Dionex ics 5000 dc system

All the transgenic lines and WT lines were grown in triplicates in BCD medium, changed every two weeks, for 6 weeks and freeze-dried for 48 h. Dried samples were ball milled in 70% EtOH in a Genogrinder (SPEX, NJ, USA) for 2 min. at 1500 rpm, centrifuged for 10 min. at 10,000g and the supernatant was discarded. The samples were first extracted with chloroform/MeOH (1:1 v/v) by adding the chloroform/MeOH, vortexing vigorously followed by centrifugation at 10,000g for 10 min. The supernatant was discarded and the pellet was extracted with 100% acetone as above. The supernatant was again discarded and the samples were then left to air dry in the fume hood. The sample weight was noted and starch was digested as described in Mosele et al., 2011 with Termamyl (Novozymes, Denmark) and amyloglucosidase (Megazyme, Ireland). The EtOH wash containing the Glc, maltose, and smaller dextrins, was dried in a speed-vac and the pellet was hydrolysed in 2 M TFA as previously described^{30 (link)} and the glucose quantified using a Dionex ICS 5000 + DC system (ThermoFisher, MA, USA) as described in Jensen et al., 2018. The pellet after starch removal was dried and analysed by both CoMPP as previously described^{31 (link),32 (link)}, and by sugar composition analysis after TFA hydrolysis as described above for starch.

Cell wall material was prepared by grinding aliquots of 50 mg K. flaccidum cells in a TissueLyser MM 200 (Qiagen, Hilden, Germany) for 2 min at 30 s⁻¹. Each sample was then extracted using 1.5 ml 70% ethanol for 5 d at 55°C, followed by four cycles of 1.5 ml 70% ethanol, one cycle of 1.5 ml acetone, and then air‐dried overnight. Monosaccharide composition was performed on cell wall material as previously described (Øbro et al., 2004) using a Dionex ICS 5000 + DC system (ThermoFisher) equipped with a  high‐performance anion exchange chromatograph with pulsed amperometric detection and a 4 μm SA‐10 column (2 × 250 mm and guard column). Run conditions were 40°C column temperature, 0.3 ml min⁻¹ eluent flow rate, 1 mM NaOH for 0–8 min, followed by 100 mM NaOH from 8 to 20 min, and subsequently 10 min equilibration at 1 mM NaOH.