Bioultra grade

As the wild type, ecotype Col-0 was used. Isolation of T-DNA knock out mutant rpt5a-4 (SALK_046321) and rpt2a-2 (SALK 005596) is described previously^{14 (link)}.
Sterilized seeds were germinated and grown on MGRL medium^{30 (link)} supplemented with 1% w/v sucrose (BioUltra grade, Sigma-Aldrich, St. Louis, MO, USA), solidified with 1% w/v agar purified (Nacalai Tesque, Kyoto, Japan). For zeocin treatment, Zeocin Selection Reagent (Thermo Fisher Scientific, Yokohama, Japan) was added at the indicated concentrations. Medium plates were horizontally placed in a growth chamber (22 °C 16-h light/8-h dark cycle).

All films were created according to the methods of Anderson and Simsek [18 (link)]. The sorbitol (BioUltra Grade) and glycerol (ACS Reagent Grade) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Film solutions were made by preparing a 26.7 g kg⁻¹ solution of AX in deionized water. The solutions were stirred for 24 h then divided into aliquots and heated at 90 °C for 15 min. Then, sorbitol or glycerol was added (100, 250 or 500 g kg⁻¹) and the samples were vortexed. After heating at 90 °C for an additional 15 min, the samples were cast onto polystyrene Petri dishes and dried at 60 °C for 8 h. The films were allowed to finish drying overnight at 23 °C. All films were stored in a Dry-Keeper (Sanplatec Corporation, Catalog No. H42056-0001, Osaka, Japan) with Boveda 49% relative humidity packs (Item No. B49-60-48). Prior to the analysis of the interactions of the AX films with water, all films were conditioned at 23 °C and 50% relative humidity for at least 48 h prior to analyses.