Klamke, M.; Maurino, V.; Grefen, C.; Desimone, M.; Tessi, T. M.

The evolution of complex plant body architectures required the refinement of hormone transport networks, yet the evolutionary origins and functional diversification of cytokinin transporters remains unclear. Here, we reconstruct the molecular evolution of the AZA-GUANINE RESISTANT (AZG) family from bacteria and fungi to land plants. We show that AZG1 represents the ancestral land plants orthologue, preserving highly conserved proton-coupling residues present in streptophyte algae. Conversely, AZG2 emerged during vascular plant diversification and displays a distinct relaxation of evolutionary constraints within the ligand-binding pocket, providing a molecular basis for its transition to a proton-independent mechanism. Structural modeling and split-ubiquitin assays further reveal that despite this ancient sequence divergence, AZG1 and AZG2 have retained the biochemical capacity to physically heterodimerize. Together, our findings uncover the stepwise evolutionary innovation of the AZG family and link structural diversification to the increasing complexity of plant hormone transport networks.