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Diverse Antioxidative Effects in Pui Vegetable (basella Alba) Induced by High Temperature Stress

M.M. Islam
Department of Biochemistry and Molecular Biology, Laboratory of Protein and Enzyme Research, University of Rajshahi, Rajshahi-6205, Bangladesh

M.S. Haque
Department of Biochemistry and Molecular Biology, Laboratory of Protein and Enzyme Research, University of Rajshahi, Rajshahi-6205, Bangladesh

M.K. Hossain
Department of Biochemistry and Molecular Biology, Laboratory of Protein and Enzyme Research, University of Rajshahi, Rajshahi-6205, Bangladesh

M.M. Hasan
Department of Biochemistry and Molecular Biology, Laboratory of Protein and Enzyme Research, University of Rajshahi, Rajshahi-6205, Bangladesh

Abstract/ Executive Summary:

Basella alba is a green vegetable and grows in both winter and summer; however the temperature sensitivity on metabolic regulation in this species is not clarified. To identify the physiological mechanisms involved in regulation of adaptive response and anti oxidative effects in leaves of Basella alba, plants grown in pot were exposed to high temperature (45^oC) for 24h, 48h and 72h periods and polyphenol oxidase (PPO) and peroxidase (POD) activities in leaves were examined. High temperature causes the higher activity of PPO and the activity was found to be potential after prolonged exposure when compared to the respective controls. Dose response characteristics of substrate on PPO activity were performed. The activity was higher at 10 mM catechol, substrate for the enzyme, than 100 mM and 200 mM concentration, however, the three doses yielded the gradual increase in activity. Conversely, POD activity in leaf was regulated reciprocally and found to be prevented up to 72h of treatment however the effects were appeared to be pronounced after 48h of exposure. The above findings demonstrate that assay of PPO and POD in response to high temperature is an index for characterization of anti oxidative effects in this species of plant and will give a new insight for adaptive response to the adverse environment.

Keywords: Temperature stress, Metabolic effects, Basella alba, Adaptive response

Location: Department of Biochemistry and Molecular Biology, Laboratory of Protein and Enzyme Research, University of Rajshahi, Rajshahi

Start Date:

End Date:

Program Area: Knowledge Management

Non-commodity: Temperature

Objectives:

The current investigation has been undertaken to find the role of high temperature exposure on the regulation of metabolic functions regarding the alteration and synthesis of PPO and POD in leaf of Basella alba and may assist in the clarification of such stress induced mechanisms.

Methodology:

Plant materials and high temperature treatment - For this experiment, two plastic pots were used; each pot size was 70 cm in diameter and 24 cm in height. An adequate amount of soil was taken in each plastic pot and the plastic pots were seeded with Basella alba. For the germination of seeds, the following points were carried out: i) the strong seeds were selected; the seeds were added to normal water and the floating seeds were discarded; ii) the seeds were kept in normal water with temperature below 37 oC for overnight; iii) the seeds which were swollen by water absorption, were expected to be effective for germination; iv) the seeds were seeded in the pots prepared with soil and the efficiency of seed germination was 65%–75%. After 20 days of germination, the two different pots were described as control and high temperature induced plant. Control pot was used for 24h, 48h and 72h treatments in room temperature, however, the temperature was maintained 30^oC by using air cooling system (AC) already fixed in the room and without giving any high temperature. The second pot was used similarly for 24h, 48h and 72h duration in the plastic chamber and was exposed to 45 oC with full aeration along with sufficient water. To maintain this temperature, electric bulbs (2×200 W) were connected to the chamber. After the treatments, the leaves were collected consecutively from each pot for 24h, 48h and 72h duration and kept in 80^oC. Assay of polyphenol oxidase (PPO) activity - The leaves of the different treatments (24h, 48h and 72h) and their respective controls were homogenized with 22 mL of distilled water in a mortar kept on ice. Approximately, 1.5 g of high temperature induced and their respective control leaves were used for homogenization. The homogenates were centrifuged at 9000 rpm for 15 min and the supernatants were used as crude extract for assay of PPO activity spectrophotometrically based on an initial rate of increase in absorbance at 495 nm where, catechol was used as substrate. One unit of enzyme activity is defined as a change in absorbance of 0.001 minute^–1 mL^–1 of enzyme extract. For determination of PPO activity in leaf, 3 mL of 0.1 M phosphate buffer (pH 6.0) and 2 mL of crude enzyme extract were taken in the test tube and kept on ice. The contents were mixed, placed in a spectrophotometer using a cuvette and the absorbance was adjusted to zero at 495 nm. The cuvette was removed, 1 mL of catechol (10 mM, 100 mM and 200 mM) was added, quickly mixed by inversion and the changes in absorbance at 495 nm were recorded for up to 3 minutes (1, 2, 3 min). In all experiments, three replicates were performed for each sample. The following calculation was used to assay PPO activity in a sample: change in A495 = Af – Ai, where, Ai = initial absorbance reading and Af = final absorbance reading. Change in A495 for each sample was used to calculate the units of PPO activity and the activity is expressed as Unit g^–1 of leaf weight. Assay of peroxidase (POD) activity - The leaves of the different treatments (24h, 48h and 72h) and their respective controls were homogenized with 15 mL of 0.5 M phosphate buffer (pH 7.0) in a mortar kept on ice. Approximately, 1.5 g of high temperature induced and their respective control leaves were used for homogenization. The homogenates were centrifuged at 9000 rpm for 15 min and the supernatants were used as crude extract for assay of POD activity. Peroxidase activity was determined spectrophotometrically at 470 nm using guaiacol as a phenolic substrate with hydrogen peroxide. The enzymatic oxidation of guaiacol changed the substrate into orange-pink product which was measured by spectrophotometer as a change in absorbance of 0.001 min^–1 and the absorbance was recorded for up to 4 min. For determination of POD activity in leaf, 2.5 mL of 0.1 M phosphate buffer (pH 7.0), 2 mL of crude enzyme extract and 0.6 mL of 1% (v/v) H₂O₂ were taken in a test tube and kept on ice. The spectrophotometer was adjusted to zero initially and the content of test tube was transferred into a cuvette and the absorbance was taken as initial reading. 0.6 mL of 4% (v/v) guaiacol was added to the cuvette, quickly mixed by inversion and the change in absorbance at 470 nm was measured for 1, 2, 3 and 4 min. In all experiments, three replicates were performed for each sample. The following calculation was used to assay peroxidase activity in a sample: change in A470 = Af – Ai, where, Ai = initial absorbance reading and Af = final absorbance reading. Change in A470 for each sample was used to calculate the units of POD activity and the activity was expressed as Unit g^–1 of leaf weight. Statistical analysis - Results of the experiments were expressed as mean and standard error of different groups based on three independent determinations. The differences between the mean values were evaluated by ANOVA followed by paired t-test using SPSS software.

Source of Information: International Journal of Agronomy and Agricultural Research (IJAAR); ISSN: 2223-7054 (Print) 2225-3610 (Online); Vol. 5, No. 5, p. 135-147, 2014

Article Link (Online Journal): http://www.innspub.net

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

It is obvious from the current investigation that high temperature induced diverse metabolic alterations regarding the enhancement of polyphenol oxidase and prevention of peroxidase activity in leaf therefore, assumes to be involved in the alteration of physiology of plants Basella alba. The adverse environment caused by high temperature produced the severe effect on the plants and thereby plants face the stress to physiological and molecular level and therefore, different regulatory metabolic alterations were observed in the circumstances. High temperature induced oxidative stress and injury is frequently observed in the critical environment and to overcome these effects, some enzymes are over expressed where PPO might be involved. Moreover denaturation of enzymes and proteins in response to high temperature is commonly observed, thereby peroxidase enzyme has been assumed to be deactivated in this species, therefore, the anti oxidative enzymes are reversibly regulated by high temperature which is an essential parameter for characterization for the prevention of the anti oxidative effects in this species of vegetables. Species adapted by natural selection to hot environments have evolved a number of physiological and morphological means to improve survival in the face of extended hot periods. During high temperature acclimation, plants possess nutritional or energy deficiency as they survive in such critical circumstances however, the complications might be due to the higher oxidative effects and the enzymes may play the critical role in this respect.

Knowledge Management: Journal