Figure 3.

Native-PAGE/SS activity staining. SP from 10 g of developing endosperm in the wild type (‘Nip’; A and D), the SSI mutant (e7−/−; B and E), and the SSIIIa mutant (ss3a-1; C and F) was fractionated by anion-exchange chromatography (HiTrapQ). Proteins in the fractions were separated by native-PAGE in gels containing 0.1% rice amylopectin (A, B, and C) or 0.8% oyster glycogen (D, E, and F). Gels were incubated overnight in a SS reaction buffer containing 0.5 m citrate (D, E, and F) or under a citrate-free condition (A, B, and C). The large white and black arrowheads indicate the positions of SSIIIa and SSI in the crude extract (CE), respectively. The small white, black, and gray arrowheads with numbers show that SS activity bands were detected in the ‘Nip’ and SSI mutant, in the ‘Nip’ and SSIIIa mutant, and in all lines, respectively. These activity bands were dependent on the addition of ADP-Glc in the incubation buffer, whereas the bands with X marks are not SS activity bands because they are also detected in the ADP-Glc-free incubation buffer (data not shown). FT, Flow through fraction; numbers 1 to 11, fraction numbers. [See online article for color version of this figure.]