conditioning has been shown to increase chilling tolerance
in different
crops Lurie,
1998. Postharvest hot-water treatment HWT over
50°C for 1 – 3 min Wild 1990; Rodov et al., 1995; Schirra and Mulas, 1995 or hot air treat-
ment HAT at 35 – 37°C at high humidity for 1 – 3 days Ben-Yehoshua et al., 1987; Lafuente
et al., 1997 improve citrus fruit resistance to CI. Both heat pretreatments reduce CI in ‘Fortune’
mandarins Mulas et al., 1995; Gonzalez-Aguilar et al., 1997; Lafuente et al., 1997. However,
HWT treatments were not able to maintain the heat-induced resistance to cold stress after pro-
longed storage in ‘Fortune’ mandarins, whereas the HAT for 3 days at 37°C did so Mulas et al.,
1995.
Chilling temperatures may induce oxidative stress in plant tissues Purvis and Shewfelt,
1993, including fruits Hariyadi and Parkin, 1991; Sala, 1998. Sala 1998 found that the
main difference between chilling-sensitive and chilling-tolerant cultivars is in the higher ability
of the chilling-tolerant to break down H
2
O
2
by CAT activity and co-operation of APX and GR
activities. Subsequently, it was reported that heating ‘Fortune’ mandarins at 37°C for 3 days
induced 2.5-, 1.4-, and 1.2-fold increases in the activities of catalase CAT, superoxide dismu-
tase SOD and ascorbate peroxidase APX, re- spectively,
and that
the differences
in the
activities produced by the heat treatment were maintained during cold storage Sala and La-
fuente, 1999. In mustard seedlings, however, a heat acclimation treatment resulted in decreased
CAT activity during the induced thermoprotec- tion period Dat et al., 1998. Aminotriazole 3-
amino-1,2,4-triazole AT is an inhibitor of CAT activity in the presence of hydrogen peroxide,
and has been used to investigate the role of CAT in animals and plants exposed to different stress
conditions
Halliwell and
Gutteridge, 1993;
Prasad, 1997. We have tested the importance of CAT, APX,
GR and SOD on the tolerance of mandarin fruits to low-temperature stress, and the effect of
AT on response of chilling-tolerant ‘Clementine’ and ‘Clemenules’ mandarins and on the heat-in-
duced tolerance to cold stress in ‘Fortune’ man- darins. The effects of short HWT on CAT
activity and protection of fruit against CI under prolonged cold storage in relation to the mainte-
nance of the heat-induced CAT activity has also been investigated.
2. Material and methods
2
.
1
. Plant material, storage and treatments Fruits of three mandarin cultivars were used.
‘Fortune’ Citrus clementina Hort. ex Tanaka × Citrus reticulata Blanco fruit were harvested at
random from 20-year-old trees grafted onto ‘Sat- suma’ mandarin Citrus unshiu Marc. and sour
orange Citrus aurantium L. rootstock and grown at Sagunto, Valencia, Spain. ‘Clementine’
Citrus
reticulata Blanco,
cv. ‘Fina’
and ‘Clemenules’
Citrus reticulata
Blanco, cv.
‘Nules’ fruits were obtained from a packing house in Almenara, Castello´n, Spain.
‘Fortune’ mandarin fruits were randomly di- vided into three lots containing three replicates
of 20 fruits to estimate chilling damage, and of ten fruits per storage period to analyse CAT
activity: 1 stored immediately for up to 28 days at 2°C and 80 – 85 RH; 2 subjected to a 3-day
heat-conditioning treatment at 90 – 95 RH and 37°C HAT and then stored under the same
conditions as the fruits of the first lot; 3 sub- merged for 3 min in a recirculating hot water
bath at 53 9 0.3°C HWT consisting of a stain- less steel chamber volume 120 l and a Honey-
well water bath controller temperature unit 9 0.1°C; model Versapak 84, UK. After HWT
all fruit were air-dried and stored as the non- treated fruit. The RH was measured using an
electronic probe Eliwell, EWS28.
Two additional experiments were conducted using AT to inhibit CAT activity. In the first
experiment, ‘Fortune’ mandarin fruits were se- lected and randomly divided into two lots. The
first lot was treated by dipping the fruits twice in an aqueous solution of 150 mM AT Sigma for
15 s, conditioned at 37°C and 90 – 95 RH for 3 days and then subdivided into two groups, which
were stored at 2 or 12°C at 80 – 85 RH for up
to 8 weeks. The second lot controls, not treated with AT, was heat-conditioned for 3 days at 37°C
and subdivided into two groups, which were stored under the same conditions as the first lot.
In the second experiment, ‘Clementine’ and ‘Clemenules’ fruits were divided into two lots, one
was used as a control and the other was treated by dipping the fruits twice in a 30-mM AT
aqueous solution for 15 s. AT-treated and non- treated fruits were stored at 2 or 12°C and 80 –
85 RH for up to 6 weeks.
In each group, fruits of the three cultivars studied were randomly divided into three repli-
cates of 27 fruits and peel damage was evaluated weekly. Three replicate samples of four fruits of
each group stored at 2°C were sampled after 2, 4, 6 and 8 weeks storage in ‘Fortune’ mandarins and
after
2 and
6 weeks
in ‘Clementine’
and ‘Clemenules’ fruits for assessment in enzyme ac-
tivities. The coloured outer layer of skin flavedo tissue was separated from the whole fruit, cut
into small pieces, frozen in liquid N
2
and stored at −
70°C for enzyme assays.
2
.
2
. CI index CI symptoms in ‘Fortune’ mandarin are small,
brown, pit-like depressions on the peel. The sever- ity of peel damage was evaluated by the method
previously described by Lafuente et al. 1997. A rating scale based on surface necrosis and inten-
sity of browning was used: 0 = no pitting; 1 = slight; 2 = medium; 3 = severe pitting.
2
.
3
. Enzyme assays CAT was extracted from 1 g fresh weight of
frozen flavedo tissue ground in 10 ml of 100 mM potassium phosphate, pH 6.8 at 4°C and then
centrifuged twice at 27 000 × g for 15 min at 4°C. The supernatant was used to determine CAT ac-
tivity by the method of Kar and Mishra 1976 in a final volume of 5 ml, which contained 1 ml of
enzyme extract 400 – 800 mg protein. The unit of CAT activity was defined as the amount of en-
zyme, which decomposes 1 mmol H
2
O
2
per minute at 25°C.
APX was extracted from 1 g of frozen flavedo tissue ground in 10 ml of 50 mM potassium
phosphate buffer, pH 7.0, containing 0.1 mM ethylenediamine tetraacetic acid EDTA, 1 mM
ascorbic acid and 1 polyvinyl-polypyrrolidone PVPP at 4°C. The homogenate was centrifu-
gated at 27 000 × g for 15 min at 4°C twice and the supernatant used to determine the APX activ-
ity by the method of Asada 1984 in a final volume of 3 ml, which contained 100 – 300 ml of
enzyme extract 40 – 240 mg protein. The unit of APX was defined as the amount of enzyme that
oxidised 1 mmol of ascorbate per minute at 25°C.
GR was extracted from 1 g of frozen flavedo tissue ground in 10 ml of 100 mM potassium
phosphate buffer, pH 7.5, containing 0.5 mM EDTA at 4°C. The homogenate was centrifuged
twice at 27 000 × g for 15 min at 4°C. The super- natant was used to determine GR activity by the
method of Smith et al. 1988 in a final volume of 3 ml, which contained 100 ml of enzyme extract
40 – 80 mg protein. The unit of GR was defined as the amount of enzyme that catalysed the oxida-
tion of 1 mmol of NADPH per minute. The activity of the GR solution used for the standard
curve was determined by the method of Carlberg and Mannervik 1985.
SOD was extracted from 1 g of frozen flavedo tissue ground in 10 ml of 50 mM potassium
phosphate buffer, pH 7.8, containing 1.33 mM diethylenetriamine pentaacetic acid at 4°C and
then centrifuged twice at 27 000 × g for 15 min at 4°C. The supernatant was used to determine SOD
activity by the method of Oberley and Spitz 1986 in a final volume of 3 ml, which contained
60 – 70 ml of enzyme extract 24 – 56 mg protein. The unit of SOD was defined as the amount of
enzyme which gave half-maximal inhibition.
Activities of all enzymes were expressed as spe- cific activities units per mg protein fresh weight.
Protein was determined by the method of Brad- ford 1976, using bovine serum albumin BSA as
a standard.
2
.
4
. Statistical design Experimental data are the mean 9 S.E. of three
replicates of the determinations for each sample.
3. Results
