Sodium thiosulfate

Human Grx1 (IMCO Corporation Ltd AB, Stockholm, Sweden) and human Grx2 (IMCO Corporation Ltd AB, Stockholm, Sweden) were adjusted to 1 mM each using 20 mM potassium phosphate buffer, pH 7.2, as a stock solution. E. coli Grx1 (IMCO Corporation Ltd AB, Stockholm, Sweden) and E. coli mutant Grx (C14S, Cys¹⁴ of E. coli Grx1 was replaced with Ser) (IMCO Corporation Ltd AB, Stockholm, Sweden) were adjusted to 3 mM each with 20 mM potassium phosphate buffer, pH 7.2. Human GRD (Sigma-Aldrich, Inc. St. Louis, MO, USA) and E. coli GRD (Novus Biologicals, Centennial, CO, USA) were 304 μM and 19.531 μM, respectively, in their original solutions. NADPH (Sigma-Aldrich, Inc. MO, USA) and GSH (Sigma-Aldrich, Inc.) were adjusted to 10 mM each with potassium phosphate buffer, pH 7.2. Sodium thiosulfate, sodium cyanide, and other chemicals were purchased from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan).

Adventitious roots (100–120 mm length) without lateral roots were cut at the root–shoot junction. The permeability of the exodermal layers at the basal parts (15–25 mm below root–shoot junction) was assessed with an apoplastic tracer, periodic acid (Soukup et al., 2002 (link); Shiono et al., 2014a (link); Pecková et al., 2016 (link)). The cut ends were covered with lanolin (Sigma-Aldrich) to prevent penetration of tracer. The roots were incubated in 0.1% (w/v) periodic acid (H₅IO₆) (Sigma-Aldrich) for 1 h, washed thoroughly with deionized water, incubated in reducing solution [1 g of potassium iodide (Wako) and 1 g of sodium thiosulfate (Wako) dissolved in 50 ml of water and acidified with 0.2 ml of 5 M hydrochloric acid (Wako)] for 1 h at room temperature, washed thoroughly with deionized water and incubated overnight at 4°C in the dark. The basal parts (17.5–22.5 mm below root–shoot junction) of adventitious roots were embedded in 5% (w/v) agar and cut in ca. 100-μm-thick cross-sections with a vibrating microtome (Leica VT1200S, Leica Biosystems). The sections were stained with Schiff’s reagent (Sigma-Aldrich) for 2 min and washed twice with 75% (v/v) glycerol (Wako). Periodic acid that penetrated into root tissue was visualized as a purple color under white light with the above microscope and camera.

The SIM medium for the H₂S production test was prepared according to past literature.⁷ The composition of the medium was as follows: 0.3% beef extract (Nacalai Tesque, Inc), 3% peptone (Nacalai Tesque, Inc), 0.005% sodium thiosulfate (FUJIFILM Wako Pure Chemical Corporation), 0.02% L‐cysteine hydrochloride monohydrate (FUJIFILM Wako Pure Chemical Corporation), and 0.05% ferric ammonium citrate (FUJIFILM Wako Pure Chemical Corporation). The medium was prepared with and without agar (ie, semi‐solid agar and broth were prepared, respectively). Each medium was aliquoted at 0.1 and 3 mL for the low‐volume and conventional methods, respectively. For the 0.1 mL volume, a 0.2 mL tube was used as the medium container. Bacteria adhered to the tip of an inoculating needle were inoculated into the medium (approximately 10⁶ CFU), and the results were determined at 2, 4, 8, and 24 hours after incubation (35°C, aerobic condition). ATCC and NITE Biological Resource Center (NBRC) strains (Proteus mirabilis ATCC 7002 with H₂S positive, Proteus vulgaris ATCC 13315 with H₂S positive, and Morganella morganii NBRC 3168 with H₂S negative) grown on CHROMagar Orientation medium were used for the examination. A sample that showed black colored medium was regarded as positive and judged by agreement of two medical technologists.