Figure 1.

Optimization of TCS. A, A concatemer of 24 repeats of the 5′-(A/G)GAT(C/T)T-3′ binding motif caused the strongest cytokinin-dependent induction of a LUC reporter in transient transfection assays. B, Reduced GFP signal in the primary root meristem of a 5-d-old transgenic TCS::GFP seedling in the fourth generation (T4) compared with a primary transformant (T1). C, Similar to mutating nucleotides essential for in vitro binding of type-B ARRs (TCS*::LUC, where the asterisk indicates point mutation G→C), the mutation of flanking nucleotides (TCSfm::LUC) abolished cytokinin-dependent response of TCS::LUC. D, Scheme representing the quantitative effects of different phasings of core 5′-(A/G)GAT(C/T)T-3′ motifs. Phasing of 11 bp results in strongest reporter gene expression. E, The frequency of 5′-(A/G)GAT(C/T)T-3′ motifs with a distance of 7 to 15 bp in cytokinin target genes (top curve) is higher than expected (dotted line), while the same motifs are not significantly higher than expected in control genes (lower curve and dashed line). F, Sequence logos (Crooks et al., 2004) generated from the alignment of clustered 5′-(A/G)GAT(C/T)T-3′ motifs in cytokinin target genes as listed in Supplemental Table S2 (top) after filtering with 5′-A(A/G)GAT(C/T)T-3′ and 5′-A (A/G)GAT(C/T)TT-3′ (middle), and the alignment based on the 12 motifs used to construct TCSn as listed in Supplemental Table S4 (bottom). Filled or empty arrowheads (A and D) or boxes (C) indicate 5′-A (A/G)GAT(C/T)TT-3′ motifs on the forward or reverse DNA strand, respectively. Bars = 20 µm. [See online article for color version of this figure.]