Evolutionary and molecular analysis of Dof transcription factors identified a conserved motif for intercellular protein trafficking.

· Cell-to-cell trafficking of transcription factors (TFs) has been shown to play an important role in the regulation of plant developmental events, but the evolutionary relationship between cell-autonomous and noncell-autonomous (NCA) TFs remains elusive. · AtDof4.1, named INTERCELLULAR TRAFFICKING DOF 1 (ITD1), was chosen as a representative NCA member to explore this evolutionary relationship. Using domain structure-function analyses and swapping studies, we examined the cell-to-cell trafficking of plant-specific Dof TF family members across Arabidopsis and other species. · We identified a conserved intercellular trafficking motif (ITM) that is necessary and sufficient for selective cell-to-cell trafficking and can impart gain-of-function cell-to-cell movement capacity to an otherwise cell-autonomous TF. The functionality of related motifs from Dof members across the plant kingdom extended, surprisingly, to a unicellular alga that lacked plasmodesmata. By contrast, the algal homeodomain related to the NCA KNOX homeodomain was either inefficient or unable to impart such cell-to-cell movement function. · The Dof ITM appears to predate the evolution of selective plasmodesmal trafficking in the plant kingdom, which may well have acted as a molecular template for the evolution of Dof proteins as NCA TFs. However, the ability to efficiently traffic for KNOX homeodomain (HD) proteins may have been acquired during the evolution of early nonvascular plants.