全 文 ：Received 26 Apr. 2004 Accepted 12 Oct. 2004
Supported by the National Natural Science Foundation of China (30371002) and Department of Science and Technology of Zhejiang
Province (021102531).
* Author for correspondence. Tel: +86 (0)13957963988; Fax: +86 (0)571 86091584; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (12): 1402-1407
1-Methylcyclopropene and CaCl2 Treatments Affect Lipolytic Enzymes
in Fresh-cut Watermelon Fruit
MAO Lin-Chun1*, QUE Fei1, Huber J DONALD2
(1. College of Biosystem Engineering and Food Science, Zhejiang University, Hangzhou 310029, China;
2. Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, USA)
Abstract: Having been held in 10 mL/L 1-methylcyclopropene (1-MCP) or air for 18 h, seedless water-
melon (Citrullus lanatus Thunb. Mansfeld, cv. Millionaire) fruit was cut to obtain pericarp cylinders (7 mm
in diameter, 40 mm thick), which were rinsed with 2% CaCl2 or deionized water and then stored at 10 ℃.
Tissue firmness, electrolyte leakage and activities of phospholipase C (PLC), phospholipase D (PLD) and
lipoxygenase (LOX) were determined. Results suggested that 2% CaCl2 stimulated activities of phospholi-
pase C (PLC), phospholipase C (PLD) and lipoxygenase (LOX), but maintained tissue firmness throughout
storage. CaCl2 alone may not be sufficient to maintain quality of fresh-cut watermelon and even would
exert negative effects for stimulating lipolytic enzymes. 1-MCP counteracted CaCl2 in regulation of PLC,
PLD and LOX. Combination of 1-MCP and CaCl2 retarded the ripening process, as illustrated by higher
firmness and lower activities of lipolytic enzymes in relative to the control.
Key words: calcium chloride; 1-methylcyclopropene (1-MCP); watermelon; phospholipase; lipoxygenase
Currently, cut watermelon accounts for about 10% of all
watermelon sales in USA (Perkins-Veazie and Collins, 2004).
However this kind of produce may deteriorate rapidly be-
cause of the physical damage caused by cutting, slicing,
peeling, chopping, shredding and other mechanical inju-
ries during processing (Watada et al., 1990). Among the
most detrimental problems in storage of fresh-cut products
are degradative enzyme reactions and microbial spoilage
(King and Bolin, 1989).
Disruption of membrane integrity has been hypothesized
to be a major contributing factor to the senescence and
stress injuries of plant tissues. One of the most characteris-
tic features in membrane deterioration is a progressive de-
cline of phospholipid levels with a relative enrichment of
free fatty acids and sterols in the membranes (Paliyath and
Droillard, 1992). Previous studies have shown that the deg-
radation of membrane lipids is achieved by the concerted
activities of a variety of membranous lipolytic enzymes such
as phospholipase D (PLD), phospholipase C (PLC), and
lipoxygenase (LOX) (Paliyath and Droillard, 1992). Activi-
ties of these phospholipases generate phosphatidyl acid
(PA) along with derivatives such as diacylglycerol and
lysoPA, which may serve as intracellular and extracellular
signaling messengers (English, 1996). Phospholipases have
been suggested to be part of the signal transduction cas-
cades in wounding (Ryu and Wang, 1996).
The aim of the present study is to examine the effects of
1-methylcyclopropene (1-MCP) application prior to cutting
and CaCl2 rinse following cutting on lipolytical and physi-
ological responses in fresh-cut seedless “Millionaire” wa-
termelon fruit during storage at 10 ℃. We provide the first
direct evidence that in fresh-cut watermelon tissue, 1-MCP
may regulate CaCl2 antagonistically in regulating PLC, PLD
and LOX activities.
1 Materials and Methods
1.1 Sample preparations
Seedless watermelon (Citrullus lanatus Thunb.
Mansfeld, cv. Millionaire) fruit at the commercial maturity
based on appearance was purchased from a local market.
Fruit was held for 18 h at 10 µL/L 1-MCP (EthyBloc®,
BioTechnologies for Horticulture, IL, USA) or air, then two
rings of 40-mm thickness were cut transversely from the
equatorial region of the fruit, discarding both calyx and
stalk ends. Cylinders of inner pericarp tissue were then
obtained from these rings using a 7-mm diameter cork borer.
For each of the triplicates, tissue cylinders corresponding
to all four treatments were obtained from the same fruit.
Cylinders from air-hold fruit were divided into two
groups. One group was rinsed with autoclaved deionized
water for 2 s (control), while another group was rinsed with
autoclaved 2% CaCl2 for 2 s (CaCl2). Cylinders from 1-MCP-
MAO Lin-Chun et al.: 1-Methylcyclopropene and CaCl2 Treatments Affect Lipolytic Enzymes in Fresh-cut Watermelon Fruit 1403
treated fruit were also rinsed with deionized water (1-MCP)
or 2% CaCl2 (1-MCP/CaCl2). Rinsed cylinders were drained
and then stored in labeled plastic boxes corresponding to
each treatment and storage time. Each plastic box has two
small holes for air ventilation. Three replicates were used in
this experiment.
Fruit, storage (processing) room, knife, cork borer, cut-
ting board, plastic boxes were washed with soap and water
and rinsed with 200 µL/L sodium hypochlorite solution prior
to use. Knife, cork borer and cutting board were rinsed and
then dried again when processing different fruit to prevent
a possible cross contamination. Cutting and cylinder stor-
age were operated at 10 ℃.
1.2 Firmness and electrolyte leakage
Firmness was determined with an Instron texture ana-
lyzer (Model 1132, Instron Corp., Mass.) using a probe of
6.35 mm in diameter and a penetrating depth of 2.5 mm with
a load cell mass of 20 kg. Electrolyte leakage from tissue
cylinders was measured with a Conductance Bridge (model
31, Yellow Springs Instruments Co., Ohio) after 1 h of incu-
bation in deionized water at 25 ℃. Total electrolyte content
was measured after boiling the sample for 30 min following
freeze, and was taken as 100% leakage.
1.3 PLC and PLD assay
PLC and PLD activities were determined as described
by Karakurt and Huber (2003). Briefly, pericarp tissue (10 g)
was homogenized in 10 mL of extraction buffer containing
50 mmol/L Tris-HCl (pH 8.0), 0.5 mol/L sucrose, 10 mmol/L
KCl, 1 mmol/L EDTA, 0.5 mmol/L PMSF and 2 mmol/L DTT.
The homogenate was centrifuged at 15 000g for 30 min, and
the supernatant was used for enzyme assay. PLC and PLD
activities were determined spectrophotometrically, using r-
nitrophenylphosphorylcholine (NPPC) as substrate, and
expressed as mmol r-nitrophenol·100 mg-1 protein·h-1.
1.4 LOX assay
Each 10 g of tissue was ground with mortar and pestle in
liquid nitrogen to a fine powder, further ground in 10 mL of
50 mmol/L Tris-HCl (pH 8.0) containing 10 mmol/L KCl, 500
mmol/L sucrose, and 0.5 mmol/L PMSF. Homogenates were
centrifuged at 15 000g for 30 min for LOX extraction. LOX
activity was measured spectrophotometrically by monitor-
ing the change in absorbance at 234 nm over 3 min. The
standard assay mixture contained 200 mL of Tween 20 and
40 mL of linoleic acid in 40 mL of 0.1 mol/L phosphate buffer
(pH 7.0). To 500 mL of 0.1 mol/L phosphate buffer (pH 7.0) in
a cuvette were added 500 mL of assay mixture and 200 mL of
LOX extract. One unit of LOX is defined as the amount of
enzyme which causes an increase in absorption at 234 nm
of 0.001 per min (3 min period) at 25 ℃.
2 Results
2.1 Changes in firmness and electrolyte leakage
Application of calcium (Ca) retarded firmness loss
in the watermelon cylinders during storage (Fig.1A).
Ca application with or without 1-MCP pre-treatment
maintained high firmness, which changed little through-
out the entire storage period. When CaCl2 was absent,
a rapid loss of firmness occurred after 4 d of storage.
Whereas, difference between the control and 1-MCP
treatment was minimal. This was also true between
CaCl2 and the combination of CaCl2 with 1-MCP. Re-
sults proved that CaCl2 provided firming effect, retard-
ing tissue softening.
The rapid increase of electrolyte leakage in fresh-cut
cylinders was found when CaCl2 was applied. However,
there was no noticeable change during storage (Fig.1B). Ca
treated samples had higher electrolyte leakages than those
without Ca application. Higher electrolyte leakages in Ca
treated cylinders could be the increased concentration in
Ca2+ resulted from the application of CaCl2. There were no
Fig.1. Firmness (A) and electrolyte leakage (B) in fresh-cut
wa t e r me lo n . Fo u r t r e a tmen t s (○ c o n t r o l , △ 1 -
methylcyclopropene (1-MCP), ■ CaCl2, ▲ 1-MCP/ CaCl2) are
described in Materials and Methods. Firmness was measured as
the force required to penetrate the tissue in six cylinders each treat-
ment to a depth of 2.5 mm. Electrolyte leakage was measured as
relative conductivity in the bath solution incubated with cylinders
for 1 h. Each data point is the average of three determinations ± SE.
Acta Botanica Sinica 植物学报 Vol.46 No.12 20041404
significant differences in electrolyte leakage between
1-MCP and control samples, indicating that 1-MCP had no
effect on electrolyte leakage.
2.2 Activities of PLC, PLD and LOX
Phospholipases including PLC, PLD and LOX were de-
termined to evaluate the effects of 1-MCP and Ca chloride
through the demonstration of membrane lipid metabolism.
Specific activities of PLC in CaCl2 treatment increased up
to much higher levels than other three treatments after 4 d
of storage (Fig.2A). Changes of PLC activities in control,
1-MCP and 1-MCP/CaCl2 treatments exhibited a similar
pattern, and indicated as a gradual decrease from the sec-
ond day after cutting to the end of 7-d storage period. There
were no noticeable differences observed among these three
treatments. By contrast, CaCl2 treatment alone retained
higher PLC activities during the late period. There was no
noticeable effect of 1-MCP on PLC.
Changes in PLD activities (Fig.2B) followed a similar
pattern as observed for PLC. Again, CaCl2 treatment re-
tained higher PLD activities than other treatments during
the most of storage period. PLD activity in the control in-
creased to a higher level than CaCl2 treatment at the sec-
ond day, then it decreased rapidly to a lower level. Whereas,
the combination of 1-MCP and CaCl2 did not show any
effect on this enzyme compared to the control.
LOX activities in fresh-cut watermelon tissues increased
rapidly within the early 2 or 4 d, and then remained at
elevated levels for CaCl2 treatment or decreased for other
three treatments (Fig.2C). Results indicated again that CaCl2
alone showed a significant effect to maintain high levels of
LOX activities as observed for PLC and PLD. Furthermore,
as observed for PLC and PLD, LOX activities in 1-MCP/
CaCl2 treatment were much lower than those in CaCl2
treatment. These results indicated that activation of PLC,
PLD and LOX by CaCl2 was greatly suppressed when
1-MCP was pre-applied. 1-MCP counteracted completely
or partially CaCl2 action with respect to the activities of
PLC, PLD and LOX.
3 Discussion
3.1 Roles of CaCl2 in fresh-cut watermelon
The firming effect of Ca chloride is confirmed in this
study (Fig.1A), which is consistent with previous reports
on numerous fresh-cut products including cantaloupe slices
(Luna-Guzmán et al., 1999; Luna-Guzmán and Barrett, 2000),
zucchini slices (Izumi and Watada, 1995), shredded carrot
(Izumi and Watada, 1994), diced tomatoes (Floros et al.,
1992), sliced strawberries and pears (Morris et al., 1985;
Rosen and Kader, 1989), kiwifruit slices (Agar et al., 1999).
This firming effect has been attributed to the crosslinking
between the carboxyl groups of adjacent polyuronide chains
and divalent Ca ions in cell wall (Morris, 1980; King and
Bolin, 1989) imparting improvement of structural integrity
and cell adhesion (Poovaiah, 1986; 1988). Relations between
Fig.2. Phospholiase C (A), phospholiase D (B), and lipoxygenase
(C) activities in fresh-cut watermelon. Four treatments (○ control, △
1-methylcyclopropene (1-MCP), ■ CaCl2, ▲ 1-MCP/ CaCl2) are
described in Materials and Methods. Phospholipase C (PLC) and
phospholipase D (PLD) were extracted from the supernatant of cylin-
ders after 15 000g centrifugation for 30 min. Activities were repre-
sented as mmol of r-ni trophenol released from r-
nitrophenylphosphorylcholine (NPPC). Lipoxygenase (LOX) activ-
ity was measured spectrophotometrically by monitoring the change in
absorbance at 234 nm in reaction mixture with linoleic acid as substrate.
Each data point is the average of three determinations ± SE.
MAO Lin-Chun et al.: 1-Methylcyclopropene and CaCl2 Treatments Affect Lipolytic Enzymes in Fresh-cut Watermelon Fruit 1405
greater turgor pressure and firmness in Ca treated tissues
were also found (Shacke et al., 1991; Mignani et al., 1995;
Picchioni et al., 1998).
Treatment with Ca results in higher electrolyte leakage
(Fig.1B), which could not be indicated as poorer membrane
integrity since tissue firmness was much higher for this
treatment throughout storage (Fig.1A). As previously ob-
served in tomatoes, soluble Ca and bound Ca levels in-
creased by 1.83 and 0.64 times, respectively, after treatment
with 1.5% CaCl2 (Njoroge et al., 1998). This would be the
same trend in watermelon cylinders when 2% CaCl2 was
applied in this experiment. More rapid increase in soluble
(free) Ca compared to bound Ca could interpret the higher
electrolyte leakage in CaCl2 treated watermelon tissues,
because the electrolyte leakage indicated as the percent-
age of leaked electrolytes (mainly soluble ions) to total
electrolytes.
In this study, hydrolysis activities of phospholipids
appeared to be increased by 2% CaCl2. Ca interferes with
enzymes probably through several mechanisms. Ca can
either directly bind to membrane, changing membrane prop-
erties and associated enzymes, or indirectly regulate nu-
merous enzymes through interaction with calmodulin. CaCl2
alone is found to retain or activate PLC, PLD, and LOX in
watermelon cylinders, as observed by Paliyath et al. (1987),
indicating that phospholipid catabolism is positively regu-
lated by Ca. These influences of CaCl2 could be concentra-
tion-dependent. Ca application may delay or even hasten,
depending on Ca2+ concentration used, senescence possi-
bly by affecting membrane lipid degradation and cell os-
motic tonicity. This postulation could be also an explana-
tion for the observations by Tsantili et al. (2002) who in-
vestigated series of concentrations of CaCl2 and found
0.09 mol/L was optimal on preventing both the softening
and yellowing in “Maglino” lemons. Based on the levels of
phospholipid intermediates, PLD and LOX were inhibited
by 0.05 mol/L CaCl2 treatment, but stimulated by 0.25 mol/L
CaCl2 treatment (Chéour et al., 1992).
CaCl2 treatment, along with previous reports, seems to
be a promising method to retard firmness loss. However, to
obtain the optimum effect of Ca and to apply it commercially,
the appropriate concentrations and treatment conditions
need further investigation in fresh-cut watermelon.
3.2 Responses of PLC, PLD and LOX to CaCl2
In 2% CaCl2 treatment, activation of LOX activity, sug-
gestive of lipid peroxidation, occurred simultaneously with
the activation of PLC and PLD, suggesting that the ob-
served phospholipid peroxidation pathway is part of the
phospholipid metabolic strategies by which cells perceive
the increase of Ca. This modulation is complex, and several
mechanisms could be proposed, including Ca2+-mediated
intracellular translocation from the cytoplasm to the target
membranes and increased membrane association (Ryu and
Wang, 1996), redistribution in plasma membrane (Young et
al., 1996), alteration of isoforms (Dyer et al., 1994; Ryu and
Wang, 1995; 1996), and G-protein activation (Munnik et al.,
1995).
High activities of PLC, PLD and LOX may be a specific
response to salt stress. Ca is likely to be involved through
stress-induced entry into the cytosol. High activity of the
lipolytic enzymes will enhance the perturbation of the plas-
malemma structure, which will in turn allow entry of more
Ca, and membrane lipid degradation will become autocata-
lytic (Chéour et al., 1992). Results suggest that CaCl2 con-
centration at 2% may be too high in aspect of phospholipid
metabolism. Further investigations on measuring kinetic
concentrations of Ca2+ in relation to applied concentra-
tions of CaCl2 and metabolic activities associated with qual-
ity could be of paramount importance to extend shelf life of
fresh-cut watermelon with CaCl2 application.
3.3 Interaction between CaCl2 and 1-MCP
1-MCP is a non-toxic compound that inhibits ethylene
perception by irreversibly binding to and hence inactivat-
ing the ethylene binding protein (EBP) (Sisler et al., 1996).
Little information on the use of 1-MCP prior to cutting of
fruit or vegetables is available (Mathooko et al., 2001; Jiang
and Joyce, 2002; Wills et al., 2002). Exposure of intact or
fresh-cut apple fruit to 1-MCP resulted in reduced respira-
tion and ethylene production rates and delayed softening
and color changes (Jiang and Joyce, 2002). In this study,
1-MCP is applied before cutting in expectations to inhibit
wound-induced ethylene biosynthesis and action. This
attempt seems partially to be realized because Ca-retention
of PLC, PLD and LOX are reduced or inhibited by 1-MCP
(Fig.2), although ethylene was not detected in fresh-cut
cylinders of different treatments.
Disappearance of CaCl2-activation of PLC, PLD and LOX
when 1-MCP was pre-applied is the interesting findings in
this study. Analysis does not clearly show any marked
activation or inhibition of PLC, PLD and LOX by 1-MCP
alone or 1-MCP/CaCl2 combination compared to the control.
To our knowledge this is the first report indicating the coun-
teraction of 1-MCP on CaCl2 in regulating lipolytic enzymes.
It is important that the signaling components in a given
cascade are present at all times so that the cell is competent
to respond to external stimuli. Because of this requirement,
it may be advantageous for certain signaling pathways to
posses a feedback mechanism to either enhance or
Acta Botanica Sinica 植物学报 Vol.46 No.12 20041406
desensitize the response of cells subjected to the constant
presence of the same stimulus (Yohiharu et al., 1998). It is
hypothesized here that 1-MCP may repress the pathways
of Ca2+ signaling mechanism through yet unknown mecha-
nisms probably involving inhibiting the Ca2+ binding
protein, calmodulin (Zielinsky, 1998), and thus effectively
shut down the Ca2+-activation systems of lipolytic enzymes.
It remained to be determined whether this antagonistic ef-
fect on Ca2+ is via interaction with calmodulin leading to
inactivation of calmodulin or inhibiting the formation of
functional complex Ca-calmodulin.
Taken together our data demonstrate that 1-MCP/CaCl2
combination retards ripening process in fresh-cut
watermelon, as illustrated by higher firmness and lower
activities of lipolytic enzymes in relative to the control. CaCl2
alone may not be sufficient to maintain quality of fresh-cut
products and even would exert negative effects such as
stimulating lipolytic enzymes. Even though no sensory
evaluation was performed, the visual quality (tissue
integrity, watery appearance, and microbial growth) of fresh-
cut watermelon with 1-MCP/CaCl2 treatment appeared to
decline at a slower rate than other samples during storage.
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