Stepwise increase in plasmodesmata during C₄evolution inFlaveria

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 C₄photosynthesis, 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 C₄plants, but how this increased cell-to-cell connectivity developed in the context of C₄evolution is not understood. Here, we examined plasmodesmata inFlaveriaspecies, including C₃, C₃-C₄intermediate, C₄-like and C₄species to represent an evolutionary gradient. Using electron microscopy, we found two distinct stepwise increases in plasmodesmata frequency along this gradient. C₃-C₄intermediate species had ∼3 fold more plasmodesmata than C₃species, and C₄-like and C₄species had >6-fold increases relative to the C₃species. In the first step between C₃and C₃-C₄intermediate 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 C₃-C₄intermediate and C₄-like/C₄species, the increase in plasmodesmata formation was predominantly observed at the M-BS cell interface, where C₄/C₃acid (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 C₄species. Thus, we propose that high cell-to-cell connectivity may facilitate “C₂metabolism” in the C₃-C₄intermediate 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 C₄metabolism. Overall, our work provides new insight into the stepwise development of cell-to-cell connectivity along the evolutionary trajectory of C₄photosynthesis.