• ancestral sequence inference;
  • coding-sequence divergence;
  • gene duplication;
  • three-dimensional structure

Abstract  APETALA1 (AP1) and CAULIFLOWER (CAL) are a pair of paralogous genes that were generated through the pre-Brassicaceae whole-genome duplication event. AP1 and CAL have both partially redundant and unique functions. Previous studies have shown that the K and C regions of their proteins are essential for the functional divergence. However, which differences in these regions are the major contributors and how the differences were accumulated remain unknown. In the present study, we compared the sequences of the two proteins and identified five gaps and 55 amino acid replacements between them. Investigation of genomic sequences further indicated that the differences in the proteins were caused by non-synonymous substitutions and changes in exon–intron structures. Reconstruction of three-dimensional structures revealed that the sequence divergence of AP1 and CAL has resulted in differences between the two in terms of the number, length, position and orientation of α-helices, especially in the K and C regions. Comparisons of sequences and three-dimensional structures of ancestral proteins with AP1 and CAL suggest that the ancestral AP1 protein experienced fewer changes, whereas the ancestral CAL protein accumulated more changes shortly after gene duplication, relative to their common ancestor. Thereafter, AP1-like proteins experienced few mutations, whereas CAL-like proteins were not conserved until the diversification of the Brassicaceae lineage I. This indicates that AP1- and CAL-like proteins evolved asymmetrically after gene duplication. These findings provide new insights into the functional divergence of AP1 and CAL genes.