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Exogenous Nitric Oxide Alleviates High Temperature Induced Oxidative Stress in Wheat (triticum Aestivum L.) Seedlings by Modulating the Antioxidant Defense and Glyoxalase System

Mirza Hasanuzzaman
Department of Agronomy, Faculty of Agriculture, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka-1207, Bangladesh; Laboratory of Plant Stress Responses, Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795, Japan

Kamrun Nahar
Department of Agricultural Botany, Faculty of Agriculture, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka-1207, Bangladesh; Laboratory of Plant Stress Responses, Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795, Japan

Md. Mahabub Alam
Laboratory of Plant Stress Responses, Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795, Japan

Masayuki Fujita
Laboratory of Plant Stress Responses, Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795, Japan

Abstract/ Executive Summary:

We investigated the protective role of exogenous nitric oxide (NO) in alleviating high temperature induced damages of wheat (Triticum aestivum L. cv. Pradip) seedlings. Heat treatment (38 °C) alone or in combination with 0.5 mM SNP (a NO donor) was applied with nutrient solution on 8-d-old hydroponically grown seedlings for a period of 24 h and 48 h. Heat stress significantly decreased the Chl content and increased the lipid peroxidation (MDA) and H₂O₂ levels in time depending manners. Ascorbate (AsA) content markedly decreased upon heat treatment but glutathione (GSH) and glutathione disulfide (GSSG) content increased. Heat treatment resulted in an increase of the activities of antioxidant enzymes - ascorbate peroxidase (APX), glutathione reductase (GR), glutathione peroxidase (GPX) and glutathione S-transferase (GST). The activities of glyoxalase enzymes also increased upon heat stress. Exogenous NO supplementation in the seedlings had little influence on the nonenzymatic and enzymatic components compared to the control. However, supplementation of heat-treated seedlings with SNP significantly reduced the high temperature induced lipid peroxidation, H₂O₂ content and increased the content of Chl, AsA and GSH as well as the GSH/GSSG ratio. Heat treated seedlings which were supplemented with SNP also upregulated the activities of APX, MDHAR, DHAR, GR, GST, CAT and Gly I. This study concludes that an exogenous supply of NO protects wheat seedlings from high temperature induced oxidative stress by upregulating antioxidant defense and methylglyoxal (MG) detoxification system.

Keywords: Abiotic stress, Glutathione, Heat stress, Reactive oxygen species, Signaling molecules, Sodium nitroprusside

Location: Laboratory of Plant Stress Responses, Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795, Japan

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Wheat

Objectives:

To evaluate the effect of exogenous NO (SNP) on the activity of the antioxidant and glyoxalase pathway enzymes as well as on the levels of oxidative stress indicators, such as lipid peroxide, H₂O₂ levels and chlorophyll (Chl) content of wheat.

Methodology:

Plant materials and stress treatments - Wheat (Triticum aestivum L. cv. Pradip) seeds of uniform size were selected and surface-sterilized with 1% sodium hypochloride solution for 10 minutes followed by washing several times with sterile distilled water. The seeds were then sown in Petri plates (9 cm) lined with 6 layers of filter paper moistened with 10 mL of distilled water for germination for two days. Germinated seedlings were then allowed to grow under controlled conditions (light, 100 μmol photon m^–2 s^–1; temperature, 25±2°C; RH, 65–70%) that contained 10,000-fold diluted Hyponex solution (Hyponex, Japan). After 8 days, seedlings were grown with 0.25 mM sodium nitroprusside (SNP, Na₂[Fe(CN)₅NO]?2H₂O – a NO donor) or without (control) at high temperatures at 38 °C for 24 and 48 h. Control plants were grown in Hyponex solution only. The experiment was repeated three times under the same conditions.
Determination of chlorophyll content - Chlorophyll (Chl) content was determined by taking fresh leaf samples (0.5 g) from randomly selected seedlings. The samples were homogenized with 10 mL of acetone (80% v/v) using pre-cooled pestle and mortar and the homogenate was centrifuged at 5,000x g for 10 min. The absorbance was measured with a UV-visible spectrophotometer at 663 and 645 nm and Chl contents were calculated. Measurement of lipid peroxidation - The level of lipid peroxidation was measured by estimating MDA, a decomposition product of the peroxidized polyunsaturated fatty acid component of the membrane lipid, using thiobarbituric acid (TBA) as the reactive material. Measurement of H₂O₂ - H₂O₂ was assayed. H₂O₂ was extracted by homogenizing 0.5 g of leaf samples with 3 mL of 50 mM potassium-phosphate (K-P) buffer (pH 6.5) at 4° C. The homogenate was centrifuged at 11,500x g for 15 min. Three mL of supernatant was mixed with 1 mL of 0.1% TiCl₄ in 20% H₂SO₄ (v/v) and kept in room temperature for 10 min. After that the mixture was again centrifuged at 11,500x g for 15 min. The optical absorption of the supernatant was measured spectrophotometrically at 410 nm to determine the H₂O₂ content (?=0.28 μM^–1cm^–1) and expressed as μmol g^–1 fresh weight. Extraction and measurement of ascorbate and glutathione - Wheat leaves (0.5 g fresh weight) were homogenized in 3 mL ice-cold acidic extraction buffer (5% meta-phosphoric acid containing 1 mM EDTA) using a mortar and pestle. Homogenates were centrifuged at 11,500x g for 15 min at 4°C and the supernatant was collected for analysis of ascorbate and glutathione. Ascorbate content was determined. The glutathione pool was assayed according to previously described methods with modifications. Standard curves with known concentrations of GSH and GSS were used. The content of GSH was calculated by subtracting GSSG from total GSH. Determination of protein - The protein concentration of each sample was determined following the method of Bradford (1976) using BSA as a protein standard. Statistical analysis - All data obtained were subjected to analysis of variance (ANOVA) and the mean differences were compared by a Duncan's multiple range test (DMRT) test using XLSTAT v.2010 software. Differences at P < 0.05 were considered significant.

Source of Information: AJCS 6(8):1314-1323 (2012); ISSN:1835-2707

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

This study concluded that an exogenous supply of NO protects wheat seedlings from high temperature induced oxidative stress by upregulating antioxidant defense and methylglyoxal (MG) detoxification system.

Knowledge Management: Journal