G1544

The cis-elements in ABI4 promoter were analyzed by PlantCARE program (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)^{19 (link)}. The chromatin immunoprecipitation (ChIP) was performed with modifications to the method originally reported^{37 (link)}. Fourteen-day-old SND1GFP seedlings were used to extract nuclear content with cross-link. An antibody against GFP (G1544, Sigma, https://www.sigmaaldrich.com/) was used for immunoprecipitation. The purified DNA was quantified by the qRT-PCR using the specific primers listed in Table S1.

Developing endosperm was ground in buffer (10 mM Tris–HCl, pH 8.0) in an automatic tissuelyser (FastPrep™ FP120, Thermo Savant) for 20 s. The samples were spun twice at 200 g for 2 min. The supernatant was collected, and it was checked with a microscope that it did not contain any starch granules. The pellet, which consisted of starch granules, was washed 10 times with buffer. Protein concentrations were determined with Bradford reagent (Bio-Rad no. 500-0006) using BSA as standard. The samples were mixed with a 6× SDS-loading buffer to reach the following final concentrations: 50 mM Tris–HCl, pH 6.8, 10% glycerol, 1% SDS, 3% β-mercaptoethanol. The samples were boiled for 10 min and the proteins were separated by SDS-PAGE using NuPAGE Novex Bis-Tris Mini Gels (4–12%, Invitrogen) and Bio-Rad Miniprotean II Multiscreen Apparatus according to the manufacturer’s instructions (Bio-Rad bulletin 1721). Proteins were transferred to polyvinylidene fluoride membranes. Rabbit antiserum raised against HvGBSSIa (Carlsberg Laboratory, Copenhagen, Denmark) was used to detect HvGBSSIa at a dilution of (1:1000). Purified antibodies from rabbit serum raised against eGFP (Sigma-Aldrich, G1544) at a dilution of (1:1000) was used to detect eGFP-tagged HvGBSSIa. The secondary antibody (1:5000) was goat anti-rabbit purified IgG coupled with alkaline phosphatase (Sigma-Aldrich, A3687).