Figure 2

Experimental tests on Motif 1. (A) Features of the promoter sequence (1kb) of CHS-D us1 allele (AF358659) in Ipomoea purpurea. The translation starting codon is shown in bold. Motif 1 and Motif 2 are large font and in italic. The underlined is the region built into pCHS-337. (B) Results of dual-luciferase transient expression activities. The firefly luciferase (LUC) was induced by a 337-nt CHSD promoter and effectors expressing the whole coding regions of MYB, bHLH2, and WDR1 of Ipomoea purpurea, with renilla luciferase (RUC) as reference. The activity of the native (orig.) promoter (pCHS-337) is compared with those of mutated promoters (C, A, C, G, T, G for Motif 1) and a negative control (−, from the tests of the native promoter driven only by the IpbHLH2 effector). All were measured in the relative fluorescent activities (LUC/RUC), with error bars based on four independent trials. (C) Probe sequences (5→3') for the subsequent EMSA binding tests. The probe CHSD-px4 hosts four dosages of the Motif-1core sequence (underlined) shown on the CHS promoter. Each of the probes (M1-0→M1-7) contains a mutation (in bold) at sites of the 5' border, the candidate cis, and the 3' border. (D) Binding activities of Ipomoea bHLH2 (IpbHLH2, EU032618) to the probes above. The upper panel showed the DNA staining of the results, with the loading order followed the probes listed in (C), and the lower panel displayed the protein staining of the same gel. In addition to the DNA marker, the binding result between CHSD-px4 and IpMYB1 was also loaded in lane c as a control for specificity. The ruler is in unit of cm. (E) Binding activities of petunia AN1 (PhAN1, AF260919) to the probes in (C) following the same procedures and annotations of (D).