Effect of abscisic acid and hydrogen peroxide on antioxidant enzymes in Syzygium cumini plant

Abstract

The present study was undertaken to study the effect of abscisic acid and hydrogen peroxide on the activities of antioxidant enzymes namely superoxide dismutase (SOD; E.C. 1.15.1.1), catalase (CAT; E.C. 1.11.1.6) and ascorbate peroxidase (APX; E.C. 1.11.1.11) in Syzygium cumini plant. The varying concentrations of ABA (2–8 mM/l) and H₂O₂ (2–8 mM/l) modulated enzyme activities differently. In general, some concentrations of the ABA and H₂O₂ stimulated the activities of all the three enzymes except that there was a dose dependent reduction in catalase activity in the plants treated with ABA.

Introduction

Antioxidants play an important role in preventing the stress-induced accumulation of reactive oxygen species (Foyer and Noctor 2005). The ROS cause rapid cell damage under stress conditions by reacting on membrane lipids, proteins and nucleic acids (Hariyadi and Parkin 1993; O’Kane et al. 1996; Prasad 1996) but also perform useful functions in cell signalling through interrelating different stimuli and respective responses (Desikan et al. 2004, Gomez et al. 2004, Rubio et al. 2009). The increased ROS content is controlled by an antioxidant system including low molecular weight antioxidant metabolites (ascorbate, tocopherol, glutathione etc.) and antioxidative enzymes such as superoxide dismutase, ascorbate peroxidase, catalase and guaiacol peroxidase (Suzuki and Mittler 2006, Turhan et al. 2008). The antioxidants thus contribute to improved stress tolerance through detoxification of ROS. Modulation of these antioxidants under various stress conditions is therefore an important adaptive response to withstand adverse condition. Indeed, maintenance of a high antioxidant capacity in cells has been related to increased tolerance against different kinds of environmental stress (Dat et al. 1998, Thomas et al. 1999).

Abscisic acid exerts prominent role in plants in response to environmental stress (Seo and Koshiba 2002). ABA induced the synthesis of hydrogen peroxide and superoxide under stressful conditions and caused oxidative stress (Guan et al. 2000). On the other hand, ABA also stimulates the antioxidant defense system in plants to yield more antioxidants that can resist the oxidative stress (Jiang and Zhang 2001). Abscisic acid is a natural growth regulator which accumulates in plants under stress conditions. However, exogenous ABA significantly decreased the yields in plants indicating a trade-off between new growth of plant and phytochemical synthesis (Zheng et al. 2010). H₂O₂ is a versatile molecule that may be involved in several cell processes under normal and stress conditions (Quan et al. 2008). Pretreatment with low concentrations of H₂O₂ molecule increases the accumulation of glutathione (Murphy et al. 2002) and antioxidant enzyme activity (Azevedo Neto et al. 2005) in plants, thereby alleviating harm from ROS (Murphy et al. 2002; Wahida et al. 2007). Syzygium cumini plant belongs to Myrtaceae family and grows naturally in clayey loamy soil in tropical as well as in sub-tropical zones of Indo-gangetic plains. These species are reported to be very rich in tannins, flavonoids, essential oils, anthocyanins and other phenolic constituents as antioxidant compounds (Deepak et al. 2011). The present investigation was under taken to study the effects of different concentrations of ABA and H₂O₂ on antioxidant enzymes in Syzygium cumini plant.

Materials and methods

Plant material

One month old seedlings of Syzygium cumini obtained from Forest Training Institute, Jaipur were grown in pots containing soil and manure under natural photoperiod in the Department of Botany, University of Rajasthan, Jaipur, Rajasthan, India. The plants of uniform height were selected and watered regularly. Two month old plants were irrigated with various concentrations of ABA (2–8 mM/l) and H₂O₂ (2–8 mM/l). After 20 days of treatment, newly growing leaves were taken and analyzed for activities of antioxidant enzyme.

Preparation of enzyme extract

Leaf tissues (0.5 g) was ground to a fine powder in 2 ml of 50 mM potassium phosphate buffer (pH 7.0), 1 mM ethylenediamine tetraacetic acid (EDTA), 1 mM ascorbic acid (AA), 2% (w/v) polyvinylpyrrolidone (PVP) and 0.05% (w/v) Triton X-100 using a chilled pestle and mortar. The homogenate was centrifuged at 10,000 g for 20 min at 4°C and the supernatants thus collected was used for the assays of catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (GPX). Protein concentrations in the enzyme extract were determined by the method of Bradford (1976) using bovine serum albumin (BSA; Sigma, fraction V) as a standard.

Catalase (EC 1.11.1.6)

Catalase (CAT) activity was determined spectrophotometrically by measuring the rate of H₂O₂ disappearance at 240 nm (Aebi 1984). The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0) and 10.5 mM H₂O₂. The reaction was run at 25°C for 2 min, after adding the enzyme extract and rate of decrease in absorbance at 240 nm (E = 39.4 mM⁻¹ cm⁻¹) was used to calculate the enzyme activity.

Ascorbate peroxidase (EC 1.11.1.11)

Ascorbate peroxidase (APX) was assayed by the method as described by Nakano and Asada (1981). The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 0.2 mM EDTA, 0.5 mM ascorbic acid and 0.25 mM H₂O₂. The reaction was started at 25°C by the addition of H₂O₂ after adding the enzyme extract. The decrease in absorbance at 290 nm for 1 min was recorded and the amount of APX was calculated from the extinction coefficient 2.8 mM⁻¹ cm⁻¹.

Superoxide dismutase (EC 1.15.1.1)

For assay of superoxide dismutase (SOD), fresh leaves (1 g) were homogenized in 8 ml potassium phosphate buffer (50 mM, pH 7.8) containing 0.1 mM Na₂–EDTA and 1% insoluble PVP with a chilled pestle and mortar. The homogenate was centrifuged at 20,000 g for 20 min. The supernatant was collected and used for the assay of SOD following the method of Beauchamp and Fridovich (1971). Reaction mixture was prepared by mixing 27 ml of 50 mM potassium phosphate, pH 7.8, 1.5 ml of L-methionine (300 mg/10 ml) and 1 ml of nitroblue tetrazolium salt (NBT) (14.4 mg/10 ml). Aliquots (2 ml) of this mixture were delivered into small glass tubes, followed by 20 μl of enzyme extract and 10 μl of riboflavin (4.4 mg/100 ml). The mixture was illuminated for 15 min in an aluminum foil-lined box, containing two 20 W fluorescent tubes. A control tube in which the sample was replaced by 20 μl of buffer was run in parallel and the absorbance at 560 nm was measured in all tubes. Under the described conditions, the increase in absorbance without the enzyme extract was taken as 100% and the enzyme activity was calculated by determining the percent inhibition per minute. 50% inhibition was taken as equivalent to 1 unit of SOD activity.

Statistical analysis

Each experiment was repeated three times. Values were expressed as means ± SE. One-way analysis of variance (ANOVA) was used for comparisons between the means. In all cases the confidence coefficient was set at 0.05.

Results and discussion

Diverse environmental stresses differentially affect plant processes that lead to loss of cellular homeostasis accompanied by ROS, which causes oxidative damage to membranes, lipids, proteins and nucleic acids (Srivalli et al. 2003). The response of ascorbate peroxidase activity was more significant at the minimum concentration of ABA (2 mM/l) as compared to control plant shown in Fig. 1(a). However no significant differences were observed with higher concentration of ABA treatment. Previous study supported the increasing activity of APX enzyme (Bellaire et al. 2000). Among the four concentrations of hydrogen peroxide tested, the 6 mm/l was most effective in increasing activity of ascorbate peroxidase enzyme as shown in Fig. 1(b).

Fig. 1.

a ABA effect on APX enzyme b H₂O₂ effect on APX enzyme c ABA effect on CAT enzyme d H₂O₂ effect on CAT enzyme e ABA effect on SOD enzyme f H₂O₂ effect on SOD enzyme

The CAT activity decreased gradually with increasing concentration of ABA, the maximum decrease being 70% under the influence of 8 mM/l of ABA concentration (Fig. 1(c). The maximum activity was observed in control plant (9.78 unit/g fr.wt.). Unlike the ABA, the increasing concentrations of hydrogen peroxide consistently increased activity of CAT shown in Fig. 1(d). The SOD activity increased with increasing ABA, the maximum being at 8 mM/l (Fig. 1(e). On the other hand, H₂O₂ treated plants showed significant increase in SOD enzyme activity reaching the maximum at 4 mm/l of H₂O₂ (Fig. 1(f)_.

It has been well documented that ABA can result in the increased generation of ROS, increased expression of antioxidant genes, and enhanced capacity of antioxidant defense systems in plants (Jiang and Zhang 2004). Previous studies showed that ABA induced the apoplastic H₂O₂ accumulation in maize leaves and increased the activities of antioxidant enzymes such as SOD and APX (Hu et al. 2005, 2006; Zhang et al. 2006). Agarwal et al. (2005) reported a decrease in oxidative stress in ABA treated in wheat cultivars.

The present study confirms that there was a drastic decrease in CAT activity but initial and overall increase in APX and SOD activity respectively in ABA treated plants. Hydrogen peroxide treatment was very effective in increasing APX, CAT and SOD enzyme activity. Previous studies suggested that coordinate function of antioxidant enzymes such as APX, CAT, SOD and glutathione reductase helps in processing of ROS and regeneration of redox ascorbate and glutathione metabolites (Halliwell 1974; Wise 1995; Asada 1996; Foyer and Nector 2000). A considerable and significant increase in the activity of SOD enzyme was recorded in both ABA and H₂O₂ treated plants.