The molecular probe H2DCF-DA was bought from Biotium (Hayward, CA, USA), while 2-(N-morpholino) ethanesulfonic acid (MES), salicylhydroxamic acid (SHAM), potassium pyruvate (C3H3KO3), aminooxy acetic acid (AOA), hypotaurine (HT), hydroxylamine (NH2OH), catalase (CAT), ammonia (NH3), diphenylene iodonium (DPI), D-cysteine, dimethyl sulfoxide (DMSO), ascorbic acid (ASA), L-cysteine, dithiothreitol (DTT) and N, N-dimethyl-p-phenylenediamine dihydrochloride were acquired from Sigma-Aldrich (Located in St Louis, MO, USA). Unless stated otherwise, the other chemicals were purchased from various Chinese suppliers with highest analytical grade.
A. thaliana ecotype Columbia (Col-0) was applied throughout this study. Seeds of L-/D-cysteine desulfhydras deletion mutants of AtL-CDes T-DNA insertion line (N541918, designated Atl-cdes), AtD-CDes T-DNA insertion line (CS853264, designated Atd-cdes), NADPH oxidase gene single mutant line (N9555, designated AtrbohD and N9557, designated AtrbohF), and homozygous transposon insertion double mutant line (N9558, designated AtrbohD/F ) were provided by Nottingham Arabidopsis Stock Centre (NASC, Nottingham, UK). The mutant Atd-cdes, Atl-cdes and AtrbohF, AtrbohD, AtrbohD/F has been respectively identified by PCR and RT-PCR [51-53].Wild-type and mutants seeds of A. thaliana. were surface-sterilized and sown on sterilized vermiculite. Seedlings were stratified in darkness for 2-4d at 4 °C. After growing 4 euphylla, they were transferred in a controlled-environment chamber with a humidity of 80%, 16-h light/8-h dark cycle, and day/night temperature cycle of 22°C/18°C with a photon flux density of 100 μmol·m−2s−1 PAR generated by cool white fluorescent tubes (Philips, New York, NY, USA). Fully expanded leaves were harvested at 4-6 weeks for immediate use.
Stomatal bioassay was performed as described by McAinsh et al. (1996) with minor modifications . The epidermal strips newly prepared were treated with MES-KCl buffer (10 mM MES, 50 mM KCl, 100 μM CaCl2, pH 6.15) alone or containing various compounds or inhibitors in light (100 μmol·m−2·s−1) or darkness. And then the stomatal apertures were recorded by an optical microscope and eyepiece graticule previously calibrated with a stage micrometer. In each treatment, 30 randomly-selected apertures were scored per replicating and the treatment was repeated three times at least. The data provided are the mean ± s.e. of 90 measurements.
Measurement of H2S emission
Measurement of H2S emission was determined by the formation of methylene blue, which was performed as described by Sekiya et al. (1982) and Hou et al. (2013) with slight modifications [34, 54]. Fully expanded leaves were utilized to measure H2S emission. Firstly, the leaves were treated with MES-KCl buffer alone or containing various scavengers or synthesis inhibitors in light (100 μmol·m− 2·s− 1) or darkness for 3h, and then 0.1 g of them was taken for grinding by adding 0.9 mL 20 mM Tris-HCl (pH 8.0) buffer. After the centrifugation, the supernatant and a trap with 1% of zinc acetate were put into a test tube, and then the tube was quickly sealed with a Parafilm at the same time. Then 100 μL 20 mM N,N-dimethyl-p-phenylenediamine dihydrochloride dissolved in 7.2 M HCl and 100 μL 30 mM FeCl3 dissolved in 1.2 M HCl were added into the trap after the absorption of H2S for 30 min at 37 °C. Finally, the absorbance was measured at 670 nm. In additional, a calibration curve was also drawn with known concentrations of Na2S solution. Each treatment was repeated three times, and all the data presented are the mean ± s.e.
L-/D-cysteine desulfhydrase activity measurements
H2S was determined to further study the activity of L-/D-cysteine desulfhydrase (L-/D-CDes), which was released from L-/D-cysteine within a certain period of time [34, 55]. The assay contained in the total volume of 1mL includes 100 μL 0.8 mM D-/L-cysteine, 400 μL 100 mM Tris-HCl, 400 μL 2.5 mM DTT, and 100 μL supernatant. Then 100 μL 20 mM N,N-dimethyl-p-phenylenediamine dihydrochloride dissolved in 7.2 M HCl and 100 μL 30 mM FeCl3 dissolved in 1.2 M HCl were added into the trap after reaction for 30 min at 37 °C. And the rate of H2S released was presented by the determination of absorbance at 670 nm. Besides, the activity of L-CDes and D-CDes was also confirmed by the same method, but the pH of Tris-HCl buffer used previously was 8, and the latter was 9. Each treatment was repeated three times, and the data presented were the mean ± s.e.
Measurement of endogenous H2O2
H2O2 levels were measured with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) by the method of Allan and Fluhr (1997) with minor modifications . In order to research the influence of H2S scavenger and synthesis inhibitors on darkness-induced H2O2 production in guard cells, the epidermal strips were incubated in MES-KCl buffer alone in light or MES-KCl buffer alone or containing ASA, CAT, DPI, and SHAM in darkness for 3 h, and then immediately loaded with 50 μM H2DCF-DA in Tris-KCl buffer (10 mM Tris, 50 mM KCl, pH 7.2) for 10 min in darkness. To study the effects of darkness on H2O2 levels in guard cells of Atl-cdes and Atd-cdes mutnts, the epidermal strips were incubated in MES-KCl buffer alone in light or MES-KCl buffer alone in darkness for 3h, and then immediately loaded with 50 μM H2DCF-DA in Tris-KCl buffer for 10 min in darkness. After that, excess dye was washed off with fresh Tris-KCl loading buffer in darkness, and the epidermal strips were immediately examined by TCS SP5 laser-scanning confocal microscopy (Leica Lasertechnik Gmbh, Heidelberg, Germany) with following settings: excitation 488 nm, emission 530 nm, power 10%, zoom about 4, normal scanning speed, and frame 512×512 pixels. Leica image software and Photoshop 7.0 (Adobe, San Jose, CA, USA) were used to analyze and process the images acquired. Each treatment was repeated at least three times. The depicted confocal images represent similar results from three replications.
The statistical importance of treatments was checked by one-way ANOVA as well as Duncan’s multiple range test. The data was considered to be statistically important when P-values were below 0.05. All the figures were plotted by Origin6.1 (Microcal Software, Nothampton, MA, USA) and processed with Photoshop 7.0 (Adobe, San Jose, CA, USA).