Ct293

Table S1 lists the twenty-five cultivars of highland barley that were used in this study: four two-rowed yellow highland barley (YT1-4), nine six-rowed yellow highland barley (SY1-9), and six-rowed purple hulless barley (PS1-12). They were planted at Lhasa, Tibet (altitude: 3658 m, oxygen content: 15.09%, mean temperature: 18.60 °C) in 2017. Whole grains were ground using a whirlwind mill (CT293, FOSS, Hillerod, Denmark) equipped with a 0.5 mm screen. Grains and grain flour were stored in double-layer self-sealing bags at 4 °C.
γ-Aminobutyric acid (GABA), putrescine (Put), spermidine (Spd), spermine (Spd), amino acid standard solution, sodium acetate, β-mercaptoethanol polyvinyl pyrrolidone (PVP), and pyridoxal 5-phosphate (PLP) were from Sigma Aldrich (Saint Louis, MO, USA). The HPLC-grade acetonitrile and methanol were purchased from Fisher Scientific (Waltham, MA, USA). All other chemicals and reagent used in the experiments were analytical grade.

Whole-grain samples were ground through a 0.5 mm grid diameter mesh using a laboratory mill (Cyclotec CT293, FOSS, Hilleroed, Denmark) to obtain a homogeneous powder. Wholemeal flour (5 g) was added to 15 mL of 1 M perchloric acid and shaken for 30 min at room temperature. The suspension was centrifuged at 5300 RCF for 3 min at 4 °C to produce a clear extract. Clear supernatant was brought to pH 8 by adding 2 M KOH and adjusted to 50 mL in a graduated cylinder. The samples were refrigerated in an iced bath for 30 min. Samples were then centrifuged at 5300 RCF for 30 min at 4 °C. Clear supernatant was filtered through a 0.45 μm syringe filter and 2 mL was transferred into a 2 mL tube and stored at −20 °C for subsequent analysis.