Abstract: Lisbon lemons [Citrus limon (L.) Brum.] harvested early in the season (green stage) were treated with hot-water dip (HWD, 53°C, 3 min) or vacuum infiltration with 1.5% CaCl2 (15°C, -33 kPa, 10 min) prior to storage at 1.5°C for up to 8 weeks, to determine the effect of these treatments on the Chilling Injury (CI) and activities of Catalase (CAT; EC 1.11.1.6.), superoxide dismutase (SOD; EC 1.15.1.1.) and Peroxidase (POD; EC 1.11.1.7.), as well as Malondialdehyde (MDA) content. The decrease of CAT and SOD, accompanied with the increase in POD activities during cold storage, might contribute to the development of CI in lemon fruits. HWD and Ca treatments similarly reduced CI and delayed the reduction of CAT, SOD and suppressed the increase in POD activities as well as MDA content compared to the control. CAT activity in fruits exposed to these treatments before cold storage increased and remained higher in the treated fruits than in the non-treated fruits over the storage period. The data indicate that CAT may be a major antioxidant enzyme involved in the defense of lemon fruits against chilling stress. Lemons harvested at the late maturity (yellow) stage had significant higher CAT, SOD and lower POD activities compared to the early maturity (green) stage. Collectively, results indicate that the chilling-tolerant property of lemon fruits may be related to the higher ability of fruits to have or maintain high CAT, SOD and low POD activities during low temperature storage.
INTRODUCTION
Many citrus cultivars, particularly lemons, are sensitive to chilling injury (CI) and may develop peel injury when exposed to cold treatment, which greatly reduces the fruit marketability (Houck et al., 1990). The beneficial effect of prestorage hot water treatments in increasing chilling tolerance has been reported in different crops (Fallik, 2004). Treatment of lemon fruits with hot water at 53°C for 3 min was found to be effective in alleviating CI after 8 week storage at 1-2°C (Rodov et al., 1995; McLauchlan et al., 1997). It was also known that calcium plays a role in the maintenance of cell stability, particularly membranes, under stress conditions such as postharvest low temperature storage (Roux and Slocum, 1982). Pre-and postharvest calcium applications have been reported to lower the rate of plant senescence, fruit ripening, sensitivity to pathogen and susceptibility to CI in different fruit and vegetables by reducing or delaying cell wall breakdown, maintaining the membrane function and prolonging the capacity for signal transduction (Poovaiah, 1986; Ferguson and Drobak, 1988). In a different trial with Fortune mandarin, calcium reduced the level of chilling injury of peel dermatosis as a field treatment or as a postharvest dipping (El-hilali et al., 2003; DAquino et al., 2005). Our previous study showed that vacuum infiltration with 1.5% CaCl2 for 10 min effectively reduced the incidence of CI in Lisbon lemon fruit following storage at 1.5°C (Safizadeh and Rahemi, 2006).
Oxidative damage is considered to be an early response of sensitive tissues to chilling (Karpinski et al., 2002). Superoxide anion, hydrogen peroxide and hydroxyl radical are Reactive Oxygen Species (ROS) that can result in oxidization of proteins, unsaturated fatty acids and DNA, causing cellular damage and eventually cell death (Mittler, 2002). Several enzymes are involved in the production and scavenging of free radicals in plant systems. Catalase (CAT; EC 1.11.1.6.) can enhance the removal of H2O2 without producing dangerously-active free radicals. Peroxidase (POD; EC 1.11.1.7.) also catalyzes the decomposition of H2O2, but the mode of action of peroxidase differs from catalase action in that peroxidase liberates free radicals instead of oxygen. These free radicals are highly phytotoxic (Burris, 1960). The damaging effects of superoxide can be modulated if the radicals are scavenged by reacting with superoxide dismutase (SOD; EC 1.15.1.1.). SOD catalyzes the dismutation of the superoxide free radical to H2O2 and O2, removes singlet oxygen and prevents formation of OH-. The combined action of SOD and CAT converts the potentially dangerous superoxide radical and hydrogen peroxide to molecular oxygen and water, thus averting cellular damage (Van Camp et al., 1994). The active oxygen detoxifying enzymes have been shown to participate in the beneficial effect of postharvest heat conditioning treatments protecting Fortune mandarins against chilling (Sala and Lafuente, 2000). However, the effect of postharvest hot-water treatment on ROS detoxifying enzymes in other citrus fruit has not been reported. There is little information available about effects of calcium on cellular protective enzyme activities in citrus fruit.
The aim of this study was to investigate the effect of hot water and calcium treatments on changes in the activities of CAT, POD and SOD in Lisbon lemon fruits during storage at chilling temperature. The activities of these enzymes in lemons harvested at the less maturity (green) stage and harvested 10 weeks later at the advanced maturity (yellow) stage was also compared.
MATERIALS AND METHODS
Plant Material, Storage and Treatments
Lisbon lemon [Citrus limon (L.) Burm. ] were harvested at the green
stage from 20-year-old trees grafted onto sour orange (Citrus aurantium
L.) rootstock and grown at Hajiabad, south of Iran.
Fruits were randomly divided into three lots containing three replicates of 15 fruits to estimate chilling damage and of 5 fruits per storage period to analyze enzyme activity. The first lot was immediately stored at 1.5±0.2°C and 85-90% Relative Humidity (RH) for up to 8 weeks. The second lot was submerged for 3 min at 53±0.1°C in a 12 L recirculating hot water bath with temperature control (Model YCM-04 M, KGIC, Germany). The third lot was treated with calcium at reduced pressure (-33 kPa; Joyce et al., 2001). The 1.5% CaCl2, 2H2O (w/v) solution, containing Tween 20 (0.2%) was applied in a 15 L capacity vacuum desiccator (Model B-1834, Gallenkamp, England) at 15°C and applying a reduced pressure of -33 kPa for 10 min. Following treatment, fruits rinsed by dipping for ≈ 2 sec in distilled water at 15±1°C before drying. After treatments of hot-water dip and vacuum infiltration with CaCl2 all fruits were air-dried and stored as the non- treated fruits.
Three replicates samples of 20 fruits of each lot were sampled after 2, 4, 6 and 8 weeks storage at 1.5°C to determine peel damages and changes in enzyme activities. The colored outer layer of skin (flavedo tissue) was separated from the whole fruit and cut into small pieces and represently 1 g samples were frozen in liquid nitrogen and stored at -70°C for enzyme assays. Frozen flavedo tissues were grounded to fine powder with a microdismembrator (Model 01M, Raz-Gol, Iran) and immediately were used for enzyme extraction and assay. All optical measurements were performed on a spectrophotometer (UV-160A; Schimadzu, Tokyo, Japan).
Chilling Injury (CI) Evaluation
CI was classified as either peel pitting or surface browning, but these
symptoms were combined as a total CI score. CI was rated as 0 (no damage), 1
(slight), 2 (moderate),and 3 (severe). CI index was determined by summing the
product of the number of fruit in each category by the score of each category
and then dividing this sum by the total number of fruits assessed (Sala, 1998).
Extraction and Assay of CAT
CAT was extracted from 1 g flavedo tissue with 10 mL of 100 mM potassium
phosphate buffer, pH 6.8 at 4°C and then centrifuged twice at 27000xg for
15 min at 4°C (Sala and Lafuente, 2000). The supernatant was used to determine
CAT activity by the method of Kar and Mishra (1976). Five mL of the assay mixture
comprised 60 mM of phosphate buffer, pH 6.8, 20 mM of H2O2
and 1 mL enzyme extract. After incubation at 25°C for 1 min, the reaction
was stopped by adding 10 mL of 2% (v/v) H2SO4 and the
residual H2O2 was titrated against 0.01N KMnO4
until a final purple color persisted for at least 15 sec. A control was run
at the same time in which the enzyme activity was stopped at “Zero”
time. The unit of CAT activity was defined as the amount of enzyme which decomposes
1 mM H2O2 per minute at 25°C.
Extraction and Assay of POD
The flavedo tissue (1 g) was homogenized in 10 mL 0.2 M phosphate buffer,
pH 5.8, containing 0.1 mM ethylenediamine tetraacetic acid (EDTA) and 1% polyvinyl
polypyrrolidone (PVPP) at 4°C. The homogenate was centrifuged at 27000xg
for 15 min at 4°C twice and the supernatant was used to determine POD activity
by the method of Siegel and Galston (1967). POD activity was determined by the
oxidation of guaiacol in the presence of H2O2 (extinction
coefficient, 26.6 mM-1 cm-1) at 470 nm. The reaction was
carried out at 25°C for 60 sec in a 3 mL reaction mixture containing 0.2
M phosphate buffer, pH 5.8, 5 mM guaiacol, 5 mM H2O2 and
25-75 μL of enzyme extract. One unit of POD activity represents the amount
of enzyme catalyzing the oxidation of 1 mM of guaiacol per minute at 25°C.
Extraction and Assay of SOD
The flavedo tissue (1 g) was homogenized in 10 mL of 50 mM potassium phosphate
buffer, pH 7.8, containing 1.33 mM Diethylenetriamine Pentaacetic Acid (DETAPAC)
at 4°C and then centrifuged twice at 27000xg for 15 min at 4°C (Sala
and Lafuente, 2000). The supernatant was used to determine SOD activity by the
method of Beauchamp and Fridovich (1971). A 3 mL reaction mixture contained
50 mM phosphate buffer(pH 7.8), 13 mM methionine, 75 μM NBT, 2 μM
riboflavin, 100 μM EDTA and 0-100 μL of enzyme extract. Riboflavin
was added last and tubes were shaken and placed in a cylindrical glass water
bath at 25°C. Two circular fluorescent lamps (40-w) were attached on the
outside wall of the water bath and the entire assembly was fitted in a box lined
with aluminium foil. The reaction was started by switching on the light and
it was allowed to run for 10 min, after which the lights were switched off and
the tubes covered with a black cloth. Absorbance by the reaction mixture was
read at 560 nm and the non-irradiated reaction mixture served as blank. The
reaction mixture lacking enzyme developed the most color and this decreased
with increasing volume of extract added. Log A 560 was plotted as
a function of the volume of enzyme extract in the reaction mixture. The volume
of enzyme extract producing 50% inhibition of the reaction was read from the
resultant graph. The unit of SOD activity was defined as that amount of enzyme
which caused 50% inhibition of the initial rate of reaction in the absence of
enzyme.
Specific activities of all enzymes were expressed as units per mg protein. Protein was determined by the method of Bradford (1976), using bovine serum albumin (BSA) as a standard.
Lipid Peroxidation
Lipid peroxidation in flavedo tissue was determined by estimating the malondialdehyde
(MDA) content following the method of Heath and Packer (1968). The supernatant
prepared for determination of POD enzyme was used. For every 1 mL of the aliquot
of the supernatant 2 mL of 20% (w/v) trichloroacetic acid (TCA) containing 0.5%thiobarbiturric
acid was added. The mixture was heated at 95°C for 30 min and then cooled
quickly on ice bath. The resulting mixture was centrifuged at 10000xg for 25
min and the absorbance of the supernatant was measured at 532 nm. Measurements
were corrected for unspecific turbidity by subtracting the absorbance at 600
nm. The concentration of MDA was calculated by using extinction coefficient
of 155 mM-1 cm-1 expressed as mM MDA g-1 Fresh
Weight (FW).
Statistical Design
Experimental data are the Mean±standard Error (SE) of three replicates
of the determinations for each sample. ANOVA analysis was performed by means
of SAS/STAT GLM procedure, using the Duncan multiple range test at 5% level
for means comparison.
RESULTS
Severity of Chilling Injury
Low-temperature storage of Lisbon lemon induced CI, peel pitting being the
primary symptoms of this physiological disorder. No pitting occurred in stored
fruit for up to 2 weeks and thereafter CI increased continuously for all samples
as storage period advanced (Fig. 1A). Slight pitting appeared
in HWD (CI index = 0.55) or Ca-treated(CI index = 0.6) fruits for up to 6 weeks
at 1.5°C, whereas in control fruits significant severe pitting was observed
(CI index = 2.37). The CI increased quickly thereafter in HWD or Ca-treated
fruits, but significantly less than control fruits up to 8 weeks storage at
chilling temperature. Therefore, the development of CI symptoms was similarly
delayed by both HWD and Ca treatments.
Activities of POD
Before storage at chilling temperature, POD activities of samples were approximate.
Low temperature storage up to 2 weeks induced a decrease in activity of POD,
thereafter increased continuously for all samples as storage period advanced
(Fig. 2A). After 8 weeks of chilling exposure at 1.5°C,
POD activity increased more than 2-fold in the control fruits. POD activity
of both HWD and Ca-treated fruits also increased during storage. However, the
rate of increase was much less in treated fruits than in the control fruits.
No significant differences in POD activity were observed between treated lemons.
POD activity in the flavedo of lemons harvested at the advanced maturity (yellow) stage was lower (2-fold) than those harvested at the less maturity (green) stage (Fig. 3A).
| Fig. 1: | Effect of a hot-water dip treatment at 53°C for 3 min and a vacuum infiltration treatment with 1.5% CaCl2 for 10 min on CI index (A) and MDA content (B) of Lisbon lemons during storage at 1.5°C. Values are the mean of three replicate samples±SE |
Activities of CAT
Calcium treatment of lemons before they were transferred to 1.5°C induced
a 2.43-fold increase in CAT activity, this increase was slightly higher than
the induced 2.23-fold by HWD treatment (Fig. 2B). The activity
of CAT increased initially for up to 2 weeks at 1.5°C and then declined
thereafter. The activity continued to decline in all samples during 4-8 weeks
of storage. In lemons treated with HWD or Ca, CAT activity also declined but
was about twice that of control fruits for up to 6 weeks of storage. In general,
the decline in CAT activity of HWD and Ca-treated fruits during storage followed
a similar pattern.
CAT activity in the flavedo of lemons harvested at the advanced maturity (yellow) stage was 2.64-fold higher than those harvested at the less maturity (green) stage (Fig. 3B).
Activities of SOD
Before storage at chilling temperature, treatment with HWD or Ca did not
affect SOD activity of lemons (Fig. 2C). SOD activity increased
initially for up to 2 weeks of storage, but declined thereafter. The activity
continued to decline in all samples throughout 4-8 weeks of exposure to low
temperature. In lemons treated with HWD or Ca the activity of SOD was higher
than those of the control lemons. The activity afforded by HWD (1-3-fold) or
Ca treatment (1-2-fold) was maintained throughout the storage period.
| Fig. 2: | Effect of a hot-water dip treatment at 53°C for 3 min and a vacuum infiltration treatment with 1.5% CaCl2 for 10 min on POD (A), CAT (B) and SOD (C) activities of Lisbon lemons during storage at 1.5°C. Values are the mean of three replicate samples±SE |
Experiment done to study the effect maturity stage on SOD activity revealed that fruit were harvest at the yellow stage had 2.33-fold more activity than those harvested at the green stage (Fig. 3C).
Malondialdehyde Content
The formation of MDA content was considered as a measure of lipid peroxidation.
As the storage period extended, MDA content of all samples increased constantly
and reached the highest value up to 8 weeks storage at 1.5°C (Fig.
1B).
| Fig. 3: | Influence of maturity stage on activities of POD (A), CAT (B) and SOD (C), as well as MDA content (D) of Lisbon lemons. Columns with different letters were significantly different by the Duncans multiple range test at p = 0.05 |
A significant difference in MDA content between the treated and control lemons was observed on the 6th week of storage and this difference was maintained for the remained of the storage. HWD and Ca treatments resulted in a significant decrease in MDA content as compared with control. No significant differences in MDA content were observed between treated lemons.
As compared to the early maturity (green) stage, lemons were harvested at the late maturity (yellow) stage had significant higher (1.5-fold) MDA content (Fig. 3D).
DISCUSSION
Free-radical processes are involved in several membrane-associated disorders including CI. Superoxide and hydrogen peroxide produced in plant tissues exposed to low temperature may cause the peroxidation of membrane lipids (Marangoni et al., 1996). The development of CI and the increase in MDA content of Lisbon lemon induced by chilling stress were restrained by both HWD and Ca treatments. Treated lemons consistently had higher SOD activities during storage than the control fruits. SOD from treated fruits may play a protective role by detoxifying free radicals. The superoxide radicals produced during chilling stress might be more efficiently dismutated to H2O2 and molecular oxygen in the treated lemons. Thus the stability of cell membranes was maintained by HWD or Ca which lead to an increase in chilling tolerance. These results agree with those for eggplant seedlings (Gao et al., 2004) and grape berries (Zhang et al., 2005). Higher content of MDA in more mature (yellow) lemons may be attributed to the chlorophyll degradation and proteolysis occurs during accelerated of aging and senescence as previously suggested (Meir et al., 1992).
Several active free-radical scavenging enzyme systems exist in plant tissues as defenses against free-radical attack. CAT uses hydrogen peroxide both as a donor of hydrogen and as a substrate in the catalytic decomposition of hydrogen peroxide to form oxygen and water (Burris, 1960). However, CAT activity was found to decrease in Lisbon lemons after 2 weeks and during subsequent chilling period. Decrease in CAT activity during chilling has also found in mandarin fruit (Sala and Lafuente, 2000), orange fruit (Sala et al., 2005) and grape berries (Zhang et al., 2005). On the other hand, POD activity increased steadily in lemons after 2 weeks of storage. Chilling temperature also enhanced POD activity in mango (Zauberman et al., 1988), Fortune mandarin (El-hilali et al., 2003) and Unshiu mandarin fruits (Lepedus et al., 2005). Peroxidases are ubiquitous enzymes that have diverse biochemical functions in higher plants and are involved in the response of plants to stress (Gaspar et al., 1981). For example in citrus fruit, an increase in POD activity has been observed in response to Penicillium digitatum infection (Ballester et al., 2006). Decreasing CAT activity and increasing POD activity could lead to slow removal H2O2 in the tissue and may contribute to the development of CI. Both HWD and Ca treatments reduced the decline of CAT activity and suppressed the increase of POD activity. These effects may contribute to increase tolerance of lemons to CI. This result is in accordance with observations that HWD (Lepedus et al., 2005) or calcium (El-hilali et al., 2003) decreased POD activity and similarly HWD or heat-conditioning treatment (Sala and Lafuente, 2000) delayed the reduction of CAT activity in mandarin fruits during chilling stress.
Previous investigations suggested that CAT may be a major antioxidant enzyme participating in the defense mechanism of citrus fruit against chilling conditions (Sala and Lafuente, 2000; Sala et al., 2005). In the study work, treating Lisbon lemon with HWD or Ca resulted the induction of CAT activity, but not involved POD and SOD activities. Furthermore, the maintenance of increased CAT activity during cold storage affects the effectiveness of these treatments. Both HWD and Ca increased chilling tolerance and CAT activity in the fruits, but after 6 weeks of cold storage CAT activity declined at the same time as resistance to chilling. Considering these results further support this idea that CAT might play a major role in the tolerance of citrus fruit to CI.
Reduction of damaging effects of chilling in resistant plants may be related to their ability to reduce and to scavenge free radicals through increase antioxidant enzyme activity, as has been reported for example in mandarin (Sala, 1998) and orange fruit (Sala et al., 2005). This further confirmed from observed increase in CAT, SOD and decrease in POD activities of Lisbon lemons during 2 weeks storage at chilling temperature, which showed no chilling peel pitting. In addition, the lemon fruit harvested at the yellow stage had higher CAT, SOD and lower POD activities than those harvested at the green stage. It is well documented that lemon fruit harvested early in the season were more susceptible to CI than fruit harvested later (Underhill et al., 1995). The overall results may indicate that the linked action of CAT, SOD and POD may contribute, at least to some extent, to the tolerance of Lisbon lemon fruit to CI and the chilling-tolerant property of fruits may be related to the higher ability of fruits to have or maintain high CAT, SOD and low POD activities during low temperature storage.
As the main objective of this study was to examine the role of HWD and Ca on CI and antioxidant enzymes activity, it is interesting to note that the pattern of alleviating chilling symptoms and the changes of antioxidant enzyme activity in the treated fruits were similar during storage. Gong et al. (1998) reported that heat stress caused a transient increase in cytosolic Ca2+ concentration and this increase may alleviate heat injury and enable plant cells to better survive. The regulatory role of calcium (Ca2+) on antioxidant enzyme to induce tolerance to various stresses has also been reported earlier (Karpinski et al., 2002; Agarwal et al., 2005). These results suggest a connection among heat, cytosolic Ca+2 and antioxidant enzymes in the reduction of chilling injury. However, further work is needed to define the exact relationship of heat and Ca on antioxidant enzymes.
ACKNOWLEDGMENTS
Thanks are due to the Darab College of Agriculture for the use of spectrophotometer instrument and to Dr. N. Razmi for the use of microdismembrator instrument.