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Roles of Glutathione S-transferease in Maize (zea Mays L.) Under Cold Stress

MA Khaleque Mian
Professor
Bangabandhu Sheikh Mujibur Rahman Gazipur 1701

Abstract/ Executive Summary:

Glutathione S-transferease (GST) activities involved in antioxidant defense and methylglyoxal detoxification were investigated in the seedlings of a Bangladeshi maize variety, BARI hybrid maize-7, to understand the protecting mechanism under cold stress condition. The activities of glutathione S-transferase (GST) increased, while the activities of catalase (CAT) decreased with the duration of stress. The western blot analysis of the dominant GST revealed that it significantly accumulated during the stress period. The continual increase in H2O2 contents along with reduced redox state and activities suggested their roles in maintaining the glutathione homeostasis. The accumulation of GST with the content of H2O2 suggested its detoxification roles for organic hydroperoxides during chilling stress. Considering all, glutathione S-transferase (GST) enzymes showed protective role in maize from oxidative damages under Chilling condition.

Keywords: Glutathione, Glutathione S-transferase, BARI hybrid maize-7, Cold stress

Location: Molecular Breeding Lab, BARI, Gazipur

Start Date: 30-11-2010

End Date: 30-06-2013

Program Area: Variety and Species

Commodity: Maize

Objectives:

Biomolecular responses of some glutathione dependent enzyme in adaptive response of BMH 7 under chilling

Methodology:

Plant material
Maize (Zea mays L.) was managed in greenhouse condition as plant material. Bangladesh Agricultural Research Institute (BARI) released hybrid maize variety BARI Hybrid Maize-7 (BMH-7), has stress tolerance capability, and was taken into consideration.Stress treatment Maize seedlings were grown in pot in greenhouse condition. After reaching two leaf stages, the seedlings were ready for stress treatment. The plants were kept at 40C temperature to induce chilling stress.Separation and purification of GSTs from crude enzyme solution of maize seedlings.Crude enzyme was extracted by homogenizing 80 g of fresh maize seedlings (except green part) in an equal volume of 25 mMTris-HCl buffer (pH 8.5), which contained 1 mM EDTA and 1% (w/v) ascorbate, with a Waring lender. The homogenate was squeezed through two layers of nylon cloth and centrifuged at 11500×g for 10 minutes, and the supernatant was used as a crude enzyme solution. Proteins were precipitated by ammonium sulfate at 65% saturation of the crude enzyme solution and centrifuged at 11500×g for 10 minutes. The proteins were dialyzed against 10 mM Tris-HCl buffer (pH 8) containing 0.01% (w/v) β-mercaptoethanol and 1 mM EDTA (buffer A) overnight to completely remove low molecular inhibitors. The dialyzate was applied to a column (1.77 cm i.d. × 20 cm) of DEAEcellulose (DE-52; Whatman, U.K.) that had been equilibrated with buffer A and eluted with a linear gradient of 0 to 0.2 M KCl in 600 ml of buffer A. The high active fractions of the highest GST peak were pooled and used to detect their inhibitory substances present in maize seedlings
extract.The pooled GST solution was directly applied on affinity column (0.76 cm i.d. × 4.0 cm) of S-hexylglutathioneagarose (Sigma, St. Louis, MO) that had been equilibrated with 10 mMTris-HCl buffer (pH 8.0) containing 0.01% (v/v) β-mercaptoethanol (buffer B). The column was washed with buffer B containing. 0.2 M KCl and eluted with buffer B containing 1.5 mM S-hexylglutathione. The high active protein fractions eluted with S-hexylglutathione were combined and dialyzed against buffer B and the dialyzate was used as the purified GST. Production of polyclonal antibodies against GST A rabbit (weighing about 2.5 kg) received subcutaneous injections of a 0.5 mg of purified GST protein in Freund’s complete adjuvant at several sites. After two weeks, the rabbit was given a first booster injection of 0.5 mg of the purified GST protein in incomplete adjuvant, and then a second booster injection of 0.5 mg of the purified protein in incomplete adjuvant was given two weeks after the first booster injection. Blood was taken from the ear vein one week after the second booster injection.SDS-PAGE and Western blotting
SDS-PAGE was done in 12.5% (w/v) gel containing 0.1% (w/v) SDS by the method of Laemmli (1970).The gel was stained with silver. Western blotting was done on the basis of the Amersham ECL detection system.Protein quantificationProtein was estimated following the method of Bradford (1976). Preparation of soluble protein extracts The plant materials were homogenized with proportional volumes of 25 mMTris–HCl buffer (pH 8.0) containing 1 mM EDTA, 1% (w/v) ascorbate and 10% (w/v) glycerol with mortar pestle. The homogenate was centrifuged at 11500 x g for 15 min, and the supernatant was used as the crude enzyme solution. All procedures were performed at 0-4°C. For H2O2 assay, Fresh seedling samples were extracted by homogenizing in 6X volume of 50 mM K-P buffer (pH 6.5). The homogenate was centrifuged at 11500 x g for15 min, and 2.5 ml supernatant was used for further centrifugation at 11500 x g for 15 min with 833 μl reaction mixture containing 5ml H2SO4 and 15 μl TiCl4. The 2nd supernatant was used for the assay of H2O2 spectrophotometrically.Enzyme assayGlutathione S-transferase (GST, EC: 2.5.1.18) activity was assayed following the methods of Rohman et al. (2010) spectrophotometrically (UV-1800, Shimadzu, Japan). The activity was calculated using the extinction coefficient of 9.6 mM-1Cm-1.Measurement of H2O2H2O2 was assayed according to the method described by Yu et al. (2000). H2O2 was extracted by homogenizing 0.5 g of leaf samples with 3 ml of 50 mM K-phosphate buffer pH (6.5) at 40C. The homogenate was centrifuged at 11,500g for 15 min. Three ml of supernatant was mixed with 1 ml of 0.1% TiCl4 in 20% H2SO4 (v/v), and the mixture was then centrifuged at 11,500g for 12 min at room temperature. The optical absorption of the supernatant was measured spectrophotometrically at 410 nm to determine the H2O2 content (= 0.28 μM-1 Cm-1) and expressed as μmol g-1 fresh weight. SDS-PAGE and Western Blotting to check GST accumulation To check the accumulation of GSTa SDS-PAGE was done in 12.5% (w/v) gel containing 0.1% (w/v) SDS by the method of Laemmli (1970) followed by western blotting.

Source of Information: Res. Agric. Livest. Fish.Vol. 2, No. 1, April 2015: 09-15

Article Link (Online Journal):

Funding Source:

1. Government

Budget: Two lacs only

Total Budget (Tk.) : Two lacs only

Achievement/Findings:

Regulation of maize GST was learnt under chilling stress to know the underlying stress tolerance mechanism.

Knowledge Management: Journal