Potassium iodide

The brown seaweed Undaria pinnatifida stem was collected from Kesennuma, Miyagi prefecture, Japan, and the harvest time is the middle of April.
High-purity carbon dioxide gas (99%) was supplied by Showa Denko Gas Products Co., Ltd. (Kawasaki, Japan). The amylose B and α-amylase from Bacillus sp. and glucoamylase for Rhizopus sp. were purchased from Nacalal Tesque, Inc. (Kyoto, Japan). Fucoxanthin standard (≥ 98% purity), sodium dihydrogen phosphate dihydrate (≥ 99% purity), disodium hydrogen phosphate 12-water (99% purity), 1 mol/L sodium hydroxide solution, iodine (99.8% purity), and potassium iodide (99.5% purity) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Milli-Q water was used in these experiments. All reagents used in this study were analytical or high-performance liquid chromatography (HPLC) grade.

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.

DMF was freshly distilled before use. All chemicals from commercial sources were used as received unless otherwise stated. Phenol, 3′-aminoacetanilide, and 1-bromobutane were purchased from TCI, 1-bromo-2,2-dimethylpropane, magnesium sulfate, sodium nitrite, potassium iodide, iodoethane, and iodomethane were purchased from FUJIFILM Wako Pure Chemical Corporation, 37% hydrochloric acid, sodium hydroxide, and potassium carbonate were purchased from KISHIDA Chemical Co., Ltd.

Chemicals used in this study were obtained from the following suppliers; DMBQ (Sigma-Aldrich, Osaka, Japan), syringic acid (Sigma-Aldrich), salicylhydroxamic acid (SHAM, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), potassium iodide (Wako Pure Chemical Industries, Ltd., Osaka, Japan), potassium benzoate (Wako Pure Chemical Industries, Ltd.), L-ascorbic acid (Wako Pure Chemical Industries, Ltd.), diphenyleneiodunium (DPI, Funakoshi Frontiers Life Science, Tokyo, Japan), umbelliferone (Tokyo Chemical Industry Co., Ltd.), 6,7-dihydroxycoumarin (esculetin, Sigma-Aldrich), phenylarsine oxide (PAO, Sigma-Aldrich), and sodium diethyldithiocarbamate trihydrate (DDC, Sigma-Aldrich). Each chemical was dissolved in DMSO or water at a stock concentration of 10 mM and stored at −20°C until use (Supplementary Table S1).
Enzymes used in this study were obtained from the indicated supplier; superoxide dismutase (SOD, Nacalai Tesque, Inc., Kyoto, Japan), catalase (Wako Pure Chemical Industries, Ltd.), and horse radish peroxidase (HRP, Wako Pure Chemical Industries, Ltd.). SOD, HRP, and catalase were dissolved in phosphate buffer (pH 7.0) at a stock concentration of 10⁴, 10⁴, and 10⁵ U/ml, respectively, and stored at −20°C until use. H₂O₂ was obtained from Nacalai Tesque.

H₂O₂ content was measured as described in [71 (link)] with some modifications. First, 0.2 g of plant leaves from WT and phy mutant plants after 8 d of water withholding and control conditions was ground in 1 mL of trichloroacetic acid (TCA) (Nacalai tesque, Kyoto, Japan) 0.1%. Then, 0.25 mL from the supernatant was added to 0.5 mL of 100 mM potassium phosphate (Wako, Osaka, Japan) buffer and 1 mL of 1 M potassium iodide (Wako, Osaka, Japan). The samples were incubated in the dark for 1 h, and the absorbance was read at 390 nm using a DU800 UV/Vis spectrophotometer (Beckman Coulter, Inc., Brea, CA, USA). Using a standard curve, the H₂O₂ concentration was calculated, with TCA 0.1% used as a blank.

Trimethyloctadecylammoniumchloride (purity >98.0%) was purchased from Tokyo Chemical Industry Co., Ltd. Nitrobenzene (purity >98.0%), potassium iodide (purity >99.9%), iodine (purity >99.8%), and agar of reagent grade were provided by Wako Chemicals Inc. All chemicals were used without further purification. Glass beads of 2 mm were provided by AS ONE Corporation. All glass beads and Petri dishes were treated by vacuum plasma so as to remove surface contamination (FEMTO Science CUTE-MP(MP/R)). After treatment, the glass surfaces were wetted with KOH aqueous solution (1 M) and rinsed with deionized water.