Plant material and growth conditions
Rice (Oryza sativa L. ‘Zhongzheyou No. 1’ hybrid indica) seedlings, obtained from the China National Rice Research Institute, were grown hydroponically in a greenhouse. Seeds were sterilized in 1% (v/v) sodium hypochlorite solution. After germination, seeds were transferred to a 0.5 mM CaCl2 solution (pH 5.5). Three days later, the seedlings were transferred to 1.5-L black plastic pots containing a solution with the following composition: NH4NO3 (0.5 mM), NaH2PO4·2H2O (0.18 mM), KCl (0.18 mM), CaCl2 (0.36 mM), MgSO4·7H2O (0.6 mM), MnCl2·4H2O (9 μM), Na2MoO4·4H2O (0.1 μM), H3BO3 (10 μM), ZnSO4·7H2O (0.7 μM), CuSO4 (0.3 μM), and FeSO4·7H2O–EDTA (ethylenediaminetetraacetic acid) (20 μM). All experiments were performed in a controlled growth room under the following conditions: 14/10 h light/dark photoperiod, 400 μmol·m− 2·s− 1 light intensity, 28/23 °C during the day and night, respectively, and 60% relative humidity. The solution pH was adjusted to 5.5 with 5 mM 2-(N-morpholino)ethanesulfonic acid (MES). The solution was replaced every 3 days.
After 6 days, seedlings of similar size were cultivated under the four following treatments: 1 mM NO3−, 1 mM NO3− + 10% PEG (PEG-6000), 1 mM NH4+ and 1 mM NH4+ + 10% PEG-6000. Water stress was induced by adding 10% PEG-6000. Eight treatments were performed in the NO donor (SNP) experiments: NH4+, NH4+ + SNP, NH4+ + PEG-6000, NH4+ + PEG-6000 + SNP, NO3−, NO3− + SNP, NO3− + PEG-6000, and NO3− + PEG-6000 + SNP. The final SNP concentration was 20 μM. Each treatment had six replicates. For each N nutrition, plants cultivated under non-water stress condition were defined as the control (Con) relative to the water stress (PEG) condition.
To determine the role of NO in the plant antioxidant defense system under water stress, rice seedlings supplied with 1 mM NO3− or 1 mM NH4+ solution were pretreated with 100 μΜ c-PTIO (as NO scavenger) for 3 h, and then subjected to non-water stress (Con treatment) or water stress (PEG) for 24 h under the same condition as those described above. Each treatment had six replicates.
To investigate the origin of the endogenous NO produced under water stress, rice seedlings supplied with 1 mM NO3− or 1 mM NH4+ solution were pretreated with the NR inhibitor (tungstate, 100 μΜ) or NOS inhibitor (L-NAME, 100 μΜ) for 3 h, and then subjected to non-water stress (Con) or water stress for 24 h under the same conditions as described above. There were eight treatments for each N nutrition: tungstate, L-NAME, tungstate + SNP, PEG-6000 + tungstate, PEG-6000 + tungstate + SNP, L-NAME + SNP, PEG-6000 + L-NAME, and PEG-6000 + L-NAME + SNP. Each treatment had six replicates.
Determination of NO and ONOO−contents
The DAF-FM DA probe was used to determine the endogenous root NO level . Root tips (1 cm) were incubated with 10 μM DAF-FM DA in the dark for 30 min, washed three times with 20 mM HEPES–KOH (pH 7.4) to remove excess fluorescence, and then observed and photographed under a Nikon Eclipse 80i fluorescence microscope (Nikon, Tokyo, Japan; excitation filter 460–500 nm, dichroic mirror 505 nm, barrier filter 510–560). The relative fluorescence intensity was measured with Photoshop v. 7.0 (Adobe Systems, Mountain View, CA, USA).
Root endogenous ONOO− was determined using the aminophenylfluorescein (APF) probe method . Root tips were incubated with 10 μM APF dissolved in 10 mM Tris–HCl (pH 7.4) in the dark for 60 min, and then washed three times with 10 mM Tris–HCl. Fluorescence images and relative fluorescence intensities were analyzed as described above for NO.
Lipid peroxidation and root cell death were detected histochemically with Schiff’s reagent and Evans blue . Root tips were incubated in Schiff’s reagent for 20 min and washed by three consecutive immersions in 0.5% (w/v) K2O3S solution. A red/purple endpoint indicated the presence of aldehydes generated by lipid peroxidation. Roots were also washed by performing three serial immersions in distilled water, then incubated in 0.25% (w/v) Evans blue for 15 min, and finally washed three times with distilled water. Roots stained with Schiff’s reagent and Evans blue were immediately photographed under a Leica S6E stereomicroscope (Leica, Solms, Germany).
The oxidative damage level, specifically expressed as membrane lipid peroxidation and protein oxidative damage, was estimated by measuring the concentrations of MDA and carbonyl group with 2,4-dinitrophenylhydrazine (DNPH) .
Determination of ROS contents
Root O2.- content was estimated using the method described in Liu et al.  with some modifications: about 0.15 g fresh root was powdered with 2 mL of 65 mM phosphate buffer saline (PBS, pH 7.8) in a pre-cooled mortar, and centrifuged at 5000 g and 4 °C for 10 min. Then, 0.9 mL of 65 mM PBS (pH 7.8) and 0.1 mL of 10 mM hydroxylammonium chloride were added to 1 mL of the root extract supernatant, thoroughly mixed, and left to react for 25 min. After this period, 1 mL of 1% (w/v) sulfanilamide and 1 mL of 0.02% (w/v) N-(1-naphthyl)-ethylenediaminedihydrochloride were added to 1 mL of root extract solution and left to react for 30 min. Absorbance was then measured at 540 nm.
Root H2O2 content was determined by the photocolorimetric method : ~ 0.15 g fresh root was powdered with 2 mL acetone in a pre-cooled mortar, and centrifuged at 5000 g and 4 °C for 10 min. Then, 0.1 mL of 5% (w/v) TiSO4 and 0.1 mL pre-cooled ammonium hydroxide were added to 1 mL of the root extract supernatant, which was re-centrifuged at 5000 g for 10 min. The supernatant was discarded and the sediment was re-dissolved in 4 mL of 2 M H2SO4. The absorbance of the root extract solution was measured at 415 nm.
Root OH− content was analyzed by the methods described in a previous study : ~ 0.1 g fresh root was powdered with 3 mL of 50 mM PBS (pH 7.0) in a mortar, and centrifuged at 10,000 g and 4 °C for 10 min. Then, 1.0 mL of 25 mM PBS (pH 7.0) containing 5 mM 2-deoxy-D-ribose and 0.2 mM NADH were added to 1 mL of the root extract supernatant, completely blended, and left to react for 60 min at 35 °C in the dark. Following this incubation, 1 mL of 1% (w/v) thiobarbituric acid and 1 mL glacial acetic acid were added to the filtrate. The mixture was heated to 100 °C for 30 min and then placed on ice for 20 min. The absorbance of the root extract solution was then measured at 532 nm, and the OH− content was inferred from the production of MDA.
Determination of enzyme activities
Fresh rice root samples (0.5 g) were homogenized in 5 mL of 10 mM phosphate buffer (pH 7.0) containing 4% (w/v) polyvinylpyrrolidone and 1 mM ethylenediaminetetraacetic acid. The supernatant was used as crude enzyme solution and collected by centrifugation at 12,000 g and 4 °C for 15 min. The activities of SOD, CAT, APX, and POD were estimated using the photocolorimetric methods described in Jiang and Zhang , and Sachadyn-Krol et al. .
Root NR and NOS activities were assayed using the methods described in previous studies [25, 26], with some modifications. Briefly, total protein was extracted using a buffer containing 100 mM HEPES–KOH (pH 7.5), 1 mM EDTA, 10% (v/v) glycerol, 5 mM 1,4-dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride, 0.1% Triton X-100 (v/v), 1% polyvinylpyrrolidone (PVP), and 20 μM flavin adenine dinucleotide. The supernatant was collected by centrifugation at 12,000 g and 4 °C for 20 min, and then used to determine the NR and NOS activities at 520 nm and 340 nm, respectively.
Specifically, the activity of NR was measured immediately by mixing 250 μL of supernatant with 250 μL pre-warmed (25 °C) assay buffer containing 50 mM HEPES–KOH (pH 7.5), 10 mM MgCl2, 1 mM DTT, 2 mM KNO3 and 200 μM NADH. The reaction was started by adding assay buffer, incubated at 30 °C for 30 min and then stopped by adding 50 μL 0.5 M Zn-acetate. The nitrite produced was measured colorimetrically at 540 nm after adding 1 mlof 1% sulfanilamide in 3 M HCl plus 1 mL of 0.02% N-(1-naphthyl)ethylenediamine in 0.2 M HCl. NOS activity was detected in 1 mL of reaction mixture containing 100 mM phosphate buffer (pH 7.0), 1 mM laevo-arginine(L-Arg), 2 mM MgCl2, 0.3 mM CaCl2, 4 μM BH4, 1 μM FAD, 1 μM flavin mononucleotide (FMN), 0.2 mM DTT, 0.2 mM NADPH, and 200 μL of protein extract. The decrease in absorbance as a result of NADPH consumption was determined at 340 nm for 5 min. NOS activity was calculated using the extinction coefficient of NADPH (ɛ = 6.22 mM− 1·cm− 1).
Determination of arginine and citrulline
Arginine and citrulline contents were estimated using the method described in Salazar et al. . Briefly, 1.0 g root samples were frozen in liquid N2 and extracted with 4 mL 80% (v/v) methanol, and then centrifuged at 10,000 g and 4 °C for 5 min. The supernatant was then used in derivatization and reaction processes. Serial concentrations of amino acid standards were prepared as described above for the derivatizing reagent, and the derivatizing samples were used to determine the arginine and citrulline contents using liquid chromatography/electrospray ionization tandem mass spectroscopy (LC-ESI-MS).
All experiments conducted in this study were performed in six replicates, at least. All data, expressed as means ± standard error (SE), were processed in SPSS v. 13.0 (IBM Corp., Armonk, NY, USA). The Least Significant Difference (LSD) test was used to determine statistical significant differences among the treatments (P < 0.05). Figures were drawn in Origin v. 8.0 (OriginLab Corporation, Northampton, MA, USA).