- Email: [email protected]
Phenylalanine ammonia-lyase and ethylene in relation to chilling injury as affected by fruit age in citrus
Postharvest Biology and Technology 29 (2003) 308 /317 www.elsevier.com/locate/postharvbio
Phenylalanine ammonia-lyase and ethylene in relation to chilling injury as affected by fruit age in citrus Marı´a T. Lafuente a,*, Lorenzo Zacarias a, Miguel A. Martı´nez-Te´llez b, Marı´a T. Sanchez-Ballesta a, Antonio Granell c a
Instituto de Agroquı´mica y Tecnologı´a de Alimentos (IATA), Consejo Superior de Investigaciones Cientı´ficas (CSIC), Apartado de Correos 73, Burjassot 46100, Valencia, Spain b Centro de Investigacio´n en Alimentacio´n y Desarrollo, A.C., CIAD, Apartado Postal 1735, Hermosillo, Sonora 83000, Mexico c Instituto de Biologı´a Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Cientı´ficas (CSIC), Universidad Polite´cnica de Valencia, Avda Tarongers, s/n Valencia, Spain Received 24 September 2002; accepted 11 March 2003
Abstract Fruit of many citrus cultivars become injured when exposed to low, non-freezing temperatures. In this study we have determined changes in ethylene production and phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in fruit of three citrus cultivars, ‘Fortune’ mandarins, and ‘Navelina’ and ‘Valencia’ late oranges, with different tolerance to chilling injury (CI) and demonstrated the influence of fruit physiological stage on those stress responses. We have shown that the increase in ethylene production and PAL are cold-induced responses which are only stimulated in fruit of citrus cultivars showing chilling damage and that both responses may occur concomitantly with the development of chilling symptoms. However, the magnitude of these responses was not indicative of the degree of tolerance of a specific cultivar to chilling. The influence of fruit age on both responses was evaluated in the most (‘Navelina’) and the least (‘Fortune’) chilling tolerant cultivars. Chilling damage was not developed in ‘Navelina’ fruit at any physiological stage, but our results in ‘Fortune’ mandarins, which always developed chilling symptoms, indicated that the induction of PAL in response to chilling was dependent on the fruit physiological stage. Interestingly, increases in both PAL mRNA and activity were barely affected by cold stress in the youngest ‘Fortune’ fruit harvested in December in spite of its noticeable CI. For a similar CI index, the older the fruit, the higher was the shift in the levels of PAL transcript and in PAL activity in response to cold. In contrast, the cold-induced ethylene production was little affected by the physiological stage of the fruit. # 2003 Elsevier B.V. All rights reserved. Keywords: Citrus fruit; Chilling tolerance; Ethylene; Low temperature; Phenylalanine ammonia-lyase; Maturity
1. Introduction * Corresponding author. Tel.: /34-96-390-0022; fax: /3496-363-6301. E-mail address: [email protected] (M.T. Lafuente).
Chilling injury (CI) is responsible for substantial postharvest losses in many citrus cultivars. Chilling induces pitting, necrosis and staining in the
0925-5214/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0925-5214(03)00047-4
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308 /317
flavedo tissue (the outer coloured part of the peel) of fruit of these cultivars. Physiological and molecular responses to low temperature are not well understood. Temperature extremes and a variety of environmental factors, including irradiation, wounding, hypoxia, heavy metals, waterlogging, drought, bending, disease and insect attacks are able to induce ethylene biosynthesis (Yang and Hoffman, 1984) and it is well known that chilling induces an increase in ethylene production in citrus fruit (Cooper et al., 1969; McCollum and McDonald, 1991) and that the cold-induced ethylene production may parallel the increase in phenylalanine ammonia-lyase (PAL) activity of cold-stored citrus fruit (Martı´nez-Te´llez and Lafuente, 1997). In turn, it has been shown that exogenous ethylene (Riov et al., 1969) or stresses that may favour ethylene biosynthesis, such as wounding (Ismail and Brown, 1979) and gamma radiation (Riov et al., 1968), may stimulate PAL activity in the flavedo. PAL is the initial rate-controlling enzyme in the phenylpropanoid pathway. It is a key enzyme in the phenolic metabolism that has been reported to protect plants against stress conditions via different phenylpropanoid products (Hahlbrock and Scheel, 1989; Dixon and Paiva, 1995). On the other hand, the ethylene-induced increase in PAL activity has been related to the development of brown necrotic tissue areas in iceberg lettuce (Hyodo et al., 1978). Inhibition of ethylene biosynthesis or action may prevent CI in horticultural crops (Ben-Amor et al., 1999) but also favour it (Lafuente et al., 2001). A large body of literature on the chilling responses of different fruit to 1MCP, an inhibitor of ethylene perception, is now established in an updated web site http:// www.hort.cornell.edu/department/faculty/watkins/ ethylene/. Previous results from our group indicate that the induction of PAL and ethylene during cold storage of ‘Fortune’ mandarin fruit may play a role in reducing the development of chilling symptoms and that the activation of PAL may be dependent on ethylene but also an independent cold signal apparently related to the cold-induced peel damage (Lafuente et al., 2001). Whether PAL or ethylene may serve as biological markers for
309
chilling sensitivity in citrus fruit or whether the ability of citrus to cope with chilling stress is directly related to its capacity to induce both responses deserves further investigation. In melon fruit, Diallinas and Kanellis (1994) showed that the ripening stage may affect changes in PAL and expression of genes for ethylene biosynthesis in response to other stress conditions, such as wounding. Young fruit showed a higher potential to increase PAL protein content than ripe fruit in response to wounding; whereas in unripe melons the expression of ethylene biosynthetic genes was not stimulated significantly by wounding. The rise in PAL activity in response to wounding or inoculation was also affected by fruit ripening in bananas (Kamo et al., 2000). In previous work we have demonstrated that the chilling susceptibility of ‘Fortune’ mandarins changed during the season (Lafuente et al., 1997; Holland et al., 2000). However, the influence of fruit maturity on the cold-induced changes in PAL activity and ethylene production has not been examined. Our research goals in the present work were to determine whether: (1) the increase in ethylene production and PAL are common responses to chilling in fruit of citrus cultivars (‘Navelina’, ‘Valencia’ late, and ‘Fortune’) showing different tolerance to this stress; (2) the constitutive levels of PAL transcript or activity, or the ability of citrus cultivars to increase PAL or ethylene production in response to cold stress could be indicative of the tolerance of a specific citrus cultivar to chilling; and (3) the cold-induced PAL activity and ethylene production were affected by the fruit physiological age.
2. Material and methods 2.1. Plant material and chilling treatments Orange fruit of the cultivars ‘Navelina’ (Citrus sinensis L. Osbeck) and ‘Valencia’ late (C. sinensis L. Osbeck), and fruit of ‘Fortune’ mandarin (Citrus clementina Hort. Ex Tanaka / Citrus reticulata , Blanco) were harvested from trees grown at Valencia, Spain (latitude: 39828?48ƒN;
310
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308 /317
longitude: 00822?52ƒW), all of them at commercial maturity. ‘Navelina’ fruit were harvested in December, those of ‘Valencia’ late in April and ‘Fortune’ mandarins in March. In the two following seasons, ‘Navelina’ or ‘Fortune’ fruit were harvested from the same orchards at different physiological ages, from November to February or from December to April, respectively. At each harvest time, three replicates of ten fruit were selected to evaluate changes in fruit colour, size and maturity index. An additional number of fruit were divided at random into two groups for each experiment and citrus cultivar. The first group was used to determine changes in PAL activity and ethylene production. Three replicates of ten fruit per temperature and storage period for PAL analysis and of three to four fruit for ethylene production measurement were included in this group. Flavedo samples were collected from the total surface of fruit to evaluate the changes in PAL gene expression and PAL activity, frozen in liquid N2 and stored at /80 8C until analysis. The second group contained three replicates of 20 fruit to determine chilling-induced peel damage. Chilling stress was applied by exposing ‘Fortune’ mandarin fruit to 2 8C and 80/85% relative humidity (RH) for at least 28 days, and ‘Valencia’ late and ‘Navelina’ oranges to 2 8C and 80/85% RH for up to 60 days in constant darkness. Control fruit were kept at 12 8C under the same conditions. 2.2. Determination of surface area, peel colour and maturity index Fruit height and diameter were measured to determine fruit surface area according to Turrell (1946). Peel colour was determined using a Hunter Lab Meter at four locations around the equatorial plane of the fruit. Hue angle: 08/red purple, 908 /yellow, 1808/bluish-green, 2708 /blue. Soluble solids (8Brix) and acids content of the juice were measured to determine the fruit internal maturity index, which was calculated by dividing the 8Brix by its acid content. The content of soluble solids was determined with an Atago/X1000 refractometer. Acids content was titrated with 0.1 N NaOH using phenolphthalein as
indicator and expressed as g of anhydrous citric acid in 100 ml of juice.
2.3. Estimation of CI index Fruit were visually scored to estimate the extent of CI development. Brown pit-like depressions of the fruit are the main symptoms of CI. A rating scale from 0 to 3, based on necrotic surface and intensity of browning, was used to evaluate CI and the average CI index determined as indicated in the following formula: CI index /a(CI scale (0 / 3) /number of fruit in each class)/total number of fruit estimated. The results are means of three replicate samples containing 20 fruit9/S.E.M.
2.4. Assay of PAL activity PAL (EC 4.3.1.5) activity was measured as described by Martı´nez-Te´llez and Lafuente (1997) in three replicate samples from flavedo acetone powder. Flavedo tissue samples were periodically collected from the total surface of fruit stored at 2 and 12 8C and ground in chilled (/20 8C) acetone. Ten ml of acetone were used per g of flavedo. The homogenate was filtered, washed twice with chilled acetone and the resulting powder dried at room temperature. PAL was extracted from 0.4 g of acetone powder with 15 ml of 0.1 M sodium borate buffer, pH 8.8, containing 0.02 M b-mercaptoethanol. Proteins were salted out with ammonium sulphate to a final saturation of 46% supernatant and thereafter dissolved in 4.5 ml of 0.1 M ammonium acetate buffer, pH 7.7, containing 0.02 M b-mercaptoethanol. PAL activity was determined by measuring the absorbance of cinnamic acid at 290 nm over a period of 2 h at 40 8C and expressed on a dry-matter basis as nanomoles of cinnamic acid per gram of acetone powder flavedo tissue per hour. The reaction mixture contained 2 ml of the purified enzyme extract and 0.6 ml L-phenylalanine 0.1 M in a total volume of 6 ml. Results are means of three replicate samples9/S.E.M.
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308 /317
311
2.5. RNA extraction and Northern analysis Total RNA was isolated from 4 g of flavedo tissue by the method of Cathala et al. (1983). Eight mg of RNA were denatured at 65 8C and separated on 1.2% agarose/formaldehyde gels and RNA loading checked on ethidium bromide-stained gels. The RNA was transferred to a Hybond-N nylon membrane (Amersham, Pharmacia, Biothech, Freiburg, Germany) using 20/SSC for at least 15 h and cross-linked using a UVC 500 Crosslinker (Amersham Pharmacia Biotech, San Francisco, CA, USA). Filters were hybridised at 65 8C in 7% (w/v) SDS, 0.33 M phosphate buffer (pH 7.2) and 1 mM EDTA (pH 8). A Fortune flavedo Fpal2 cDNA (Sanchez-Ballesta et al., 2000a) was used as probe. Probes were labelled with [a-32P]dCTP by the random primer labelling method. Membranes were washed twice in 2/ SSC, 0.1% SDS at room temperature, and twice in 0.1 /SSC, 0.1% SDS at 65 8C, and exposed to Kodak X-Omat SX film with intensifying screens at /80 8C. 2.6. Measurement of ethylene production Three replicate samples of four ‘Fortune’ mandarin fruit were incubated in 1 l glass jars, or of three ‘Valencia’ late or ‘Navelina’ orange fruit in 1.5 l glass jars, for 4 h at 2 or 12 8C to determine the ethylene production in chilled (2 8C) or nonchilled control (12 8C) fruit. Three replicate samples of 1 ml gas sample were withdrawn from the head space of each jar and injected in a gas chromatograph equipped with a flame ionisation detector. An activated alumina column was used. Results are means of three replicate samples9/ S.E.M.
3. Results 3.1. Changes in PAL activity and ethylene production during cold exposure of fruit from citrus cultivars with different tolerance to CI As shown in Fig. 1, ‘Fortune’ mandarins developed more CI than ‘Valencia’ late or ‘Navelina’
Fig. 1. Changes in CI index of ‘Fortune’ mandarin (m) and ‘Valencia’ late (j) and ‘Navelina’ orange fruit (') held at 2 8C. No CI was detected in fruit of these cultivars stored at 12 8C. ‘Fortune’ mandarins were stored for up to 28 days and ‘Valencia’ late and ‘Navelina’ oranges for up to 60 days. Results of CI index are means of three replicate samples of 20 fruit9/S.E.M.
oranges. No pitting or rind staining was observed in fruit of any of these citrus cultivars stored at 12 8C (data not shown). ‘Fortune’ fruit were stored at 2 8C (chilling temperature) for up to 28 days since severe damage occurred after this time. The two other cultivars showed a much greater tolerance to chilling and were stored for up to 60 days. After this period, we did not continue the experiment since senescence of control fruit kept at 12 8C became important. CI appeared by 14 days storage at 2 8C on ‘Fortune’ mandarins. The CI index of fruit of this cultivar was 1.1 and 2.7 by 14 and 28 days cold exposure, respectively. However, in ‘Valencia’ late oranges, slight peel damage (CI index 0.4) was observed after 42 days storage. Peel damage was not induced in fruit of the ‘Navelina’ cultivar even after 60 days storage at 2 8C (Fig. 1). This behaviour was confirmed in further experiments. PAL activity increased concomitantly with the development of chilling-induced peel damage during storage of ‘Fortune’ mandarins (Fig. 2A). The activation of PAL in ‘Valencia’ late oranges also
312
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308 /317
was similar to that of ‘Fortune’ mandarins and ‘Valencia’ late oranges in freshly harvested fruit, but it did not increase in response to the chilling temperature (Fig. 2C). A similar trend in the coldinduced ethylene production was found in fruit of these cultivars (Fig. 2). Negligible differences between the ethylene production of ‘Navelina’ fruit stored at the chilling and non-chilling temperature were observed (Fig. 2C). As in the case of PAL, the increase in ethylene production paralleled the development of chilling-induced peel damage in ‘Fortune’ mandarins and ‘Valencia’ late oranges (Fig. 2A, B). The amount of ethylene produced by ‘Valencia’ late oranges in response to chilling was, however, higher than that of the least chilling tolerant cultivar.
3.2. Influence of fruit age on cold-induced responses
Fig. 2. Changes in PAL activity (j, I) and ethylene production (', ^) of ‘Fortune’ mandarin fruit (A), ‘Valencia’ late (B) and ‘Navelina’ oranges (C) held at 2 8C (j, ') or 12 8C (I, ^). ‘Fortune’ mandarins were stored for up to 28 days and ‘Valencia’ late and ‘Navelina’ oranges for up to 60 days. PAL activity was determined in flavedo tissue of fruit. Values of PAL activity are means of three replicate samples of 10 fruit9/S.E.M. and those of ethylene production means of three replicate samples of three to four fruit9/S.E.M.
paralleled the development of CI (Fig. 2B). In this cultivar, the cold-induced PAL activity increased after 42 days (Fig. 2B) and was considerably lower than that induced in ‘Fortune’ mandarins after 28 days exposure at 2 8C (Fig. 2A). In the most chilling-tolerant cultivar (‘Navelina’), PAL activity
‘Navelina’ oranges did not show CI or an increase in PAL activity or ethylene production during low temperature storage (60 days at 2 8C) independently of the fruit physiological age (data not shown). However, the tolerance of ‘Fortune’ mandarins to CI changed during the season (Fig. 3 (season 1) and Fig. 4 (season 2)). Changes in size and colour, as well as in the maturity index of fruit of this cultivar are shown in Table 1 as indicators of the external and internal degree of fruit physiological age. The induction of PAL activity in response to chilling increased with fruit age (Figs. 3 and 4). In the first experiment in which we evaluated the effect of fruit physiological stage (Fig. 3), we found that the effect of cold stress in increasing PAL activity was clearly higher in fruit harvested in February than in those harvested in January although they showed a similar CI index over the whole storage period. By 28 days at 2 8C, PAL activity in fruit from January was about 30% that of fruit from February. In fruit harvested in December, PAL activity was barely affected by cold-stress despite CI being noticeable (Fig. 3). In the following season, it was confirmed that, for a similar CI index, the cold-induced PAL activity was higher in fruit harvested later in the season (Fig. 4). After 28 days storage at 2 8C, PAL activity of fruit from April was about six times
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308 /317
313
that of fruit from December despite the initial activity being similar (Fig. 4). To investigate whether the effect of fruit physiological age on the cold-induced PAL activity is linked to the accumulation of PAL transcript, total mRNA extracted from flavedo tissue from freshly harvested ‘Fortune’ fruit and from fruit stored for 21 days at 2 8C was analysed by Northern hybridisation using a Fpal2 cDNA probe. As shown in Fig. 5, the age at which the fruit were harvested had a clear effect on the coldinduced PAL mRNA accumulation in the flavedo. As in the case of PAL activity (Fig. 3), no difference was found before cold storage in the
Fig. 4. Influence of fruit age on CI index, PAL activity (nmol g 1 h 1) and ethylene production (pmol g 1 h 1) of ‘Fortune’ mandarins harvested during season 2 and exposed for up to 42 day at 2 8C. Values of CI index are means of three replicate samples of 20 fruit9/S.E.M. Results of ethylene production are means of three replicate samples of four fruit9/S.E.M. and those of PAL activity means of three replicate samples of ten fruit9/S.E.M.
Fig. 3. Changes in CI index and in PAL activity in ‘Fortune’ mandarins harvested during season 1 and exposed to chilling stress. Fruit were harvested in December (m), January (j), and February (') and held at 2 8C for up to 28 days. Values of CI index are means of three replicate samples of 20 fruit9/S.E.M. Results of PAL activity are the means of three replicate samples of ten fruit9/S.E.M.
levels of PAL transcript in fruit harvested during the season, whereas the accumulation of PAL mRNA in response to chilling increased with fruit age. It is to be noted that no increase in transcript levels occurred in fruit harvested in December. In the following citrus season (Fig. 4), we demonstrated that ethylene production in response to cold stress was, however, barely affected by fruit age. Ethylene production from freshly harvested fruit was always very low (0.9 /1.5 pmol g1 h1).
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308 /317
314
Table 1 Seasonal changes in fruit size, peel colour, and maturity index (soluble solids (8Brix)/acids content (g citric acid per 100 ml juice)) of ‘Fortune’ mandarin fruit harvested in two consecutive seasons Season
Month
Fruit surface area (cm2)
Colour index (h8)
Maturity index
1
December January February December January February April
96.469/1.97 104.149/1.34 108.289/0.94 97.209/1.66 106.049/0.92 114.079/0.59 122.099/1.91
76.949/3.26 43.269/1.12 38.269/1.16 68.649/2.87 44.919/0.81 37.989/0.76 37.209/1.21
4.279/0.14 6.469/0.21 8.629/0.16 3.789/0.17 5.579/0.11 7.539/0.26 10.519/0.33
2
Values are means of three replicate samples of ten fruit9/S.E.M.
Cold-stress always increased ethylene production, as measured at 2 8C, but the rate of ethylene production of fruit from January and February was higher than that of fruit from April. Fruit harvested in April showed a similar chilling tolerance and a higher cold-induced PAL activity than that of fruit harvested earlier in the season (January and February) (Fig. 4). However, the increase in ethylene production in fruit harvested in December was similar (42 days) or higher (28 days) than that of the more mature fruit from April. This behaviour was further confirmed in the following citrus season.
4. Discussion In a previous work we found that constitutive levels of PAL in a mandarin cultivar which showed
Fig. 5. Changes in PAL mRNA levels in ‘Fortune’ mandarins harvested during season 1 and stored for 21 days at 2 8C. Fruit were harvested in December (D), January (J), and February (F). Eight micrograms of total RNA extracted from flavedo tissue was fractionated, blotted, and hybridised with Fpal2 probe. Equivalence of RNA loading was demonstrated by ethidium bromide staining.
high tolerance to chilling were higher than those in a mandarin showing very low tolerance (SanchezBallesta et al., 2000b). This result could suggest that PAL serves as a biological marker for chilling sensitivity in citrus fruit. However, we have found in the present study that constitutive levels of PAL activity in the flavedo of three other citrus cultivars do not correlate with their tolerance to chilling. Therefore, these results indicate that high constitutive levels of PAL in the flavedo at harvest are not indicative of chilling tolerance of different citrus species. We have also shown that ethylene production and PAL activity are common coldinduced physiological responses only stimulated in fruit of those citrus cultivars, which developed chilling symptoms (Figs. 1 and 2) and that both responses occurred concomitantly with the appearance of symptoms. Interestingly, the least tolerant cultivar showed the earliest and most pronounced increase in ethylene production and PAL activity. However, the magnitude of these responses was not indicative of the degree of tolerance of a specific cultivar to chilling. Thus, for a similar CI index, the cold-induced increase in ethylene production or PAL activity was higher in ‘Valencia’ late than in ‘Fortune’ fruit. It is to be noted that stress-induced changes in PAL and ethylene described in this experiment may be due to differences in the age of the peel of the fruit (Diallinas and Kanellis, 1994; Kamo et al., 2000). These cultivars were harvested at commercial maturity, as determined by the ratio of total soluble solids/ acidity of the juice, since citrus are non-climacteric fruit and there is not a good physiological marker
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308 /317
which allows us to select different citrus cultivars with the same peel physiological stage (Alonso et al., 1995). Therefore, these results could not rule out the influence of peel fruit age on the different behaviour of the citrus cultivars examined in this experiment. It is to be noted that ‘Navelina’ oranges did not show CI or an increase in PAL activity or ethylene production during storage at 2 8C independently of the fruit physiological age (data not shown), while ‘Fortune’ mandarins (the least tolerant cultivar) developed CI at any physiological stage examined (Figs. 3 and 4). PAL may be induced during development and ripening in some climacteric (Diallinas and Kanellis, 1994; Assis et al., 2001) and non-climacteric fruit (Given et al., 1988). Our results in ‘Fortune’ mandarins indicating that PAL activity does not change with the fruit physiological stage (Figs. 3 and 4) are in agreement with those reported by Lisker et al. (1983) in grapefruit. Furthermore, we have demonstrated that changes in PAL mRNA levels (Fig. 5) paralleled those in PAL activity in fruit harvested during the season (Fig. 3). Increased PAL activity and ethylene production have been reported to occur in citrus to protect fruit from CI (Lafuente et al., 2001). One of the objectives of the present paper was to determine whether the participation of the cold-induced PAL and ethylene in protecting citrus fruit against chilling were dependent on fruit age and, if so, whether these responses were related to the chilling susceptibility of the fruit. Since ‘Fortune’ mandarins always developed chilling symptoms, changes in CI index, PAL levels and ethylene production during cold storage of fruit of this cultivar harvested during the seasons were compared. During a first citrus season (Fig. 3) we observed that the susceptibility to CI of fruit harvested in January and February was similar and higher than that of fruit harvested earlier in the season (December). However, the older the fruit, the higher was the shift in the cold-induced PAL activity. Interestingly, the activity of the enzyme was barely affected by cold stress in fruit harvested in December despite of CI being noticeable. Development of PAL activity in citrus can be repressed by phenols (Dubery, 1990). In addition, de novo synthesis of a PAL-inactivating factor has
315
been suggested to be involved in the loss of ethylene-induced PAL activity in lettuce (Ritenour and Saltveit, 1996). Changes in PAL mRNA levels in response to cold stress (Fig. 5) were tightly linked to changes in PAL activity in ‘Fortune’ fruit harvested at different physiological stages (Fig. 3). Data shown in the present paper correspond to fruit harvested after colour break. However, this result was confirmed in other experiments using fruit harvested from November (before colour break) until April (data not shown). Therefore, the effect of fruit age on the cold-induced changes in PAL activity could be due to transcript levels and does not need to invoke any PAL-inactivating factors. Considering these results and previous results from our group indicating that the cold-induced ethylene production and PAL activity may be related to the severity of cold-induced peel damage (Sanchez-Ballesta et al., 2000a; Zacarı´as et al., 2002), we further investigated the effect of fruit age on cold-induced PAL activity and whether or not changes observed in PAL activity paralleled those in ethylene production in the following citrus season (Fig. 4). The chilling tolerance of ‘Fortune’ mandarins harvested during this season was higher than that of the first citrus season, probably because their different preharvest daily temperature history (Gonzalez-Aguilar et al., 2000). In order to reach a CI index comparable to that of season 1, we prolonged the storage period until 42 days in season 2. It was confirmed that the ability of fruit to increase PAL activity in response to cold stress increased with fruit age. Thus, PAL activity in ‘Fortune’ fruit harvested in December and stored for up to 42 days at 2 8C was about 24% that of fruit from April (Fig. 4). However, the cold-induced ethylene production appeared to be little affected by the age of the fruit. The lack of correlation between the rate of increase in both responses in fruit harvested at different physiological stages demonstrates that ethylene may be a triggering factor for PAL but other factors may also contribute to the changes in PAL activity occurring in the flavedo in response to chilling. This would support the idea that cold-induced PAL activity in citrus fruit may have different inducers, dependent and independent on ethylene
316
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308 /317
(Lafuente et al., 2001). Interestingly, in grapefruit it was found that the ethylene-induced PAL activity in the flavedo portion of the peel was higher in the more developed fruit and that treating the fruit with ethylene did not induce PAL activity as long as the fruit were not completely yellow (Lisker et al., 1983). In response to other stress conditions, such as mechanical wounding, controversial results have been found since PAL activity may be dependent but also independent on ethylene production (Ke and Saltveit, 1989; Diallinas and Kanellis, 1994). In addition, the increases in ethylene production and PAL activity have been shown to be independent responses to pathogenic inoculation (Boller, 1991; Meravy et al., 1991). To our knowledge, this is the first report showing the influence of fruit physiological age on the induction of PAL activity and on changes in mRNA levels in response of citrus fruit to cold stress. The increase in PAL activity in response to other stress conditions, such as wounding or inoculation with conidia, appeared to be also influenced by fruit ripening. Thus, the rise in PAL activity in wounded and/or inoculated ripe banana fruit was lower than that in unripe fruit (Kamo et al., 2000). Ripe melon fruit showed also a lower increase in PAL activity than unripe fruit in response to wounding. In this climacteric fruit, PAL gene expression followed the kinetics of expression of the ethylene biosynthetic genes during fruit development. In contrast, ethylene biosynthetic genes were scarcely induced in young unripe fruit but significantly activated in ripe fruit by wounding (Diallinas and Kanellis, 1994). The results reported in the present paper add new information to our understanding of the role of PAL in CI of citrus fruit and its relationship with ethylene. Previous results indicate that the induction of PAL in the cold-exposed ‘Fortune’ mandarins could be a protective response of the fruit to repair the damage originated by chilling (Lafuente et al., 2001). Other results appeared to suggest that the accumulation of PAL transcript could serve as a molecular marker for chilling tolerance in citrus fruit (Sanchez-Ballesta et al., 2000b). From the results reported here, PAL still stands as a defensive response of citrus against
chilling stress. Nevertheless, taking into consideration the data presented in this work, PAL induction appears not to be directly related to the chilling tolerance of different citrus fruit. This response was even not induced in fruit harvested earlier in the season showing chilling symptoms. Therefore, PAL appears not to be a good biochemical marker for chilling tolerance in citrus fruit. By contrast, chilling-induced ethylene was little affected by the fruit physiological stage. In summary, from the overall results obtained in this work we can conclude that: (1) the induction of PAL activity and ethylene production occurred concomitantly with the appearance of CI in the chilling-sensitive citrus cultivars; (2) the increase in PAL activity and PAL mRNA levels, but not in ethylene, stimulated by CI is affected by the age of the fruit.
Acknowledgements This work was supported by research grants ALI-93-117 and ALI-96-0506-CO3 from the CICYT, Spain and by FAIR-CT98-4096 from the EU. The technical assistance of D. Arocas is also acknowledged.
References Alonso, J.M., Chamarro, J., Granell, A., 1995. Evidence for the involvement of ethylene in the expression of specific RNAs during maturation of the orange, a non-climacteric fruit. Plant Mol. Biol. 29, 385 /390. Assis, J.S., Maldonado, R., Mun˜oz, T., Escribano, M.I., Merodio, C., 2001. Effect of high carbon dioxide concentration on PAL activity and phenolic contents in ripening cherimoya fruit. Postharvest Biol. Technol. 23, 33 /39. Ben-Amor, M., Flores, B., Latche´, A., Bouzayen, M., Pech, J.C., Romojaro, F., 1999. Inhibition of ethylene biosynthesis by antisense ACC oxidase RNA prevents chilling injury in Charentais cantaloupe melons. Plant Cell Environ. 22, 1579 /1586. Boller, T., 1991. Ethylene and pathogenesis and disease resistance. In: Mattoo, A.T., Suttle, J.C. (Eds.), The Plant Hormone Ethylene. CRC Press, Florida, USA, pp. 293 / 314. Cathala, G., Savouret, J.F., Mendez, B., West, B.L., Karin, M., Martial, J.A., Baxter, D., 1983. A method for isolation of
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308 /317 intact, translationally active ribonucleic acid. DNA 2, 329 / 335. Cooper, W.C., Rasmussen, E.S., Waldon, E.S., 1969. Ethylene evolution stimulated by chilling in citrus and Persea sp. Plant Physiol. 44, 1194 /1196. Diallinas, G., Kanellis, A.K., 1994. A phenylalanine ammonialyase gene from melon fruit: cDNA cloning, sequence and expression in response to development and wounding. Plant Mol. Biol. 26, 473 /479. Dixon, R.A., Paiva, N.L., 1995. Stress-induced phenylpropanoid metabolism. Plant Cell 7, 1085 /1097. Dubery, J.A., 1990. Effect of hydroxylated and methoxylated coumarins on the regulatory properties of phenylalanine ammonia-lyase from Citrus sinensis . Phytochemistry 29, 2107 /2108. Given, N.K., Venis, M.A., Grierson, D., 1988. Purification and properties of phenylalanine ammonia-lyase from strawberry fruit and its synthesis during ripening. J. Plant Physiol. 133, 31 /37. 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. Hahlbrock, K., Scheel, D., 1989. Physiology and molecular biology of phenylpropanoid metabolism. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40, 347 /369. Holland, N., Sala, J.M., Menezes, H.C., Lafuente, M.T., 2000. Carbohydrate content and metabolism as related to maturity and chilling sensitivity of Cv. Fortune mandarins. J. Agric. Food Chem. 47, 2513 /2518. Hyodo, H., Kuroda, H., Yang, S.F., 1978. Induction of phenylalanine ammonia-lyase and increase in phenolics in lettuce leaves in relation to the development of russet spotting caused by ethylene. Plant Physiol. 62, 31 /35. Ismail, M.A., Brown, G.E., 1979. Postharvest wound healing in citrus fruit: Induction of phenylalanine ammonia-lyase in injured ‘Valencia’ orange flavedo. J. Am. Soc. Hort. Sci. 104, 126 /129. Kamo, T., Hirai, N., Tsuda, M., Fujikoda, D., Ohigashi, H., 2000. Changes in the content and biosynthesis of phytoalexins in banana fruit. Biosci. Biotech. Biochem. 64, 2089 / 2098. Ke, D., Saltveit, M.E., Jr, 1989. Wound-induced ethylene production, phenolic metabolism and susceptibility to russet spotting in iceberg lettuce. Physiol. Plant. 76, 412 /418. Lafuente, M.T., Martı´nez-Te´llez, M.A., Zacarias, Z., 1997. Abscisic acid in the response of ‘Fortune’ mandarin to chilling. Effect of maturity and high temperature conditioning. J. Sci. Food Agric. 73, 494 /502. Lafuente, M.T., Zacarias, L., Martı´nez-Te´llez, M.A., SanchezBallesta, M.T., Dupille, E., 2001. Phenylalanine ammonia-
317
lyase as related to ethylene in the development of chilling symptoms during cold storage of citrus fruits. J. Agric. Food Chem. 49, 6020 /6025. Lisker, N., Cohen, L.E., Chalutz, E., Fuchs, Y., 1983. Fungal infections suppress ethylene-induced phenylalanine ammonia-lyase activity in grapefruits. Physiol. Plant Pathol. 22, 331 /338. Martı´nez-Te´llez, M.A., Lafuente, M.T., 1997. Effect of high temperature conditioning on ethylene, phenylalanine ammonia-lyase, peroxidase and polyphenol oxidase activities in flavedo of chilled ‘Fortune’ mandarin fruit. J. Plant Physiol. 150, 674 /678. McCollum, T.G., McDonald, E.E., 1991. Electrolyte leakage, respiration, and ethylene production as indices of chilling injury in grapefruit. HortScience 26, 1191 /1192. Meravy, L., Machackova, I., Cvikrova, M., Eder, J., 1991. The effect of aminoethoxyvinylglicine and silver on ethylene synthesis and activity of phenylalalnine ammonia-lyase in alfalfa cell suspension culture. Plant Physiol. Biochem. 29, 165 /170. Riov, J., Monselise, S.P., Kahan, R.S., 1968. Effect of gamma radiation on phenylalanine ammonia-lyase activity and accumulation of phenolic compounds in citrus fruit peel. Radiat. Bot. 8, 463 /466. Riov, J., Monselise, S.P., Kahan, R.S., 1969. Ethylene-controlled induction of phenylalanine ammonia-lyase. Plant Physiol. 44, 631 /635. Ritenour, M.A., Saltveit, M.E., 1996. Identification of phenylalanine ammonia-lyase inactivating factor in harvested head lettuce (Lactuca sativa). Physiol. Plant. 97, 327 /331. Sanchez-Ballesta, M.T., Lafuente, M.T., Zacarias, L., Granell, A., 2000a. Involvement of phenylalanine ammonia-lyase in the response of Fortune mandarin fruits to cold temperature. Physiol. Plant. 108, 382 /389. Sanchez-Ballesta, M.T., Zacarias, L., Granell, A., Lafuente, M.T., 2000b. 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. Turrell, F.M., 1946. Tables of Surfaces and Volumes of Spheres and of prolate and Oblate Spheroids, and Spheroidal Coefficients. University of California Press, Berkeley, CA. Yang, S.F., Hoffman, N.E., 1984. Ethylene biosynthesis and its regulation in higher plants. Annu. Rev. Plant Physiol. 35, 155 /189. Zacarı´as, L., Lafuente, M.T., Marcos, J.F., Saladı´e, M., Dupille, E., 2002. Regulation of ethylene biosynthesis during cold storage of the cold-sensitive Fortune mandarin fruit. Biology and Biotechnology of the Plant Hormone Ethylene. NATO. Advanced Research Workshop, Murcia, Spain. Abstract S2-06.
Phenylalanine ammonia-lyase and ethylene in relation to chilling injury as affected by fruit age in citrus Marı´a T. Lafuente a,*, Lorenzo Zacarias a, Miguel A. Martı´nez-Te´llez b, Marı´a T. Sanchez-Ballesta a, Antonio Granell c a
Instituto de Agroquı´mica y Tecnologı´a de Alimentos (IATA), Consejo Superior de Investigaciones Cientı´ficas (CSIC), Apartado de Correos 73, Burjassot 46100, Valencia, Spain b Centro de Investigacio´n en Alimentacio´n y Desarrollo, A.C., CIAD, Apartado Postal 1735, Hermosillo, Sonora 83000, Mexico c Instituto de Biologı´a Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Cientı´ficas (CSIC), Universidad Polite´cnica de Valencia, Avda Tarongers, s/n Valencia, Spain Received 24 September 2002; accepted 11 March 2003
Abstract Fruit of many citrus cultivars become injured when exposed to low, non-freezing temperatures. In this study we have determined changes in ethylene production and phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in fruit of three citrus cultivars, ‘Fortune’ mandarins, and ‘Navelina’ and ‘Valencia’ late oranges, with different tolerance to chilling injury (CI) and demonstrated the influence of fruit physiological stage on those stress responses. We have shown that the increase in ethylene production and PAL are cold-induced responses which are only stimulated in fruit of citrus cultivars showing chilling damage and that both responses may occur concomitantly with the development of chilling symptoms. However, the magnitude of these responses was not indicative of the degree of tolerance of a specific cultivar to chilling. The influence of fruit age on both responses was evaluated in the most (‘Navelina’) and the least (‘Fortune’) chilling tolerant cultivars. Chilling damage was not developed in ‘Navelina’ fruit at any physiological stage, but our results in ‘Fortune’ mandarins, which always developed chilling symptoms, indicated that the induction of PAL in response to chilling was dependent on the fruit physiological stage. Interestingly, increases in both PAL mRNA and activity were barely affected by cold stress in the youngest ‘Fortune’ fruit harvested in December in spite of its noticeable CI. For a similar CI index, the older the fruit, the higher was the shift in the levels of PAL transcript and in PAL activity in response to cold. In contrast, the cold-induced ethylene production was little affected by the physiological stage of the fruit. # 2003 Elsevier B.V. All rights reserved. Keywords: Citrus fruit; Chilling tolerance; Ethylene; Low temperature; Phenylalanine ammonia-lyase; Maturity
1. Introduction * Corresponding author. Tel.: /34-96-390-0022; fax: /3496-363-6301. E-mail address: [email protected] (M.T. Lafuente).
Chilling injury (CI) is responsible for substantial postharvest losses in many citrus cultivars. Chilling induces pitting, necrosis and staining in the
0925-5214/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0925-5214(03)00047-4
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308 /317
flavedo tissue (the outer coloured part of the peel) of fruit of these cultivars. Physiological and molecular responses to low temperature are not well understood. Temperature extremes and a variety of environmental factors, including irradiation, wounding, hypoxia, heavy metals, waterlogging, drought, bending, disease and insect attacks are able to induce ethylene biosynthesis (Yang and Hoffman, 1984) and it is well known that chilling induces an increase in ethylene production in citrus fruit (Cooper et al., 1969; McCollum and McDonald, 1991) and that the cold-induced ethylene production may parallel the increase in phenylalanine ammonia-lyase (PAL) activity of cold-stored citrus fruit (Martı´nez-Te´llez and Lafuente, 1997). In turn, it has been shown that exogenous ethylene (Riov et al., 1969) or stresses that may favour ethylene biosynthesis, such as wounding (Ismail and Brown, 1979) and gamma radiation (Riov et al., 1968), may stimulate PAL activity in the flavedo. PAL is the initial rate-controlling enzyme in the phenylpropanoid pathway. It is a key enzyme in the phenolic metabolism that has been reported to protect plants against stress conditions via different phenylpropanoid products (Hahlbrock and Scheel, 1989; Dixon and Paiva, 1995). On the other hand, the ethylene-induced increase in PAL activity has been related to the development of brown necrotic tissue areas in iceberg lettuce (Hyodo et al., 1978). Inhibition of ethylene biosynthesis or action may prevent CI in horticultural crops (Ben-Amor et al., 1999) but also favour it (Lafuente et al., 2001). A large body of literature on the chilling responses of different fruit to 1MCP, an inhibitor of ethylene perception, is now established in an updated web site http:// www.hort.cornell.edu/department/faculty/watkins/ ethylene/. Previous results from our group indicate that the induction of PAL and ethylene during cold storage of ‘Fortune’ mandarin fruit may play a role in reducing the development of chilling symptoms and that the activation of PAL may be dependent on ethylene but also an independent cold signal apparently related to the cold-induced peel damage (Lafuente et al., 2001). Whether PAL or ethylene may serve as biological markers for
309
chilling sensitivity in citrus fruit or whether the ability of citrus to cope with chilling stress is directly related to its capacity to induce both responses deserves further investigation. In melon fruit, Diallinas and Kanellis (1994) showed that the ripening stage may affect changes in PAL and expression of genes for ethylene biosynthesis in response to other stress conditions, such as wounding. Young fruit showed a higher potential to increase PAL protein content than ripe fruit in response to wounding; whereas in unripe melons the expression of ethylene biosynthetic genes was not stimulated significantly by wounding. The rise in PAL activity in response to wounding or inoculation was also affected by fruit ripening in bananas (Kamo et al., 2000). In previous work we have demonstrated that the chilling susceptibility of ‘Fortune’ mandarins changed during the season (Lafuente et al., 1997; Holland et al., 2000). However, the influence of fruit maturity on the cold-induced changes in PAL activity and ethylene production has not been examined. Our research goals in the present work were to determine whether: (1) the increase in ethylene production and PAL are common responses to chilling in fruit of citrus cultivars (‘Navelina’, ‘Valencia’ late, and ‘Fortune’) showing different tolerance to this stress; (2) the constitutive levels of PAL transcript or activity, or the ability of citrus cultivars to increase PAL or ethylene production in response to cold stress could be indicative of the tolerance of a specific citrus cultivar to chilling; and (3) the cold-induced PAL activity and ethylene production were affected by the fruit physiological age.
2. Material and methods 2.1. Plant material and chilling treatments Orange fruit of the cultivars ‘Navelina’ (Citrus sinensis L. Osbeck) and ‘Valencia’ late (C. sinensis L. Osbeck), and fruit of ‘Fortune’ mandarin (Citrus clementina Hort. Ex Tanaka / Citrus reticulata , Blanco) were harvested from trees grown at Valencia, Spain (latitude: 39828?48ƒN;
310
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308 /317
longitude: 00822?52ƒW), all of them at commercial maturity. ‘Navelina’ fruit were harvested in December, those of ‘Valencia’ late in April and ‘Fortune’ mandarins in March. In the two following seasons, ‘Navelina’ or ‘Fortune’ fruit were harvested from the same orchards at different physiological ages, from November to February or from December to April, respectively. At each harvest time, three replicates of ten fruit were selected to evaluate changes in fruit colour, size and maturity index. An additional number of fruit were divided at random into two groups for each experiment and citrus cultivar. The first group was used to determine changes in PAL activity and ethylene production. Three replicates of ten fruit per temperature and storage period for PAL analysis and of three to four fruit for ethylene production measurement were included in this group. Flavedo samples were collected from the total surface of fruit to evaluate the changes in PAL gene expression and PAL activity, frozen in liquid N2 and stored at /80 8C until analysis. The second group contained three replicates of 20 fruit to determine chilling-induced peel damage. Chilling stress was applied by exposing ‘Fortune’ mandarin fruit to 2 8C and 80/85% relative humidity (RH) for at least 28 days, and ‘Valencia’ late and ‘Navelina’ oranges to 2 8C and 80/85% RH for up to 60 days in constant darkness. Control fruit were kept at 12 8C under the same conditions. 2.2. Determination of surface area, peel colour and maturity index Fruit height and diameter were measured to determine fruit surface area according to Turrell (1946). Peel colour was determined using a Hunter Lab Meter at four locations around the equatorial plane of the fruit. Hue angle: 08/red purple, 908 /yellow, 1808/bluish-green, 2708 /blue. Soluble solids (8Brix) and acids content of the juice were measured to determine the fruit internal maturity index, which was calculated by dividing the 8Brix by its acid content. The content of soluble solids was determined with an Atago/X1000 refractometer. Acids content was titrated with 0.1 N NaOH using phenolphthalein as
indicator and expressed as g of anhydrous citric acid in 100 ml of juice.
2.3. Estimation of CI index Fruit were visually scored to estimate the extent of CI development. Brown pit-like depressions of the fruit are the main symptoms of CI. A rating scale from 0 to 3, based on necrotic surface and intensity of browning, was used to evaluate CI and the average CI index determined as indicated in the following formula: CI index /a(CI scale (0 / 3) /number of fruit in each class)/total number of fruit estimated. The results are means of three replicate samples containing 20 fruit9/S.E.M.
2.4. Assay of PAL activity PAL (EC 4.3.1.5) activity was measured as described by Martı´nez-Te´llez and Lafuente (1997) in three replicate samples from flavedo acetone powder. Flavedo tissue samples were periodically collected from the total surface of fruit stored at 2 and 12 8C and ground in chilled (/20 8C) acetone. Ten ml of acetone were used per g of flavedo. The homogenate was filtered, washed twice with chilled acetone and the resulting powder dried at room temperature. PAL was extracted from 0.4 g of acetone powder with 15 ml of 0.1 M sodium borate buffer, pH 8.8, containing 0.02 M b-mercaptoethanol. Proteins were salted out with ammonium sulphate to a final saturation of 46% supernatant and thereafter dissolved in 4.5 ml of 0.1 M ammonium acetate buffer, pH 7.7, containing 0.02 M b-mercaptoethanol. PAL activity was determined by measuring the absorbance of cinnamic acid at 290 nm over a period of 2 h at 40 8C and expressed on a dry-matter basis as nanomoles of cinnamic acid per gram of acetone powder flavedo tissue per hour. The reaction mixture contained 2 ml of the purified enzyme extract and 0.6 ml L-phenylalanine 0.1 M in a total volume of 6 ml. Results are means of three replicate samples9/S.E.M.
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308 /317
311
2.5. RNA extraction and Northern analysis Total RNA was isolated from 4 g of flavedo tissue by the method of Cathala et al. (1983). Eight mg of RNA were denatured at 65 8C and separated on 1.2% agarose/formaldehyde gels and RNA loading checked on ethidium bromide-stained gels. The RNA was transferred to a Hybond-N nylon membrane (Amersham, Pharmacia, Biothech, Freiburg, Germany) using 20/SSC for at least 15 h and cross-linked using a UVC 500 Crosslinker (Amersham Pharmacia Biotech, San Francisco, CA, USA). Filters were hybridised at 65 8C in 7% (w/v) SDS, 0.33 M phosphate buffer (pH 7.2) and 1 mM EDTA (pH 8). A Fortune flavedo Fpal2 cDNA (Sanchez-Ballesta et al., 2000a) was used as probe. Probes were labelled with [a-32P]dCTP by the random primer labelling method. Membranes were washed twice in 2/ SSC, 0.1% SDS at room temperature, and twice in 0.1 /SSC, 0.1% SDS at 65 8C, and exposed to Kodak X-Omat SX film with intensifying screens at /80 8C. 2.6. Measurement of ethylene production Three replicate samples of four ‘Fortune’ mandarin fruit were incubated in 1 l glass jars, or of three ‘Valencia’ late or ‘Navelina’ orange fruit in 1.5 l glass jars, for 4 h at 2 or 12 8C to determine the ethylene production in chilled (2 8C) or nonchilled control (12 8C) fruit. Three replicate samples of 1 ml gas sample were withdrawn from the head space of each jar and injected in a gas chromatograph equipped with a flame ionisation detector. An activated alumina column was used. Results are means of three replicate samples9/ S.E.M.
3. Results 3.1. Changes in PAL activity and ethylene production during cold exposure of fruit from citrus cultivars with different tolerance to CI As shown in Fig. 1, ‘Fortune’ mandarins developed more CI than ‘Valencia’ late or ‘Navelina’
Fig. 1. Changes in CI index of ‘Fortune’ mandarin (m) and ‘Valencia’ late (j) and ‘Navelina’ orange fruit (') held at 2 8C. No CI was detected in fruit of these cultivars stored at 12 8C. ‘Fortune’ mandarins were stored for up to 28 days and ‘Valencia’ late and ‘Navelina’ oranges for up to 60 days. Results of CI index are means of three replicate samples of 20 fruit9/S.E.M.
oranges. No pitting or rind staining was observed in fruit of any of these citrus cultivars stored at 12 8C (data not shown). ‘Fortune’ fruit were stored at 2 8C (chilling temperature) for up to 28 days since severe damage occurred after this time. The two other cultivars showed a much greater tolerance to chilling and were stored for up to 60 days. After this period, we did not continue the experiment since senescence of control fruit kept at 12 8C became important. CI appeared by 14 days storage at 2 8C on ‘Fortune’ mandarins. The CI index of fruit of this cultivar was 1.1 and 2.7 by 14 and 28 days cold exposure, respectively. However, in ‘Valencia’ late oranges, slight peel damage (CI index 0.4) was observed after 42 days storage. Peel damage was not induced in fruit of the ‘Navelina’ cultivar even after 60 days storage at 2 8C (Fig. 1). This behaviour was confirmed in further experiments. PAL activity increased concomitantly with the development of chilling-induced peel damage during storage of ‘Fortune’ mandarins (Fig. 2A). The activation of PAL in ‘Valencia’ late oranges also
312
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308 /317
was similar to that of ‘Fortune’ mandarins and ‘Valencia’ late oranges in freshly harvested fruit, but it did not increase in response to the chilling temperature (Fig. 2C). A similar trend in the coldinduced ethylene production was found in fruit of these cultivars (Fig. 2). Negligible differences between the ethylene production of ‘Navelina’ fruit stored at the chilling and non-chilling temperature were observed (Fig. 2C). As in the case of PAL, the increase in ethylene production paralleled the development of chilling-induced peel damage in ‘Fortune’ mandarins and ‘Valencia’ late oranges (Fig. 2A, B). The amount of ethylene produced by ‘Valencia’ late oranges in response to chilling was, however, higher than that of the least chilling tolerant cultivar.
3.2. Influence of fruit age on cold-induced responses
Fig. 2. Changes in PAL activity (j, I) and ethylene production (', ^) of ‘Fortune’ mandarin fruit (A), ‘Valencia’ late (B) and ‘Navelina’ oranges (C) held at 2 8C (j, ') or 12 8C (I, ^). ‘Fortune’ mandarins were stored for up to 28 days and ‘Valencia’ late and ‘Navelina’ oranges for up to 60 days. PAL activity was determined in flavedo tissue of fruit. Values of PAL activity are means of three replicate samples of 10 fruit9/S.E.M. and those of ethylene production means of three replicate samples of three to four fruit9/S.E.M.
paralleled the development of CI (Fig. 2B). In this cultivar, the cold-induced PAL activity increased after 42 days (Fig. 2B) and was considerably lower than that induced in ‘Fortune’ mandarins after 28 days exposure at 2 8C (Fig. 2A). In the most chilling-tolerant cultivar (‘Navelina’), PAL activity
‘Navelina’ oranges did not show CI or an increase in PAL activity or ethylene production during low temperature storage (60 days at 2 8C) independently of the fruit physiological age (data not shown). However, the tolerance of ‘Fortune’ mandarins to CI changed during the season (Fig. 3 (season 1) and Fig. 4 (season 2)). Changes in size and colour, as well as in the maturity index of fruit of this cultivar are shown in Table 1 as indicators of the external and internal degree of fruit physiological age. The induction of PAL activity in response to chilling increased with fruit age (Figs. 3 and 4). In the first experiment in which we evaluated the effect of fruit physiological stage (Fig. 3), we found that the effect of cold stress in increasing PAL activity was clearly higher in fruit harvested in February than in those harvested in January although they showed a similar CI index over the whole storage period. By 28 days at 2 8C, PAL activity in fruit from January was about 30% that of fruit from February. In fruit harvested in December, PAL activity was barely affected by cold-stress despite CI being noticeable (Fig. 3). In the following season, it was confirmed that, for a similar CI index, the cold-induced PAL activity was higher in fruit harvested later in the season (Fig. 4). After 28 days storage at 2 8C, PAL activity of fruit from April was about six times
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308 /317
313
that of fruit from December despite the initial activity being similar (Fig. 4). To investigate whether the effect of fruit physiological age on the cold-induced PAL activity is linked to the accumulation of PAL transcript, total mRNA extracted from flavedo tissue from freshly harvested ‘Fortune’ fruit and from fruit stored for 21 days at 2 8C was analysed by Northern hybridisation using a Fpal2 cDNA probe. As shown in Fig. 5, the age at which the fruit were harvested had a clear effect on the coldinduced PAL mRNA accumulation in the flavedo. As in the case of PAL activity (Fig. 3), no difference was found before cold storage in the
Fig. 4. Influence of fruit age on CI index, PAL activity (nmol g 1 h 1) and ethylene production (pmol g 1 h 1) of ‘Fortune’ mandarins harvested during season 2 and exposed for up to 42 day at 2 8C. Values of CI index are means of three replicate samples of 20 fruit9/S.E.M. Results of ethylene production are means of three replicate samples of four fruit9/S.E.M. and those of PAL activity means of three replicate samples of ten fruit9/S.E.M.
Fig. 3. Changes in CI index and in PAL activity in ‘Fortune’ mandarins harvested during season 1 and exposed to chilling stress. Fruit were harvested in December (m), January (j), and February (') and held at 2 8C for up to 28 days. Values of CI index are means of three replicate samples of 20 fruit9/S.E.M. Results of PAL activity are the means of three replicate samples of ten fruit9/S.E.M.
levels of PAL transcript in fruit harvested during the season, whereas the accumulation of PAL mRNA in response to chilling increased with fruit age. It is to be noted that no increase in transcript levels occurred in fruit harvested in December. In the following citrus season (Fig. 4), we demonstrated that ethylene production in response to cold stress was, however, barely affected by fruit age. Ethylene production from freshly harvested fruit was always very low (0.9 /1.5 pmol g1 h1).
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308 /317
314
Table 1 Seasonal changes in fruit size, peel colour, and maturity index (soluble solids (8Brix)/acids content (g citric acid per 100 ml juice)) of ‘Fortune’ mandarin fruit harvested in two consecutive seasons Season
Month
Fruit surface area (cm2)
Colour index (h8)
Maturity index
1
December January February December January February April
96.469/1.97 104.149/1.34 108.289/0.94 97.209/1.66 106.049/0.92 114.079/0.59 122.099/1.91
76.949/3.26 43.269/1.12 38.269/1.16 68.649/2.87 44.919/0.81 37.989/0.76 37.209/1.21
4.279/0.14 6.469/0.21 8.629/0.16 3.789/0.17 5.579/0.11 7.539/0.26 10.519/0.33
2
Values are means of three replicate samples of ten fruit9/S.E.M.
Cold-stress always increased ethylene production, as measured at 2 8C, but the rate of ethylene production of fruit from January and February was higher than that of fruit from April. Fruit harvested in April showed a similar chilling tolerance and a higher cold-induced PAL activity than that of fruit harvested earlier in the season (January and February) (Fig. 4). However, the increase in ethylene production in fruit harvested in December was similar (42 days) or higher (28 days) than that of the more mature fruit from April. This behaviour was further confirmed in the following citrus season.
4. Discussion In a previous work we found that constitutive levels of PAL in a mandarin cultivar which showed
Fig. 5. Changes in PAL mRNA levels in ‘Fortune’ mandarins harvested during season 1 and stored for 21 days at 2 8C. Fruit were harvested in December (D), January (J), and February (F). Eight micrograms of total RNA extracted from flavedo tissue was fractionated, blotted, and hybridised with Fpal2 probe. Equivalence of RNA loading was demonstrated by ethidium bromide staining.
high tolerance to chilling were higher than those in a mandarin showing very low tolerance (SanchezBallesta et al., 2000b). This result could suggest that PAL serves as a biological marker for chilling sensitivity in citrus fruit. However, we have found in the present study that constitutive levels of PAL activity in the flavedo of three other citrus cultivars do not correlate with their tolerance to chilling. Therefore, these results indicate that high constitutive levels of PAL in the flavedo at harvest are not indicative of chilling tolerance of different citrus species. We have also shown that ethylene production and PAL activity are common coldinduced physiological responses only stimulated in fruit of those citrus cultivars, which developed chilling symptoms (Figs. 1 and 2) and that both responses occurred concomitantly with the appearance of symptoms. Interestingly, the least tolerant cultivar showed the earliest and most pronounced increase in ethylene production and PAL activity. However, the magnitude of these responses was not indicative of the degree of tolerance of a specific cultivar to chilling. Thus, for a similar CI index, the cold-induced increase in ethylene production or PAL activity was higher in ‘Valencia’ late than in ‘Fortune’ fruit. It is to be noted that stress-induced changes in PAL and ethylene described in this experiment may be due to differences in the age of the peel of the fruit (Diallinas and Kanellis, 1994; Kamo et al., 2000). These cultivars were harvested at commercial maturity, as determined by the ratio of total soluble solids/ acidity of the juice, since citrus are non-climacteric fruit and there is not a good physiological marker
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308 /317
which allows us to select different citrus cultivars with the same peel physiological stage (Alonso et al., 1995). Therefore, these results could not rule out the influence of peel fruit age on the different behaviour of the citrus cultivars examined in this experiment. It is to be noted that ‘Navelina’ oranges did not show CI or an increase in PAL activity or ethylene production during storage at 2 8C independently of the fruit physiological age (data not shown), while ‘Fortune’ mandarins (the least tolerant cultivar) developed CI at any physiological stage examined (Figs. 3 and 4). PAL may be induced during development and ripening in some climacteric (Diallinas and Kanellis, 1994; Assis et al., 2001) and non-climacteric fruit (Given et al., 1988). Our results in ‘Fortune’ mandarins indicating that PAL activity does not change with the fruit physiological stage (Figs. 3 and 4) are in agreement with those reported by Lisker et al. (1983) in grapefruit. Furthermore, we have demonstrated that changes in PAL mRNA levels (Fig. 5) paralleled those in PAL activity in fruit harvested during the season (Fig. 3). Increased PAL activity and ethylene production have been reported to occur in citrus to protect fruit from CI (Lafuente et al., 2001). One of the objectives of the present paper was to determine whether the participation of the cold-induced PAL and ethylene in protecting citrus fruit against chilling were dependent on fruit age and, if so, whether these responses were related to the chilling susceptibility of the fruit. Since ‘Fortune’ mandarins always developed chilling symptoms, changes in CI index, PAL levels and ethylene production during cold storage of fruit of this cultivar harvested during the seasons were compared. During a first citrus season (Fig. 3) we observed that the susceptibility to CI of fruit harvested in January and February was similar and higher than that of fruit harvested earlier in the season (December). However, the older the fruit, the higher was the shift in the cold-induced PAL activity. Interestingly, the activity of the enzyme was barely affected by cold stress in fruit harvested in December despite of CI being noticeable. Development of PAL activity in citrus can be repressed by phenols (Dubery, 1990). In addition, de novo synthesis of a PAL-inactivating factor has
315
been suggested to be involved in the loss of ethylene-induced PAL activity in lettuce (Ritenour and Saltveit, 1996). Changes in PAL mRNA levels in response to cold stress (Fig. 5) were tightly linked to changes in PAL activity in ‘Fortune’ fruit harvested at different physiological stages (Fig. 3). Data shown in the present paper correspond to fruit harvested after colour break. However, this result was confirmed in other experiments using fruit harvested from November (before colour break) until April (data not shown). Therefore, the effect of fruit age on the cold-induced changes in PAL activity could be due to transcript levels and does not need to invoke any PAL-inactivating factors. Considering these results and previous results from our group indicating that the cold-induced ethylene production and PAL activity may be related to the severity of cold-induced peel damage (Sanchez-Ballesta et al., 2000a; Zacarı´as et al., 2002), we further investigated the effect of fruit age on cold-induced PAL activity and whether or not changes observed in PAL activity paralleled those in ethylene production in the following citrus season (Fig. 4). The chilling tolerance of ‘Fortune’ mandarins harvested during this season was higher than that of the first citrus season, probably because their different preharvest daily temperature history (Gonzalez-Aguilar et al., 2000). In order to reach a CI index comparable to that of season 1, we prolonged the storage period until 42 days in season 2. It was confirmed that the ability of fruit to increase PAL activity in response to cold stress increased with fruit age. Thus, PAL activity in ‘Fortune’ fruit harvested in December and stored for up to 42 days at 2 8C was about 24% that of fruit from April (Fig. 4). However, the cold-induced ethylene production appeared to be little affected by the age of the fruit. The lack of correlation between the rate of increase in both responses in fruit harvested at different physiological stages demonstrates that ethylene may be a triggering factor for PAL but other factors may also contribute to the changes in PAL activity occurring in the flavedo in response to chilling. This would support the idea that cold-induced PAL activity in citrus fruit may have different inducers, dependent and independent on ethylene
316
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308 /317
(Lafuente et al., 2001). Interestingly, in grapefruit it was found that the ethylene-induced PAL activity in the flavedo portion of the peel was higher in the more developed fruit and that treating the fruit with ethylene did not induce PAL activity as long as the fruit were not completely yellow (Lisker et al., 1983). In response to other stress conditions, such as mechanical wounding, controversial results have been found since PAL activity may be dependent but also independent on ethylene production (Ke and Saltveit, 1989; Diallinas and Kanellis, 1994). In addition, the increases in ethylene production and PAL activity have been shown to be independent responses to pathogenic inoculation (Boller, 1991; Meravy et al., 1991). To our knowledge, this is the first report showing the influence of fruit physiological age on the induction of PAL activity and on changes in mRNA levels in response of citrus fruit to cold stress. The increase in PAL activity in response to other stress conditions, such as wounding or inoculation with conidia, appeared to be also influenced by fruit ripening. Thus, the rise in PAL activity in wounded and/or inoculated ripe banana fruit was lower than that in unripe fruit (Kamo et al., 2000). Ripe melon fruit showed also a lower increase in PAL activity than unripe fruit in response to wounding. In this climacteric fruit, PAL gene expression followed the kinetics of expression of the ethylene biosynthetic genes during fruit development. In contrast, ethylene biosynthetic genes were scarcely induced in young unripe fruit but significantly activated in ripe fruit by wounding (Diallinas and Kanellis, 1994). The results reported in the present paper add new information to our understanding of the role of PAL in CI of citrus fruit and its relationship with ethylene. Previous results indicate that the induction of PAL in the cold-exposed ‘Fortune’ mandarins could be a protective response of the fruit to repair the damage originated by chilling (Lafuente et al., 2001). Other results appeared to suggest that the accumulation of PAL transcript could serve as a molecular marker for chilling tolerance in citrus fruit (Sanchez-Ballesta et al., 2000b). From the results reported here, PAL still stands as a defensive response of citrus against
chilling stress. Nevertheless, taking into consideration the data presented in this work, PAL induction appears not to be directly related to the chilling tolerance of different citrus fruit. This response was even not induced in fruit harvested earlier in the season showing chilling symptoms. Therefore, PAL appears not to be a good biochemical marker for chilling tolerance in citrus fruit. By contrast, chilling-induced ethylene was little affected by the fruit physiological stage. In summary, from the overall results obtained in this work we can conclude that: (1) the induction of PAL activity and ethylene production occurred concomitantly with the appearance of CI in the chilling-sensitive citrus cultivars; (2) the increase in PAL activity and PAL mRNA levels, but not in ethylene, stimulated by CI is affected by the age of the fruit.
Acknowledgements This work was supported by research grants ALI-93-117 and ALI-96-0506-CO3 from the CICYT, Spain and by FAIR-CT98-4096 from the EU. The technical assistance of D. Arocas is also acknowledged.
References Alonso, J.M., Chamarro, J., Granell, A., 1995. Evidence for the involvement of ethylene in the expression of specific RNAs during maturation of the orange, a non-climacteric fruit. Plant Mol. Biol. 29, 385 /390. Assis, J.S., Maldonado, R., Mun˜oz, T., Escribano, M.I., Merodio, C., 2001. Effect of high carbon dioxide concentration on PAL activity and phenolic contents in ripening cherimoya fruit. Postharvest Biol. Technol. 23, 33 /39. Ben-Amor, M., Flores, B., Latche´, A., Bouzayen, M., Pech, J.C., Romojaro, F., 1999. Inhibition of ethylene biosynthesis by antisense ACC oxidase RNA prevents chilling injury in Charentais cantaloupe melons. Plant Cell Environ. 22, 1579 /1586. Boller, T., 1991. Ethylene and pathogenesis and disease resistance. In: Mattoo, A.T., Suttle, J.C. (Eds.), The Plant Hormone Ethylene. CRC Press, Florida, USA, pp. 293 / 314. Cathala, G., Savouret, J.F., Mendez, B., West, B.L., Karin, M., Martial, J.A., Baxter, D., 1983. A method for isolation of
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308 /317 intact, translationally active ribonucleic acid. DNA 2, 329 / 335. Cooper, W.C., Rasmussen, E.S., Waldon, E.S., 1969. Ethylene evolution stimulated by chilling in citrus and Persea sp. Plant Physiol. 44, 1194 /1196. Diallinas, G., Kanellis, A.K., 1994. A phenylalanine ammonialyase gene from melon fruit: cDNA cloning, sequence and expression in response to development and wounding. Plant Mol. Biol. 26, 473 /479. Dixon, R.A., Paiva, N.L., 1995. Stress-induced phenylpropanoid metabolism. Plant Cell 7, 1085 /1097. Dubery, J.A., 1990. Effect of hydroxylated and methoxylated coumarins on the regulatory properties of phenylalanine ammonia-lyase from Citrus sinensis . Phytochemistry 29, 2107 /2108. Given, N.K., Venis, M.A., Grierson, D., 1988. Purification and properties of phenylalanine ammonia-lyase from strawberry fruit and its synthesis during ripening. J. Plant Physiol. 133, 31 /37. 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. Hahlbrock, K., Scheel, D., 1989. Physiology and molecular biology of phenylpropanoid metabolism. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40, 347 /369. Holland, N., Sala, J.M., Menezes, H.C., Lafuente, M.T., 2000. Carbohydrate content and metabolism as related to maturity and chilling sensitivity of Cv. Fortune mandarins. J. Agric. Food Chem. 47, 2513 /2518. Hyodo, H., Kuroda, H., Yang, S.F., 1978. Induction of phenylalanine ammonia-lyase and increase in phenolics in lettuce leaves in relation to the development of russet spotting caused by ethylene. Plant Physiol. 62, 31 /35. Ismail, M.A., Brown, G.E., 1979. Postharvest wound healing in citrus fruit: Induction of phenylalanine ammonia-lyase in injured ‘Valencia’ orange flavedo. J. Am. Soc. Hort. Sci. 104, 126 /129. Kamo, T., Hirai, N., Tsuda, M., Fujikoda, D., Ohigashi, H., 2000. Changes in the content and biosynthesis of phytoalexins in banana fruit. Biosci. Biotech. Biochem. 64, 2089 / 2098. Ke, D., Saltveit, M.E., Jr, 1989. Wound-induced ethylene production, phenolic metabolism and susceptibility to russet spotting in iceberg lettuce. Physiol. Plant. 76, 412 /418. Lafuente, M.T., Martı´nez-Te´llez, M.A., Zacarias, Z., 1997. Abscisic acid in the response of ‘Fortune’ mandarin to chilling. Effect of maturity and high temperature conditioning. J. Sci. Food Agric. 73, 494 /502. Lafuente, M.T., Zacarias, L., Martı´nez-Te´llez, M.A., SanchezBallesta, M.T., Dupille, E., 2001. Phenylalanine ammonia-
317
lyase as related to ethylene in the development of chilling symptoms during cold storage of citrus fruits. J. Agric. Food Chem. 49, 6020 /6025. Lisker, N., Cohen, L.E., Chalutz, E., Fuchs, Y., 1983. Fungal infections suppress ethylene-induced phenylalanine ammonia-lyase activity in grapefruits. Physiol. Plant Pathol. 22, 331 /338. Martı´nez-Te´llez, M.A., Lafuente, M.T., 1997. Effect of high temperature conditioning on ethylene, phenylalanine ammonia-lyase, peroxidase and polyphenol oxidase activities in flavedo of chilled ‘Fortune’ mandarin fruit. J. Plant Physiol. 150, 674 /678. McCollum, T.G., McDonald, E.E., 1991. Electrolyte leakage, respiration, and ethylene production as indices of chilling injury in grapefruit. HortScience 26, 1191 /1192. Meravy, L., Machackova, I., Cvikrova, M., Eder, J., 1991. The effect of aminoethoxyvinylglicine and silver on ethylene synthesis and activity of phenylalalnine ammonia-lyase in alfalfa cell suspension culture. Plant Physiol. Biochem. 29, 165 /170. Riov, J., Monselise, S.P., Kahan, R.S., 1968. Effect of gamma radiation on phenylalanine ammonia-lyase activity and accumulation of phenolic compounds in citrus fruit peel. Radiat. Bot. 8, 463 /466. Riov, J., Monselise, S.P., Kahan, R.S., 1969. Ethylene-controlled induction of phenylalanine ammonia-lyase. Plant Physiol. 44, 631 /635. Ritenour, M.A., Saltveit, M.E., 1996. Identification of phenylalanine ammonia-lyase inactivating factor in harvested head lettuce (Lactuca sativa). Physiol. Plant. 97, 327 /331. Sanchez-Ballesta, M.T., Lafuente, M.T., Zacarias, L., Granell, A., 2000a. Involvement of phenylalanine ammonia-lyase in the response of Fortune mandarin fruits to cold temperature. Physiol. Plant. 108, 382 /389. Sanchez-Ballesta, M.T., Zacarias, L., Granell, A., Lafuente, M.T., 2000b. 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. Turrell, F.M., 1946. Tables of Surfaces and Volumes of Spheres and of prolate and Oblate Spheroids, and Spheroidal Coefficients. University of California Press, Berkeley, CA. Yang, S.F., Hoffman, N.E., 1984. Ethylene biosynthesis and its regulation in higher plants. Annu. Rev. Plant Physiol. 35, 155 /189. Zacarı´as, L., Lafuente, M.T., Marcos, J.F., Saladı´e, M., Dupille, E., 2002. Regulation of ethylene biosynthesis during cold storage of the cold-sensitive Fortune mandarin fruit. Biology and Biotechnology of the Plant Hormone Ethylene. NATO. Advanced Research Workshop, Murcia, Spain. Abstract S2-06.