In this project, we have investigated the proteome of the plasmodesmata (PD) enriched membrane fraction from poplar cell suspension cultures and quantitatively compared its protein composition with other membrane fractions. We build on the previously published literature on the PD proteome by using semiquantitative methods and, for the first time, demonstrate in vitro callose synthase activity from Populus PD proteins. To our knowledge, this study is the first proteomic investigation of the PD performed on a tree species.