Isolation and characterization of a floral homeotic gene in Fraxinus nigra causing earlier flowering and homeotic alterations in transgenic Arabidopsis

Reproductive sterility, which can be obtained by manipulating floral organ identity genes, is an important tool for gene containment of genetically engineered trees. In Arabidopsis, AGAMOUS (AG) is the only C-class gene responsible for both floral meristem determinacy and floral organ identity, and its mutations produce sterility. As a first step in an effort to develop transgenic sterile black ash (Fraxinus nigra), an AG ortholog in black ash (FnAG) was isolated using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. Analysis of the deduced amino acid sequence showed a typical MIKC structure of type II plant MADS-box protein with a highly conserved MADS-domain. Phylogenetic analysis revealed that FnAG had a close relationship with AG orthologs from other woody species. FnAG transcript was mainly expressed in reproductive tissues, but rarely detected in the vegetative tissues, consistent with the ABC model for floral development. A functional analysis was performed by ectopic expression of FnAG driven by the CaMV 35S promoter in transgenic Arabidopsis. Transformed plants showed homeotic conversions of carpeloid sepals and stamenoid petals. Curled leaves, reduced plant size, and earlier flowering were also observed in transgenic Arabidopsis. These data indicated that the FnAG functions in the same way as AG in Arabidopsis. These results provide the framework for targeted genome editing of black ash, an ecologically and economically important wetland species.