Nitric Oxide Enhances Photosynthetic Nitrogen and Sulfur-Use Efficiency and Activity of Ascorbate-Glutathione Cycle to Reduce High Temperature Stress-Induced Oxidative Stress in Rice (Oryza sativa L.) Plants

The effects of nitric oxide (NO) as 100 µM sodium nitroprusside (SNP, NO donor) on photosynthetic-nitrogen use efficiency (NUE), photosynthetic-sulfur use efficiency (SUE), photosynthesis, growth and agronomic traits of rice (Oryza sativa L.) cultivars, Taipie-309 (high photosynthetic-N and SUE) and Rasi (low photosynthetic-N and SUE) were investigated under high temperature stress (40 °C for 6 h). Plants exposed to high temperature stress caused significant reduction in photosynthetic activity, use efficiency of N and S, and increment in H2O2 and thiobarbituric acid reactive substance (TBARS) content. The drastic effects of high temperature stress were more pronounced in cultivar Rasi than Taipie-309. However, foliar spray of SNP decreased the high temperature induced H2O2 and TBARS content and increased accumulation of proline and activity of ascorbate–glutathione cycle that collectively improved tolerance to high temperature stress more effectively in Taipie-309. Exogenously applied SNP alleviated the high temperature induced decrease in photosynthesis through maintaining higher photosynthetic-NUE and photosynthetic-SUE, activity of ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco), and synthesis of reduced glutathione (GSH). The use of 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxy-3-oxide (cPTIO, NO scavenger) substantiated the study that in the absence of NO oxidative stress increased, while NO increased photosynthetic-NUE and photosynthetic-SUE, net photosynthesis and plant dry mass. Taken together, the present investigation reveals that NO increased heat stress tolerance and minimized high temperature stress adversaries more effectively in cultivar Taipie-309 than Rasi by enhancing photosynthetic-NUE and SUE and strengthening the antioxidant defense system.


Estimation of proline
Fresh leaf tissue (300 mg) was homogenized in 3 ml of 3% sulphosalicylic acid using mortar and pestle. 1 ml each of acid ninhydrin and glacial acetic acid was added to the homogenate filtrate. Then, reaction was carried for 1 h in a test tube placed in a water bath at 100°C, then the solution was cooled and the reaction was terminated. The mixture was extracted using 4 ml toluene mixed vigorously for few seconds and the OD value was measured in a spectrophotometer (UV-vis L164, Elico, New Delhi) for colored components at 520 nm using L-proline as a standard.

Determination of TBARS
Fresh leaf tissue (500 mg) was homogenized in 0.25% 2-thiobarbituric acid in 10% trichloroacetic acid using mortar and pestle. Homogenized mixture was heated at 95°C for 30 min and then the mixture was cooled on ice bath and centrifuged at 10,000 × g for 10 min. The absorbance of the supernatant was read at 532 nm and corrected for nonspecific turbidity by subtracting the absorbance of the same at 600 nm. The amount of thiobarbituric acid reactive substances (TBARS) was calculated using an extinction coefficient (155 mM -1 cm -1 ).

Determination of H2O2
Fresh leaf tissue (500 mg) was homogenized in pre-cooled 200 mM perchloric acid (HClO4) using mortar and pestle, followed by centrifugation at 1200 × g for 10 min. Centrifuged supernatant containing perchloric acid was neutralized with 4 M KOH, followed by further centrifugation at 500 × g for 3 min to remove insoluble potassium perchlorate (KClO4). In a final volume of 1.5 ml, the content of reaction mixture were 1 ml of eluate, 400 µl of 12.5 mM 3-(dimethylamino) benzoic acid in 0.375 M phosphate buffer (pH 6.5), 80 µl of 3-methyl-2benzothiazoline hydrazone and 20 µl of peroxidase (0.25 unit). Further peroxidase was added at 25°C to start reaction mixture and the increase in absorbance was recorded at 590 nm for 3 min. Later, H2O2 content was calculated using an extinction coefficient (0.28 µmol -1 cm -1 ).

Assay of antioxidant enzymes activities
Activity of APX (EC 1.11.1.11) was determined according to the method described by Nakano and Asada (1981) by recording the decrease in absorbance of ascorbate at 290 nm. The assay mixture contained phosphate buffer (50 mM, pH 7.0), 0.1 mM EDTA, 0.5 mM ascorbate, 0.1 mM H2O2 and the enzyme extract. The activity of APX was calculated by using the extinction coefficient 2.8 mM -1 cm -1 . One unit of enzyme was the amount necessary to decompose 1 µmol of substrate per min at 25°C. Activity of SOD (EC 1.15.1.1) was determined according to the methods of Beyer and Fridovich (1987) and Giannopolitis and Ries (1977) measured on the basis of potential of the enzyme to prevent the photochemical reduction of nitro blue tetrazolium chloride (NBT). A (5.0 ml) of reaction mixture containing 5.0 mM HEPES (pH 7.6), 50 mM Na2CO3 (pH 10.0), 0.1 mM EDTA, 0.025% (v/v) Triton X-100, 13 mM methionine, 1.3 µmol riboflavin and 63 µmol NBT. The enzyme extract was illuminated for 15 min (360 µmol m -2 s -1 ) and a control set was not illuminated to correct the turbidity of background absorbance. A unit of SOD was defined as the amount of enzyme required to cause 50% inhibition of the reaction of NBT at 560 nm.
Activity of GR (EC 1.6.4.2) was determined by the method of Foyer and Halliwell (1976) by monitoring the glutathione dependent oxidation of nicotinamide adenine dinucleotide phosphate (NADPH) at 340 nm. The assay mixture contained phosphate buffer (25 mM, pH 7.8), 0.5 mM oxidized glutathione (GSSG), 0.2 mM NADPH, and the enzyme extract. The activity of GR was calculated by using extinction coefficient 6.2 mM -1 cm -1 . One unit of enzyme is the amount necessary to decompose 1 µmol of NADPH per min at 25°C.

Nitrate reductase activity
Fresh leaf tissue (1.0 g) was grinded in liquid N2 using chilled mortar and pestle and then powder was stored at -80°C. The powder was thawed for 10 min. at 4°C and homogenized in Tris-HCl buffer (250 mM and pH 8.5) containing 1.0 mM DTT, 1.0 mM EDTA, 20 mM FAD, 10 mM cysteine and 10% (v/v) glycerol. Homogenate was centrifuged at 10,000 × g for 30 min. at 4°C. Using the procedure of (Nakagawa et al., 1984) NR activity was measured as the rate of nitrite production at 28 C. The assay mixture contained 10 mM KNO3, HEPES (0.065 M, pH 7.0), 0.5 mM NADH in phosphate buffer (0.04 mM, pH 7.2) and the enzyme extract in the final volume of 1.5 ml. NADH was added for initiating the reaction, incubated for 15 min. and the reaction was terminated by adding 1.0 ml of 1.0 N HCl solution containing 1% sulphanilamide followed by the addition of 1.0 ml of 0.02% aqueous N-1-napthylethylene-di-amine-dihydrochloride. The absorbance was read at 540 nm after 10 min.