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Improvement of Salt Tolerance in Rice (oryza Sativa L.) by Increasing Antioxidant Defense Systems Using Exogenous Application of Proline

Dhiman Bhusan
Department of Soil Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Debasish Kumar Das
Department of Soil Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Mahmud Hossain
Department of Soil Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Yoshiyuki Murata
Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan

Md. Anamul Hoque
Department of Soil Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Proline accumulation contributes to the protection of plants against salt stress by inducing antioxidant defense systems. To investigate the protective effects of proline against salt stress, salt-sensitive (BRRI dhan29) and moderately salt-tolerant (BRRI dhan47) rice cultivars were grown in pots. Rice plants were exposed to different concentrations of NaCl at active tillering stage, and proline solutions (0, 25 and 50 mM) were sprayed on the leaves. Salt stress caused a significant reduction in growth and yield of both rice cultivars. Growth was drastically reduced and both cultivars failed to produce grains at 50 and 100 mM NaCl stresses, and even application of proline failed to compensate the adverse effects at those high salt stresses. Exogenous proline showed a significant increase in plant growth of both cultivars at 25 mM NaCl stress, and that proline increased grain yield of only salt-sensitive rice at same salt stress. Salt stress significantly decreased chlorophyll and ascorbate contents, straw K+/Na+ ratio and activity of antioxidant enzyme guaiacol peroxidase (POX) in both cultivars. Intracellular proline content increased in salt-sensitive rice but decreased in salt-tolerant rice under salt stress. Exogenous proline increased chlorophyll, intracellular proline and ascorbate contents, K+/Na+ ratio and activities of antioxidant enzymes in salt-sensitive rice at 25 mM NaCl stress although most of those data were not affected in salt-tolerant rice. The present study; therefore, suggests that exogenous proline confers tolerance to salt stress in salt-sensitive rice by maintaining higher K+/Na+ ratio and enhancing proline accumulation and antioxidant defense systems.

Keywords: Antioxidant enzymes, Chlorophyll, Proline, Rice growth, Salt stress

Location: Net house of the Department of Soil Science, Bangladesh Agricultural University, Mymensingh

Start Date:

End Date:

Program Area: Risk Management in Agriculture

Commodity: Rice

Objectives:

To investigate the effects of exogenous proline on the growth, chlorophyll, intracellular proline and ascorbate contents, and activity of antioxidant enzymes in two contrasting rice genotypes exposed to salt stress.

Methodology:

Soil collection and pot preparation - Pot experiments were carried out in the net house of the Department of Soil Science, Bangladesh Agricultural University, Mymensingh. Soils were collected from 0-15 cm depth from Bangladesh Agricultural University farm. A total of 80 plastic pots were prepared with 8 kg soils in each pot. The soil was silt loam having pH 6.15, electrical conductivity 0.17 dS m^-1, exchangeable Na 0.35 meq 100 g^-1 soil, total N 0.11% and organic matter 1.90%. Plant materials and treatments - Two rice (Oryza sativa L.) cultivars viz. BRRI dhan29 (salt-sensitive) and BRRI dhan47 (moderately salt-tolerant) were used in the experiment. Ten treatment combinations viz. control (no NaCl or proline), 25 mM NaCl, 25 mM NaCl+25 mM proline, 25 mM NaCl+50 mM proline, 50 mM NaCl, 50 mM NaCl+25 mM proline, 50 mM NaCl+50 mM proline, 100 mM NaCl, 100 mM NaCl +25 mM proline, and 100 mM NaCl +50 mM proline were used for the two rice cultivars. Three healthy seedlings of 30-day-old were transplanted in a single hill in each pot. NaCl was used as per treatments for developing salinity. Rice plants were exposed to different concentrations of NaCl at 30 days after transplanting (active tillering stage). Active tillering refers to a stage when tillering rate is high with leaf emergence at regular intervals. On the same day, different doses of proline containing 0.1% Tween-20 were sprayed on the leaves at a volume of 25 mL per plant as per treatments. The experiment was laid out in a completely randomized design with four replications. Management practices, crop harvesting and data recording - Normal water was used as irrigation. Fertilization and other management practices were performed as and when required. At 15 days after proline application, healthy green leaves were collected from rice plants to determine chlorophyll, proline and ascorbate contents, and activity of antioxidant enzymes. The crop was harvested at full maturity. Grain and straw yields and plant parameters were recorded. The K and Na contents from grain and straw samples were analyzed. Assay of chlorophyll contents - Chlorophyll content was measured. An aliquot amount of fresh green leaf was suspended in 10 mL of 80% acetone, mixed well and kept at room temperature in the dark for 7 days. The supernatant was collected after centrifugation at 5000 rpm for 15 min. The absorbance was recorded at 645 nm and 663 nm using a spectrophotometer (model 336001, Spectronic Instruments, USA). Assay of intracellular proline contents - Proline content was determined following the standard method. An aliquot amount of fresh green leaf was homogenized in 10 mL of 3% sulfosalicylic acid and then the homogenate was centrifuged at 5000 rpm for 15 min. Other details of the assay were followed. Assay of ascorbate contents - Ascorbate content was determined by 2,6-dichlorophenolindophenol visual titration method where ascorbate stoichiometrically reduced the dye 2,6-dichlorophenolindophenol to colorless compound. An aliquot amount (0.5 g) of green leaf was blended in 10 mL of 3% metaphosphoric acid using a blender to yield homogenous extract. The whole extract was filtered through a piece of cheese cloth and washed with 3% metaphosphoric acid solution. Ten mL of aliquot of the filtrate was titrated against the standardized dye. Preparation of enzyme extract and assay of antioxidant enzymes - An aliquot amount of fresh green leaf was homogenized with 5 mL of 50 mM Tris–HCl buffer (pH 8.0) for CAT, and 50 mM KH₂PO₄ buffer (pH 7.0) for POX and APX. The homogenate was centrifuged at 5000 rpm for 20 min and the supernatant was then used as enzyme extract. CAT (EC: 1.11.1.6) activity was assayed. POX (EC: 1.11.1.7) and APX (EC: 1.11.1.11) activities were assayed. Digestion of plant samples for K and Na determination - Grain and straw samples were dried in an oven at about 65°C for 48 h and then ground in a grinding machine to pass through a 20-mesh sieve. Ground sample (0.5 g) was taken into a 100 mL digestion vessel. Ten mL of diacid mixture (HNO₃:HClO₄= 2:1) was added into the vessel. After leaving for a while, the vessels were heated at a temperature slowly raised to 200°C. Heating was stopped when the dense white fume of HClO₄ occurred. After cooling, the content was transferred into a 50 mL volumetric flask and the volume was made with distilled water. The digest was diluted to make a desired concentration. The K and Na contents in the digest were determined using a flame photometer (Jencon PFP 7, JENCONS-PLS, UK). Statistical analysis - Data were analyzed statistically using analysis of variance with the help of software package MSTAT-C. The significant differences between mean values were compared by Duncan’s Multiple Range Test. Differences at P≤0.05 were considered significant.

Source of Information: AJCS 10(1):50-56 (2016); ISSN:1835-2707

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Funding Source:

Budget:

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Achievement/Findings:

Salt-tolerant rice maintained higher antioxidant defense systems without elevated levels of proline accumulation than salt-sensitive rice during salt stress. On the contrary, exogenous proline improved the growth and grain yield of salt-sensitive rice under salt stress by increasing proline accumulation and antioxidant defense systems as well as maintaining higher K+/Na+ ratio. However, the biomolecular mechanisms of proline in plant responses to salinity remain to be elucidated. Further studies are required to elucidate the biomolecular mechanisms and signaling pathways underlying the roles of proline in salt tolerance of plants.

Knowledge Management: Journal