Figure 6.

Transient Promoter Activity Assay of WLHS1 Promoters and Distributions of Methylated Cytosine in the 5′ Region of WLHS1.

(A) Transient expression of GUS in immature inflorescences of wheat. The upstream region of WLHS1-A, WLHS1-B, or WLHS1-D was ligated to the reporter gene GUS and introduced into immature inflorescences by particle bombardment. Arrows indicate the GUS spot stained by X-gluc solution.

(B) The methylation status of CpG/CpNpG sites in the 5′ regions of WLHS1 homoeologous genes. Comparison of the distribution of methylated cytosines in the 5′ regions shows that the WLHS1-B promoter is hypermethylated compared with WLHS1-A or WLHS1-D. Genomic DNA was treated by sodium bisulfite and used for PCR with primers that amplify the first exon and promoter regions of the three WLHS1 homoeologous genes. The sequences were determined in >10 clones of each gene, and the ratios of methylated cytosines are indicated by percentages. Closed circles, methylated; open circles, unmethylated.