Phenylalanine ammonia-lyase and ethylene in relation tochilling injury as affected by fruit age in citrus
Marıa T. Lafuente a,*, Lorenzo Zacarias a, Miguel A. Martınez-Tellez b,Marıa T. Sanchez-Ballesta a, Antonio Granell c
a Instituto de Agroquımica y Tecnologıa de Alimentos (IATA), Consejo Superior de Investigaciones Cientıficas (CSIC),
Apartado de Correos 73, Burjassot 46100, Valencia, Spainb Centro de Investigacion en Alimentacion y Desarrollo, A.C., CIAD, Apartado Postal 1735, Hermosillo, Sonora 83000, Mexico
c Instituto de Biologıa Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Cientıficas (CSIC),
Universidad Politecnica 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
Chilling injury (CI) is responsible for substantial
postharvest losses in many citrus cultivars. Chil-
ling induces pitting, necrosis and staining in the
* Corresponding author. Tel.: �/34-96-390-0022; fax: �/34-
96-363-6301.
E-mail address: [email protected] (M.T. Lafuente).
Postharvest Biology and Technology 29 (2003) 308�/317
www.elsevier.com/locate/postharvbio
0925-5214/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0925-5214(03)00047-4
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 iswell 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-Tellez 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 differ-ent 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 bio-
synthesis 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 1-
MCP, 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 coldstorage 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
chilling sensitivity in citrus fruit or whether theability 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 higherpotential to increase PAL protein content than
ripe fruit in response to wounding; whereas in
unripe melons the expression of ethylene biosyn-
thetic 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 thechilling 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 ethyleneproduction 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;
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308�/317 309
longitude: 00822?52ƒW), all of them at commercialmaturity. ‘Navelina’ fruit were harvested in De-
cember, those of ‘Valencia’ late in April and
‘Fortune’ mandarins in March. In the two follow-
ing 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 wereselected 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 ethyleneproduction 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 ‘For-tune’ 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. So-
luble solids (8Brix) and acids content of the juicewere 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/X-
1000 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 Martınez-Tellez 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, contain-
ing 0.02 M b-mercaptoethanol. PAL activity was
determined by measuring the absorbance of cin-
namic acid at 290 nm over a period of 2 h at 40 8Cand 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�/317310
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 labelledwith [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’ man-
darin 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 non-
chilled control (12 8C) fruit. Three replicate sam-
ples of 1 ml gas sample were withdrawn from thehead 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 devel-
oped more CI than ‘Valencia’ late or ‘Navelina’
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 toler-
ance to chilling and were stored for up to 60 days.
After this period, we did not continue the experi-
ment since senescence of control fruit kept at 12 8Cbecame 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 experi-
ments.PAL activity increased concomitantly with the
development of chilling-induced peel damage dur-
ing storage of ‘Fortune’ mandarins (Fig. 2A). The
activation of PAL in ‘Valencia’ late oranges also
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.
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308�/317 311
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
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 cold-
induced 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 tem-
perature were observed (Fig. 2C). As in the case ofPAL, the increase in ethylene production paral-
leled 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
‘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 bycold-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
Fig. 2. Changes in PAL activity (j, I) and ethylene produc-
tion (', ^) 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.
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308�/317312
that of fruit from December despite the initial
activity being similar (Fig. 4).
To investigate whether the effect of fruit phy-
siological 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 cold-
induced PAL mRNA accumulation in the flavedo.
As in the case of PAL activity (Fig. 3), no
difference was found before cold storage in the
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 g�1 h�1).
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.
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.
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308�/317 313
Cold-stress always increased ethylene production,
as measured at 2 8C, but the rate of ethyleneproduction 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 (28days) 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
high tolerance to chilling were higher than those in
a mandarin showing very low tolerance (Sanchez-
Ballesta 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 cold-
induced 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 appear-
ance 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 pro-
duction 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 de-
scribed in this experiment may be due to differ-
ences 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
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 96.469/1.97 76.949/3.26 4.279/0.14
January 104.149/1.34 43.269/1.12 6.469/0.21
February 108.289/0.94 38.269/1.16 8.629/0.16
2 December 97.209/1.66 68.649/2.87 3.789/0.17
January 106.049/0.92 44.919/0.81 5.579/0.11
February 114.079/0.59 37.989/0.76 7.539/0.26
April 122.099/1.91 37.209/1.21 10.519/0.33
Values are means of three replicate samples of ten fruit9/S.E.M.
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.
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308�/317314
which allows us to select different citrus cultivarswith 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 phy-
siological stage examined (Figs. 3 and 4).
PAL may be induced during development and
ripening in some climacteric (Diallinas and Ka-
nellis, 1994; Assis et al., 2001) and non-climacteric
fruit (Given et al., 1988). Our results in ‘Fortune’
mandarins indicating that PAL activity does notchange 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 protectfruit 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’ mandar-
ins always developed chilling symptoms, changesin 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, thehigher 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
been suggested to be involved in the loss ofethylene-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 usingfruit 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-inducedethylene production and PAL activity may be
related to the severity of cold-induced peel damage
(Sanchez-Ballesta et al., 2000a; Zacarıas 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 tempera-
ture 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 coldstress 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 physiolo-gical 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
M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308�/317 315
(Lafuente et al., 2001). Interestingly, in grapefruitit 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 foundsince 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 reportshowing 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 ripebanana 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 youngunripe 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 thefruit 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 considera-tion the data presented in this work, PAL induc-
tion 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 bio-
chemical marker for chilling tolerance in citrus
fruit. By contrast, chilling-induced ethylene waslittle 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 ofthe fruit.
Acknowledgements
This work was supported by research grants
ALI-93-117 and ALI-96-0506-CO3 from the CI-
CYT, Spain and by FAIR-CT98-4096 from theEU. The technical assistance of D. Arocas is also
acknowledged.
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