Improved mesophyll-bundle sheath connectivity is achieved via different mechanisms in C₂ vs C₄ Alternanthera

Connectivity between mesophyll (M) and bundle sheath (BS) cells must improve during the evolution of C₄ photosynthesis to facilitate large metabolite fluxes between these cell types, but the trait combinations that enhance M-BS connectivity and the points at which these enhancements occur along the C₃ to C₄ evolutionary trajectory remain unknown. We investigated shifts in M-BS connectivity that accompanied photosynthetic diversification within the eudicot genus Alternanthera from the agriculturally important Amaranthaceae family. We combined transmission electron microscope and confocal microscopy to reconstruct three-dimensional internal leaf structures of C₃, C₂, and C₄ Alternanthera species to reveal differences in the M-BS interface and cell-to-cell communication capacity. We found that M-BS connectivity increases stepwise from C₃ to C₂ and to C₄ species, but these shifts are achieved via different combinations of underlying traits in C₂ vs C₄ species. Specifically, increases to vein density were unique to C₂ whilst enhancements to BS cell, pit field, and plasmodesmata densities were unique to C₄ Alternanthera species. Both C₂ and C₄ Alternanthera shorten BS cell length, which may facilitate tighter BS packing to enhance M-BS connectivity. Our findings highlight the diverse mechanisms through which photosynthetic innovation can emerge, providing valuable insight for evolutionary biology and agricultural bioengineering efforts.