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

Md. Mahfuzur Rahman
Bangabandhu Sheikh Mujibur Rahman Agricultural University, Salna, Gazipur-1706,

MA Khaleque Mian
Bangabandhu Sheikh Mujibur Rahman Agricultural University, Salna, Gazipur-1706, Bangladesh

Asgar Ahmed
Breeding Division, Bangladesh Agricultural Research Institute, Joydevpur, Gazipur-1701, Bangladesh

Md. Motiar Rohman*
Breeding Division, Bangladesh Agricultural Research Institute, Joydevpur, Gazipur-1701, Bangladesh

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) de creased 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) enzymessh owed protective role in maize from oxidative damages under Chilling condition.

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

Location: Bangladesh Agricultural Research Institute

Start Date:

End Date:

Program Area: Seed Technology

Commodity: Maize

Objectives:

To investigate the biomolecular responses of some glutathione dependent enzyme in adaptive response of BMH 7 under chilling in seedling sage in green house condition.

Methodology:

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.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 4ºC temperature to induce chilling stress.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. Protei ns 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 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 Shexylglutathioneagarose (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. 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. H₂O₂ was assayed according to the method. H₂O₂ was extracted by homogenizing 0.5 g of leaf samples with 3 ml of 50 mM K-phosphate buffer pH (6.5) at 4ºC. 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 spectr ophotometrically at 410 nm to determine the H₂O₂ content (= 0.28 µM ^-1 Cm ^-1 ) and expressed as µmol g ^-1 fresh weight.

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

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The activity level and Western blotting of GSTa in maize under chilling stress suggested their protective role of maize under cold. The catalase activity showed the decreasing trend over time. The induction of CAT over control was maximum at 4 day (232.48%) followed by 7 day (45.82%) and 10 day (34.98%) of stress treatment. Lee and Lee (2000) supported the result as they also found that under chilling stress CAT activity decreased in cucumber over time. The reduction of CAT activity is supposedly due to the inhibition of enzyme synthesis or change in the assembly of enzyme subunits under chilling stress condition.

Knowledge Management: Journal