) non-conditioned fruit stored at 2 °C ( ); (2) conditioned fruit stored at 2 °C (); (3) non-conditioned fruit stored at 12 °C ( ); (4) conditioned fruit stored at 12 °C ( ). Values are the mean of three replicate samples containing 10 fruit 9SE.
to heating 12 °C (Fig. changes in holding of
or exposure of the fruit to 2 or 3). No clear trend in the pattern of these hexoses was observed during the non-conditioned fruit at 2 or
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12 °C when the effect of chilling temperature was compared in fruit harvested at different maturity stages. Both, glucose and fructose markedly decreased at 2 or 12 °C in fruit from January but not in fruit harvested early or later in the season (Fig. 3). Except in the less mature fruit, levels of reducing sugars were lower in fruit treated at 37 °C for 3 days (Fig. 3). In contrast to sucrose, heat-conditioned fruit stored at 2 °C had
the lowest levels of glucose and fructose. The hexose levels of fruit stored at the chilling temperature (2 °C) were considerably lower in conditioned than in non-conditioned fruit harvested at the three maturity stages. The levels of these sugars in the heat-conditioned fruit were, in general, lower in fruit kept at 2 than at 12 °C. The starch content was immediately reduced by holding the fruit at either 2 or 12 °C and by heating the fruit at 37 °C at all maturity stages
Fig. 3. Glucose (left panel) and fructose (right panel) content in the flavedo of ‘Fortune’ mandarin fruit stored immediately after harvest (non-conditioned fruit) or after 3 days of conditioning at 37 °C and 90 – 95% RH (conditioned fruit), at a chilling (2 °C) or a non-chilling (12 °C) temperature. Fruit were exposed to the following treatments: (1) non-conditioned fruit stored at 2 °C ( ); (2) conditioned fruit stored at 2 °C (); (3) non-conditioned fruit stored at 12 °C ( ); (4) conditioned fruit stored at 12 °C ( ). The harvest dates were: December 9 (A and B), January 27 (C and D), and March 3 (E and F). Values are the mean of three replicate samples containing 10 fruit 9 SE.
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decrease in starch during conditioning. In general, after 10 days storage, no significant differences in this carbohydrate were found between conditioned and non-conditioned fruit stored at 2 or 12 °C.
3.3. Effect of chilling and high temperature conditioning on the respiratory rate of fruit
Fig. 4. Starch content in the flavedo of ‘Fortune’ mandarin fruit harvested at different maturity stages and stored immediately after harvest (non-conditioned fruit) or after 3 days of conditioning at 37 °C and 90 –95% RH (conditioned fruit), at a chilling (2 °C) or a non-chilling (12 °C) temperature. Fruit were exposed to the following treatments: (1) non-conditioned fruit stored at 2 °C ( ); (2) conditioned fruit stored at 2 °C (); (3) non-conditioned fruit stored at 12 °C ( ); (4) conditioned fruit stored at 12 °C ( ). Values are the mean of three replicate samples containing 10 fruit 9 SE.
(Fig. 4). The levels of starch in non-conditioned fruit kept for 10 days at 2 or 12 °C were similar to those of fruit exposed for 3 days at 37 °C. Starch levels of the conditioned fruit did not decrease after transfer to low temperatures, except in the more mature fruit, which had a relative
The respiratory rate of the non-conditioned fruit always decreased during the 1st week of holding at 2 and 12 °C but thereafter showed only slight changes (Fig. 5). The CO2 production of freshly harvested fruit at the later stage of maturity was higher than that of fruit harvested early in the season. However, after storage at both temperatures, it was similar for fruit harvested at different maturity stages, and significantly higher levels of CO2 were consistently found in fruit kept at 12 °C than in those kept at 2 °C (P5 0.05) (Fig. 5, inset panels). Heating ‘Fortune’ mandarins at 37 °C and 90–95% RH considerably increased the respiratory rate of the fruit (Table 1). After 1 day, the CO2 production increased 3.7-fold in fruit harvested in December, three-fold in those from January, and 2.3-fold in the more mature fruit harvested in March. Fruit from all maturity stages produced similar levels of CO2 after 1 day of conditioning. These increases in the respiratory rises were followed by a decline during the 2nd and 3rd day of heating. The transfer of the 3 day heat-conditioned mandarins to 2 and 12 °C resulted in an immediate and noticeable decline in the respiratory rate, reaching levels similar to those of the non-conditioned mandarins (Fig. 5). This response was uniform for the fruit harvested at the three different maturity stages. As in non-heated fruit, the respiration rate remained nearly constant during storage of the fruit and was significantly lower in fruit kept at the chilling temperature (2 °C).
4. Discussion Sugars may have several beneficial effects in protecting plants against stresses (Ingram and Bartels, 1996). In the present work we have shown
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N. Holland et al. / Posthar6est Biology and Technology 25 (2002) 181–191 Table 1 Respiration of ‘Fortune’ mandarin fruit harvested at different maturity stages and exposed for up to 3 days at 37 °C and 90–95% RH Harvest date
Days at 37 °C
mmol CO2 kg−1 min−1
December 9
0 1 2 3
9.7 9 0.3 35.3 9 1.9 32.2 94.2 25.7 94.7
January 27
0 1 2 3
10.9 90.4 33.1 9 2.1 31.1 9 1.0 22.0 91.0
March 3
0 1 2 3
14.9 9 1.0 34.1 93.3 27.8 9 2.9 20.2 9 1.9
Values are mean of three replicate samples containing four fruit9 SE.
Fig. 5. Respiration of ‘Fortune’ mandarin fruit harvested at different maturity stages and stored at a chilling (2 °C) or non-chilling temperature (12 °C) immediately after harvest (non-conditioned fruit) or after 3 days of conditioning at 37°C and 90 – 95% RH (conditioned fruit). Fruit were exposed to the following treatments: (1) non-conditioned fruit stored at 2 °C ( ); (2) conditioned fruit stored at 2 °C (); (3) non-conditioned fruit stored at 12 °C ( ); (4) conditioned fruit stored at 12 °C ( ). Inset panels show values in a wider scale to better illustrate the differences between the respiratory rates of fruit held at 2 and 12 °C. Values are the mean of three replicate samples containing four fruit 9SE.
that sucrose levels declined in the flavedo of non-conditioned (non-heated) ‘Fortune’ fruit, harvested at any maturity stage, during storage at the chilling temperature (2 °C) (Fig. 2). However, levels of reducing sugars suffered little change in fruit harvested in December and March and decreased in fruit from January (Fig. 3). Interestingly, we found in previous work that the decrease in sucrose does not occur in the flavedo of fruit attached to the tree during their exposure to the more extreme minimum field temperatures, while the hexoses glucose and fructose increased (Holland et al., 1999). Purvis and Yelenosky (1983) reported that sucrose does not interchange between different tissues of the fruit at low temperature but is translocated into the fruit from other parts of the plant. Therefore, the loss of ability to maintain sucrose levels and to increase reducing sugars in detached mandarins held at 2 °C could be explained by the consumption of carbohydrate reserves for respiration in conjunction with the lack of sucrose transport from the plant, which in turn would be necessary to continuously increase reducing sugars in the fruit exposed to cold stress. Reducing sugars may be accumulated, however, in the flavedo tissue of
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detached grapefruit exposed to cold stress (Purvis and Rice, 1983). Although, anomalous high respiratory activity may occur during chilling in chilling-sensitive species (Lyons, 1973), we found that the respiratory rate of non-conditioned mandarins stored at 2 °C, and showing cold-induced peel damage, was lower than that of the fruit stored at 12 °C (Fig. 5). A significant positive correlation between respiratory rates and the sugar content has been shown in some plant tissues (Saglio and Pradet, 1980). Interestingly, the rate of decrease in sucrose content during the storage of the non-conditioned ‘Fortune’ fruit at 2 °C was always considerably lower than in fruit stored at 12 °C but less noticeable differences in the levels of reducing sugars or starch were found, in general, between fruit stored at both temperatures. This may suggest that sucrose appears to be the more accessible sugar as a respiratory substrate in the non-conditioned fruit. Our data also suggest that cold stress may not stimulate the increase in reducing sugars in nonconditioned detached fruit and that carbohydrate changes occurring in the flavedo of fruit harvested at different maturity stages in response to cold stress appear not to be related to variations in their susceptibility to CI. This result appears to reinforce our previous idea that the increase in reducing sugars in citrus fruit attached to the tree was more likely related to osmotic adjustment occurring during development and maturation, to assist the fruit in cell expansion, than to acclimation at low temperature to cope with chilling stress during the winter (Holland et al., 1999). High temperatures may result in a decrease (Savin and Nicolas, 1996) but also in an increase in carbohydrate levels in plants, which may be related to a shift in partitioning from one part of the plant to another (Lorenzen and Lafta, 1996). The beneficial effect of heat conditioning on increasing chilling resistance of plants is well known (Lurie, 1998), but the potential role of carbohydrates is still unclear. Heating detached ‘Fortune’ mandarin fruit for 3 days at 37 °C always avoided the development of CI symptoms in fruit stored at 2 °C. Although, fruit maturity affected changes in soluble sugars during heating, this treatment always avoided the decline of sucrose
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during storage of the fruit at the chilling temperature (2 °C) (Fig. 2). Heating muskmelons at 45 °C for 3 h prior to storage at 4 °C also inhibited the decline in sucrose content at the end of the cold storage period (Lingle and Dunlap, 1987). As these authors suggested, some of the processes responsible for sucrose degradation in the peel may have been heat-sensitive, but heat conditioning did not avoid the decline in sucrose in ‘Fortune’ fruit stored at the non-chilling temperature (12 °C). In contrast to sucrose, heated fruit kept at the chilling temperature presented the lowest levels of reducing sugars (Fig. 3). The increase in the respiratory rate occurring in ‘Fortune’ fruit during the high temperature conditioning treatment was, in general, accompanied by a decline in soluble sugars. Heat treatment of lemons at 55 °C for 5 min, however, significantly increased the sucrose content of the flavedo, while the glucose and fructose contents decreased slightly (Aung et al., 1988). The different patterns of change observed in response to heat treatment between these citrus species might be related in part to the severity of the stress imposed on them and therefore to the higher consumption of sucrose as a consequence of the higher heat-induced increase in the respiratory rate of ‘Fortune’ mandarins (3 days at 37 °C). Additionally, changes observed in the soluble sugars in the flavedo of ‘Fortune’ mandarins may be related to the effect of the different temperature regimes imposed on the fruit on the balance between sucrose synthesis and degradation. Thus, sucrose decreased while the reducing sugars increased in conditioned fruit from January and March stored at 12 °C. This metabolic change appears to require the conjunction of both thermal conditions (37+ 12 °C), since it did not occur either at 37+ 2 °C or in the unheated fruit stored at 12 °C (Figs. 3 and 4), and is also dependent on fruit maturity. This is in concordance with previous results from Lorenzen and Lafta (1996) showing that high temperature induced a significant increase in activities of enzymes involved in sucrose degradation in young potato leaves but not in mature leaves. Starch appears to be also used as a respiratory metabolite during heating of ‘Fortune’ fruit. This carbo-
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hydrate dramatically decreased at 37 °C and suffered smaller changes during storage of the fruit at 2 or 12 °C, which presented a lower respiratory rate than fruit held at 37 °C. Although, soluble sugars and starch may play a physiological role in defending plant tissues against chilling (Levitt, 1980; King et al., 1988), no relationship between the susceptibility of ‘Fortune’ mandarins to CI and changes in carbohydrates during cold storage was apparently found in non-conditioned fruit. The heat-conditioning treatment avoided the decrease in sucrose, favoured the loss of glucose, fructose and starch during holding of the fruit at the chilling temperature and greatly increased the tolerance of ‘Fortune’ mandarins to chilling. Therefore, sucrose but not glucose, fructose or starch could have a role in the heat-induced chilling tolerance of citrus fruit detached from the tree. Considering that the effect of the heat treatment on sucrose changes during storage of the fruit at 2 °C was scarcely relevant in the less mature fruit and that the heat treatment was at least as effective as in the more mature fruit increasing their chilling tolerance, it seems that the heat-induced changes in sucrose would not be a limiting factor in heat-induced chilling tolerance in citrus fruit.
Acknowledgements This work was supported by research grants (Project ALI 93-0117 and project ALI 96-0506C03-C1) of the Comisio´ n Interministerial de Ciencia y Tecnologı´a (CICYT), Spain. Holland was the recipient of a fellowship from the CNPq, Brazil and this study is part of her doctoral thesis. We gratefully acknowledge the technical assistance of D. Arocas.
References Aung, L.H., Obenland, D.M., Houck, L.G., 1988. Conditioning and heat treatments influence flavedo soluble sugars of lemon. J. Hort. Sci. Biotech. 73, 399 –402. Gonzalez-Aguilar, G.A., Zacarias, L., Perez-Amador, M.A., Carbonell, J., Lafuente, M.T., 2000. Polyamine content and chilling susceptibility are affected by seasonal
changes in temperature and by conditioning temperature in cold-stored ‘Fortune’ mandarin fruit. Physiol. Plant 108, 140 – 146. Holland, N., Sala, J.M., Menezes, H.C., Lafuente, M.T., 1999. Carbohydrate content and metabolism as related to maturity and chilling sensitivity of cv. ‘Fortune’ mandarins. J. Agric. Food Chem. 47, 2513 – 2518. Ingram, J., Bartels, D., 1996. The molecular basis of dehydration tolerance in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 377 – 403. Keller, E., Steffen, K.L., 1995. Increased chilling tolerance and altered carbon metabolism in tomato leaves following application of mechanical stress. Physiol. Plant 93, 519 – 525. King, A.I., Joyce, D.C., Reid, M.S., 1988. Role of carbohydrates in diurnal chilling sensitivity of tomato seedlings. Plant Physiol. 86, 764 – 768. Lafta, A.M., Lorenzen, J.H., 1995. Effect of high temperature on plant growth and carbohydrate metabolism in potato. Plant Physiol. 109, 637 – 643. Lafuente, M.T., Martı´nez-Te´ llez, M.A., Zacarias, L., 1997. Abscisic acid in the response of ‘Fortune’ mandarins to chilling. Effect of maturity and high-temperature conditioning. J. Sci. Food Agric. 73, 494 – 502. Levitt, J., 1980. Responses of plants to environmental stresses. In: Chilling, Freezing and High Temperature Stresses, vol. 1. Academic Press, New York, NY, p. 497. Lingle, S.E., Dunlap, J.R., 1987. Sucrose metabolism in netted muskmelon fruit during development. Plant Physiol. 84, 386 – 389. Lorenzen, J.H., Lafta, A.M., 1996. Effect of heat stress on enzymes that affect sucrose levels in potato shoots. J. Am. Soc. Hort. Sci. 121, 1152 – 1156. Lurie, S., 1998. Postharvest heat treatments. Postharvest Biol. Technol. 14, 257 – 269. Lyons, J.M., 1973. Chilling injury in plants. Annu. Rev. Plant Physiol. 24, 445 – 466. MacRae, E., Quick, W.P., Benker, C., Stitt, M., 1992. Carbohydrate metabolism during postharvest ripening in kiwifruit. Planta 188, 314 – 323. Mertens, E., Marcellin, P., Van Schaftingen, E., Hers, H.G., 1987. Effect of ethylene treatment on the concentration of fructose-2,6-bis-phosphate and on the activity of phosphofructokinase 2/fructose-2,6-biphosphatase in banana. Eur. J. Biochem. 167, 579 – 583. Palonen, P., Buszard, D., Donnelly, D., 2000. Changes in carbohydrates and freezing tolerance during cold acclimation of raspberry cultivars grown in vitro and in vivo. Physiol. Plant 110, 393 – 401. Purvis, A.C., Grierson, W., 1982. Accumulation of reducing sugar and resistance of grapefruit peel to chilling injury as related to winter temperatures. J. Am. Soc. Hortic. Sci. 107, 139 – 142. Purvis, A.C., Kawada, K., Grierson, W., 1979. Relationship between mid-season resistance to chilling injury and reducing sugar level in grapefruit peel. HortScience 14, 227 – 229.
N. Holland et al. / Posthar6est Biology and Technology 25 (2002) 181–191 Purvis, A.C., Rice, J.D., 1983. Low-temperature induction of invertase activity in grapefruit flavedo tissue. Phytochemistry 22, 831 – 834. Purvis, A.C., Yelenosky, G., 1983. Translocation of carbohydrates and proline in young grapefruit trees at low temperatures. Plant Physiol. 73, 877 – 880. Saglio, P.H., Pradet, A., 1980. Soluble sugars, respiration, and energy charge during aging of excised maize root tips. Plant Physiol. 6, 516 –519. Sala, J.M., Lafuente, M.T., 1999. Catalase in the heat-induced chilling tolerance of cold-stored hybrid ‘Fortune’ mandarin fruits. J. Agric. Food Chem. 47, 2410 –2414.
191
Sanchez-Ballesta, M.T., Zacarias, L., Granell, A., Lafuente, M.T., 2000. Accumulation of PAL Transcript and PAL activity as affected by heat-conditioning and low-temperature storage and its relation to chilling sensitivity in mandarin fruits. J. Agric. Food Chem. 48, 2726 – 2731. Savin, R., Nicolas, M.E., 1996. Effects of short periods of drought and high temperature on grain growth and starch accumulation of two malting barley cultivars. Aust. J. Plant Physiol. 23, 201 – 210. Wang, F., Sanz, A., Brenner, M.L., Smith, A., 1993. Sucrose synthase, starch accumulation, and tomato fruit sink strength. Plant Physiol. 101, 321 – 327.