Journal of Experimental Botany, Vol. 49, No. 325, pp. 1339–1347, August 1998
Effect of high CO level on the titres of c-aminobutyric
2
acid, total polyamines and some pathogenesis-related
proteins in cherimoya fruit stored at low temperature
Carmen Merodio1, Maria Teresa Muñoz, Begoña Del Cura, Dolores Buitrago and
Marı́a Isabel Escribano
Departamento de Ciencia y Tecnologı́a de Productos Vegetales, Instituto del Frı́o, Consejo Superior de
Investigaciones Cientı́ficas, Ciudad Universitaria, E-28040-Madrid, Spain
Received 31 October 1997; Accepted 31 March 1998
Abstract
In this study the effect of treatment with 20% CO
2
plus 20% O for 3 d on c-aminobutyric acid (GABA) was
2
analysed and total polyamine titres and the production
of some pathogenesis-related proteins (PR-proteins) in
cherimoya (Annona cherimola Mill.) fruit stored at low
temperature. In immunoassays with anti-PR-Q and
PR-2 protein sera, high CO levels were found to pro2
voke the co-ordinated accumulation of a chitinase-like
protein and 1,3-b-glucanase. Chitinase activity was
higher in treated than in untreated fruit. At the end of
CO treatment, total polyamine and c-aminobutyric
2
acid content and uptake of O were observed to be
2
higher in treated compared to untreated fruit, but the
accumulation of these compounds decreased when
the fruit was transferred to air. Since this treatment
effectively retains the fruit quality (Escribano et al.,
1997), high CO levels may have a direct effect on the
2
activation of the above-mentioned specific responses
that enables cherimoya fruit to overcome chilling temperature. The relationship between the activation of
the defence system and the capacity to regulate cytoplasmic pH in CO -treated fruits was also addressed.
2
Key words: Chilling temperature, stress/defence system,
pH, fruit.
Introduction
Low temperature storage is the most effective post-harvest
technology to prolong the shelf-life of fruit and vegetables.
Unfortunately, for chilling injury-sensitive commodities
such as tropical and subtropical fruit, low temperature
storage is often responsible for substantial post-harvest
losses. Several technologies, such as modified (MA) or
controlled atmospheres (CA), with high CO and low O
2
2
concentrations, have been shown to control some physiological disorders, thereby extending the storage period of
fruit and vegetables ( Kader, 1986; Wang, 1990). In cherimoya (Annona cherimola Mill. cv. ‘Fino de Jete’), CA
storage and pretreatments with high levels of CO are
2
beneficial for maintaining fruit quality (Merodio and De
La Plaza, 1997). In addition to being a competitive postharvest technology, CA is an alternative to chemical
fumigation ( Ke and Kader, 1992; Yahia and VazquezMoreno, 1993). No reports have been published, however,
about the induction of defence mechanisms involved in
the control of abiotic/biotic damage in CO -treated fruit
2
and vegetables.
Hypersensitive reaction to pathogen attack, one of the
most effective and most common defence mechanisms,
leads to the accumulation of PR-proteins ( Van Loom
and Van Kammen, 1970). However, at least some members of the different PR families are induced under a
number of conditions in which pathogens are not involved
(Hon et al., 1995; Hincha et al., 1997). PR-Q protein,
for instance, which represent a class of acidic chitinases
(Legrand et al., 1987), is also induced in plants as a
response to different kinds of stress as well as to treatment
with elicitors, abiotic factors such as heavy metals and
plant hormones (Collinge et al., 1993). Some of these
treatments are known to be able to provoke the
co-ordinated co-induction of chitinase and 1,3-b-glucanase ( Vögeli et al., 1988). Moreover, PR production has
also been shown to be developmentally regulated in
1 To whom correspondence should be addressed. Fax: +34 1 5493627. E-mail: [email protected]
© Oxford University Press 1998
1340
Merodio et al.
healthy plants and high levels of some PR-proteins have
been reported in root and senescent leaves, during flowering and seed germination and recently in fruit ripening
( Robinson et al., 1997).
In connection with the adaptive responses to stress
conditions, several authors have reported rises in
c-aminobutyric acid (GABA) levels ( Wallace et al., 1984;
Crawford et al., 1994) and total polyamine content
( Reggiani et al., 1989) in different plant systems.
Specifically, a preconditioning treatment ( Kramer and
Wang, 1989) and infiltration with methyl jasmonate
( Wang and Buta, 1994), which reduce and prevent chilling
injury in several kinds of fruit, may be associated with
increased polyamine levels. Also, treatment with low
oxygen ( Kramer et al., 1989) or carbon dioxide-rich
atmospheres (Philosoph-Hadas et al., 1993) has been
related to increased polyamine levels. In the case of CO 2
rich atmospheres, some beneficial effects may be due to
a mechanism that offsets cellular acidification caused by
the high concentration of CO , as observed in various
2
plant systems (Guern et al., 1986; Lange and Kader,
1997).
Cherimoya fruit is susceptible to chilling injury at low
temperatures. The critical temperature for cherimoya cv.
‘Fino de Jete’ is believed to be about 8 °C, but long
exposure at chilling temperatures is required before cells
show signs of serious injury. In a previous paper it was
shown that treatment with 20% CO plus 20% O for 3 d
2
2
effectively retains the quality of cherimoya fruit during
storage at chilling temperature ( Escribano et al., 1997).
The aim of the study discussed here was to evaluate if
high CO levels have a direct effect on the activation of
2
adaptive responses to stress caused by storage at low
temperature. This study evaluated the effect of high CO
2
treatment on GABA and total polyamine titres and the
production of some PR-proteins, using antibodies against
chitinase and 1,3-b-glucanase. Ethylene production, respiration rate and titratable acidity during storage at
low temperature were also determined to obtain more
information regarding the physiological effect of this
treatment. The results indicate that, at the end of the
short-term high CO treatment, the levels of GABA and
2
polyamines titres and oxygen consumption were greatly
increased compared with those of untreated fruit. High
CO treatment is also associated with a characteristic
2
peak in chitinase protein content with a parallel rise in
1,3-b-glucanase. It may be that the above responses
observed in CO -treated fruit may be considered as part
2
of the general protective mechanism of such a technology
to improve tolerance to low temperature storage in cherimoya fruit. The sum of these protective events in treated
fruit, possibly as result of a controlled acidification
brought on by high CO levels, may be what enables
2
cherimoya fruit to overcome chilling temperature.
Materials and methods
Plant material
Cherimoyas (Annona cherimola Mill. cv. ‘Fino de Jete’) were
harvested in Almuñecar (Granada, Spain). One day after
harvest, fruit was selected so as to obtain a healthy uniform
population. Treated fruit was stored in two 20 l respiration
chambers at 6 °C and ventilated with a 180 ml min−1 continuous
flow of 20% CO 520% O . After 3 d, treated fruit were
2
2
transferred to a continuous humidified air flow. Untreated
(control ) fruit were stored at 6 °C in a respiration chamber
under a continuous flow of air. During low temperature storage,
untreated and CO -treated cherimoyas of identical chronological
2
ages were periodically collected, peeled, chopped, and frozen in
liquid nitrogen. The mesocarp tissues were stored at −80 °C
for further assays. Three individual fruit replicates per day were
evaluated.
Measurement of physiological parameters
Respiration and ethylene production rates: Concentrations of O ,
2
CO and ethylene were determined immediately after removal
2
of the fruit from the air or controlled atmosphere by introducing
individual pieces into an air-tight glass container and measuring
gas concentration in the head-space after 1 h. 1 ml aliquots
were injected into a gas chromatograph (model 3700; Varian,
Walnut Creek, CA). Oxygen and carbon dioxide were detected
by thermal conductivity on a molecular sieve (2 m×3.2 mm)
and Porapak Q (4 m×3.2 mm) columns, respectively, and
ethylene was detected by a flame ionization detector on Porapak
Q, with He as the carrier gas (30 ml min−1). Quantification was
performed using external standards and results were expressed
in ml O kg−1 h−1 and ml ethylene kg−1 h−1.
2
Titratable acidity: Titratable acidity was determined in a
mesocarp homogenate (10 g fresh weight) in 20 ml of distilled
water (heated to eliminate volatile acids and cooled to room
temperature) by titration with 0.1 N sodium hydroxide to
pH 8.1. Results are expressed in meq 100 g−1 fresh weight.
Protein extraction
Frozen mesocarp tissues (10 g fresh weight) were ground in a
precooled coffee grinder and homogenized at 4 °C in 30 ml of
ice-cold extraction buffer (50 mM TRIS-HCl, pH 7.4, 0.4 M
NaCl, 20 mM NaHCO , 20 mM MgSO , 10 mM sodium
3
4
ethylene-diamino tetraacetic acid, 5 mM b-mercaptoethanol,
0.5 mM phenylmethylsulphonyl-fluoride, 0.01 mM leupeptin,
10% (v/v) glycerol, and 1% (v/v) Triton X-100). The resulting
homogenate was centrifuged at 20 000 g for 20 min at 4 °C, the
pellet was discarded and the supernatant was filtered through
eight layers of cheesecloth. Thereafter, soluble protein was
purified using the phenol–ammonium acetate–methanol method
described by Montero et al. (1995).
Electrophoresis and immunoblotting
Dried soluble purified proteins were dissolved in electrophoresis
sample buffer containing 63 mM TRIS-HCl, pH 6.8, 10% (v/v)
glycerol, 2% (w/v) SDS, 5% (v/v) b-mercaptoethanol, and
0.002% (w/v) bromophenol blue. Samples were heated at 95 °C
for 4 min and separated by SDS-PAGE under denaturing
conditions on 14% polyacrylamide slab gels (15×11 cm and
1 mm thick) as described by Laemmli (1970). Duplicate gels
were prepared, one for transferring and the other for staining
with Coomassie brilliant blue. Total protein concentration was
measured by the method of Bradford (1976).
Adaptative responses to low temperature
Proteins separated on SDS-PAGE gels were transferred on
to nitrocellulose filters (0.22 mm) after the method described by
Towbin et al. (1979). The membranes were immunorevealed
with anti-PR-Q (dilution:1/20 000) and PR-2 (dilution:1/4000)
sera which were detected with rabbit antiserum against IgG
horseradish peroxidase conjugate (Biorad ) and developed with
chemiluminescent substrates and exposure to X-ray films
following factory (Amersham) recommendations. Depending
on the compatibility of epitopes, some filters could be stripped
by incubating them in a stripping buffer consisting of 100 mM
b-mercaptoethanol, 2% (w/v) SDS and 62.5 mM TRIS-HCl,
pH 6.7 and further reprobed with other sera. Levels of antigen
on the nitrocellulose filter were quantified using an image
analyser (BioImage and Visage, Millipore Corporation, Ann
Arbor, Michigan).
Chitinase extraction and activity
Protein extract was obtain by homogenizing ground frozen
mesocarp cherimoya tissues (1 g fresh weight) at 4 °C in 5 ml
of 100 mM sodium acetate buffer, pH 5.0 and 2% (w/v)
polyvinylpyrrolidone. The homogenate was centrifuged at
27 000 g for 30 min at 4 °C and the clarified supernatant was
recovered. Chitinase activity was assayed using a commercial
blue enzyme substrate, CM-Chitin-RBV solution (Loewe),
based on the precipitability of a non-degraded, highly polymerized substrate when acid is added. Enzyme activity was assayed
by incubating a standard reaction mixture containing 30 ml of
crude enzyme extract, 200 ml of aqueous CM-chitin-RBV
(2 mg ml−1) and 100 mM sodium acetate buffer, pH 5.0 to yield
a final reaction volume of 0.8 ml, in a gently shaken Eppendorf
tube for 15 min at 37 °C; the reaction was stopped by adding
200 ml of 2 N HCl and cooling on ice for 10 min. The nondegraded substrate was precipitated by centrifuging at 10 000 g
for 5 min. The supernatant containing degraded polymers was
diluted (151, v/v) with nanopure water and absorbance was
measured at 550 nm against a blank reaction (incubation
mixture with HCl-treated crude extract). Specific enzyme
activity was defined as ‘absorbance at 550 nm h−1 mg−1 of total
protein’. Total protein content was analysed in the 5% (v/v)
ice-cold perchloric acid (PCA) precipitated extract. The pellet,
resuspended in 1 N NaOH, was centrifuged and the protein
content measured with the method of Lowry et al. (1951) using
BSA as a standard.
c-Aminobutyric acid extraction and determination
The procedure described by Crawford et al. (1994), with minor
modifications, was used to prepare an aqueous fraction for
determination of GABA. Ground frozen mesocarp tissues (1 g
fresh weight) were treated with 3 ml of 100% methanol to
inactivate enzymes as recommended by Wallace et al. (1984).
Chloroform (6 ml ) and water containing 5% (w/v) polyvinylpolypyrrolidone (3 ml ) were added one after another while
stirring. The samples were centrifuged at 3500 g for 20 min to
separate the aqueous and organic phases. The aqueous phase
was dried under a stream of air at 40 °C and the precipitate
redissolved in a 0.1 M, pH 8.6 potassium pyrophosphate buffer.
Prior to GABA analysis, extracts were centrifuged at 15 000 g
for 10 min to precipitate insoluble material. GABA was
determined on the basis of the increase in A
after 30 min
340
following supplier recommendations for commercially available
GABAse (Sigma), a spectrophotometric-coupled enzyme assay
system for GABA. No interference with spectrophotometric
measurements was detected in the cherimoya extract. The assay
was linear for 0.05 to 0.4 mmol of GABA in a reaction volume
1341
of 3.02 ml, with a recovery rate of 90% as measured against a
standard solution.
Polyamine analysis
The method described by Escribano and Merodio (1994) was
used to perform polyamine analysis. Briefly, extracts for
polyamine analysis were prepared by homogenizing frozen
tissues in 5% (v/v) ice-cold PCA. 1,6-hexanediamine was added
as an internal standard before the tissue was ground. After 1 h
on ice the homogenate was centrifuged at 27 000 g for 30 min
at 4 °C. The supernatant was removed and used for polyamine
analysis. Polyamines were derivatized with dansyl chloride and
analysed using high performance liquid chromatography
(HPLC ). Samples were injected onto a reverse phase C
18
column (150×4.6 mm, 5 mm particle diameter) and eluted from
the column with a programmed water:methanol (v/v) solvent
gradient at a flow rate of 0.9 ml min−1. Dansyl-polyamines
were detected by a fluorescence spectrophotometer (excitation
wavelength, 350 nm; emission wavelength, 495 nm; Kontron
Instruments, model SFM 25) and the peaks, areas and retention
times were computer-recorded and calculated with Integration
Pack Program software ( Kontron Instruments).
Results
Effect of high CO treatment on physiological parameters
2
To eliminate any possible interference by CO that accu2
mulated in the fruit tissue as a result of CO treatment,
2
both CO emission and O consumption were analysed
2
2
to estimate respiration. The values obtained, shown in
Table 1, confirm that treatment with the 20% CO atmos2
phere provoked a transient increase in the respiration
rate (O consumption) of treated fruit not observed in
2
untreated fruit. Higher CO production was also observed
2
(data not shown). After 3 d of exposure to a CO -rich
2
atmosphere, the fruit exhibited high respiration rates
(57.85 ml O kg−1 h−1 versus 12.63 ml O kg−1 h−1 in
2
2
control fruit) that subsequently declined sharply to levels
similar to those obtained in untreated fruit ( Table 1).
Although ethylene production was very low in general
during storage at 6 °C, levels were lower in fruit treated
with 20% CO than in fruit under ambient atmospheric
2
conditions. The increases in ethylene production and
respiration rate recorded on the ninth day of storage at
low temperature were not observed in treated fruit
( Table 1). A similar pattern of delayed rises due to high
CO levels was observed in titratable acidity content, one
2
of the most traditional and characteristic physiological
parameters of ripening in cherimoya fruit ( Table 1).
Pathogenesis-related proteins in cherimoya tissues during
storage at low temperature: effect of high CO treatment
2
Figure 1A shows the results of SDS-PAGE electrophoresis
of protein extracted from mesocarp of untreated and
treated fruit after 5 d and 9 d at 6 °C. Whereas no relevant
differences were found between protein patterns of treated
and untreated fruit, Westhern blot analyses revealed
1342
Merodio et al.
Table 1. Physiological parameters in untreated and CO -treated fruit
2
Cherimoya fruit was stored in two 20 l respiration chambers at 6 °C and either ventilated with a 180 ml min−1 continuous flow of 20% CO –20%
2
O (treated fruit) or a continuous flow of CO -free air (untreated fruit). After 3 d, the treated fruit was transferred to a continuous air flow. Oxygen
2
2
consumption and ethylene production were determined daily in three individual fruit kept at 6 °C and enclosed for 1 h in an air-tight glass container.
Samples of head-space air were taken with a 1 ml syringe and injected into a gas chromatograph. Titratable acidity was measured in a fresh
mesocarp homogenate in distilled water by titration with 0.1 N sodium hydroxide to pH 8.1.
Days
3
5
9
Oxygen consumption (ml kg−1 h−1)
Ethylene production ( ml kg−1 h−1)
Titratable acidity (meq 100 g−1 FW )
Untreated
Treated
Untreated
Treated
Untreated
Treated
12.63±5.66a
8.05±0.87
44.22±9.40
57.85±1.08
11.55±1.96
13.99±3.50
0.05±0.01
0.05±0.01
0.22±0.05
2.77±0.07
2.69±0.05
3.41±0.06
2.64±0.09
2.82±0.19
2.36±0.12
0.63±0.20
0.55±0.27
3.88±0.31
aData are averages±SE of two separate experiments (n=6).
Fig. 2. Histograms representing the quantification of changes in (A)
Chitinase-like proteins and (B) 1,3-b-glucanase protein levels, in
untreated (air) and CO -treated fruit during storage at 6 °C, as described
2
in the legend to Fig. 1. The immunoblots were quantified by densitometry and data are given as peak areas relative to initial value at
prestored fruit.
Fig. 1. High CO -induced PR-protein content in cherimoya tissues
2
during storage at low temperature. (A) Coomasie blue-stained SDSPAGE separated proteins extracted as described in ‘Materials and
methods’. Lane 0 corresponds to prestored fruit [(fruit after harvest
(0 d)]; the others lanes correspond to untreated (air) and treated (3 d
20% CO –20% O ) fruit after 5 d and 9 d of storage at 6 °C. 20 mg of
2
2
total protein were loaded in each lane. Resolved protein was transferred
to nitrocellulose sheets and the immunoblots probed with anti-PR-Q
(B) and anti-PR-2 (C ) sera. Three replicates revealed similar patterns
of protein variation in cherimoya fruit.
different accumulation kinetics of the two immunodetected chitinase-like proteins (38 and 41 kDa) (Fig. 1B).
In untreated fruits these protein levels gradually decreased
all through the storage period, achieving final values 50%
lower than those detected in prestored fruits ( Fig. 2A).
High CO treatment led to significant increases in protein
2
level; 2 d after the end of the treatment, detected levels
Adaptative responses to low temperature
were almost 100% higher than initial values, and three
times as high as those in fruits of the same chronological
age stored in air. A subsequent decrease led to an
immunodetected signal similar to that of prestored fruits,
but still higher than untreated samples.
Having determined the accumulation of PR-Q protein
in fruit treated with high CO levels, the induction of
2
PR-2 protein by 1,3-b-glucanase activity was examined
( Kauffmann et al., 1987). When SDS-polyacrylamide gels
were blotted and probed with PR-2 protein antibodies,
positive immunodectection was observed ( Fig. 1C ).
Antibodies against PR-2 identified a predominant 54 kDa
protein and reacted to a lesser extent at the 66 kDa band;
these polypeptides did not cross-react with antiserum
against PR-Q. The immunoblots showed that 1,3-bglucanase antigen content increased in response to CO
2
treatment ( Fig. 2B). A substantial accumulation was
observed in pretreated fruit by the fifth day, after which
a significant decrease (50%) to values similar to untreated
values was detected. A large accumulation of this polypeptide was also detected in untreated fruits. By the fifth day
values 10 times higher than initial values were achieved,
and were maintained until the end of the storage time.
All changes reported were statistically significant
according to the Bonferroni test at P=0.01.
Changes in chitinase activity
It is generally believed that the formation of reaction
products in the chitinase assay is not proportional to
enzyme concentration, and some evidence points to structural heterogeneity of the susbstrate as the cause of this
behaviour (Molano et al., 1977). The kinetics of the
cherimoya chitinase assay were studied. Plots of the
amount of product formed versus the volume of crude
enzymatic extract did not yield a straight line (Fig. 3,
insert). It was, however, possible to obtain reasonably
accurate results by using a small amount of the diluted
crude enzyme extract ( Fig. 3). Moreover, plots of product
formed versus reaction time were linear in a range of
5–180 min for a 30 ml-diluted crude enzyme extract assay
(between 9.6 and 16.8 mg soluble protein in the 1 ml
reaction mixture) showing that cherimoya chitinase maintained linearity for a longer time in the standard assay
(described in Materials and methods) (data not shown).
Changes in chitinase activity, expressed as per cent of
the activity present in freshly harvest fruit (19.53±0.4
A
h−1 mg−1 total protein), were determined in
550 nm
untreated and high CO -treated fruit during storage at
2
6 °C (Fig. 4). At the end of the CO treatment, chitinase
2
activity was found to be considerably higher than in fruit
just harvested as well as in untreated fruit. These results
indicate that the increase in chitinase activity observed in
response to high CO treatment was the result of a net
2
accumulation of the enzyme. Moreover, while enzyme
1343
activity dropped continually in treated fruit, the levels of
activity in CO -treated fruit were higher than in untreated
2
control fruit.
c-Aminobutyric acid titres
Figure 5 shows GABA levels in untreated and CO 2
treated fruit during storage at low temperature. Whereas
insignificant variations in GABA titres were observed in
untreated control fruit during the first 3 d of storage at
6 °C, in treated fruit the levels of GABA increased 9-fold
by the end of CO treatment. After transfer to air, the
2
GABA content dropped sharply to levels similar to those
observed in untreated fruit on the ninth day of storage
( Fig. 5).
Effect of high CO on polyamine levels
2
The levels of free putrescine, spermidine and spermine
content were determined. Figure 6 shows the effect of
CO treatment on total polyamine content in cherimoya
2
mesocarp tissues, which was higher in fruit treated for
3 d with CO . The effect of CO treatment on all poly2
2
amines was virtually identical, with significantly similar
increases (33%) being observed for putrescine, spermidine
and spermine (data not shown). A progressive decrease
in the total polyamine titre was observed after transfer to
air, dropping to the same levels as in the control fruit
after 9 d of storage. These results show a correlation
between CO treatment and higher polyamine levels in
2
mesocarp tissues.
Discussion
In general, post-harvest technologies applied to reduce
chilling injury to fruit and vegetables involve either
retarding the development of injury symptoms or increasing the tolerance to low temperature storage. It is possible
that to improve tolerance to low temperature storage it
could be necessary to activate the defence systems in the
fruit. Hence, analysing the initial presence or absence of
specific defence responses might make it possible to
anticipate the advent of chilling injury symptoms. Several
hypotheses have been put forward to account for the
nature of chilling injury, most of which are based on lipid
phase changes and loss of membrane integrity (Lyons,
1973). The alteration of membrane fluidity directly affects
membrane-bound metabolic processes. Of these processes,
respiration is known to be affected in chilling-sensitive
species, many of which show clear signs of anaerobic
metabolism at low temperature. The results confirm that,
contrary to untreated fruit, CO -treated fruit is able to
2
maintain an aerobic metabolism (higher O consumption)
2
during the first days of storage at low temperatures.
Moriguchi and Romani (1995) observed that exposure of
avocado fruit to CO -rich atmospheres enhanced the
2
1344
Merodio et al.
Fig. 3. Product formation versus volume of diluted crude enzyme extract. Inset: Curved plots obtained in terms of volume of crude enzyme extract.
Data are averages of two separate experiments (n=6).
Fig. 4. Chitinase activity patterns in untreated (&) and CO -treated
2
(+) cherimoya fruit during storage at 6 °C. Data are averages of two
separate experiments (n=6) and SE are shown by vertical bars where
they exceed the size of the respective symbol.
capacity of their mitochondria to restore energy-linked
functions. This increase in the respiration rate might
strengthen the defence system set off to alleviate the wellknown oxidative stress caused by chilling temperatures
( Hariyadi and Parkin, 1991). Relationships between poly-
Fig. 5. Changes in GABA levels in untreated (&) and CO -treated (+)
2
cherimoya fruit during storage at low temperature. Data are averages
of two separate experiments (n=6) and SE are shown by vertical bars.
FW, fresh weight.
Adaptative responses to low temperature
Fig. 6. Total polyamine levels in untreated and CO -treated cherimoya
2
fruit during storage at low temperature. Data are averages of two
separate experiments (n=6) and SE are shown by vertical bars. FW,
fresh weight.
amines and protection against oxidative damage to membranes caused by chilling injury has been observed
( Kramer and Wang, 1989). Furthermore, an increase in
polyamine content as a function of intracellular pH has
also been associated with acidic and other kinds of stress
( Reggiani et al., 1989; Turano and Kramer, 1993). These
results confirm that CO affects polyamine levels, with
2
higher levels in treated than in control fruit. The pattern
of evolution of polyamine levels over time would seem to
confirm that the initial effect of CO is lost when fruit is
2
transferred to normal atmospheric conditions. Also, polyamine levels (mainly spermidine and spermine) have been
observed to rise in fruit and vegetables subjected to
controlled atmospheres and other kinds of treatments
applied to reduce chilling injury ( Kramer et al., 1989;
Wang and Buta, 1994).
Chitinases and b-1,3-glucanases have been intensively
studied in relation to plant defence reactions, with most
work focusing on their roles as pathogenesis-related proteins. However, recent studies (Hon et al., 1995; Hincha
et al., 1997) address the protective role of some
PR-proteins in cold acclimation of plants. Immunological
cross-reaction of cherimoya mesocarp protein with PR-Q
antibodies identified two bands of 41 and 38 kDa and
antibodies against PR-2 identified a predominant protein
of a molecular mass of 54 kDa. In general, PR-proteins
found in plants are small, sometimes glycosylated, with
molecular weights ranging from 25 000 to 35 000 (Stintzi
et al., 1993). The immunological relationship between
tobacco PR and cherimoya proteins would indicate structural features in common. These results confirmed that
during storage at low temperature, high CO treatment
2
effectively triggered PR-Q and PR-2 protein accumulation. The highest content for these type of proteins was
consistently found in CO -treated fruit. The co-ordinated
2
1345
induction of chitinase and 1,3-b-glucanase proteins, in
response to high CO levels, and the absence of such
2
induction in untreated fruit, seem to confirm that CO 2
rich atmospheres activate their synthesis in cherimoya
fruit. Furthermore, mesocarp chitinase activity patterns
concurred with this immunoserological results. Induction
of chitinase is often co-ordinated with the induction of
specific b-1,3-glucanases and other pathogenesis-related
proteins ( Vögeli et al., 1988). PR-protein accumulation
may be via ethylene (Boller et al., 1983), although GABA
has also been shown to induce accumulation of chitinases
and 1,3-b-glucanases in a similarly co-ordinated fashion
(Lotan and Fluhr, 1990). Moreover, these authors
observed that in GABA eliciting of PR-proteins, PR-Q
accumulation was only partially inhibited by aminoethoxyvinylglycine, an inhibitor of ethylene biosynthesis.
These data seem to provide support for these authors’
hypothesis that at least one of the induction pathways
regulating PR-proteins synthesis does not involve ethylene. It was observed that untreated and treated fruit
whose chitinase content differed had very low levels of
ethylene production, with the lowest values corresponding
to treated fruit. Furthermore, in a number of different
kinds of fruit exposure to CO -rich (5–30%) atmospheres
2
results in a considerable reduction in ethylene production
(Rothan and Nicolas, 1994). Induction of some PRproteins has been observed in grape, a non-climacteric
fruit ( Tattersall et al., 1997). Although, it is difficult to
understand the role that PR-proteins may play in cherimoya fruit, the induction of these proteins may be related
with CO -activation of an adaptative mechanism to stress2
ful conditions.
With further reference to the activation of responses
against stress conditions, this study analysed GABA titre
patterns in CO -treated and untreated fruit stored at low
2
temperature. This compound has been reported ( Wallace
et al., 1984) to accumulate rapidly in soybean leaves
transferred abruptly to a lower temperature and in
response to mechanical damage. Rapid GABA accumulation has also been shown to be involved in the defence
against phytophagous insects (Ramputh and Bown, 1996)
and as an adaptative response to stress-related cytosolic
acidification (Crawford et al., 1994). The results obtained
confirmed that GABA content, which had accumulated
by the end of CO treatment, plummeted after transfer
2
to air. Although the precise mechanism is not known, it
is clear that high CO treatment causes large and transient
2
increases in GABA and polyamine levels that are not
observed in fruit stored in air.
Bearing in mind the role attributed to GABA and
polyamines in pH regulation (Carroll et al., 1994) and
the modification of cytoplasmic pH by high CO treat2
ments (Siriphanich and Kader, 1986; Guern et al., 1986),
it is possible that the beneficial effect of high CO levels
2
in preventing low temperature stress may be related to
1346
Merodio et al.
the capacity to regulate cytoplasmic pH in CO -treated
2
fruits.
In conclusion, the activation of specific responses
such as increased respiration, GABA, polyamines and
co-induction of chitinase and 1,3-b-glucanase are in fact
involved in the effect that high CO levels have on
2
cherimoya fruit to overcome chilling temperature.
Nevertheless, further analysis of the homeostatic potential
of this fruit and its capacity to recover from external
(biotic/abiotic stress) or endogenous metabolic perturbation (changes in cytoplasmic pH ) is required and may
provide useful insight into ways to improve post-harvest
technologies.
Acknowledgements
The authors are grateful to Professor Fritig for his generosity
in providing PR-Q and PR-2 antisera. This work was supported
by grants from the European Union ( TS3-CT93–0205) and
Spain, CICYT (ALI96–0506-CO3-O2).
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