Uv 1202

The concentration of sulfide ions in liquid phase was monitored spectrophotometrically at 660 nm using Shimadzu UV-1202 (Shimadzu, Kyoto, Japan) [31 (link)].
The control concentrations of sulfones and the purity of the starting sulfone materials were analyzed by gas chromatography using a “Crystal-2000M” chromatograph with a flame ionization detector, column–Zebron L = 30 m, d = 0.32 mm, liquid phase ZB-1, while programming the temperature from 100 °C to 250 °C. Chromatograms were recorded and analyzed on a computer using the Chromatech Analytic 1.5 program.

Col-0 wild-type, ANN5-OE_2 and ANN5_OE1 seeds were surface-sterilized with 75% ethanol for 2 min and then with 10% sodium hypochlorite for 10 min. Next, the seeds were washed three times with sterile water and spread onto agar-solidified (1% w/v) MS media supplemented or not with 1.5% (w/v) sucrose. After 2 days stratification at 4 °C, the plates were placed in the growth chamber under a photon flux density of 220 μE m^− 2 s^− 1 at the shelf level. Seedlings were grown under short-day conditions (8 h light) for 10 days. Aerial part of 10 day old seedlings were harvested, weighed and kept at − 80 °C. Samples were mechanically ground in 2 ml microfuge tubes with two stainless-steel beads by a bead mill (TissueLyser II, Qiagen, Hilden, Germany). After extraction with 1 ml cold 80% acetone, the samples were centrifuged 6000 rpm for 5 min at 4 °C. Extraction was repeated two times with fresh solvent. Absorbance of the pooled extracts was measured at 664 and 647 nm with a spectrophotometer (UV-1202, Shimadzu, Kyoto, Japan). Chlorophyll content was calculated using equations described previously [22 ].

The NADH–ferricyanide reductase activity was measured with minor modification, as recently published [31 (link)]. The medium of the cells was exchanged against 1 mL buffer (50 mM Tris-HCl pH 8.0, 500 μM β-NADH) providing 500 μM potassium ferricyanide (III) (K₃Fe(CN)₆) (Riedel-de-Haen—Honeywell Specialty Chemicals Seelze GmbH, Seelze, Germany) per 35 mm dish and the cells were incubated for 60 min at 37 °C and 5% CO₂. Ferricyanide was reduced to ferrocyanide. Subsequently, an aliquot of the buffer was drawn to measure the absorbance at 420 nm with a photometer (UV1202, Shimadzu, Kyoto, Japan). The NADH–ferricyanide reductase activity was expressed as reduced ferricyanide in nmol/min/4 × 10⁵ cells. Per the experiment, each condition was measured in duplicates.