Stepwise increase in plasmodesmata during C ₄ evolution in Flaveria

Plant cells are interconnected via plasmodesmata that enable the exchange of mobile molecules such as metabolites between neighbouring cells. In leaves of plants that conduct C ₄ photosynthesis, carbon fixation is separated between mesophyll (M) and bundle sheath (BS) cells, and the shuttling of metabolites between these cells is thought to be crucial for running the carbon concentrating mechanism. Higher numbers of plasmodesmata for metabolite exchange between M and BS cells have been observed in multiple lineages of C ₄ plants, but how this increased cell-to-cell connectivity developed in the context of C ₄ evolution is not understood. Here, we examined plasmodesmata in Flaveria species, including C ₃ , C ₃ -C ₄ intermediate, C ₄ -like and C ₄ species to represent an evolutionary gradient. Using electron microscopy, we found two distinct stepwise increases in plasmodesmata frequency along this gradient. C ₃ -C ₄ intermediate species had ∼3 fold more plasmodesmata than C ₃ species, and C ₄ -like and C ₄ species had >6-fold increases relative to the C ₃ species. In the first step between C ₃ and C ₃ -C ₄ intermediate species, plasmodesmata numbers were higher in the intermediate species, but equal in frequency between M-M and M-BS cell interfaces. However, in a second step between C ₃ -C ₄ intermediate and C ₄ -like/C ₄ species, the increase in plasmodesmata formation was predominantly observed at the M-BS cell interface, where C ₄ /C ₃ acid (malate/alanine) shuttles are active. The higher physical cell-to-cell connectivity along the evolutionary gradient occurred without significant changes in BS cell size, but coincided with the formation of increased chloroplast area/coverage in BS cells of C ₄ species. Thus, we propose that high cell-to-cell connectivity may facilitate “C ₂ metabolism” in the C ₃ -C ₄ intermediate species, which also requires metabolite transfer between cell types (e.g. glycine shuttle), but full polar enrichment of plasmodesmata at the M-BS interface accompanies C ₄ metabolism. Overall, our work provides new insight into the stepwise development of cell-to-cell connectivity along the evolutionary trajectory of C ₄ photosynthesis.