Conservation and divergence of candidate class B genes in Akebia trifoliata (Lardizabalaceae)

There is evidence that gene duplication and diversification within the MADS-box gene family had significant impact on floral architecture. In this study, we report the isolation of four class B homologous genes from Akebia trifoliata, termed AktAP3_1, AktAP3_2, AktAP3_3, and AktPI. Phylogenetic analysis indicates that the three AktAP3 paralogs were produced by two gene duplication events and AktAP3_2 and AktAP3_3 are recent paralogs, which are yielded by the duplication before the origin of the genus Akebia. In situ hybridization demonstrates that these genes are mainly expressed in the stamens and carpels of A. trifoliata, but in differential patterns, similar to those in other basal eudicot and basal angiosperm species. AktAP3_3 and AktPI are expressed in the developing petaloid perianth, suggesting that the petaloidy of the perianth is caused by the expression of class B genes. Reverse transcriptase polymerase chain reaction analyses indicate that these genes are expressed in both male and female flowers, but at different levels. We explore the interaction behavior of the class B proteins in the basal eudicots using yeast two-hybrid system for the first time. The AktAP3_1/2/3 proteins and the AktPI protein can form obligate heterodimers, but at different strength. From the mRNA expression and protein interaction patterns of the duplicated copies of the AktAP3 genes, we conclude that subfunctionalization very likely contributes to the maintenance of multiple AP3-like gene copies in A. trifoliata.