Abstract: Floral organ identity genes, most of which are MADS-box genes, play key roles in flower development and floral organ identity determination. To specify the identities of different floral organ types, proteins of the floral MADS-box genes need to form dimers and higher-level complexes before they bind to the control regions of downstream genes and regulate their expression. Previous studies have shown that understanding the evolution of the interactions among proteins of the floral MADS-box genes may be an excellent step towards uncovering the underlying mechanisms of the origin of the flower. Yet, due to the lack of such information in early-branching angiosperm lineages, it has been difficult to determine the evolutionary changes of the protein–protein interactions (PPIs) before and after the origin of the flower. In this study, we first isolated counterparts of the floral MADS-box genes from Nuphar pumila (Timm) D.C., a representative of the basalmost angiosperms Nymphaeales, and investigated the interactions among their proteins by carrying out yeast two-hybrid assays. We then estimated the PPIs in the most recent common ancestor of extant angiosperms by using two different methods: ancestral character state reconstruction and ancestral sequence reconstruction followed by yeast two-hybrid assay. Based on these results, we examined the evolutionary transitions of the PPIs before and after the occurrence of extant angiosperms, and discussed their contributions to the origin of the flower. We found that duplication and diversification of floral MADS-box genes, as well as non-random losses of some once-existed PPIs, have been the driving force of the origin of the flower.

Key words: ancestral genes, floral MADS-box genes, Nuphar pumila, protein–protein interaction (PPI)