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Exogenous Proline and Glycine Betaine Mediated Up Regulation of Antioxidant Defense and Glyoxalase Systems Provides Better Protection Against Salt-induced Oxidative Stress in Two Rice (oryza Sativa L.) Varieties

Mirza Hasanuzzaman
Department of Agronomy, Faculty of Agriculture, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka 1207, Bangladesh

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

Anisur Rahman
Department of Agronomy, Faculty of Agriculture, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka 1207, Bangladesh

Md. Hasanuzzaman
Department of Agronomy, Faculty of Agriculture, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka 1207, Bangladesh

Kamrun Nahar
Department of Agricultural Botany, Faculty of Agriculture, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka 1207, Bangladesh

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:

The present study investigates the roles of exogenous proline (Pro, 5 mM) and glycine betaine (GB, 5 mM) in improving salt stress tolerance in salt-sensitive (BRRI dhan49) and salt-tolerant (BRRI dhan54) rice (Oryza sativaL.) varieties. Salt stresses(150 and 300 mM NaCl for 48 h) significantly reduced leaf relative water (RWC) and chlorophyll (chl) content and increased endogenous Pro and increased lipid peroxidation and H2O2levels. Ascorbate (AsA), glutathione (GSH) and GSH/GSSG, ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione peroxidase (GPX), catalase (CAT), and glyoxalase I (Gly I) activities were reduced in sensitive variety and these were increased intolerant variety due to salt stress. The glyoxalase II (Gly II), glutathione S-transferase (GST), and superoxide dismutase (SOD) activities were increased in both cultivars by salt stress. Exogenous Pro and GB application with salt stress improved physiological parameters and reduced oxidative damage in both cultivars where BRRI dhan54 showed better tolerance. The result suggests that exogenous application of Pro and GB increased rice seedlings’ tolerance to salt-induced oxidative damage by upregulating their antioxidant defense system where these protectants rendered better performance to BRRI dhan54 and Pro can be considered as better protectant than GB.

Keywords: Exogenous Proline and Glycine, Salt-Induced Oxidative Stress, Rice

Location:

Start Date:

End Date:

Program Area: Risk Management in Agriculture

Commodity: Rice

Objectives:

We investigated the protective effects of these osmoprotectants on the antioxidant defense and glyoxalase systems in rice seedlings grown under saline media. We also investigated the comparative performance of two modern rice varieties differing in their salt tolerance to know their actual adaptive mechanisms under salt stress with or without osmoprotectants.

Methodology:

2.1. Plant Materials and Stress Treatments.Seeds of two rice (Oryza sativaL.) cultivars, cv. BRRI dhan49 (salt-sensitive) and cv. BRRI dhan54 (salt tolerant) was collected from Bangladesh Rice Research Institute (BRRI) and surface-sterilized with 70% ethanol for 10 min followed by washing several times with sterilized distilled water. Seeds were then soaked for 24 h in the dark. Seeds were sown on plastic nets upon plastic beakers containing distilled water and kept in the dark at 28±2⁰C for germination. After 48 h, uniformly germinated seeds were transferred to the growth chamber with controlled conditions (light intensity, 100mol m⁻² s⁻¹; temperature, 25±2⁰C; relative humidity, 65–70%) and during the growing period hyponex solution (Hyponex, Japan) was used as a nutrient. Two levels of salt stresses (150 and 300 mMNaCl) were imposed on fourteen-day-old rice seedlings with, without 5 mM proline [l (-) Proline, Wako, Japan] and betaine (Betaine, Wako, Japan), and where these protectants were sprayed twice a day mixing with the wetting agent 0.02%Tween 20 (Tween 20, Wako, Japan). Control plants were grown with Hyponex solution only. Data were taken after 48 h of NaCl treatment. The experiment was repeated three times under the same conditions. 2.2. Measurement of Relative Water Content.Relative water content (RWC) was measured according to Barrs and Weath-erley 33]. Leaf laminas were weighed (fresh wt, FW) and then immediately floated on distilled water in a Petri dish for 8 h in the dark. Turgid weights (TW) were obtained after drying excess surface water with paper towels. Dry weights (DW)were measured after drying at 80⁰C for 48 h. 2.3. Determination of Chlorophyll Content. Chlorophyll content was determined by homogenizing leaf samples (0.5 g) BioMed Research International 3 with 10 mL of acetone (80% v/v) followed by centrifuging at 5,000×g for 10 min. The absorbance was measured with a UV-visible spectrophotometer at a specified wavelength and chl contents were calculated using the equations proposed by Arnon. 2.4. Determination of Proline Content.Free proline in leaf tissues was appraised following the protocol of Bates et al. Fresh leaf tissue (0.5 g) was homogenized in 10 mL of 3% sulfosalicylic acid in ice. The homogenate was centrifuged at11,500×g for 15 min. Two mL of the filtrate was mixed with 2mL of acid ninhydrin and 2mLof glacial acetic acid. After incubation at 100⁰C for 1h it was cooled and4mLoftoluenewas added. The optical density of the chromophore containing toluene was read spectrophotometrically at 520 nm using toluene as a blank. The amount of Pro was determined by comparison with a standard curve. of 2.5. Measurement of Lipid Peroxidation.The level of lipid peroxidation was measured by estimating MDA, using thiobarbituric acid (TBA) as the reactive material following the method of Heath and Packer with slight modifications. The leaf samples (0.5 g) were homogenized in 3 mL5% (w/v) trichloroacetic acid (TCA) and the homogenate was centrifuged at 11,500×gfor10min. One mL supernatant was mixed with 4m L of TBAreagent (0.5% of TBA in 20% TCA). The reaction mixture was heated at 95⁰C for 30 min in a water bath and then quickly cooled in an ice bath and centrifuged at 11,500×g for 15min. The absorbance of the colored supernatant was measured at 532 nm and was corrected for nonspecific absorbance at 600 nm. The concentration of MDA was calculated by using the extinction coefficient of 155 mM⁻¹ cm⁻¹ and expressed as nmol of MD Ag⁻¹ fresh weight. 2.6. Measurement of H2O2. H2O2 was assayed according to the method described by Yu et al.H2O2 was extracted by homogenizing 0.5 g of leaf samples with 3 mL of 50 m M potassium-phosphate (K-P) buffer (pH 6.5) at 4⁰C. The homogenate was centrifuged at 11,500×g for 15 min. Three milliliters of supernatant were mixed with 1 mL of 0.1% TiCl4in20% H2SO4 (v/v) and kept in room temperature for 10 min.After that the mixture was again centrifuged at 11,500×g for 15 min. The optical absorption of the supernatant was measured spectrophotometrically at 410 nm to determine the H2O2 content (L=0.28 M⁻¹cm⁻¹) and expressed as nmol g⁻¹fresh weight. 2.7. Extraction and Measurement of Ascorbate and Glutathione. Rice 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 11,500×g for15 min at 4⁰C and the supernatant was collected for analysis of ascorbate and glutathione. Ascorbate content was determined following the method of Huang et al. with some modifications. The supernatant was neutralized with 0.5 M K-P buffer (pH 7.0). The AsAwas assayed spectrophotometrically at 265 nm in 100 mM K-P buffer (pH 7.0) with 0.5 units of ascorbate oxidase (AO). A specific standard curve with As A was used for quantification. Based on enzymatic recycling, GSH is oxidized by 5,5-dithio-bis (2-nitrobenzoicacid) (DTNB) and reduced by NADPH in the presence of GR, and glutathione content is evaluated by the rate of absorption changes at 412 nm of 2-nitro-5-thiobenzoicacid (NTB) generated from the reduction of DTNB. GSSGwas determined after removal of GSH by 2-vinyl pyridine derivatization. Standard curves with known concentrations of GSH and GSSG were used. The content of GSH was calculated by subtracting GSSG from total GSH.2.8. Determination of Protein.The protein concentration of each sample was determined following the method of Bradford [40]usingBSA as a protein standard.

Source of Information: BioMed Research International Volume 2014, Article ID 757219, 17 pages

Article Link (Online Journal): http://dx.doi.org/10.1155/2014/757219

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Achievement/Findings:

Considering the above results we, therefore, conclude that exogenous Pro and GB are effective protectants to improve short-term salt tolerance both in salt-sensitive BRRI dhan49and salt-tolerant BRRI dhan54 as Pro and GB effectively maintained better physiological conditions and significantly alleviated oxidative damages of rice seedlings by enhancing the antioxidant and glyoxalase systems. In all the cases BRRI dhan54 was a better performer under salt stress. Although the studied antioxidant and glyoxalase enzymes were improved by both the osmoprotectants; the GB could not restore the nonenzymatic antioxidants of salt-sensitive cultivar BRRI dhan49 in some cases. Considering these facts it can be assumed that Pro is good for salt-sensitive cultivar present studied condition. From the comparative studies of salt-tolerant and sensitive cultivars, it can be said that enhancement of tolerance by Pro and GB even in the salt-sensitive cultivar is an interesting point and this result deserves further intrinsic researches. The dose duration-dependent study with Pro and GB in different salinity levels might be elucidated. Mechanisms of Pro and GB as osmoprotectants, free radical scavengers, enzyme activators, or as regulators of other physiological processes were stated in many articles but merely studied under different stress conditions. Studies on their protective mechanisms and signaling cascades are the further scope of research.

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