Hydrogen sulfide quantification was performed as described by Nashef et al. (1977) (link). Briefly, strawberry leaf tissue was ground into fine powder with a mortar and pestle under liquid nitrogen and ~0.3g of frozen tissue were homogenized in 1ml of 100mM potassium phosphate buffer (pH 7) containing 10mM EDTA. The homogenate was centrifuged at 15,000 g for 15min at 4 °C and 100 μl of the supernatant was used for the quantification of H2S, in an assay mixture containing also 1880 μl extraction buffer and 20 μl of 20mM 5,5’-dithiobis(2-nitrobenzoic acid), in a total volume of 2ml. The assay mixture was incubated at room temperature for 2min and the absorbance was read at 412nm. Hydrogen sulfide was quantified based on a standard curve of known concentrations of NaHS.
Leaf hydrogen peroxide content was assayed as described by Loreto and Velikova (2001 (link)). Frozen leaf material (~0.1g) was homogenized on ice with 0.1% (w/v) TCA. The homogenate was centrifuged at 15,000 g for 15min at 4 °C and 0.5ml of the supernatant was added to 0.5ml of 10mM potassium phosphate buffer (pH 7.0) and 1ml of 1M KI. The absorbance of assay mixture was read at 390nm and the content of H2O2 was calculated based on a standard curve of known concentrations of H2O2.
Nitric oxide content was determined according to Zhou et al. (2005) (link). Briefly, frozen leaf material (~0.1g) was homogenized in 50mM cool acetic acid (pH 3.6) containing 4% zinc acetate and centrifuged at 10,000 g for 15min at 4 °C. The supernatant was collected and the pellet was washed with 0.5ml extraction buffer and centrifuged again. The two supernatants were combined and 0.1g charcoal was added. The mixture was agitated and centrifuged at 15,000 g for 15min at 4 °C. To 1ml of clear supernatant, 1ml Griess reagent was added and the mixture was incubated at room temperature for 30min. The absorbance of the mixture was read at 540nm and NO content was calculated by comparison to a standard curve of NaNO2.
Leaf hydrogen peroxide content was assayed as described by Loreto and Velikova (2001 (link)). Frozen leaf material (~0.1g) was homogenized on ice with 0.1% (w/v) TCA. The homogenate was centrifuged at 15,000 g for 15min at 4 °C and 0.5ml of the supernatant was added to 0.5ml of 10mM potassium phosphate buffer (pH 7.0) and 1ml of 1M KI. The absorbance of assay mixture was read at 390nm and the content of H2O2 was calculated based on a standard curve of known concentrations of H2O2.
Nitric oxide content was determined according to Zhou et al. (2005) (link). Briefly, frozen leaf material (~0.1g) was homogenized in 50mM cool acetic acid (pH 3.6) containing 4% zinc acetate and centrifuged at 10,000 g for 15min at 4 °C. The supernatant was collected and the pellet was washed with 0.5ml extraction buffer and centrifuged again. The two supernatants were combined and 0.1g charcoal was added. The mixture was agitated and centrifuged at 15,000 g for 15min at 4 °C. To 1ml of clear supernatant, 1ml Griess reagent was added and the mixture was incubated at room temperature for 30min. The absorbance of the mixture was read at 540nm and NO content was calculated by comparison to a standard curve of NaNO2.
