Membrane trafficking is a fundamental cellular process that is conserved across diverse eukaryotic lineages. Conversely, in evolutionary contexts, highly diversified and specialized membrane trafficking systems play pivotal roles in the distinctive biological functions acquired by individual organisms. For example, within plant systems, membrane trafficking is deeply involved in phenomena such as cell plate formation and pollen tube elongation, which are uniquely characteristic of plants. The diversification of membrane trafficking pathways and organelle functions is closely linked to the diversification of key membrane trafficking factors, including SNARE proteins. SNARE proteins, which execute fusion between two membrane compartments, are integral to all trafficking pathways connecting single-membrane organelles. Therefore, it is postulated that the proliferation of SNARE genes through gene duplication and subsequent functional differentiation was a prerequisite for the emergence of novel membrane trafficking pathways and, by extension, novel organelles. However, empirical evidence elucidating the mechanisms underlying functional diversification and neofunctionalization of SNAREs remains elusive. We aim to address this question, with a particular focus on SNARE molecules that have been uniquely acquired during plant evolution. In land plants, functional diversification is particularly evident in the VAMP7 group; three types of VAMP7 with distinct functions are conserved in seed plants. I would like to present the recent results from our research aimed at reconstructing the evolutionary processes underlying the neofunctionalization of VAMP727, the plant-unique VAMP7 member involved in multivesicular endosome-vacuole fusion.