OXIDATIVE STRESS AND ANTIOXIDANT SYSTEMS IN TOMATO FRUITS DURING STORAGE

OXIDATIVE STRESS AND ANTIOXIDANT SYSTEMS IN TOMATO FRUITS DURING STORAGE

K. MONDAL, N.S. SHARMA, S.P. MALHOTRA, K. DHAWAN and R. SINGH'

Plant Biochemistry and Molecular Biology Laboratory Department of Biochemistry

CCS Haryana Agricultural University Hisar - 125 004. India

Received for Publication December 20, 2002 Accepted for Publication April 18, 2003

ABSTRACT

Tomato varieties ARTH-3 (long shelflife; 14-15 days) and Sel-7 (short shelf-life; 5-7days), harvested at color turning stage, were stored in open trays at 10, 25 and 35Cand sampled at two day intervals until complete deterioration. Variety ARTH-3 could be stored at all the temperatures for ten days, while Sel-7 could tolerate 35C only for four days. However, at 10 and 25C, it could be stored for six days. In both varieties, lipoxygenase (LOX) activity, malondialde- hyde (MDA) value and H,O, content increased during storage. Increase in storage temperature further enhanced the activity of LOX, and also increased MDA value and H202 content. Sel-7 had higher values for these parameters than ARTH-3. Activities of enzymes responsible for scavenging reactive oxygen species (ROS) viz., superoxide dismutase, peroxidase, ascorbate peroxidase, glutathione reductase and catalase decreased continuously during storage. With increase in temperature, the activities of these enzymes further decreased significantly in both varieties. Sel-7 had significantly lower activities of ROS scavenging enzymes than ARTH-3 throughout the storage period. n e s e results suggest that fruits stored at higher temperature are subjected to severe oxidative damage leading to extensive membrane damage and loss oftissue structure.

Author for correspondence. TEL: 01662-232825; FAX: +91-1662-234952; EMAIL: [email protected]

Journal of Food Biochemishy 27 (2003) 515-527. All Rights Reserved. BCopyrigkt 2003 by Food C? Nutrition Press, Inc., Trumbull. Connecticut. 515

516 K. MONDAL, N.S. SHARMA, S.P. MALHOTRA, K. DHAWAN and R. SINGH

INTRODUCTION

Fruits deteriorate during periods of prolonged storage. However, the degree and duration of deterioration depend to a large extent on storage conditions. Though the exact cause of deterioration is still not fully understood, the fruits undergo peroxidative changes during storage. This free radical-induced nonenzymatic peroxidation, which has the potential to damage membranes, seems to be a primary cause of deterioration of stored fruits. In addition, the production of reactive forms of oxygen including superoxide radicals (O;), the hydroxyl radical (OH-) and hydrogen peroxide (H,OJ is stimulated by a variety of environmental stresses such as exposure to high light (Foyer et al. 1997), drought (Loggini et al. 1999), heavy metals (Rao and Sresty 2000), high salt concentrations (Meneguzzo et al. 1999), extremes of temperature (Rao et al. 1995), UV radiations (Murphy and Huerta 1990), water stress (Boo and Jung 1999), herbicides (Miyagawa et al. 2000), mechanical and physical stresses (Legendre er al. 1993) and also in response to biotic stresses such as invasion by various pathogens (Low and Merida 1996). Membrane damage due to active oxygen species increases lipid peroxidation during the oxidative stress (Galliard and Matthew 1977; Wise 1995). Since a number of environmental factors have been associated with oxidative damage, it seems possible that a common response of plants to any type of stress is associated with active oxygen species.

Protective mechanisms that could scavenge the peroxidatively produced free radicals and peroxide have been evolved within the plants to keep these deleterious compounds to a minimum. This protective mechanism involves a combination of antioxidants, i.e., reduced glutathione and ascorbate, and a regeneration cycle involving superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), and dehydro- and monodehydro-ascorbate reductases (DHAR and MDHAR). Alterations in the activities of these enzymes have been observed under the influence of various types of environmental stresses (Mehdy 1994) leading to increased senescence. Many studies have also linked chilling tolerance to antioxidant capacity in maize (Pastori et al. 2000) and tomato (Bruggemann et at. 1999). How temperature influences the antioxidant ability of tomato fruits during storage was the main focus of the present studies.

MATERIALS AND METHODS

Plant Material

The present experiments were conducted with detached tomato fruits of ARTH-3 (a variety with long shelf-life, 14-15 days) and Selection-7 (Sel-7; a variety with short shelf-life, 5-7 days). The plants of these cultivars were field

OXIDATIVE STRESS IN TOMATO DURING STORAGE 517

grown at the Experimental Farms of the Department of Vegetable Crops, CCS Haryana Agricultural University, Hisar. Fruits were harvested at the color turning stage, when the pink color developed around the apical end of the fruit and spread over the entire surface. Freshly harvested fruits free of any visible defects were surface sterilized with 0.2% mercuric chloride, rinsed in tap water and then in distilled water and air dried. The fruits were kept in open trays, stored at three different temperatures viz., 10, 25 and 35C. Samples were taken at two day intervals until complete decay. Sel-7, a shorter shelf-life variety, could be stored for 6 days at 10 and 25C, while at 35C, it deteriorated completely just after four days of storage. ARTH-3 could tolerate all temperatures for 10 days. However, the extent of deterioration was greater at higher temperatures.

Analytical Methods

Rate of lipid peroxidation was measured by the formation of malondialde- hyde (MDA) as described by Heath and Packer (1968). One gram tissue was homogenized in 5 mL of 0.1 % (w/v) trichloroacetic acid (TCA) and centrifuged at 8,000 x g for 15 min. One milliliter of the supernatant was thoroughly mixed with 4 mL of 20% (w/v) TCA containing 0.5% (w/v) 2-thiobarbituric acid (TBA) and the mixture was incubated at 95C for 30 min. The samples were cooled in an ice bath, centrifuged at 8,OOO x g for 15 min and the absorbance of the supernatant taken at 532 nm. Nonspecific absorbance of the sample at 600 nm was subtracted from the absorbance at 532 nm. The concentration of MDA was calculated using the molar extinction coefficient of 155 mM-' cm-' (Dipierro and Leonardis 1997). In a modified procedure for MDA estimation, Du and Bramlage (1992) have advocated measuring absorbance at 532,600 and 440 nm. They further prepared a standard curve for sucrose (2.5 - 10 pmol mL') and used it to rectify the interference of soluble sugars in samples. However, the tomato samples at turning red stage used in the present investigations contained 0.9 mg sucrose g-' dry weight (Singh et al. 2000), indicating that the concentration of sucrose in the present case was lower than the interfering range as suggested by the above workers.

H202 was extracted by homogenizing 4 g tissue in 5 mL of ice cold 0.01 M phosphate buffer (pH 7.0) and centrifuging the homogenate at 8,000 X g for 10 min (Sinha 1972). The sample (50 pL) was added to 1.95 mL of 0.01 M phosphate buffer (pH 7.0), then 2 mL of 5% potassium dichromate and finally glacial acetic acid (1:3, Y/V) was added. The A was read at 570 nm against the reagent blank without sample extract. The quantity of H202 was determined by comparing with standards (10 to 160 pmoles).


Enzyme Extraction and Assays

Extraction conditions were standardized with respect to type, molarity, pH of the buffer, concentration(s) of stabilizing agent@) and other constituents of the extraction medium to achieve maximum extraction of the enzymes. Extraction medium for SOD, CAT, APOX, GR and LOX consisted of 0.1 M phosphate buffer (pK 7.5) containing 5 % (wh) polyvinylpolypyrrolidone (PVPP), 1 mM EDTA, and 10 mM 0-mercaptoethanol. For POX, however, the extraction buffer consisted of 0.01 M phosphate buffer (pH 7.0) containing 4% (wiv) PVPP. The homogenate was prepared by grinding 4 g (fresh weight) of tomato fruits in 5 mL of ice cold extraction medium in precooled mortar and pestle. The homogenate thus prepared was centrifuged at 10,000 x g for 15 min at 4C.

LOX activity was measured at 30C by monitoring the formation of conjugated dienes from linoleic acid at 234 nm (Chen and Whitaker 1986). SOD activity was determined by quantifying the ability of the enzyme extract to inhibit light induced conversion of nitroblue tetrazolium (NBT) to formazan (Beauchamp and Fridovich 1971). CAT and POX activities were assayed at 37C as described by Sinha (1972) and Dias and Costa (1983), respectively. The method of Nakano and Asada (1981) was employed to assay APOX. GR activity was determined at 30C by adding 100 pL of enzyme extract to 1 mL of 0.2 M phosphate buffer (pH 7.0) containing 1 mM EDTA, 0.75 mL distilled water, 0.1 mL of 20 mM oxidized glutathione (GSSG) and 0.1 mL of 2 mM NADPH. Oxidation of NADPH by GR was monitored at 340 nm and the rate (nmol min I)

was calculated using the extinction coefficient of 6.2 mM-' cm-' (Dipierro and Leonardis 1997).

Data were analyzed statistically as explained by Cochran and Cox (1957).

RESULTS AND DISCUSSION

As shown in Table 1, both ARTH-3 and Sel-7 showed an increase in lipoxygenase activity during storage. Temperature further increased LOX activity in both varieties. The data revealed that ARTH-3 at 35C after 10 days of storage had similar LOX activity as Sel-7 after 6 and 4 days of storage at 25 and 35C, respectively. This suggests that Sel-7, being a variety with short shelf- life, had higher rate of deterioration because of higher lipid peroxidation. Data show a significant increase in MDA value in both varieties during storage of tomato fruits. Fruits stored at higher temperatures exhibited a considerable increase in MDA value. Sel-7 showed significantly higher values for MDA than ARTH-3 at all days of storage and at all temperatures. The enhanced lipid peroxidation, as indicated by MDA values at higher temperatures during storage,

TAB

LE 1

. EF

FEC

T O

F TE

MPE

RA

TUR

E O

N L

IPO

XY

GEN

ASE

AC

TIV

ITY

, MD

A V

ALU

E A

ND

H20

2 CO

NTE

NT

IN

TOM

ATO

FR

UIT

S D

UR

ING

STO

RA

GE

0 t4 3 2 m

v) i! v) 52

C.D

at 5

% l

evel

: Fo

r lip

oxyg

enas

e:

Vb=

0.88

; TC

=1.0

8;

Dd=

0.88

; V

T=1.

52;

VD

=1.2

4;

VTD

=2.1

5 Fo

r MD

A v

alue

: V

=0.0

2;

T=0.

03;

D=0

.02;

V

T=0.

04;

VD

=0.0

3;

VTD

=0.0

6 Fr

o HzOz co

nten

t: V

=4.8

5;

T=5.

93;

D=4

.85;

V

T=8.

39;

VD

=6.8

5;

VTD

=11.

87

a.

Day

s afte

r sto

rage

b.

V

arie

ty

c.

Tem

pera

ture

d.

D

ays a

fter s

tora

ge

U

C n B


was indirectly related to the increased LOX activity. These results are supported by the findings of Rao and Watkins (1998), Sung and Chiu (1995) and Pinto et al. (2001), who found LOX activity and MDA values to increase during storage of apple, soybean and pear fruits, respectively, at different temperatures. An increase in storage temperature enhanced membrane deterioration due to higher LOX activity and higher content of MDA. These results taken in conjunction with increased H,O, content during storage (Table 1) might partially explain the enhanced rate of deterioration for Sel-7 than ARTH-3 during storage. H,02 content increased throughout storage, however, in ARTH-3, it decreased only slightly on the 4th day of storage. At higher temperatures, its content was significantly higher in both varieties. Sel-7 had significantly higher H202 content than ARTH-3 at all storage temperatures. The increase in H202 content at elevated temperatures had been reported by Foyer ez al. (1997) in tobacco seedlings. However, maize leaves grown at low temperatures were found to contain higher amounts of H20, than those grown at higher temperatures (Hodges ef al. 1997).

As shown in Tables 2 and 3, all the peroxide scavenging enzymes examined here had maximum activities after 2 days of storage in both varieties and under all storage temperatures. Peroxidase activity in ARTH-3 exhibited a decline in activity up to 6 days of storage and then increased, whereas in Sel-7, the activity decreased continuously during storage. With increase in temperature, the activity further decreased significantly in both varieties. The decline in activity was more rapid in Sel-7 than in ARTH-3 as the later retained higher activity (percent of original) at all temperatures during storage. A decrease in peroxidase activity at higher temperature has been reported earlier by Lee and Lee (2000). An increase in peroxidase activity later during storage of ARTH-3 might reflect fungus infestation in stored fruits (Johnson and Cunningham 1972). Ascorbate peroxidase activity declined in both varieties during storage and increasing storage temperature further decreased the activity substantially. As was true for peroxidase, the decrease in APOX activity was more rapid in Sel-7. No information is available in the literature as to how high temperature affects ascorbate peroxidase activity. However, low temperature during storage of potato tubers (Dipierro and Leonardis 1997), tomato fruits (Bruggemann et al. 1999) and maize leaves (Pastori er ai. 2000) increased the activity of ascorbate peroxidase. However, Hull ef al. (1997) reported lower ascorbate peroxidase activity in maize leaves stored at lower temperature.

TA

BL

E 2

. EF

FEC

T O

F T

EM

PER

AT

UR

E O

N S

UPE

RO

XID

E D

ISM

UT

ASE

, PE

RO

XID

ASE

AN

D C

AT

AL

ASE

AC

TIV

ITIE

S IN

TO

MA

TO

FRU

ITS

DU

RIN

G S

TOR

AG

E

0

U

3 2 m v)

2 3 C

.D a

t 5%

leve

l: z

V=0

.98;

T

=1.2

0;

D=0

.98;

V

T=1

.70;

V

D=1

.39;

V

TD

=2.4

1 U

b. V

arie

ty

0

For s

uper

oxid

e dism

utas

e:

Vb=

0.74

; Y

=0.9

1;

Dd=

0.74

; V

T=1

.29;

V

D=1

.05;

V

TD

=1.8

2 5 ti 0

For p

erox

idas

e:

For c

atal

ase:

V

=1.4

1;

T=1

.73;

D

=1.4

1;

VT

=2.4

5;

VD

=2.0

0;

VT

D=3

.46

a.

Day

s afte

r sto

rage

c. Te

mpe

ratu

re

d. D

ays a

fter s

tora

ge

z g $


Catalase activity increased transiently and slightly on the 4th day after storage and then decreased continuously in ARTH-3. Increasing storage temperature resulted in a decrease in the activity. However in Sel-7, the activity was quite low as compared to ARTH-3 and it remained almost constant during storage. Temperature also had no effect on catalase activity in Sel-7. Low activity of catalase in Sel-7 might explain the higher content of H202 (Table 1) in this variety as compared to ARTH-3. A decrease in catalase activity during storage has been supported by the findings of Pinto et at. (2001), who observed a decrease in catalase activity during storage of core brown affected pear fruits. Dipierro and Leonardis (1997) have reported an increase in catalase activity in potato tubers throughout storage. Apple fruits stored at lower temperature exhibited higher catalase activity than those stored at higher temperature (Du and Bramlage 1995; Rao and Watkins 1998). Superoxide dismutase (Table 2) activity declined with increasing storage duration in both varieties. SOD activity was low in ARTH-3 than in Sel-7. Similarly, glutathione reductase (GR) also showed a decrease in activity during storage but the level was greater in ARTH-3 than in Sel-7 until day 6 at 10 and 25C and until day 4 at 35C. Higher SOD activity at lower storage temperature has also been reported in maize (Pastori et al. 2000; Kingston-Smith and Foyer 2000) and cucumber (Lee and Lee 2000) leaves. However, pear fruits exhibited an increase in SOD activity upon storage. Maize leaves have also been reported to exhibit higher GR activity when stored at lower temperature (Hull et at. 1997). However, Bruggemann et al. (1999) reported a decrease in GR activity during low temperature treatment of tomato leaves. In potato tubers (Dipierro and Leonardis 1997), GR activity was affected by temperature and remained almost constant throughout storage.

From the results discussed above, it is apparent that the two cultivars of tomato varying in length of shelf-life responded to storage temperature in an identical fashion except that the cv Sel-7 deteriorated faster and could tolerate a storage temperature of 35C for only four days. Further, it was clear that during storage, lipoxygenase activity, MDA value and H,O, content showed linear increases. As a result, temperature had significant effect on lipid peroxidation, reflecting that the fruit senescence was mainly the result of high temperature stress. Thus, the loss of membrane integrity and tissue structure during storage of tomato fruits could be correlated well with increased lipid peroxidatin caused by build-up of free radicals. The cv with longer shelf-life showed significant (P = 0.05) increase in the activities of CAT, GR, APOX and POX during storage. Sel-7, on the other hand, exhibited higher activity of SOD. Higher activity of SOD (H,O,-producing activity) and lower activity of catalase (H,O,-scavenging activity) in this variety simultaneously might be responsible


for higher H202 content during storage. This imbalance in enzyme activities has been proposed to cause accumulation of H202 in leaves exhibiting natural and artificially induced senescence (Pastori and del Rio 1997). The hypothesis that oxidative damage caused by generation of H202 results in membrane deterioration during senescence (del Rio et al. 1998) seems to be true of the deterioration during storage as well. Tomato fruits during storage showed the accumulation of H,02 and the rate of H202 accumulation was significantly higher in Sel-7, a variety with short shelf-life than ARTH-3, a variety with long shelf-life. This suggests that deterioration of tomato fruits during storage is akin to the process of deterioration characterized by increased oxidative stress during senescence.

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OXIDATIVE STRESS AND ANTIOXIDANT SYSTEMS IN TOMATO FRUITS DURING STORAGE

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