A histochemical staining procedure was used to detect superoxide and hydrogen peroxide in situ as described (Fryer et al. 2002 (link)). Rosette leaves sampled from seedlings with or without ABA or NaCl treatment were infiltrated with nitroblue tetrazolium (NBT) (Amresco, 1 mg ml−1 NBT in 10 mM sodium azide and 10 mM phosphate buffer, pH 7.8) or 3, 3′-diaminobenzidine (DAB) solutions (Sigma–Aldrich, 1 mg ml−1 DAB-HCl, pH 3.8), respectively. The stained leaves were imaged after removal of chlorophylls by boiling in 96% ethanol for 10 min. Cell death was examined by Evans Blue staining as described (Kim et al. 2003 (link)) with minor modifications. Briefly, detached leaves from plants untreated or treated with 50 mM NaCl for 1 h were vacuum-infiltrated in 0.1% Evans Blue (w/v; Sigma–Aldrich) for 15 min and then kept under vacuum for 6 h, then washed three times (15 min each) with a phosphate-buffered saline containing 0.05% (v/v) Tween 20 followed by boiling in 96% ethanol for 10 min to remove chlorophylls.
ROS production in intact guard cells was detected using 2, 7-dichlorofluorescin diacetate (H2DCF-DA, Fluka) as described (Pei et al. 2000 (link)). Epidermal peels were floated in an incubation buffer (30 mM KCl, 10 mM Mes-KOH, pH 6.15) for 2 h under the light of 150 μmol photos m−2 s−1 at 22°C to induce stomatal opening and then loaded with 50 μM H2DCF-DA. After 10 min of dye loading, the epidermal strips were washed three times with the incubation buffer. For ABA treatment, 10 μM ABA was added to the incubation buffer and the epidermal strips were further incubated for 2 h before dye loading. Guard cells were visualized by confocal laser microscope (Leica TCS SP2) with excitation at 488 nm and emission at 525 nm and images were acquired using the Leica Confocal Software version 2.6 Build 1537. The DCF fluorescence emission of guard cells was quantified using the Leica Qwin software.
Determination of cellular levels of ROS by Electron Spin Resonance (ESR) spectroscopy was performed essentially according to the protocol as described (Capani et al. 2001 (link)) with minor modifications. Briefly, plants of the same developmental stage grown aseptically on 1/2 MS agar medium were collected, weighed and homogenized on ice in 600 μl ice-cold spin trap solution containing 100 mM α-phenyl-N-tert-butylnitron (PBN, Sigma–Adrich) and 2 mM diethylenetriamine-pentacetic acid (DPTA) in phosphate-buffered saline (PBS, pH 7.4). After homogenization, 250 μl ethyl acetate were added, vortexed for 30 s and centrifuged at 12,000 rpm for 15 min at 4°C for complete phase separation. Then the ethyl acetate phase was transferred to a clean tube for ESR measurement. The Brucker 200D SRC ESR spectrometer was set as followings: center field, 3385 Gauss; scan width, 400 Gauss; modulation amplitude, 3.2 G; microwave power, 20 mW; receiver gain, 4.0 × 105.
ROS production in intact guard cells was detected using 2, 7-dichlorofluorescin diacetate (H2DCF-DA, Fluka) as described (Pei et al. 2000 (link)). Epidermal peels were floated in an incubation buffer (30 mM KCl, 10 mM Mes-KOH, pH 6.15) for 2 h under the light of 150 μmol photos m−2 s−1 at 22°C to induce stomatal opening and then loaded with 50 μM H2DCF-DA. After 10 min of dye loading, the epidermal strips were washed three times with the incubation buffer. For ABA treatment, 10 μM ABA was added to the incubation buffer and the epidermal strips were further incubated for 2 h before dye loading. Guard cells were visualized by confocal laser microscope (Leica TCS SP2) with excitation at 488 nm and emission at 525 nm and images were acquired using the Leica Confocal Software version 2.6 Build 1537. The DCF fluorescence emission of guard cells was quantified using the Leica Qwin software.
Determination of cellular levels of ROS by Electron Spin Resonance (ESR) spectroscopy was performed essentially according to the protocol as described (Capani et al. 2001 (link)) with minor modifications. Briefly, plants of the same developmental stage grown aseptically on 1/2 MS agar medium were collected, weighed and homogenized on ice in 600 μl ice-cold spin trap solution containing 100 mM α-phenyl-N-tert-butylnitron (PBN, Sigma–Adrich) and 2 mM diethylenetriamine-pentacetic acid (DPTA) in phosphate-buffered saline (PBS, pH 7.4). After homogenization, 250 μl ethyl acetate were added, vortexed for 30 s and centrifuged at 12,000 rpm for 15 min at 4°C for complete phase separation. Then the ethyl acetate phase was transferred to a clean tube for ESR measurement. The Brucker 200D SRC ESR spectrometer was set as followings: center field, 3385 Gauss; scan width, 400 Gauss; modulation amplitude, 3.2 G; microwave power, 20 mW; receiver gain, 4.0 × 105.
