Ed50 pad system

A 10mg aliquot of flour samples was used for analysis. Soluble sugars were removed by washing the powder in 80% (v/v) ethanol, in a boiling water bath for 30min and once for 10min, and 96% ethanol at room temperature for a final wash. The supernatant was removed at the end of every step after centrifugation at 16 000 g (Heraeus biofuge pico) for 5min and the pellet was dried at 40 °C. Complete hydrolysis of BGs was conducted using lichenase and β-glucosidase from the mixed-linkage BG kit (Megazyme International Ltd) following the method provided by the manufacturer (AOAC Method 995.16 AACC Method 32-23/ICC Standard Method 32-23 No. 168; the reaction volumes were scaled down according to the smaller sample mass). Samples treated in that way were centrifuged, filtered, and the quantification of the resulting glucose units was performed by a high performance anion exchange system (Thermo Fisher Scientific Inc.) with an AS50 autosampler, GS50 gradient pump, and ED50 PAD system equipped with a CarboPac^® PA1 column using the run program described before (Lunde et al., 2008 (link)).

Barley pre-germinated dry grain and seedlings germinated for 1, 2, 3, 4, 8, and 12 d were used for analysis. Coleoptiles and radicles (if present) were cut off and residual grains were freeze-dried. Freeze-dried residual grain samples were ground in a tissue lyser (TissueLyser, Retsch, Qiagen) operating at 30 Hz for 15 s and passed through a 0.5mm screen. A 10mg aliquot of each flour sample was extracted in 80% (v/v) ethanol at 80 °C for 2h in a thermomixer. The samples were centrifuged for 5min at 13 000 g and the supernatant collected. The samples were then re-extracted in 80% (v/v) ethanol for 30min, centrifuged, and the supernatant was collected and pooled with the one from the previous extraction, and dried in a spin-vac. The samples were then reconstituted in water to the correct dilutions for analysis. Soluble sugars were identified and quantified using a gradient high performance anion exchange system (Thermo Fisher Scientific Inc.) with an AS50 autosampler, GS50 gradient pump, and ED50 PAD system equipped with a CarboPac^® PA1 column.