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Calcium Mitigates Arsenic Toxicity in Rice Seedlings by Reducing Arsenic Uptake and Modulating the Antioxidant Defense and Glyoxalase Systems and Stress Markers

Anisur Rahman
Laboratory of Plant Stress Responses, Department of Applied Biological Science, Faculty of Agriculture, Kagawa University,Miki-cho, Kita-gun, Kagawa 761-0795, Japan

Mohammad Golam Mostofa
Laboratory of Plant Stress Responses, Department of Applied Biological Science, Faculty of Agriculture, Kagawa University,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,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 Nagor,Dhaka 1207, Bangladesh

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

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

Abstract/ Executive Summary:

The effect of exogenous calcium (Ca) on hydroponically grown rice seedlings was studied under arsenic (As) stress by investigating the antioxidant and glyoxalase systems. Fourteen-day-old rice (Oryza sativa L. cv. BRRI dhan29) seedlings were exposed to 0.5and 1 mM Na2HAsO4 alone and in combination with 10 mM CaCl2(Ca) for 5 days. Both levelsofAscausedgrowthinhibition, chlorosis, reduced leaf RWC and increased As accumulation in the rice seedlings. Both doses of As in growth medium induced oxidative stress through the overproduction of reactive oxygen species (ROS) by disrupting the antioxidant defense and glyoxalase systems. Exogenous application of Ca along with both levels of As significantly decreased As accumulation and restored plant growth and water loss. Calcium supplementation in the As-exposed rice seedlings reduced ROS production, increased ascorbate(AsA) content, and increased the activities of monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT), glutathione peroxidase (GPX), superoxide dismutase (SOD), and the glyoxalase I (Gly I) and glyoxalase II (GlyII) enzymes compared with seedlings exposed to As only. These results suggest that Ca supplementation improves rice seedling tolerance to As-induced oxidative stress by reducing As uptake, enhancing their antioxidant defense and glyoxalase systems, and also improving growth and physiological condition

Keywords: Calcium Mitigates, Arsenic Toxicity, Rice Seedlings, Glyoxalase Systems, Stress Markers

Location:

Start Date:

End Date:

Program Area: Risk Management in Agriculture

Commodity: Arsenic

Objectives:

The present study was designed to investigate the influential role of exogenous Ca in alleviating As toxicity by regulating the antioxidant defense and glyoxalase systems along with Asaccumulation and other physiological processes.

Methodology:

Plant Materials and Treatments. Rice (Oryza sativaL. cv. BRRI dhan29) seeds were surface-sterilized with 70% ethanol for 8–10 min followed by washing several times with sterilized distilled water and soaked in distilled water in a dark place for 48 h. The imbibed seeds were then sown on plastic nets floating on distilled water in 250 mL plastic beakers and kept in the dark at 28±2⁰C for 48 h. Uniformly germinated seeds were then transferred to a growth chamber (light, 350 mol photon m−2s−1; temperature, 25±2⁰C; and relative humidity,65–70%) with the same pot providing a diluted (7500 times)commercial hydroponics nutrient solution (Hyponex, Japan).The nutrient solution contained 8% N, 6.43% P, 20.94% K,11.8% Ca, 3.08% Mg, 0.07% B, 0.24% Fe, 0.03% Mn, 0.0014%Mo, 0.008% Zn, and 0.003% Cu. The nutrient solutions were renewed twice a week. Fourteen-day-old rice seedlings were exposed to Ca (10 mM CaCl2) and As (0.5 mM and1mM Na2HAsO4) separately and in combination. Control plants were grown in Hyponex solution only. Therefore, our experiments consisted of six treatments as follows: control,10 mM CaCl2(Ca), 0.5 mM Na2HAsO4(As0.5), 0.5 mMNa2HAsO4+ 10 mM CaCl2,1mMNa2HAsO4(As1), and1mM Na2HAsO4+ 10 mM CaCl2. The experiment was repeated three times under the same conditions. Data were taken after 5 days of treatment. 2.2. Observation of Seedling Growth. Seedling growth of the As-treated rice seedlings was determined by measuring dry weight (DW). For DW, seedlings were oven-dried at 80⁰C for 48 h. Dry weight is expressed as g ten seedlings⁻¹. 2.3. Determination of Leaf Relative Water Content. Relative water content (RWC) of the leaf was measured according to Barrsand Weatherley [19]. Fresh leaf laminas were weighed (fresh weight, FW), then placed immediately between two layers of filter paper, and immersed in distilled water in a petri dish for24 h in a dark place. Turgid weight (TW) was measured after gently removing excess water with a paper towel. Dry weight (DW) of leaf laminas was measured after 48 h oven-drying at 80⁰C. .4. Determination of As Content. Arsenic content was determined by using an atomic absorption spectrophotometer (Hitachi Z-5000; Hitachi, Japan). The plant samples were oven-dried at 80⁰C for 72 h. The dried samples from roots and shoots (0.1 g) were ground and digested separately with an acid mixture at 80⁰C for 48 h. The acid mixture consisted ofHNO3:HClO4 (5: 1 v/v).2.5. Determination of Chlorophyll Content. Chlorophyll (chl)content was measured according to Arnon by homogenizing leaf samples (0.5 g) with 10 mL of acetone (80% v/v)followed by centrifuging at 9,000×g for 10 min. 2.6. Determination of Pro Content. Proline content was determined according to Bates et al. [21]. Leaf samples (0.5 g)were homogenized in 5 mL 3% sulfosalicylic acid and the homogenate was centrifuged at 11500×g for 12 min. The supernatant (1 mL) was mixed with 1 mL glacial acetic acid and1 mL acid ninhydrin. After 1 h incubation at 100⁰C, the mixture was cooled. The developed color was extracted with 2mL toluene and the optical density of the chromophore was observed spectrophotometrically at 520 nm. Proline content was determined by comparing with a standard curve of known concentration of Pro.2.7. Determination of Lipid Peroxidation. The level of lipid peroxidation was measured by estimating malondialdehyde (MDA, a product of lipid peroxidation) following the method of Heath and Packer. Leaf samples (0.5 g) were homogenized in 3 mL 5% (w/v) trichloroacetic acid (TCA)and the homogenate was centrifuged at 11500×g for 15 min. The supernatant (1 mL) was mixed with 4 mL thiobarbituric acid (TBA) reagent (0.5% of TBA in 20% TCA), heated in a water bath at 95⁰C for 30 min, and then quickly cooled by transferring to an ice bath. MDA content was measured by observing the difference in absorbance at 532 nm using an extinction coefficient of 155 mM−1cm−1and expressed as nmol of MDA g−1FW.2.8. Determination of H2O2 Content. Hydrogen peroxide content was determined according to Yu et al. by extracting 0.5 g leaf in a potassium-phosphate buffer (K-P buffer)(pH 6.5). The homogenized leaf tissues were centrifuged at11500×g for 15 min and then treated with a mixture of TiCl4 in 20% H2SO4.H2O2 content was measured by observing the absorbance at 410 nm using an extinction coefficient.

Source of Information: BioMed Research InternationalVolume 2015, Article ID 340812, 12 pages

Article Link (Online Journal): http://dx.doi.org/10.1155/2015/340812

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Achievement/Findings:

Considering the above results, the present study suggests that As exposure in the growth medium disrupts the antioxidant defense system by overproducing ROS, which induces oxidative stress. Arsenic in the growth medium also negatively changed other physiological conditions, including DW, RWC, Proaccumulation, chl content, and the glyoxalase system. Excess As in the growth medium also caused higher As accumulation in the plants along with other physiological changes that ultimately arrested plant growth. Arsenic-induced dam-age in the rice seedlings increased with increasing As concentration in the growth medium. However, supplementation with Ca in the As-treated rice seedlings reduced As uptake, enhanced the antioxidant defense and glyoxalase systems, and resulted in other physiological changes that positively modulated As-induced damage in the rice seedlings.

Knowledge Management: Journal