Figure 9.

In Vivo Function of a GAMYB Binding-Like Motif in KAR.

(A) Schematic representation of the 5′-flanking region of KAR. The closed circles show GAMYB binding-like motifs. Fragments 1 and 2 (Frg. 1, bp −332 to −123; Frg. 2, bp −143 to −4) were used as probes or competitors for gel-shift assays in (B) and (C), respectively.

(B) Gel-shift assay with the recombinant GAMYB protein and ³²P-labeled Fragments 1 and 2 presented in (A).

(C) Competitive gel-shift assay. The interaction between GAMYB and ³²P-labeled RAmy1A probe was efficiently competed by Fragment 2 but not by Fragment 1, whereas mutagenized Fragment 2 (mFrg. 2), which contains displaced nucleotides at the GAMYB binding-like motif, did not show effective competitive activity. Competition experiments were performed using increasing molar amounts (×25, ×50, and ×100) of the indicated unlabeled fragment. The sequences of the oligonucleotides used as Frg. 2 and mFrg.2 are shown at the bottom of the panel. The putative binding site is underlined, and the mutations are indicated in lowercase letters.

(D) and (E) Whole flowers at the VP stage. Bars = 1 mm.

(F) and (G) Close-up view of stamens at the VP stage. Bars = 300 μm.

(D) and (F) Flower transformed with the intact ProKAR:GUS (line #8).

(E) and (G) Flower transformed with the mutagenized ProKAR (mFrg. 2):GUS (line #12). We analyzed 10 T0-independent lines for ProKAR:GUS and 12 lines for mFrg. 2.