Improved mesophyll–bundle sheath connectivity is achieved via different mechanisms in C 2 vs C 4 Alternanthera

Summary: 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.