Figure 5.

GhWRKY15 transcriptionally represses GhCalS4 and GhCalS8 during anther development. A, RT-qPCR analysis of expression of CalS4, CalS8, and WRKY15 in wild-type cotton anthers. Total RNA isolated from anthers of 15-, 20-, and 25-d-old plants and relative values of CalS4, CalS8, and WRKY15 expression in cotton are shown as a percentage of GhUBI1 (EU604080) expression activity. Data are means ± sd for three replicates. B, RT-qPCR analysis of expression of CalS4, CalS8, and WRKY15 in 25-d-old anthers of PSP231 RNAi cotton lines L3, L5, and L8. Expression level of CalS4, CalS8, and WRKY15 in the wild type (WT) is set to 1. Data are means ± sd for three replicates. Dunnett's t test demonstrated that there was very significant difference (**P < 0.01) in gene expression level between the PSP231 RNAi plants and the wild type. C, Schematic diagram of the GhCalS4 and GhCalS8 promoters showing relative positions of the W-box elements (p1–p3). D, Yeast one-hybrid assay showed interaction of GhWRKY15 with the GhCalS4 and GhCalS8 promoter fragments. Transformants were grown on SD/-Leu nutritional selection medium with 600 ng mL⁻¹ AbA (top), and SD/-Leu medium served as the control (bottom; see “Materials and Methods”). E and F, EMSA of GhWRKY15 binding to the W-box elements of the GhCalS4 (E) and GhCalS8 (F) promoters. MBP-GhWRKY15 protein was incubated with a biotin-labeled probe, using 1×, 10×, 100×, and 1,000× unlabeled probe as competitor. Biotin-labeled probes incubated with MBP protein or without any protein served as the negative control.