Insight into the petunia Dof transcription factor family reveals a new regulator of male-sterility

Abstract Petunia hybrida is a hybrid ornamental that serves as one of the most popular bedding plants in the world. Numerous petunia seeds are distributed on an annual basis, which requires labor-intensive hybrid-breeding. The Dof (DNA-binding with one finger) transcription factor family is a plant-specific member of the zinc finger superfamily. Dofs play diverse roles in regulating plant developmental processes; however, their functions during flower development are not well characterized, especially in ornamental species. In this study, we used genome-wide analysis to identify 35 Dof genes that are encoded in the P. inflata reference genome. These Petunia Dofs (PiDofs) can be organized into five distinct phylogenetic groups, based on the sequences of their highly conserved Dof domains. Expression analysis using quantitative-RT-PCR identified several PiDofs that exhibit unique expression patterns during reproductive development. Many of these reproductive PiDofs were expressed in all four floral whorls throughout flower development, while other PiDofs exhibited developmental stage and/or organ specific expression patterns. RNAi was used to knock-down the expression level of PiDof14, an anther-specific Dof. Reduced expression of PiDof14 promoted tapetum degradation and depressed meiotic divisions in the microsporocytes, ultimately leading to a male-sterile phenotype. The organ- and stage-specific expression patterns of many of the other PiDof genes indicate that additional members of this family may play specific roles in regulating male and female fertility. This work provides insight into a novel regulator of male-fertility in Petunia, and lays the foundation for targeted genome engineering of the larger Dof family in order to generate an efficient hybrid breeding system for P. hybrida.