Starch Biosynthetic Enzyme Activities

Soluble proteins were extracted from developing barley grains at 13–15 DAA using 2 volumes (3 volumes for BE) of grinding solution [50 mM imidazole–HCl (pH 7.4), 8 mM MgCl₂, 500 mM 2-mercaptoethanol, 0.1% (w/v) acarbose, 12.5% (v/v) glycerol] relative to grain fresh weight. Developing grains (10–15 DAA) of rice (Oryza sativa subspecies japonica “Nipponbare”) grown in a paddy field of Akita Prefectural University during the summer months under natural condition was used as control. SS activity staining was performed using a gel containing 0.8% (w/v) oyster glycogen (Sigma-Aldrich, G8751) as primer (Miura et al. 2018 (link)). BE activity staining was assessed using a gel containing 0.0001% oyster glycogen (Yamanouchi and Nakamura 1992 (link)). DBE activity staining was performed using gels containing 0.4% (w/v) potato amylopectin (Sigma-Aldrich, A8515) as described in Fujita et al. (1999 (link)). PUL activity staining was performed using a gel including 1.34% (w/v) red pullulan (Megazyme) as described in Fujita et al. (2009 (link)).