Functional divergence of two duplicated D-lineage MADS-box genes BdMADS2 and BdMADS4 from Brachypodium distachyon

MADS-box genes are core members of the ABCDE model for flower development where D-lineage genes play essential roles in ovule identity determination. We report here the cloning and functional characterization of two duplicated MADS-box genes, BdMADS2 and BdMADS4 from Brachypodium distachyon, the model plant of temperate grasses. BdMADS2 and BdMADS4 were highly similar to grass D-lineage MADS-box genes on the protein level and they fell in a distinctive clade on the phylogenetic tree, with conserved intron/exon structures to their rice and maize orthologues. Quantitative real time PCR revealed comparable expression levels were detected in all floral organs of Brachypodium for both genes, except for the carpel where the expression level of BdMADS2 was five times higher than that of BdMADS4. Over expression of these two genes in Arabidopsis caused curly rosette leaves, small sepals and petals, and early flowering. However, BdMADS4 showed stronger phenotypic effects than BdMADS2, suggesting functional divergence between the two genes. Cis-regulatory element prediction showed that the promoter region (including the first intron) of BdMADS4 possesses much less class I BPC protein binding motifs than that of BdMADS2 which may be responsible for the specific expression in carpels. Yeast two-hybrid assays showed that both BdMADS2 and BdMADS4 can interact with BdSEP3, but BdMADS2 can additionally interact with the putative APETALA1 orthologue (BdAP1), suggesting a deviation in their protein interaction patterns. Taken together, our data demonstrate a significant divergence between the two Brachypodium D-lineage MADS-box genes and provide evidences for their sub-functionalization.