The Journal of Horticultural Science and Biotechnology

ISSN: 1462-0316 (Print) 2380-4084 (Online) Journal homepage: http://www.tandfonline.com/loi/thsb20

Relationships between storage disorders and fruit

calcium contents, lipoxygenase activity, and rates
of ethylene evolution and respiration in ‘Royal
Delicious’ apple (Malus × domestica Borkh.)

R. R. Sharma, R.K. Pal, D. Singh, J. Singh, M. R. Dhiman & M. R. Rana

To cite this article: R. R. Sharma, R.K. Pal, D. Singh, J. Singh, M. R. Dhiman & M. R. Rana (2012)
Relationships between storage disorders and fruit calcium contents, lipoxygenase activity,
and rates of ethylene evolution and respiration in ‘Royal Delicious’ apple (Malus × domestica
Borkh.), The Journal of Horticultural Science and Biotechnology, 87:4, 367-373

To link to this article: http://dx.doi.org/10.1080/14620316.2012.11512878

Published online: 07 Nov 2015.

Submit your article to this journal

View related articles

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=thsb20

Download by: [McMaster University] Date: 28 January 2016, At: 22:40

 Journal of Horticultural Science & Biotechnology (2012) 87 (4) 367–373

Relationships between storage disorders and fruit calcium contents,

lipoxygenase activity, and rates of ethylene evolution and respiration
in ‘Royal Delicious’ apple (Malus
domestica Borkh.)

By R. R. SHARMA1*, R. K. PAL1, D. SINGH2, J. SINGH1, M. R. DHIMAN3 and M. R. RANA4

1
Division of Post-Harvest Technology, Indian Agricultural Research Institute, New Delhi-110 012,
India
2
Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi-110 012, India
3
Indian Agricultural Research Institute, Regional Research Station, Katrain, Dist. Kullu 175 129
(H.P.), India
4
Apple Grower, Baragaon, Katrain, Dist. Kullu 175 129 (H.P.), India
(e-mail: rrs_fht@rediffmail.com) (Accepted 31 March 2012)

SUMMARY
Downloaded by [McMaster University] at 22:40 28 January 2016

Experiments were conducted to determine the relationships between the occurrence of storage disorders in ‘Royal
Delicious’ apple (Malus
domestica Borkh.) and fruit Ca2+ ion contents, rates of ethylene evolution and respiration,
and lipoxygenase (LOX) activity. Apples were stored at 0º ± 1ºC and 90 – 95% relative humidity and sampled each
month, up to 6 months, for storage disorders, fruit Ca2+ ion contents, rates of ethylene evolution and respiration, and
LOX activity. Storage disorders such as bitter pit, cork spot, and brown core appeared after 3 months and increased
in frequency at subsequent samplings. Regardless of the presence or absence of storage disorders, fruit showed a
decline in Ca2+ ion concentration during storage. Ca2+ ion concentrations (means of 3, 4, 5, and 6 month samples) were
significantly higher in disorder-free fruit [0.419 mg g–1 dry weight (DW)] than in fruit showing bitter pit (0.329 mg g–1
DW) or cork spot (0.340 mg g–1 DW), but were not significantly higher than in fruit with brown core (0.393 mg g–1 DW).
Conversely, LOX activity was significantly lower in disorder-free fruit [0.168 µmoles linolenic acid oxidised min–1 g–1
fresh weight (FW)] compared to symptomatic fruit. LOX activity increased with the duration of storage in all fruit
samples. The rates of ethylene evolution (58.5 µl C2H4 kg–1 FW fruit h–1) and respiration (16.0 ml CO2 kg–1 FW fruit
h–1) were significantly lower in disorder-free fruit than in symptomatic fruit. The rates of ethylene evolution and
respiration increased between 3 – 5 months in storage. After 5 months in storage, they remained stable or declined in
all fruit. The correlations (R2) between Ca2+ ion content vs. the presence of physiological disorders such as bitter pit
(–0.77), cork spot (–0.75), and brown core (–0.64), and between Ca2+ ion contents vs. LOX activity were strongly
negative (–0.94). In contrast; the correlation between LOX activity vs. physiological disorders was strongly positive.
Our study concluded that an inverse relationship existed between fruit Ca2+ ion content and the occurrence of bitter
pit, cork spot, and/or brown core, and between fruit Ca2+ ion content and LOX activity. Moreover, the relationship
between LOX activity and the presence of storage disorders was strongly positive.

pple (Malus
domestica Borkh.) is considered to

A be the most important temperate fruit crop in the
World. Several cultivars are grown commercially, but
‘Royal Delicious’ is a popular cultivar in almost every
country. In India, apple orchards account for 75% of the
land area used for temperate fruit production, of which
60% is covered by ‘Royal Delicious’ (Chadha and
Awasthi, 2005). However, apple productivity in India is
low in comparison with other countries. During storage,
apple fruit suffer from physiological disorders such as
bitter pit, brown core, and cork spot, which cause high
post-harvest losses. In bitter pit, small sunken areas
develop on the peel and flesh of the fruit. In cork spot,
the spots are similar to bitter pit, except they are solid in
nature and occur more frequently in the flesh than on the
peel (Chadha and Awasthi, 2005). In brown core, the area
surrounding the seed becomes brown and may rot. Due
to a lack of flesh firmness, such fruit become susceptible
*Author for correspondence.
to fruit rot during storage or transportation, and so
become unfit for human consumption.
Calcium (Ca2+) ions are considered to be one of the
most important nutrient elements controlling the
metabolism of plant cells and have a role in preventing
various physiological disorders (Faust and Shear, 1972;
Shear, 1975; Bangerth, 1979; Sharma and Singh, 2009).
Ca2+ ions also act to retard fruit ripening and senescence
(Ferguson, 1984). Although, the mechanism by which
Ca2+ ions prevent physiological disorders is not well-
understood, it is clear that they act principally on the
middle lamella of cell walls, which plays a role in cross-
linking (Poovaiah, 1985; 1988; Marcelle, 1989), where
they may influence membrane-bound enzymes such as
lipoxygenase (LOX; E.C. 1.13.11.12), which converts
unsaturated fatty acids such as linoleic and linolenic acid
to their hydroperoxide derivatives (Wardale and
Lambert, 1980; Leshem et al., 1982; Lieten and Marcelle,
1993; Perkins-Veazie, 1995; Perez et al., 1999; Sharma and
Singh, 2008). Ca2+ ions also play a role in maintaining
 368 Calcium content and lipoxygenase activity in apple fruit
membrane structure and integrity by binding to
phospholipids and proteins in the plasma membrane.
Many studies have shown that a low total Ca2+ ion
content in fruit leads to higher plasma membrane
leakage, which can potentially affect lipid metabolism
through effects on LOX activity. Hence, the breakdown
of linolenic acid may also be responsible for the
development of some physiological disorders in fruit
(Feys et al., 1980). Furthermore, it has been
demonstrated that if the Ca2+ ion content of fruit is low
during storage, LOX activity is also likely to be low, and
vice versa (Marcelle, 1989; Sharma and Singh, 2008). This
demonstrates the causal role of Ca2+ in fruit senescence.
Fruit Ca2+ ion content also has a close relationship with
respiration (Faust and Shear, 1972).
Thus, the primary aim of this study was to determine
the relationship between fruit Ca2+ ion content, LOX
activity, the rates of respiration and ethylene evolution,
and the occurrence of storage disorders such as bitter pit,
cork spot, and brown core in ‘Royal Delicious’ apples
kept in cold storage for 6 months.
Downloaded by [McMaster University] at 22:40 28 January 2016
MATERIALS AND METHODS
Studies were conducted at the Division of Post-
Harvest Technology, Indian Agricultural Research
Institute, New Delhi, India between 2008 – 2010. ‘Royal
Delicious’ apples were harvested at full maturity from a
well maintained, private orchard located at Kullu
(Himachal Pradesh) and transported to New Delhi for
experimentation.
After sorting and grading, all apples (n = 1,200) were
placed in cold storage at 0º ± 1ºC and 90 – 95% relative
humidity for 6 months. During storage, observations on
the incidence of physiological disorders including bitter
pit, cork spot, and brown core, LOX activity, the rates of
respiration and ethylene evolution, and fruit Ca2+ ion
contents were recorded at monthly intervals.
To determine the incidence of the storage disorders,
fruit were divided into four lots, each of 100 fruit, with
three replicates. However, to determine LOX activities,
the rates of ethylene evolution and respiration, and fruit
Ca2+ ion contents, 20 fruit were sampled at random at
monthly intervals with three replicates. At harvest, LOX
activities, the rates of ethylene evolution and respiration,
and fruit Ca2+ ion contents were also determined in 20
randomly selected fruit per lot with three replicates, but
these data are not shown because, at the time of harvest,
all apples were healthy and showed no symptoms of any
disorder.
Incidence of physiological disorders
The incidence of various physiological disorders such
as bitter pit, cork spot, and brown core was determined
by counting the number of symptomless (disorder-free)
fruit and those affected by each specific disorder in each
100-fruit lot, replicated three times. The incidence of
each of the different disorders was then represented as a
percentage (%).
Estimation of fruit Ca2+ ion contents
Fruit Ca2+ ion contents during storage were determined
in symptomless (disorder-free) and symptomatic fruit
(bitter-pitted, cork-spotted, and/or brown-cored) as
described by Sharma and Singh (2008). A sample (150 g
FW) from each stored fruit (a 3 – 4 mm-thick longitudinal
slices from the fruit flesh) was reduced to 50 – 60 g total
dry weight (DW) by ashing (Sharma and Singh, 2008).
After ashing, the residue was dissolved in 0.1 M nitric
acid. The Ca2+ ion content of each fruit sample was
determined by atomic absorption spectrophotometry
(AAS 4141; ECO Ltd., New Delhi, India).
Determination of the rates of respiration and ethylene
evolution
The rates of ethylene production and respiration were
measured using the static headspace technique (Pal,
1998). Two fruit were randomly selected from each lot at
each time point and enclosed for 2 h in a 1 l hermetically-
sealed container, fitted with a silicon rubber septum. The
concentrations of O2 and CO2 were recorded in the
headspace of the container using a gas analyser
(Checkmate 9900 O2/CO2; PBI Dansensor, Ringsted,
Denmark) and the rate of respiration was expressed in
ml CO2 kg–1 FW fruit h–1.
To determine ethylene contents, 1.0 ml of headspace
gas was withdrawn using a gas-tight syringe and injected
into a gas chromatograph (HP 5890; Hewlett Packard,
Avondale, PA, USA) which was calibrated using
standard ethylene gas (Laser Gases, New Delhi, India).
The gas chromatograph was equipped with a 80 - 100
mesh Porapak-N column and a flame ionisation detector
(FID). Nitrogen was used as the carrier gas at a flow rate
of 30 ml min–1, while hydrogen and air were the fuel gases
with flow rates of 25 ml min–1 and 250 ml min–1,
respectively. The temperatures of the injector, the
column, and the detector were maintained at 110ºC, 60ºC
and 275ºC, respectively. The rate of ethylene production
was expressed as µl C2H4 kg–1 FW fruit h–1.
Lipoxygenase (LOX) activity
Preparation of substrate: The substrate was prepared as
described by Feys et al. (1980), for apple, with slight
modifications. Pure linolenic acid (0.1 ml; Merck India
Ltd., New Delhi, India) was dissolved in 1.0 ml 0.1 M
NaOH, and 150 µl Triton-X-100 was added. The solution
was emulsified in an Ultra-Turrax (Panacea Instruments
Pvt. Ltd., New Delhi, India) for 2 min, then diluted to
50 ml with distilled water. A blank was prepared
similarly, but without adding linolenic acid. The substrate
and the blank were stored in the dark at 4ºC until use.
Preparation of crude enzyme extract: Each crude enzyme
extract was prepared at 4°C following the method of
Axelrod et al. (1981) with minor modifications. Diced
apple fruit (1.0 g) was homogenised in a pre-chilled
pestle and mortar by mixing it with 10 ml ice-cooled 0.2
M EDTA. The homogenate was centrifuged at 15,000

g for 20 min at 4ºC and the supernatant was used to assay
for LOX activity.
Measurement of lipoxygenase activity: The LOX assay
was carried out as described by Axelrod et al. (1981) with
minor modifications. Fifty µl of crude enzyme extract was
added to 2.5 ml of substrate solution in a cuvette, then
mixed thoroughly and the absorbance at 234 nm was
recorded at 30 s intervals in a spectrophotometer
(Perkin-Elmer UV-VIS Lambda-25; San Jose, CA, USA)
 R. R. SHARMA, R. K. PAL, D. SINGH, J. SINGH, M. R. DHIMAN and M. R. RANA 369

for 3 min. LOX activity was expressed as µmoles
Disorder-free (healthy) fruit  Bitter-pitted fruit
 Cork-spotted fruit Brown-cored fruit
linolenic acid oxidised g–1 FW min–1.

Statistical design and analysis of data

The experiment was laid out in a completely
randomised factorial design (CRFD) with four lots, each
lot consisting of 100 fruit with three replications, for
measuring the incidence of physiological disorders, and
20 fruit per lot with three replicates for all other
biochemical observations. The data from 2 years were
averaged because there was no significant variation
between years, then analysed using the SAS statistical
package (SAS Institute Inc., Cary, NC, USA).
The effect of storage interval on the incidence of
various physiological disorders, on changes in the rates of
respiration and ethylene production, and on Ca2+ ion
contents were compared by ANOVA by calculating the
least square difference (LSD) at the 5% level of
significance (P ≤ 0.05). Correlations (R2) between Ca2+
ion concentrations and the incidence of physiological
disorders, between Ca2+ ion contents and LOX activity,
Downloaded by [McMaster University] at 22:40 28 January 2016

between LOX activity and physiological disorders, and

between LOX activity and the rates of ethylene
production and respiration were determined. These
correlations were calculated on the basis of 60 samples
(n = 20, with three replicates).

RESULTS
Development of physiological disorders in apples during
cold storage
No storage disorders appeared in any apple fruit until
month-3 of storage, at which time 2.2% of the fruit were
affected by bitter pit, 0.8% by cork spot, and 0.2% by
brown core. Between month-3 and month-6 of storage,
bitter pit had the highest incidence (11.1%), followed by
FIG. 1
brown core (3.3%) and cork spot (2.8%; Table I). The Changes in lipoxygenase (LOX) activity (µmoles linolenic acid oxidised
mean incidence of all storage disorders thus increased –1 –1
g FW min ; Panel A) and the rates of ethylene production (µl C2H4
from 1.1% in month-3 to 12.2% in month-6 of storage. kg–1 FW h–1; Panel B) and respiration (ml CO2 kg–1 FW h–1; Panel C) in
disorder-free (healthy) fruit (
) or in bitter-pitted (), cork-spotted
(), or brown-cored (solid line) ‘Royal Delicious’ apple fruit during
Ca2+ ion contents and LOX activities in symptomatic and storage at 0º ± 1ºC and 90 – 95% RH for 6 months. Each datum point
disorder-free (healthy) apple fruit during cold storage represents the mean of 20 fruit samples with three replications (n = 60),
recorded at monthly intervals. Bars represent standard deviation (SD).
Fruit Ca2+ ion concentrations were notably higher in Significant at P ≤ 0.05 after factorial analysis of variance.
disorder-free fruit (0.419 mg g–1 DW) compared with
cork-spotted (0.347 mg g–1 DW), brown-cored (0.393 mg (0.329 µmoles linolenic acid oxidised g–1 FW min–1),
g–1 DW), or bitter-pitted fruit (0.329 mg g–1 DW; LSD = brown-cored (0.356 µmoles linolenic acid oxidised g–1
0.03 at P ≤ 0.05). Ca2+ ion levels decreased with the FW min–1) or bitter-pitted fruit (0.468 µmoles linolenic
duration of storage in both symptomatic and disorder- acid oxidised g–1 FW min–1; LSD = 0.003 at P ≤ 0.05), and
free fruit (Table II). Conversely, LOX activity was the activity increased with the duration of storage in both
significantly lower in disorder-free fruit (0.168 µmoles disorder-free and symptomatic fruit (Figure 1).
linolenic acid oxidised g–1 FW min–1) than in cork-spotted TABLE II
Changes in Ca2+ ion concentrations (mg g–1 DW) in disorder-free and
symptomatic ‘Royal Delicious’ apple fruit during storage at 0º C and
TABLE I 90 – 95% RH for 6 months
Incidence (%) of physiological disorders in ‘Royal Delicious’ apples
during cold storage Symptomatic fruit
Duration of
Duration of storage (months) storage Disorder- Bitter- Cork- Brown- Mean
Physiological (months) free fruit pitted spotted cored value
disorder 3 4 5 6 Mean value
3 0.442 0.413 0.417 0.422 0.424
Bitter pit (%) 2.2 7.5 12.3 22.2 11.1 4 0.430 0.378 0.384 0.392 0.396
Cork spot (%) 0.8 1.3 2.5 6.5 2.8 5 0.412 0.332 0.354 0.388 0.372
Brown core (%) 0.2 0.6 4.6 7.8 3.3 6 0.392 0.194 0.233 0.360 0.295
Mean 1.1 3.1 6.5 12.2 – Mean 0.419 0.329 0.347 0.393 –
LSD0.05 LSD0.05
Physiological disorder (P) = 2.2 Physiological disorder (P) = 0.03
Storage period (S) = 0.8 Storage period (S) = 0.07
P
S = 2.9 P
S = 0.11
 370 Calcium content and lipoxygenase activity in apple fruit
Rates of ethylene production and respiration in disorder-
free and symptomatic apple fruit during cold storage
The rates of ethylene production were significantly
lower in disorder-free fruit (58.5 µl C2H4 kg–1 FW h–1;
LSD = 2.8 at P < 0.05) than in cork-spotted, bitter-pitted,
or brown-cored fruit, and increased with the duration of
storage in both disorder-free and symptomatic fruit up to
month-5, then declined in month-6 (Figure 1B).
Likewise, the rates of respiration were significantly lower
in disorder-free fruit (16.0 ml CO2 kg–1 FW h–1; LSD = 1.8
at P < 0.05) than in cork-spotted, bitter-pitted or brown-
cored fruit, and increased with the duration of storage in
both disorder-free and symptomatic fruit, up to month-5,
then declined in month-6 (Figure 1C).
Correlations between Ca2+ ion content and the incidence
of storage disorders, between LOX activity and storage
disorders, and between Ca2+content and LOX activity
Correlations (R2) between Ca2+ ion contents and
physiological disorders such as bitter pit (–0.77), cork
spot (-0.75), and brown core (–0.64), and between Ca2+
Downloaded by [McMaster University] at 22:40 28 January 2016
ion contents and LOX activity (–0.94) were strongly
negative (Figure 2A–D). Furthermore, the correlations
(R2) between LOX activity and physiological disorders
such as bitter pit (+0.87), cork spot (+0.77), and brown
core (+0.59) were strongly positive (Figure 2E–G).
However, the correlations (R2) between the rates of
ethylene production or respiration and the incidence of
physiological disorders were non-significant (data not
shown).
DISCUSSION
Symptoms of various physiological disorders such as
bitter pit, cork spot, and brown core in ‘Royal Delicious’
apples did not become evident until month-3 of cold
storage, and increased progressively thereafter,
indicating that such disorders can cause significant losses
during storage. It is difficult to market apples that are
affected by bitter pit or cork spot because the symptoms
of these disorders appear on the peel, whereas brown-
cored apples may be sold because the affected portion is
internal and may not be visible to the consumer. A
substantial proportion of the annual ‘Royal Delicious’
apple crop is lost due to these disorders, which usually
occur during storage. Although, it is difficult to explain
why these disorders appeared only after a certain period
of storage (month-3), Ca2+ ion contents may have
dropped below a critical limit during storage, which
might have resulted in the development of these
disorders (Chadha and Awasthi, 2005). In a similar study,
Winska-Krysiak and Lata (2010) reported that Ca2+ ion
contents at fruit harvest were higher (571 – 705 µg g–1
DW) in bitter pit-resistant cultivars (‘Gloster’, ‘Gala’)
than in bitter pit-susceptible cultivars (‘Ligol’, ‘Mutsu’)
and, after prolonged storage (4 months), there was a
decline in fruit Ca2+ ion contents in some cultivars
(‘Idared’, ‘Gala’, ‘Sampion’, ‘Mutsu’). They also reported
that Ca2+ ion contents in ‘Sampion’, ‘Ligol’, ‘Mutsu’ and
‘Cortland’ fruit with bitter pit were reduced to 38%
compared with fruit of the same cultivars without bitter
pit. Duranni et al. (2010) also observed a reduction in
Ca2+ ion contents from 9.76 to 3.62 µg g–1 DW (by approx.
63%) during storage of apples for 3 months. Similarly,
Oluwaliana et al. (2006) reported a decline in fruit Ca2+
contents in plantains during storage under ambient
tropical conditions. Ca2+ ions are highly immobile in
fruit, yet their decline in fruit flesh during storage may be
due to a redistribution or movement towards the fruit
core (Perring, 1984; Perring and Pearson, 1986; 1987). In
addition, the fruit may have undergone some senescence
during storage, which might have increased LOX activity
and the rates of respiration and ethylene production,
thereby increasing the symptoms of these disorders. A
progressive loss in fruit Ca2+ content during storage also
resulted in fruit softening and senescence (Stow, 1993),
which may have indirectly increased LOX activity. Apple
fruit softening results from a loss of Ca2+ due to ionic
linkages between pectin molecules in the middle-lamella
which cause a loss of Ca2+ (Stow, 1993). Ortiz et al. (2011)
reported that a pre-harvest application of Ca2+ preserved
the middle lamellae of cell walls, improved cell-to-cell
adhesion, and partially suppressed the activities of those
enzymes involved in senescence.
The variable occurrence of these physiological
disorders can be correlated with fruit Ca2+ ion contents
and LOX activities in disorder-free and symptomatic
fruit. Our study indicated that fruit Ca2+ ion contents
were notably higher in disorder-free fruit, which had a
lower average LOX activity than fruit with storage
disorders, suggesting that disorder-affected fruit were
more senescent that disorder-free (healthy) fruit at
different storage times. Ca2+ ions have been considered
to be a major factor in preventing various physiological
disorders in different fruit (Faust and Shear, 1972;
Bangerth, 1979; Sharma and Singh, 2009), and also as
retardants of fruit ripening and senescence (Ferguson,
1984). Ca2+ ion levels could influence the permeability of
membranes and modify the activities of membrane-
bound enzymes such as LOX (Leshem et al., 1982)
which are involved in the development of several
physiological disorders in apple (Feys et al., 1980). Thus,
higher LOX activities in symptomatic apple fruit are
indicative of such apples being more senescent than
disorder-free apples. For example, strawberries
exhibiting albinism, malformation, or buttoning
disorders were more senescent than disorder-free
berries (Sharma and Singh, 2008). Winska-Krysiak and
Lata (2010) demonstrated a negative correlation
between Ca2+ ion levels and LOX activities, and
between Ca2+ ion contents and the incidence of bitter pit
in apple. They reported that bitter pit was accompanied
by a 40% increase in LOX activity. Apple cultivars with
higher LOX activities (e.g., ‘Mutsu’, ‘Cortland’, and
‘Sampion’) had a lower incidence of bitter pit, and vice
versa. There are no reports in the literature to support
our findings on the relationships between Ca2+ ion
contents and cork spot or brown core. Similarly, a
decrease in Ca2+ content and an increase in LOX activity
in fruit during storage indicated that the apples became
more senescent during storage, as LOX catalyses the
hydroperoxidation of polyunsaturated fatty acids
(Leshem et al., 1982; Perez et al., 1999; Sharma and
Singh, 2008). Such unsaturated fatty acids occur in high
concentrations in apple fruit at harvest, then decrease
during storage due to increased activity of LOX in
stored fruit. Thus, fruit Ca2+ ion contents and LOX
activities showed strong negative correlations with the
 R. R. SHARMA, R. K. PAL, D. SINGH, J. SINGH, M. R. DHIMAN and M. R. RANA 371
Downloaded by [McMaster University] at 22:40 28 January 2016

FIG. 2
Correlations between apple fruit Ca2+ ion content and bitter pit (Panel
A), Ca ion content and cork spot (Panel B), Ca2+ ion content and
2+

brown core (Panel C), Ca2+ ion content and LOX activity (Panel D),
bitter pit and LOX activity (Panel E), cork spot and LOX activity (Panel
F), and brown core and LOX activity (Panel G) during storage of ‘Royal
Delicious’ apple fruit at 0º ± 1ºC and 90 – 95% RH for 6 months. Each
datum point is the mean of 20 fruit samples with three replications
(n = 60), recorded at monthly intervals.
 372 Calcium content and lipoxygenase activity in apple fruit
occurrence of bitter pit, cork spot, and brown core in
‘Royal Delicious’ apple fruit (Figure 2A –D).
Several reports exist on bitter pit and cork spot and
their relationship with Ca2+ ion contents, but limited
information is available on the relationship between Ca2+
contents and brown core. It is believed that the
development of brown core is triggered not only by low
Ca2+ ion levels in fruit, but also by low storage
temperatures (Meheriuk et al., 1994). Our study also
revealed brown core to be a physiological disorder, as we
observed a relationship with Ca2+ ion content (R2 = –0.64),
although not as strong a correlation as for bitter pit or
cork spot.
At the end of the experiment, several pathogens
invaded the fruit, primarily through the calyx end, and
entered the fruit core, resulting in the development of
brown core rot disease. Thus, higher Ca2+ ion contents
may minimise the occurrence of all storage disorders by
strengthening fruit structures and maintaining sub-
cellular compartmentalisation, thereby delaying the
enzyme-mediated reactions required for expression of
Downloaded by [McMaster University] at 22:40 28 January 2016
the symptoms of the different disorders (Stow, 1993;
Winska-Krysiak and Lata, 2010; Ortiz et al., 2011).
De Freitas et al. (2010) showed increased de-
esterification of pectin, therefore more Ca2+-binding
sites, in the cell wall, and a higher fraction of total cortical
tissue Ca2+ bound to the cell walls in pitted fruit
compared with non-pitted fruit. Cells of the outer
cortical tissue of pitted fruit had consistently higher
membrane permeability than the outer cortical cells of
REFERENCES
AXELROD, B., CHEESBROUGH, T. M. and LEAKSO, S. (1981).
Lipoxygenase from soybeans. Methods in Enzymology, 7,
443–451.
BANGERTH, F. (1979). Calcium related physiological disorders of
plants. Annual Review of Phytopathology, 17, 97–122.
CHADHA, K. L. and AWASTHI, R. P. (2005). The Apple. Malhotra
Publishing House, New Delhi, India. 23–42.
DE FREITAS, S. T., DO AMARANTE, C. V., LABAVITCH, J. M. and
MITCHAM, E. J. (2010). Cellular approach to understand bitter
pit development in apple fruit. Postharvest Biology and
Technology, 57, 6–13.
DURRANI, Y., AYUB, M., ALI, M. and ALI, A. (2010). Physicochemical
response of apple pulp to chemical preservatives and antioxi-
dant during storage. Internet Journal of Food Safety, 12, 20–28.
FAUST, M. and SHEAR, C. B. (1972). The effect of calcium on respi-
ration of apples. Journal of the American Society for
Horticultural Science, 97, 437–439.
FERGUSON, I. (1984). Calcium in plant senescence and fruit ripen-
ing. Plant Cell and Environment, 7, 477–489.
FEYS, M., NAESENS, W., TOBBACK, P. and MAES, E. (1980).
Lipoxygenase in apples in relation to storage and physiological
disorders. Phytochemistry, 19, 1009–1011.
LESHEM, Y. Y., WURZBURGER, Y., FRIMER, A. A., BARNESS, G. and
FERGUSON, I. B. (1982). Calcium and calmodulin metabolism in
senescence: inter-relation of lipoxygenase and superoxide dis-
mutase with ethylene and cytokinin. In: Plant Growth
Substances. (Waring, P.F., Ed.). Academic Press, New York, NY,
USA. 569–578.
LIETEN, F. and MARCELLE, R. D. (1993). Relationships between fruit
mineral content and the ‘albinism’ disorder in strawberry.
Annals of Applied Biology, 123, 433–439.
non-pitted fruit. Thus, Ca2+ ion accumulation in storage
organelles (e.g., vacuoles) and Ca2+ ion binding to cell
walls are important contributors to the development of
bitter pit in apple fruit (De Freitas et al., 2010).
Increased rates of ethylene production and respiration
indicate the onset of the climacteric stage and the
beginning of fruit senescence. In our study, the rates of
ethylene production and respiration were lower in
disorder-free fruit than in disorder-affected fruit. This
may have been in response to tissue damage (Feys et al.,
1980). Between month-5 and month-6, the rates of
respiration and ethylene production began to decline,
indicating that all biological activities had started
declining. However, the rates of ethylene production and
respiration had no significant correlation with the
occurrence of the storage disorders, suggesting that,
although senescent apples produced ethylene and/or
respired at a higher rate, this did not have any
relationship with the occurrence of storage disorders in
‘Royal Delicious’ apple fruit (Marcelle, 1989).
Our studies revealed negative relationships between
fruit Ca2+ ion contents and the occurrence of storage
disorders such as bitter pit, cork spot, and brown core,
and between Ca2+ ion contents and LOX activity. Thus,
technologies that maintain Ca2+ ion levels in apple fruit,
either by pre-harvest spraying with calcium chloride or
calcium nitrate, or post-harvest dips, may be useful for
suppressing LOX activity and preventing the occurrence
of a variety of physiological disorders, thereby reducing
the severe post-harvest losses caused by these disorders.
MARCELLE, R. D. (1989). Ethylene formation, lipoxygenase activity
and calcium content in apple cv. Jonagold. Acta Horticulturae,
258, 61–68.
MEHERIUK, M., PRANGE, R. K., LIDSTER, P. A. and PORITT, S. W.
(1994). Post-harvest Disorders of Apples and Pears. Agriculture
Canada Publication 1737/E. Communications Branch,
Agriculture Canada, Ottawa, Ontario, Canada. 1–67.
OLUWALIANA, L. B., OLUWAMUKOM, M. O. and OLAJIDE, S. T. (2006).
Physicochemical changes in ripening plantain stored at tropical
ambient conditions. Journal of Food Technology, 4, 253–254.
ORTIZ, A., GRAELL, J. and LARA, I. (2011). Preharvest calcium appli-
cations inhibit some cell wall-modifying enzymes activities and
delay cell wall disassembly at commercial harvest of ‘Fuji Kiku-
8’ apples. Postharvest Biology and Technology, 62, 161–167.
PAL, R. K. (1998). Influence of ethrel and calcium chloride on eth-
ylene evolution, respiration rate, electrolyte leakage and firm-
ness of ‘Dashehari’ mango (Mangifera indica L.). Indian
Journal of Agricultural Sciences, 68, 201–203.
PANSE, V. G. and SUKHATME, P. V. (1984). Statistical Methods for
Agricultural Workers. 3rd Edition. Indian Council of
Agricultural Research, New Delhi, India. 312–324.
PEREZ, A. G. M., SANZ, C., OLIAS, R. and OLIAS, J. M. (1999).
Lipoxygenase and hyperoxide lyase activities in ripening straw-
berry fruit. Journal of Agricultural and Food Chemistry, 47,
249–253.
PERKINS-VEAZIE, P. (1995). Growth and ripening of strawberry
fruit. Horticultural Reviews, 17, 267–297.
PERRING, M. E. (1984). Redistribution of minerals in apple fruit
during storage, preliminary investigations with the variety
Spartan. Journal of the Science of Food and Agriculture, 35,
182–190.
 R. R. SHARMA, R. K. PAL, D. SINGH, J. SINGH, M. R. DHIMAN and M. R. RANA
PERRING, M. E. and PEARSON, K. (1986). Incidence of bitter pit in
relation to calcium content of apple: calcium distribution in the
fruit. Journal of the Science of Food and Agriculture, 37, 709–718.
PERRING, M. E. and PEARSON, K. (1987). Redistribution of minerals
in apple fruit during storage: the effect of storage atmosphere
on calcium concentration. Journal of the Science of Food and
Agriculture, 40, 37–42.
POOVAIAH, B. W. (1985). Role of calcium and calmodulin in plant
growth and development. HortScience, 20, 347–352.
POOVAIAH, B. W. (1988). Molecular and cellular aspects of calcium
action in plants. HortScience, 23, 267–271.
SHARMA, R. R. and SINGH, R. (2008). Fruit nutrient content and
LOX activity in relation to the production of malformed and
button berries in strawberry (Fragaria
ananassa Duch.).
Scientia Horticulturae, 119, 28-31.
Downloaded by [McMaster University] at 22:40 28 January 2016
373
SHARMA, R. R. and SINGH, R. (2009). The fruit pitting disorder – a
physiological anomaly in mango (Mangifera indica L.) due to
deficiency of calcium and boron. Scientia Horticulturae, 119,
388–391.
SHEAR, C. B. (1975). Calcium related disorders in fruit and vegeta-
bles. HortScience, 10, 361-365.
STOW, J. (1993). Effect of calcium ions on apple fruit softening
during storage and ripening. Postharvest Biology and
Technology, 3, 1-9.
WARDALE, D. A. and LAMBERT, E. A. (1980). Lipoxygenase from
cucumber fruit: localization and properties. Phytochemistry, 19,
1013-1016.
WINSKA-KRYSIAK, M. and LATA, B. (2010). Influence of lipoxyge-
nase activity and calcium contents on bitter pit occurrence in
commercial apple cultivars. Folia Horticulturae, 22, 13-17.