Chemicals
The molecular probe H2DCF-DA was obtained from Biotium (Hayward, CA, USA). 2-(N-morpholino) ethanesulfonic acid (MES), hypotaurine (HT), aminooxy acetic acid (AOA), hydroxylamine (NH2OH), potassium pyruvate (C3H3KO3), ammonia (NH3), ascorbic acid (ASA), catalase (CAT), diphenylene iodonium (DPI), salicylhydroxamic acid (SHAM), L-cysteine, D-cysteine, dimethyl sulfoxide (DMSO), N,N-dimethyl-p-phenylenediamine dihydrochloride and dithiothreitol (DTT) were obtained from Sigma-Aldrich (St Louis, MO, USA). Unless stated otherwise, the remaining chemicals were of the highest analytical grade available from various Chinese suppliers.
Plant materials
A. thaliana ecotype Columbia (Col–0) was used 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),, and NADPH oxidase gene single mutant lines (N9555, designated AtrbohD) and (N9557, designated AtrbohF) and of the homozygous transposon insertion double mutant line (N9558, designatedAtrbohD/F ) were obtained from the Nottingham Arabidopsis Stock Centre (NASC, Nottingham, UK). The mutant Atl-cdes, Atd-cdes and AtrbohD, AtrbohF, AtrbohD/F has been identified by PCR and RT-PCR [52–54].Wild-type and mutants seeds of A. thaliana. were surface-sterilized and sown on sterilized vermiculite. Seedlings were stratified in darkness for 2–4 d at 4 °C. After growing 4 euphylla, seedlings were grown in controlled-environment chamber with a humidity of 80%, a 16-h light/8-h dark cycle, and a day/night temperature cycle of 22 °C/ 18 °C with a photon flux density of 100 μmol·m−2·s−1 PAR generated by cool white fluorescent tubes (Philips, New York, NY, USA). Fully expanded leaves were harvested for immediate use at 4–6 weeks.
Stomatal bioassays
Stomatal bioassay was performed as described by McAinsh et al. (1996) with minor modifications [41]. The freshly prepared epidermal strips 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 with a light microscope and an eyepiece graticule previously calibrated with a stage micrometer. In each treatment, we scored 30 randomly-selected apertures per replicating and treatments were repeated at least three times. The data presented are the mean ± s.e. of 90 measurements.
Measurement of H2S emission
Measurement of H2S emission was determined by formation of methylene blue, which was performed as described by Sekiya et al. (1982) and Hou et al. (2013) with slight modifications [34, 55]. Fully expanded leaves were used 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 took 0.1 g of them for grinding by addition 0.9 mL 20 mM Tris-HCl (pH 8.0) buffer. After centrifugation, the supernatant and a trap with 1% of zinc acetate was put in a test tube, meanwhile the test tube was sealed quickly with a Parafilm. After H2S was absorbed for 30 min at 37 °C, 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 was added into the trap. Finally, the absorbance was measured at 670 nm. A calibration curve was made with known concentrations of Na2S solution. Each treatment was repeated 3 times, and the data presented are the mean ± s.e.
L-/D-cysteine desulfhydrase activity measurements
To further investigate the L-/D-cysteine desulfhydrase (L-/D-CDes) activity, we determined the H2S which was released from L-/D-cysteine within a certain period of time [34, 56]. The assay contained in a total volume of 1mL: 100 μL 0.8 mM L-/D-cysteine, 400 μL 2.5 mM DTT, 400 μL 100 mM Tris-HCl, and 100 μL supernatant. Then added 100 μL 20 mM N,N-dimethyl-p-phenylenediamine dihydrochloride dissolved in 7.2 M HCl and 100 μL 30 mMFeCl3 dissolved in 1.2 M HCl into the trap after reaction for 30 min at 37 °C. And the rate of H2S released was showed by the determined of absorbance at 670 nm. The activity of L-CDes and D-CDes was determined by the same method, but the pH of Tris-HCl buffer that the former used was 9, and the latter was 8. Each treatment was repeated 3 times, and the data presented are 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 [30]. To study the effects 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 in MES-KCl buffer alone or containing ASA, CAT, DPI, and SHAM in darkness for 3 h, and then were 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 in MES-KCl buffer alone in darkness for 3 h , and then were immediately loaded with 50 μM H2DCF-DA in Tris-KCl buffer for 10 min in darkness. Then 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 the following settings: excitation 488 nm, emission 530 nm, power 10%, zoom about 4, normal scanning speed, and frame 512×512 pixels. Images thus acquired were analyzed with Leica image software and processed with Photoshop 7.0 (Adobe, San Jose, CA, USA). Each treatment was repeated at least 3 times. The confocal images depicted here represent similar results from all three replications.
Statistical analyses
The statistical significance of treatments was checked using one-way ANOVA followed by Duncan’s multiple range test. The data were considered statistically significant when P-values were below 0.05. The figures were plotted by Origin6.1 (Microcal Software, Nothampton, MA, USA) and processed with Photoshop 7.0 (Adobe, San Jose, CA, USA).