Changes in ethylene production, carbohydrase activity and antioxidant status in pepper fruits during ripening

Scientia Horticulturae 142 (2012) 23–31

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Changes in ethylene production, carbohydrase activity and antioxidant status in pepper fruits during ripening Chung Keat Tan a , Zainon Mohd Ali a , Zamri Zainal a,b,∗ a b

School of Bioscience and Biotechnology, National University of Malaysia, 43600 Bangi, Selangor, Malaysia Institute of System Biology (INBIOSIS), National University of Malaysia, 43600 Bangi, Selangor, Malaysia

a r t i c l e

i n f o

Article history: Received 24 February 2012 Received in revised form 25 April 2012 Accepted 26 April 2012 Keywords: Capsicum annuum Ethylene production Carbohydrase activity Antioxidant status Ripening

a b s t r a c t The aim of the present study was to evaluate the changes in ethylene production, carbohydrase activities and antioxidant status in pepper Kulai at different ripening stages. This cultivar of pepper fruits exhibits a peak in ethylene production during stage 3 of ripening. The production of 1-aminocyclopropane-1carboxylic acid (ACC) and ACC oxidase activity also peaked during stage 3, whereas ACC synthase activity increased gradually during ripening. In the carbohydrase activity study, the ␤-galactosidase activity increased significantly, whereas the ␣-mannosidase and ␣-galactosidase activities remained low and fairly constant throughout the ripening process. The lipoxygenase (LOX) activity, lipid peroxidation level and hydrogen peroxide content were also examined to evaluate changes in oxidation status. The LOX activity and hydrogen peroxide concentration decreased during ripening, whereas lipid peroxidation increased. However, the total antioxidant potential also increased, most likely as a defensive response towards oxidative stress. The activities of ascorbate peroxidase (APX) and glutathione reductase (GR) and the total phenolic content significantly increased during ripening. Furthermore, ascorbic acid levels greatly increased, and the ratio of reduced glutathione (GSH) to oxidised glutathione (GSSG) remained constant. However, the activities of superoxide dismutase (SOD) and catalase (CAT) declined. The overall results indicated that pepper Kulai is a climacteric fruit and that ␤-galactosidase activity increases as the fruit ripens and softens, suggesting a role for this enzyme in cell wall modification. In addition, the results presented here also revealed that the antioxidant capacity is enhanced during the ripening process and is accompanied by an increase in oxidative stress. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Peppers have been a part of the human diet in the Americas since at least 7500 BC, and now they are widely used as a spice, food and medicine throughout the world. The widespread use of pepper is benefited by its natural colours and antioxidant compounds (Howard et al., 2000; Antonious et al., 2006). Epidemiological studies consistently indicate that there is an inverse correlation between the consumption of fruits containing antioxidants and the risk of human cancers, cardiovascular disease, diabetes and age-related declines in cognition (Knekt et al., 2002). Pepper fruits are harvested and consumed at different stages of maturity, mainly the red and green stages, and they are sometimes

∗ Corresponding author at: School of Bioscience and Biotechnology, National University of Malaysia, 43600 Bangi, Selangor, Malaysia. Tel.: +60 3 8925 5951; fax: +60 3 8921 2698. E-mail addresses: jacktan0808 [email protected] (C.K. Tan), [email protected] (Z.M. Ali), [email protected] (Z. Zainal). 0304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2012.04.030

consumed before they are completely ripe. Previously, peppers were harvested at the mature green stage when the pericarp becomes thick and the fruits reach their maximum sizes. However, in recent years, the mature red stage has become more favourable due to its improved flavour and nutritional aspects (Frank et al., 2005). During the ripening process, carbohydrates and vitamin C accumulate, and changes in the levels of total phenolic compounds and carotenoids have also been reported in some papers (Zhang and Hamauzu, 2003). Phenolic compounds are well known for their antioxidant qualities and their contributions to the sensory and nutritive qualities of the fruit, particularly the colour, taste, aroma and flavour (Tomás-Barberán and Espín, 2001). The ripening of fruits is always accompanied by endogenous ethylene production and by the ability to respond to exogenous ethylene. In general, peppers are classified as a non-climacteric fruit based on the patterns of carbon dioxide and ethylene production, in addition to a transcriptome analysis (Lee et al., 2010). However, this finding is not conclusive and is limited to certain cultivars of Capsicum. Biles et al. (1993) have reported that the concentration of internal ethylene in New Mexican peppers is high enough to

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initiate ripening. In addition, some cultivars of hot pepper have also demonstrated a peak in ethylene production during the ripening process (Gross et al., 1986). Apart from ethylene production, fruit softening is also another common physiological change occur during ripening process. The fruit softening and textural changes are mainly due to differences in the cell wall thickness and composition (Harker et al., 1997). Previous studies have shown that pectin depolymerisation mediated by endo-polygalacturonase activity alone is not sufficient to significantly impact texture (Giovannoni et al., 1989). Other enzymes that are involved in cell wall metabolism have been identified in ripening fruit and, in some cases, have been tested for function. ␤galactosidase (EC 3.2.1.23) may be one of the key enzymes involved. Gene expression study has indicated that there is a remarkable increase in ␤-galactosidase expression during the ripening process of tomato fruit (Smith and Gross, 2000). Down-regulation of tomato ␤-galactosidase results in decreased fruit softening (Smith et al., 2002). In addition to these and other enzymes, non-enzymatic factors such as reactive oxygen species (ROS) have also been identified that may play a role in cell wall degradation (Miller, 1986; Fry et al., 2001). The pepper fruit has been identified as a potential solanaceous crop with extremely high antioxidant activity (Ou et al., 2002). A wide spectrum of antioxidant compounds, namely phenolic compounds, ascorbic acid, reduced glutathione (GSH)/oxidised glutathione (GSSG) and antioxidant enzymes, such as ascorbate peroxidase (APX; EC 1.11.1.11), superoxide dismutase (SOD; EC 1.15.1.1), glutathione reductase (GR; EC 1.8.1.7) and catalase (CAT; 1.11.1.6), are present in pepper fruits (Namiki, 1990). Recently, more attention has been paid to the role of natural antioxidants, particularly phenolic compounds. These compounds have shown positive but non-significant correlations (R = 0.41–0.55) with antioxidant activities (Al-Mamary, 2002) and may have more antioxidant activity than vitamins C and E and ␤-carotene (Vinson et al., 1995). Phenolic compounds reduce lipid autoxidation by inhibiting the initiation or propagation of oxidising chain reactions (Namiki, 1990). The glutathione and ascorbate regulation cycle, along with SOD and CAT, constitute the major non-enzymatic and enzymatic systems that scavenge free radicals and H2 O2 (Sala, 1998). Similarly to senescence, fruit ripening is accompanied by the deterioration of cell membranes (Ferrie et al., 1994). Lipoxygenase (LOX; EC 1.13.11.12) has been shown to have a central role in senescence-induced membrane deterioration by peroxidising free polyunsaturated fatty acids (Paliyath and Droillard, 1992). H2 O2 may also be involved in the oxidative processes required in the initiation and promotion of fruit ripening (Brennan and Frenkel, 1977). Formal studies involving pepper fruit have not been fully comprehensive and mostly concentrated on biophysical changes that occur during ripening. In this study, we examined the main physiological changes, which are ethylene production and carbohydrase activity involved in cell wall modifications during the ripening process. In addition, the evolution of the antioxidant status and the oxidative stress of peppers at different stages of maturity were also studied to improve the management and harvesting of this crop and to obtain fruit with higher nutritional values. 2. Materials and methods 2.1. Materials Hot pepper fruits (Capsicum annuum cv. Kulai) were harvested at the Bukit Lancong Farm (Selangor, Malaysia). The selection was based on 5 different maturity stages: considerably mature green (stage 1; fully developed fruit with thick pericarp and dark green

skin), 25% red (stage 2; skin of the fruit starting to turn red), 50% red (stage 3; approximately one-half green skin and the other half red), 75% red (stage 4; approximately 75% of the skin had turned red) and red (stage 5; fully ripe fruit). Overripe and damaged fruits were discarded. Fifty uniform fruits were selected for each stage of maturity and cut into small pieces, and the seeds were discarded. The samples were immediately frozen in liquid nitrogen and stored at −80 ◦ C until use. Unless otherwise stated, all solvents, salts and acids were purchased from Sigma Chemical Co. (St. Louis, USA). All of the reagents were of HPLC grade and were of the highest purity available. All aqueous solutions were prepared with distilled water.

2.2. Ethylene production 2.2.1. Ethylene measurement Fresh fruits were used in this study. The ethylene production was measured by enclosing fresh fruits in an airtight container for 2 h at room temperature, withdrawing 1 mL of the headspace gas, and injecting this gas into an AutoSystem gas chromatograph (GC, PerkinElmer) fitted with a flame ionisation detector (FID).

2.2.2. ACC, ACC synthase and ACC oxidase assays 1-Aminocyclopropane-1-carboxylic acid (ACC) was extracted using the method of Sitrit et al. (1986) with slight modifications. The frozen tissues were homogenised with distilled water and centrifuged at 7000 rpm for 10 min. The supernatant was then used for determination of the ACC content based on the method described by Lizada and Yang (1979). A 100 ␮L aliquot of the aqueous pepper extract was added to a test tube with 100 ␮L of 5 ␮M HgCl2 and 500 ␮L of distilled water. The test tubes were then sealed with serum caps, and 0.2 mL of an ice-cold mixture (2:1, v/v) of commercial bleach (5% NaOCl) and saturated NaOH was injected. The assay tubes were then vortexed and incubated in an ice bath for 15 min. After incubation, 1 mL of gas was withdrawn for the measurement of ACC content using gas chromatography. The efficiency of the conversion of ACC to ethylene in each sample was determined by adding a known amount of ACC as an internal standard to a replicate assay tube. The ACC content was defined as nmol per gram of fresh weight. The ACC synthase (ACS; EC 4.4.1.14) activity was determined using the method of Nakatsuka et al. (1997) with some modifications. Five grams of frozen tissue was homogenised with 0.1 M phosphate buffer (pH 8.5) that contained 30 mM ascorbic acid, 4 mM dithiothreitol (DTT), 30 ␮M pyridoxal phosphate (PLP), 2% polyvinylpyrrolidone (PVP) and 10% glycerol. The homogenate was then filtered using cheesecloth and centrifuged at 11,000 rpm for 20 min. A 2-mL aliquot of the aqueous extract was desalted and subjected to gel filtration using Sephadex G-25 (1.5 cm × 6 cm). One millilitre of the desalted eluent was added to 0.2 mL of 0.5 mM Sadenosylmethionine (S-AdoMet or SAM). The reaction mixture was incubated at 30 ◦ C for 30 min. The ACC production was then assayed according to the method of Lizada and Yang (1979), as described above. The enzyme activity was defined as nmol of ACC produced in 1 h/g of fresh weight. The ACC oxidase (ACO, EC 1.14.17.4) activity was also assayed according to the method of Nakatsuka et al. (1997) with slight changes. The eluent collected from the gel filtration was mixed with 50 ␮L of 20 mM ACC, 5 ␮L of 2 mM FeSO4 and 100 ␮L of 30 mM NaHCO3 in a test tube. The test tube was then sealed with a serum cap. The test tube was incubated at 30 ◦ C with shaking for 30 min, and the amount of ethylene formed was determined as described above. The ACO activity was defined as nmol of ethylene produced per hour per gram of fresh weight.

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2.3. Cell wall modification enzymes The extraction of the cell wall enzymes was modified from the method proposed by Ali et al. (1998). Five grams of the tissue sample was homogenised with 10 mL of 0.1 M citrate buffer (pH 4.6) that contained 1 M NaCl, 13 mM EDTA, 1% PVP and 10 mM ␤mercaptoethanol. The homogenate was centrifuged at 17,000 rpm for 30 min. The supernatant was then used for enzymatic assays and protein determination. The glycosidase activities were assayed according to the method of Pressey (1983). The assay mixture, which consisted of 0.52 mL of assay buffer, 0.40 mL of BSA (0.1%) and 0.40 mL of the corresponding ␳-nitrophenyl derivatives as substrates (13 mM), was incubated at 37 ◦ C for 10 min. Next, 80 ␮L of the supernatant was added and incubated for 15 min at the same temperature. The reaction was then quenched by the addition of 2 mL of Na2 CO3 (0.2 M), and the absorbance at 412 nm was determined using a UV–visible spectrophotometer (Shimadzu, UV-160A). One unit of enzyme was defined as the amount of enzyme needed to produce 1 ␮mol of ␳nitrophenol in 1 min. The specific activities are expressed in units per gram of fresh weight.

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The H2 O2 content of the pepper samples is expressed in ␮mol per gram of fresh weight. 2.5. Non-enzymatic antioxidants 2.5.1. Total antioxidant potential The potential of the total antioxidants was measured using the ferric-reducing antioxidant power assay (FRAP) according to the procedure of Wong et al. (2006) with slight modifications. Five grams of pepper tissue was ground and extracted with 10 mL of 80% methanol at 40 ◦ C for 24 h. The homogenate was then centrifuged at 4500 rpm and 4 ◦ C for 15 min. The FRAP reagent was prepared from ten parts of acetate buffer (0.3 M, pH 6.5), 1 part of a 10 mM tripyridyltriazine (TPTZ) solution in 40 mM hydrochloric acid (HCl) and 1 part of a 20 mM ferric chloride solution (v/v). The reaction mixture consisting of 50 ␮L of the supernatant and 1.5 mL of the FRAP reagent was incubated for 3 min. The absorbance of the reaction mixture was then measured at 593 nm using a UV–visible spectrophotometer (Shimadzu, UV-160A). A standard curve was prepared using the same procedure with a ferrous sulphate solution (100–2000 ␮M). The results are expressed in ␮mol Fe(II) per gram of fresh weight.

2.4. Oxidative stress 2.4.1. Lipid peroxidation Malondialdehyde (MDA) is the main product of lipid peroxidation. The extraction of MDA was performed by grinding 5 g of tissue with 25 mL of cold 5% trichloroacetic acid (TCA). The homogenate was then centrifuged at 12,000 rpm for 15 min at 4 ◦ C. The MDA content was measured according to the method of Heath and Packer (1968). A 2 mL aliquot of the supernatant was added to 2 mL of 0.67% 2-thiobarbituric acid (TBA) in 20% TCA. The solution was heated at 100 ◦ C for 15 min and quickly cooled in an ice bath for 5 min. The solution was then centrifuged at 15,000 rpm for 15 min to clarify the solution. The absorbance was monitored at 532 nm using a UV–visible spectrophotometer (Shimadzu, UV-160A). The value of the non-specific absorption at 600 nm was subtracted. The MDA content was calculated from the extinction coefficient of 155 mM−1 cm−1 and is expressed as nmol per gram of fresh weight. The LOX activity was determined using the method of Anese and Sovrano (2006) with minor changes. Five grams of pepper tissue was homogenised with 10 mL of phosphate buffer (0.1 M, pH 7.0). The homogenate was then centrifuged at 9700 rpm and 4 ◦ C for 20 min. Each reaction mixture contained 0.05 mL of enzymatic extract, 0.15 mL of a linoleic acid solution (2 mM) and 1.30 mL of sodium phosphate buffer (0.05 M, pH 6.5). The LOX activity was assayed by measuring the change in absorbance at 234 nm using a UV–visible spectrophotometer (Shimadzu, UV-160A). One unit of LOX was defined as the amount of enzyme needed to form 1 ␮mol hydroperoxide under the standard conditions. The specific activity is expressed as units per gram of fresh weight. 2.4.2. Hydrogen peroxide (H2 O2 ) content The hydrogen peroxide content was determined using a FOX II method based on the principles of Jiang et al. (1990) with slight modifications suggested by Bou et al. (2008). H2 O2 was extracted from 5 g of tissue using 10 mL of cold 95% methanol. The homogenate was then centrifuged at 5000 rpm for 10 min. The FOX II reagent (0.95 mL), consisting of 100 ␮M xylenol orange, 250 ␮M ammonium ferrous sulphate, 4 mM butylated hydroxytoluene (BHT) and 25 mM sulphuric acid dissolved in a methanolic solution (90%, v/v), was mixed with 0.05 mL of enzyme extract. The mixture was then allowed to react at room temperature for 10 min. The colour development was measured at 560 nm with a UV–visible spectrophotometer (Shimadzu, UV-160A). A standard curve was prepared using the same procedure with H2 O2 (0–5 ␮M).

2.5.2. Total phenolic content The total phenolic content in the pepper fruits was measured using a method adapted from Camacho-Cristóbal et al. (2002). The pepper tissue (5 g of fresh weight) was pulverised using a mortar and pestle with 20 mL of cold 100% methanol. The homogenate was centrifuged at 3000 rpm and 4 ◦ C for 15 min. The extract was diluted 10 times and then oxidised with 100 ␮L of freshly diluted 50% Folin–Ciocalteau reagent. After 3 min, this reaction was neutralised by adding 2 mL of 2% (w/v) sodium carbonate. After incubation for 30 min at room temperature, the absorbance of the resulting bluecoloured solution was determined at 750 nm using a UV–visible spectrophotometer (Shimadzu, UV-160A). A standard curve was prepared using the same procedure with gallic acid (10, 20, 50 and 100 mg/L). The total phenolic content of the pepper samples is expressed in mg of gallic acid equivalent (GAE) per gram of fresh weight. 2.5.3. Ascorbic acid content Five grams of the pepper sample was extracted using 25 mL of HPLC-grade water. The homogenate was centrifuged at 13,000 rpm for 15 min. The supernatant was then filtered using a syringe filter (nylon membrane, 0.02 ␮m). The quantification of the sample was achieved using an external standard method adapted from Lim et al. (2007) with slight modifications. The HPLC analysis of ascorbic acid was performed using a Shimadzu (Tokyo, Japan) mode HPLC with a diode array detector. The samples (20 ␮L) were separated at 40 ◦ C on a Waters Symmetry C18 column (3.9 mm × 150 mm id; 5 ␮m particle size) (Milford, MA) using a mobile phase of 5% acetic acid at flow rate of 1 mL/min. The amount of ascorbic acid was calculated from the absorbance at 254 nm using ascorbic acid (20, 40, 60, 80 and 100 mg/L) as a standard. The results are expressed as mg per gram of fresh weight. 2.5.4. Total glutathione contents Five grams of the sample was homogenised in a cold mortar using 15 mL of 5% 5-sulphosalicilic acid. The homogenate was then centrifuged at 9000 rpm for 15 min. The assay of the total glutathione content was based on the method of Anderson (1985) with slight modifications. The reaction mixture was composed of 700 ␮L of 0.2 mM NADPH, 100 ␮L of 6 mM DTNB and 180 ␮L of 0.143 M potassium phosphate buffer (pH 7.5). The mixture was incubated in a water bath at 30 ◦ C for 5 min before the addition of 20 ␮L of the supernatant and 1.5 units of glutathione reductase.

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The change in the absorbance at 412 nm during 1 min was monitored using a UV–visible spectrophotometer (Shimadzu, UV-160A). A standard curve was prepared using the same procedure with GSH equivalents (50, 100, 150, and 200 ␮M). The total glutathione content of the pepper samples is expressed in ␮mol per gram of fresh weight. For the GSSG determination, the supernatant was first diluted 10 times with 0.5 M potassium phosphate buffer (pH 6.5). Then, 20 ␮L of 2-vinylpyridine was added to 1 mL of the mixture, and the solution was vigorously mixed. After 1-h incubation at room temperature, 20 ␮L of the mixture was removed and used for the glutathione assay described above. A standard curve was plotted using the amounts of GSSG (25, 50, 75 and 100 ␮M), and the results are expressed in ␮mol per gram of fresh weight.

2.6. Enzymatic antioxidants The APX and CAT assays both used the same extraction procedure. For 5 g of sample tissue, 10 mL of phosphate buffer (0.1 M, pH 7.0), which contained 1.0 mM EDTA and 1% polyvinylpolypyrrolidone (PVP), was used as the extraction buffer. The method used for the analysis of the APX activity was adapted from Nakano and Asada (1981). The reaction mixture contained 1.91 mL of phosphate buffer (50 mM, pH 7.0), 0.05 mL of ascorbate (0.5 mM), 0.01 mL of H2 O2 (0.1 mM) and 30 ␮L of enzyme extract. The specific activity of APX was determined by monitoring the decline in the absorbance at 290 nm using a UV–visible spectrophotometer (Shimadzu, UV160A) and is expressed as units per gram of fresh weight. One unit of APX was defined as the amount of enzyme that oxidised 1 ␮mol of ascorbate per min at room temperature. The CAT activity was estimated according to the method of Beers and Sizer (1952). The reaction mixture consisted of 0.1 mL of enzyme extract, 50 mM phosphate buffer (pH 7.0) and 15 mM H2 O2. The depletion of H2 O2 was determined by measuring the change in the absorbance at 240 nm using a UV–visible spectrophotometer (Shimadzu, UV-160A). One unit of CAT was defined as the amount of enzyme needed to reduce 1 ␮mol H2 O2 in 1 min. The specific activity is expressed as units per gram of fresh weight. The SOD activity assay was performed according to the method of Constantine and Stanley (1977). Five grams of pepper tissue was homogenised with 10 mL of phosphate buffer (0.05 M, pH 7.8) that consisted of 1% PVP and 0.025% Triton X-100. The homogenate was then centrifuged at 15,000 rpm for 20 min at 4 ◦ C. The reaction mixture (3 mL) contained 2.31 mL of sodium phosphate buffer (0.05 M, pH 7.8 and 0.025% Triton X-100), 0.30 mL of l-methionine (0.13 M), 0.30 mL of nitro blue tetrazolium (NBT) (0.63 mM), 0.04 mL of riboflavin (0.15 mM) and 0.05 mL of enzyme extract. The reaction mixtures were vortexed and illuminated with fluorescent light for 10 min. The absorbance of the mixture was then measured at 560 nm using a UV–visible spectrophotometer (Shimadzu, UV160A). One unit of SOD activity was defined as the amount of enzyme that inhibited the nitro blue tetrazolium (NBT) photoreduction by 50% under standard conditions. The specific activity is expressed as units per gram of fresh weight. GR was extracted from 5 g samples using 20 mL of cold potassium phosphate extraction buffer, which contained 1 mM EDTA, 0.5% Triton X-100, 0.1 mM 2-mercaptoethanol and 2% PVP. The assay was adapted from the method of Bergmeyer et al. (1987) with slight modifications. The reaction mixture consisted of 0.2 M potassium phosphate buffer (pH 7.5), 2 mM NADPH, 20 mM GSSG and 80 ␮L of enzyme extract. The change in absorbance was monitored at 340 nm using a UV–visible spectrophotometer (Shimadzu, UV-160A). One unit of GR was defined as the amount of enzyme needed to oxidise 1 ␮mol of NADPH in 1 min. The specific activity is expressed as units per gram of fresh weight.

Fig. 1. Changes in the ACO activity (), ethylene level (), ACS activity () and ACC content () of the pepper Kulai during ripening. The values are the means of six replicate samples ± S.E.

2.7. Statistical analysis All of the experiments were conducted with six replicates in a completely randomised design. Analysis of variance (ANOVA) was performed using the Statistical Analysis System program version 6.12 (SAS Institute Inc., Cary, NC, USA). The means were compared using the least significant differences (LSD) test at a significance level of 0.05. 3. Results and discussion 3.1. Ethylene production In the current study, the pepper fruit had a very low rate of ethylene production. However, the ethylene production in the pepper gradually increased at the beginning of ripening and reached a climacteric peak of 0.49 ± 0.021 ␮L/h/kg at stage 3, at which point the ethylene production decreased until the end of ripening (Fig. 1A). Such changes have also been observed in other cultivars of hot pepper (Gross et al., 1986). Unlike many other climacteric fruits, the production of ethylene was low in hot peppers, a behaviour that has been previously reported in sweet peppers (Tadesse et al., 2002). The ACO activity increased in parallel with the ethylene production and reached a peak activity of 1.3 ± 0.043 nmol ethylene/h/g at stage 3 of ripening (Fig. 1A). This finding is in agreement with a previous report from Zhang et al. (2006). The increased ACO activity enhanced the rates of conversion of ACC to ethylene, respiration and ethylene production. The establishment of S-adenosylmethionine (S-AdoMet) and ACC as precursors of ethylene was a major finding in the ethylene

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Fig. 2. Changes in the activity of ␤-galactosidase (), ␣-manosidase () and ␣galactosidase () in the pepper Kulai during ripening. The values are the means of six replicate samples ± S.E.

biosynthesis pathway. In the present study, the ACC content increased 75%, in parallel with the ethylene production, but returned to 0.79 ± 0.035 nmol/g at the end of the ripening process, whereas the ACS activity increased gradually throughout the ripening process (Fig. 1B). The sudden increase in the ACC content concomitant with a burst of ethylene production due to the newly synthesised ACS at the onset of ripening is a wellknown phenomenon in climacteric fruits (Lelievre et al., 1997). This observation indicated that the pepper exhibited an autocatalytic stimulation of ethylene synthesis and suggested that C. annuum cv. Kulai is a climacteric fruit that exhibits an ethylene peak during the ripening process. 3.2. ˇ-Galactosidase, ˛-mannosidase and ˛-galactosidase activity The most active of the examined pepper fruit carbohydrase enzymes is ␤-galactosidase. The ␤-galactosidase activity increased significantly (P < 0.05) during the course of ripening and had increased 7-fold by the end of the ripening process. The ␣-mannosidase activity increased gradually during the preclimacteric stage and reached a maximum at stage 3, which was followed by a gradual decline. The activity of the last carbohydrase enzyme, ␣-galactosidase, remained almost unchanged throughout the ripening process (Fig. 2). The increase in the ␤-galactosidase throughout the ripening process suggests that this enzyme may play a role in the softening of the pepper fruit. This finding is in agreement with a previous study performed on sweet peppers (Ogasawara et al., 2007) in which the softening of the peppers was accompanied by the loss of a considerable amount of galactose residues from the cell wall. ␤-Galactosidase has been shown to be correlated with the depolymerisation of matrix glycan in pepper fruits, either moderately (Harpster et al., 2002) or extensively (Gross et al., 1986). In addition, ␤-galactosidase also proved to be important in assisting pectin-modifying enzymes, such as polygalacturonase, pectin methylesterase and pectate lyase, in pectin depolymerisation (Tieman et al., 1992). The hydrolysis of the galactose residues in pectin substances appears to be the first event in the ripening process of bell pepper fruits (Ogasawara et al., 2007). As a consequence, the pectin solubility increases, and non-glucose neutral sugars, especially galactose, are lost (Redgwell et al., 1997). This finding is supported by previous study done by Brummell and Harpster (2001), who pointed out that the removal of pectin

Fig. 3. Changes in the MDA content (), LOX activity () and H2 O2 content () of the pepper Kulai during ripening. The values are the means of six replicate samples ± S.E.

galactan side-chains, is an important factor in the cell wall changes leading to ripening-related firmness loss. The significance of the role of ␤-galactosidase in modifying the cell wall has been specifically highlighted in previous studies (Chin et al., 1999). Therefore, it is reasonable to suggest that these enzymes might have played a certain role in cell wall modification which happens during ripening process. 3.3. Oxidative stress Membrane lipids and free fatty acids are highly susceptible to oxidation and, therefore, are often used as markers in oxidative stress development. The present study showed that the activity of LOX and MDA evolved antagonistically during the entire ripening process. The MDA content increased significantly (P < 0.05), up to 2fold difference was observed when the pepper fruit reached stage 5 of the ripening process, whereas the LOX activity decreased significantly (P < 0.05) during the ripening process (Fig. 3A). The apparent inverse relationship between MDA accumulation in peppers and LOX activity suggests that these two phenomena may operate independently. Similar findings have been previously reported; LOX is considered to be partially involved in lipid peroxidation (Gardner, 1995) or totally independent of MDA accumulation (Boonsiri et al., 2007). Lipoxygenase has been shown to play a central role in triggering lipid peroxidation during the early stage of ripening (Riley et al., 1996). The main products of LOX action are the fatty acid hydroperoxides, which can attack additional membrane lipids and fatty acids. In addition, hydroperoxides also act as ionophores by admitting extracellular Ca2+ , which in turn increases

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phospholipase activity, enhancing the cascade of oxidative deterioration (Hildebrand, 1989). As such, a high activity of LOX during the early stages of ripening is important for the generation of an autoperoxidation system during the later stages. The interaction of activated oxygen species at the membrane may also contribute to the lipid peroxidation during the later stages of ripening (Mckersie et al., 1988). This finding is in agreement with a previous study on bell pepper fruits (Luning et al., 1995). The gradual rise in MDA content implied that the oxidative stress in pepper fruits gradually increased during ripening. The overproduction of H2 O2 may be a causative factor for oxidative stress, which damages plants by lipid peroxidation, protein degradation and DNA fragmentation, ultimately leading to cell death (Ashraf, 2009). The ability to adjust the antioxidant systems in response to changes in the ROS concentrations is vital to the ability of all species to combat oxidative stress (Foyer et al., 2002). The measured H2 O2 content had declined significantly (P < 0.05) upon reaching the end of ripening and the decrement is about 2-fold difference with the content at early stage (Fig. 3B). These results suggested that the pepper fruit may have developed an active oxygen-scavenging system in response to elevated oxidative stress. It was apparent that the accumulation of hydrogen peroxide contents in pepper fruit appeared prior to the time of cell wall modification and accumulation of MDA. This phenomenon suggested that high levels of hydrogen peroxide could have participated in the oxidative products formation, which required for the initiation of fruit softening (Yang et al., 2008). 3.4. Antioxidant status In the present study, the total antioxidant potential of the pepper increased during the ripening process and appeared to reach a maximum level of 7.4 ± 0.31 ␮mol Fe(II) per gram of fresh weight during stage 4 of the ripening process (Fig. 4A). A similar trend has been observed in other cultivars of pepper fruit (Howard et al., 2000). This result suggested that the enhanced antioxidant potential might be a self-defensive response against the effects of oxidative stress during the ripening process (Smirnoff, 1995). The current study showed that the phenolic compounds reached a maximum concentration of 1.9 ± 0.081 mg/g FW at stage 3 and remained almost unchanged during the remainder of the ripening process (Fig. 4A). This result demonstrates that the phenolic content in peppers is highly correlated with the antioxidant capacity. This finding is similar to the finding of Howard et al. (2000), which stated that the total phenolic content increases as the pepper reaches maturity. The present study also showed that the elevation of the phenolic content was closely correlated with the total antioxidant capacity. This result is supported by a study performed with other tropical fruits (Avila et al., 2008), which stated that the phenolic content was the main contributor to the total antioxidant capacity. The trend of an increase in the phenolic content in parallel with ethylene production suggests that the biosynthesis of phenol may be induced by ethylene production. A similar finding has been previously reported regarding the biosynthesis of light-independent fat-soluble antioxidants (Hornero-Mendez et al., 2000). Ascorbic acid was first isolated and purified from Capsicum fruit because of interest in the high amount of ascorbate present in this fruit (Govindarajan, 1985). The roles of ascorbate in plant growth and in the cell division, maturity, fruit ripening and abscission phenomena have been recognised in other plants (Davey et al., 2000). The result in Fig. 4B showed that the content of ascorbic acid increased remarkably and significantly (P < 0.05) from 0.14 ± 0.0047 mg/g FW to 1.3 ± 0.054 mg/g FW during the ripening process. The large increase suggests that ascorbic acid may be actively involved in the removal of H2 O2 in response to the elevated oxidative stress (Buettner and Jurkiewicz, 1996). This

Fig. 4. Changes in the total antioxidant potential (), total phenolic content (), ascorbic acid content (), GSH () and GSSG content () of the pepper Kulai during ripening. The values are the means of six replicate samples ± S.E.

result is consistent with other reports for other cultivar of pepper, which demonstrated an increase in ascorbate levels during ripening (Osuna-Garcia et al., 1998). The role of glutathione-mediated radical scavenging was also examined as a potential system in the defence against oxidative stress. The glutathione system is directly involved in the maintenance of a low redox potential and a highly reduced intracellular environment (Tanaka et al., 1994). The total glutathione and GSSG content in peppers gradually decreased as the fruit progressed through maturity stages 1–5 (Fig. 4C). Although the declination was significant (P < 0.05), but the GSH/GSSG ratio was maintained at approximately the same level throughout the ripening process. These results show that the tolerance towards oxidative stress remained high during ripening, although the oxidative stress gradually increased. The GSH/GSSG ratio may be more important in the regulation of defence-related gene expression than the absolute amounts of either form (Mehdy, 1994).

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of Mondal et al. (2004) regarding tomatoes. However, the accumulation of other ROS would eventually lead to oxidative stress as a result of the loss of CAT activity during the later stages of ripening. Furthermore, this trend of change in the CAT activity was believed to be correlated with the change in ethylene production. This correlation has been previously observed during the on-tree ripening of apples (Masia, 1998). The agreement of these findings suggests the onset of oxidative stress at an earlier stage of ripening in the pepper fruit. Elevated lipid peroxidation as one of the normal ripening phenomena suggests that the oxidative stress was increased during the ripening process. However, this phenomenon was tightly regulated by the antioxidant system of the pepper fruit. The free-radical scavenging system was up-regulated until the end of the ripening process as a self-defensive response against oxidative stress. A similar finding has been previously observed in a study regarding berry fruits (Rogiers et al., 1998). 4. Conclusion

Fig. 5. Changes in the activity of GR (), APX (), SOD () and CAT () in the pepper Kulai during ripening. The values are the means of six replicate samples ± S.E.

In addition to antioxidants such as ascorbate and GSH, plants are also protected against the effects of ROS by enzymatic antioxidants such as APX, GR, SOD and CAT (Sala, 1998). In the pepper Kulai, the activity of APX and GR increased during the ripening process. However, the GR activity increased more significantly (P < 0.05) and had increased 3-fold by the end of ripening (Fig. 5A). In the APX–GR system, APX acts together with ascorbate to remove H2 O2 , which escapes CAT activity and is generated during respiration (Foyer et al., 1994), whereas GR helps to regenerate sufficient ascorbate for the detoxification of H2 O2 (Saruyama and Tanida, 1995). In addition, GR keeps the cellular environment reduced in the presence of NADPH by maintaining a high GSH/GSSG ratio (Gamble and Burke, 1984). The detoxification of cellular H2 O2 via the activation of this system is an important element of the defence mechanism against ROS (Lee and Lee, 2000). A similar result has been observed when hot pepper seeds are treated with solutions of different salinity levels (Supanjani and Lee, 2006) and during bell pepper ripening (Schantz et al., 1995). Therefore, the elevation of APX–GR activity may be the main reason for the decline in H2 O2 during ripening. In contrast to the activity of the APX–GR system, the activity of SOD decreased (P < 0.05) 2-fold by the end of ripening. The CAT activity increased during the early stage of ripening but declined after stage 2 (Fig. 5B). Both CAT and SOD are important active free radical scavenging enzymes (Lee and Lee, 2000). SOD detoxifies O2 − free radicals by converting them to O2 and H2 O2 (Monk et al., 1989). CAT catalyses the breakdown of H2 O2 (Bowler et al., 1992) and removes electrons, which can lead to the production of free radicals (Singh and Kesavan, 1992). The results in the present study suggested that the decrease in H2 O2 could be attributed to the decrease in SOD activity and high CAT activity during the early stage of the ripening process. This result is in agreement with the study

The pepper fruit (C. annuum cv. Kulai) was found to be a climacteric fruit with a peak in ethylene production during ripening. Considering the function of ␤-galactosidase and ␣-mannosidase and the observation of increasing activities of these enzymes throughout the ripening process, it is likely that these enzymes play an important role in cell wall depolymerisation, which appeared to be an important process during ripening. The antioxidant status of the pepper fruit was enhanced towards the end of the ripening process, likely in response to the elevated oxidative stress levels, and the total phenolic content may be the main contributor to this elevated total antioxidant capacity. The effectiveness of the APX–GR system was demonstrated by the consistent removal of H2 O2 . However, the elevated lipid peroxidation and the loss of CAT activity implicated the onset of oxidative stress during the second stage of ripening. The LOX activity in the pepper appeared to be independent of the lipid peroxidation. The overall results indicate that the antioxidant status in the pepper was maturity dependent and that the interactions among the various antioxidants are important for overcoming the elevated stresses and maintaining the fruit quality during ripening. Such a finding is of considerable importance in the pepper industry when determining the best timing to harvest peppers with high nutritional value that are capable of withstanding prolonged postharvest storage. This finding is also helpful for minimising the senescence that occurs during pepper production. Further studies may be carried out to further evaluate the antioxidant status of the pepper Kulai after harvest to develop an effective postharvest handling system for the pepper Kulai. Acknowledgements This work was supported by Research University Grant Scheme (OUP) through UKM-GUP-KPB-08-33-135 and UKM-OUP-KPB-33169/2011. Chung Keat Tan is one of the recipient of MyBrain15 Sponsorship Program (MyPhD) provided by High Education Ministry. References Ali, Z.M., Ng, S.Y., Othman, R., Goh, L.Y., Lazan, H., 1998. Isolation, characterization and significance of papaya ␤-galactosidase to cell wall modification and fruit softening during ripening. Physiol. Plant. 104, 105–115. Al-Mamary, A., 2002. Antioxidant activity of commonly consumed vegetables in Yemen. M’sia J. Nutr. 8, 179–189. Anderson, M.E., 1985. Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol. 113, 548–555. Anese, M., Sovrano, M., 2006. Kinetics of thermal inactivation of tomato lipoxygenase. Food Chem. 95, 131–137.

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C.K. Tan et al. / Scientia Horticulturae 142 (2012) 23–31

Antonious, G.F., Kochhar, T.S., Jarret, R.L., Snyder, J.C., 2006. Antioxidants in hot pepper: variation among accessions. J. Environ. Sci. Health 41, 1237–1243. Ashraf, M., 2009. Biotechnological approach of improving plant salt tolerance using antioxidants as markers. Biotechnol. Adv. 27, 84–93. Avila, P.A., Toledo, F., Park, Y.S., Jung, S.T., Kang, S.G., Heo, B.G., Lee, S.H., Sajewicz, M., Kowalska, T., Gorinstein, S., 2008. Antioxidant properties of durian fruit as influenced by ripening. Food Sci. Technol. 41, 2118–2125. Beers, R.F., Sizer, I.W., 1952. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195, 133–140. Bergmeyer, H.U., Gawehn, K., Grassl, M., 1987. Glutathione reductase. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis. Academic Press, New York, pp. 465–466. Biles, C.L., Wall, M.M., Blackstone, K., 1993. Morphological and physiological changes during maturation of New Mexican type peppers. J. Am. Soc. Hortic. Sci. 118, 476–480. Boonsiri, K., Ketsa, S., Doorn, W.G., 2007. Seed browning of hot peppers during low temperature storage. Postharvest Biol. Technol. 45, 358–365. Bou, R., Codony, R., Tres, A., Decker, E.A., Guardiola, F., 2008. Determination of hydroperoxides in foods and biological samples by the ferrous oxidation–xylenol orange method: a review of the factors that influence the method’s performance. Anal. Biochem. 377, 1–15. Bowler, C., Montague, M.V., Oxborough, K., 1992. Superoxide dismutase and stress tolerance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, 83–116. Brennan, T., Frenkel, C., 1977. Involvement of hydrogen peroxide in the regulation of senescence in pear. Plant Physiol. 59, 411–416. Brummell, D.A., Harpster, M.H., 2001. Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants. Plant Mol. Biol. 47, 311–339. Buettner, G.R., Jurkiewicz, B.A., 1996. Chemistry and biochemistry of ascorbic acid. In: Cadenas, E., Packer, L. (Eds.), Handbook of Antioxidants. Marcel Dekker Inc., New York, pp. 91–115. Camacho-Cristóbal, J.J., Anzelotti, D., González-Fontes, A., 2002. Changes in phenolic metabolism of tobacco plants during short-term boron deficiency. Plant Physiol. Biochem. 40, 997–1002. Chin, L.H., Ali, Z.M., Lazan, H., 1999. Cell wall modifications, degrading enzymes and softening of carambola fruit during ripening. J. Exp. Bot. 50, 767–775. Constantine, N.G., Stanley, K.R., 1977. Superoxide dismutases. Plant Physiol. 59, 309–314. Davey, M.W., Van, M.W., Sanmartin, M., Kanellis, A.K., Smirnoff, N., Benzie, E.F., Strain, J.J., Fletcher, J., Favell, F.D., 2000. Plant l-ascorbic acid: chemistry, function, metabolism, bioavailability and processing. J. Sci. Food Agric. 80, 825–860. Ferrie, B.J., Beaudoin, N., Burkhart, W., Bowsher, C.G., Rothstein, S.J., 1994. The cloning of two tomato lipoxygenase genes and their differential expression during tomato fruit ripening. Plant Physiol. 106, 109–118. Foyer, C.H., Descourvières, P., Kunert, K.J., 1994. Protection against oxygen radicals: an important defense mechanism studied in transgenic plants. Plant Cell Environ. 17, 507–523. Foyer, C.H., Vanacker, H., Gornez, L.D., Harbinson, J., 2002. Regulation of photosynthesis and antioxidant metabolism in maize leaves at optimal and chilling temperatures: review. Plant Physiol. Biochem. 40, 659–668. Frank, C.A., Nelson, R.G., Simonne, E.H., Behe, B.K., Simonne, A.H., 2005. Consumer preferences for color, price and vitamin C content of bell peppers. HortScience 36, 795–800. Fry, S.C., Dumville, J.C., Miller, J.G., 2001. Fingerprinting of polysaccharides attacked by hydroxyl radicals in vitro and in the cell walls of ripening pear fruit. Plant Mol. Biol. 44, 245–253. Gamble, P.E., Burke, J.J., 1984. Effect of water stress on the chloroplast antioxidant system. Alterations in glutathione reductase activity. Plant Physiol. 76, 615–621. Gardner, H.W., 1995. Biological roles and biochemistry of the lipoxygenase pathway. HortScience 30, 197–205. Giovannoni, J., DellaPenna, D., Bennett, A., Fischer, R., 1989. Expression of a chimeric polygalacturonase gene in transgenic rin (ripening inhibitor) tomato fruit results in polyuronide degradation but not fruit softening. Plant Cell 1, 53–63. Gross, K., Watada, A.E., Kang, M.S., Kim, S.D., Kim, K.S., Lee, S.W., 1986. Biochemical changes associated with the ripening of hot pepper fruit. Physiol. Plant. 66, 31–36. Govindarajan, V.S., 1985. Capsicum-production, technology, chemistry and quality. Part I. History, botany, cultivation and primary processing. Crit. Rev. Food Sci. Nutr. 22, 109–176. Harker, F.R., Redgwell, R.J., Hallett, I.C., Murray, S.H., Carter, G., 1997. Texture of fresh fruit. In: Janik, J. (Ed.), Horticultural Reviews, vol. 20. John Wiley & Sons Inc., New York, pp. 121–224. Harpster, M.H., Brummell, D.A., Dunsmuir, P., 2002. Suppression of a ripeningrelated endo-1,4-␤-glucanase in transgenic pepper fruit does not prevent depolymerization of cell wall polysaccharides during ripening. Plant Mol. Biol. 50, 345–355. Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125, 189–198. Hildebrand, D.F., 1989. Lipoxygenase. Physiol. Plant. 76, 249–253. Hornero-Mendez, D., Gomez-Ladron, R., Minguez-Mosquera, M.I., 2000. Carotenoid biosynthesis changes in five red pepper (Capsicum annuum L.) cultivars during ripening. Cultivar selection for breeding. J. Agric. Food Chem. 48, 3857–3864. Howard, L.R., Talcott, S.T., Brenes, C.H., Villalon, B., 2000. Changes in phytochemical and antioxidant activity of selected pepper cultivars (Capsicum species) as influenced by maturity. J. Agric. Food Chem. 48, 1713–1720.

Jiang, Z.Y., Woollard, A.C.S., Wolff, S.P., 1990. Hydrogen peroxide production during experimental protein glycation. FEBS Lett. 268, 69–71. Knekt, P., Kumpulainen, J., Jarvinen, R., 2002. Flavonoid intake and risk of chronic diseases. Am. J. Clin. Nutr. 76, 560–568. Lee, D.H., Lee, C.B., 2000. Chilling stress-induced changes of antioxidant enzymes in the leaves of cucumber: in gel enzyme activity assays. Plant Sci. 159, 75–85. Lee, S.H., Chung, E.J., Joung, Y.H., Choi, D., 2010. Non-climacteric fruit ripening in pepper: increased transcription of EIL-like genes normally regulated by ethylene. Funct. Integr. Genomics 10, 135–146. Lelievre, J.M., Latche, A., Jones, B., Bouzayen, M., Pech, J.C., 1997. Ethylene and fruit ripening. Physiol. Plant. 101, 727–739. Lim, C.S., Kang, S.M., Cho, J.L., 2007. Bell pepper (Capsicum annuum L.) fruits are susceptible to chilling injury at the breaker stage of ripeness. HortScience 42, 1659–1664. Lizada, M.C.C., Yang, S.F., 1979. A simple and sensitive assay for 1aminocyclopropane-l-carboxylic acid. Anal. Biochem. 100, 140–145. Luning, P.A., Carey, A.T., Roozen, J.P., Wichers, H.J., 1995. Characterization and occurrence of lipoxygenase in bell peppers at different ripening stages in relation to the formation of volatile compounds. J. Agric. Food Chem. 43, 1493–1500. Masia, A., 1998. Superoxide dismutase and catalase activities in apple fruit during ripening and postharvest and with special reference to ethylene. Physiol. Plant. 104, 668–672. Mckersie, B.D., Senaratna, T., Walker, M.A., Kendall, E.J., Hetherington, P.R., 1988. Deterioration of membranes during aging in plants: evidence for free radical mediation. In: Nooden, L.D., Leopold, A.C. (Eds.), Senescence and Aging in Plants. Academic Press, New York, pp. 441–464. Mehdy, M.C., 1994. Active oxygen species in plant defense against pathogens. Plant Physiol. 105, 467–542. Miller, A.R., 1986. Oxidation of cell wall polysaccharides by hydrogen peroxide: a potential mechanism for cell wall breakdown in plants. Biochem. Biophys. Res. Commun. 141, 238–244. Mondal, K., Sharma, N.S., Malhotra, S.P., Dhawan, K., Singh, R., 2004. Antioxidant systems in ripening tomato fruits. Biol. Plant. 48, 49–53. Monk, L.S., Fagerstedt, K.V., Crawford, R.M.M., 1989. Oxygen toxicity and superoxide dismutase as an antioxidant in physiological stress. Physiol. Plant. 76, 456–459. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867–880. Nakatsuka, A., Shiomi, S., Kubo, Y., Inaba, A., 1997. Expression and internal feedback regulation of ACC synthase and ACC oxidase genes in ripening of tomato fruit. Plant Cell Physiol. 38, 1103–1110. Namiki, M., 1990. Antioxidants/antimutagens in food. Crit. Rev. Food Sci. Nutr. 29, 273–300. Ogasawara, S., Abe, K., Nakajima, T., 2007. Pepper ␤-galactosidase 1 (PBG1) plays a significant role in fruit ripening in bell pepper (Capsicum annuum). Biosci. Biotechnol. Biochem. 71, 309–322. Osuna-Garcia, J.A., Wall, M.M., Waddell, C.A., 1998. Endogenous levels of tocopherols and ascorbic acid during fruit ripening of New Mexican-type chile (Capsicum annuum L.). J. Agric. Food Chem. 46, 5093–5096. Ou, B., Huang, D., Hampsch-Woodill, M., Flanagan, J.A., Deemer, E.K., 2002. Analysis of antioxidant activities of common vegetables employing oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays: a comparative study. J. Agric. Food Chem. 50, 3122–3128. Paliyath, G., Droillard, M.J., 1992. The mechanisms of membrane deterioration and disassembly during senescence. Plant Physiol. Biochem. 206, 331–336. Pressey, R., 1983. ␤-Galactosidase in ripening tomatoes. Plant Physiol. 7, 132–135. Redgwell, R.J., Fischer, M., Kendall, M., 1997. Galactose loss and fruit ripening: highmolecular-weight arabinogalactans in the pectin polysaccharides of fruit cell walls. Planta 203, 174–181. Riley, J.C.M., Willemo, C., Thompson, J.E., 1996. Lipoxygenase and hydroperoxide lyase activities in ripening tomato fruit. Postharvest Biol. Technol. 7, 97–107. Rogiers, S.Y., Kumar, G.N., Knowles, N.R., 1998. Maturation and ripening of Amelanchier alnifolia Nutt. are accompanied by increasing oxidative stress. Ann. Bot. 81, 203–211. Sala, J.M., 1998. Involvement of oxidative stress in chilling injury in cold-stored mandarin fruits. Postharvest Biol. Technol. 13, 255–261. Saruyama, H., Tanida, M., 1995. Effect of chilling on activated oxygen-scavenging enzymes in low temperature-sensitive and -tolerant cultivars of rice (Oryza sativa L.). Plant Sci. 109, 105–113. Schantz, M.L., Schreiber, H., Guilemaut, P., Schantz, R., 1995. Changes in ascorbate peroxidase activities during fruit ripening in Capsicum annuum. FEBS Lett. 358, 149–152. Singh, S.P., Kesavan, P.C., 1992. Post-irradiation modification of oxygen, nitrogen and nitrous oxide-mediated damage in dry barley seeds by catalase and superoxide dismutase: Influence of post-hydration temperature. Environ. Exp. Bot. 32, 329–342. Sitrit, Y., Rior, J., Blumenfeld, A., 1986. Regulation of ethylene biosynthesis in avocado fruit during ripening. Plant Physiol. 81, 130–135. Smith, D.L., Gross, K.C., 2000. A family of at least seven ␤-galactosidase genes is expressed during tomato fruit softening. Plant Physiol. 123, 1173–1183. Smith, D.L., Abbott, J.A., Gross, K.C., 2002. Down-regulation of tomato ␤galactosidase results in decreased fruit softening. Plant Physiol. 129, 1755–1762. Smirnoff, N., 1995. Antioxidant systems and plant response to the environment. In: Smirnoff, N. (Ed.), Environment and Plant Metabolism: Flexibility and Acclimation. BIOS Scientific, Oxford, pp. 217–243. Supanjani, T., Lee, K.D., 2006. Hot pepper response to interactive effects of salinity and boron. Plant Soil Environ. 52, 227–233.

C.K. Tan et al. / Scientia Horticulturae 142 (2012) 23–31 Tadesse, T., Hewett, E.W., Nichols, M.A., Fisher, K.J., 2002. Changes in physiochemical attributes of sweet pepper cv. Domino during fruit growth and development. Sci. Hortic. 93, 91–103. Tanaka k. Sano, T., Tshizuka, K., Kitta, K., Kawamura, Y., 1994. Comparison of properties of leaf and root glutathione reductases from spinach. Physiol. Plant. 91, 353–358. Tieman, D.M., Harriman, R.W., Ramamohan, G., Handa, A.K., 1992. An antisense pectin methylesterase gene alters pectin chemistry and soluble solids in tomato fruit. Plant Cell 4, 667–679. Tomás-Barberán, F.A., Espín, J.C., 2001. Phenolic compounds and related enzymes as determinants of quality in fruits and vegetables. J. Sci. Food Agric. 81, 853–876. Vinson, J.A., Dabbag, Y.A., Serry, M.M., Jang, J., 1995. Plant flavonoids, especially tea flavonols, are powerful antioxidants using an in vitro oxidation model for heart disease. J. Agric. Food Chem. 43, 2800–2802.

31

Wong, C.C., Li, H.B., Cheng, K.W., Chen, F., 2006. A systematic survey of antioxidants activity of 30 Chinese medicinal plants using the ferric reducing antioxidant power assay. Food Chem. 97, 705–711. Yang, S., Su, X., Prasad, K.N., Yang, B., Cheng, G., Chen, Y., Yang, E., Jiang, Y., 2008. Oxidation and peroxidation of postharvest banana fruit during softening. Pak. J. Bot. 40, 2023–2029. Zhang, D.L., Hamauzu, Y., 2003. Phenolic compounds, ascorbic acid, carotenoids and antioxidant properties of green, red and yellow bell peppers. J. Food Agric. Environ. 1, 22–27. Zhang, X., Koo, J., Eun, J.B., 2006. Antioxidant activities of methanol extracts and phenolic compounds in Asian pear at different stages of maturity. Food Sci. Biotechnol. 15, 44–50.