Figure 1.

Zymogram analysis of mutants affected for DBE activities. A to C, Samples of soluble proteins were prepared from leaves of different wild-type and mutant plants as described in “Materials and Methods,” loaded onto polyacrylamide gels impregnated with glycogen (A), potato soluble starch (B), and maize β-limit dextrins (C), and subsequently submitted to electrophoresis. After migration, the gels were incubated in the suitable buffer to allow enzymes to modify the substrate present into the polyacrylamide matrix. Enzymatic activities were then developed by soaking the gel for several minutes in lugol solution. Pictures were taken immediately after the gels were briefly rinsed in deionized water to remove iodine in excess. A, The gel was incubated in an ADP-Glc-containing buffer that is specific to soluble starch synthases. SS1 and SS3 are indicated in the picture. B and C, Gels were incubated in a Tris buffer that allows glucan-modifying enzymes to act on the substrate available within the polyacrylamide matrix. Bands corresponding to various DBEs (Iso1, ISA3, and PU1) are indicated in the picture. Lane 1, Ws; lane 2, isa1-1; lane 3, isa3-1; lane 4, pu1-1; lane 5, isa1-1 isa3-1; lane 6, isa3-1 pu1-1; lane 7, isa1-1 isa3-1 pu1-1; lane 8, Col-0.