Multiple mechanisms for enhanced plasmodesmata density in disparate subtypes of C4 grasses

High plasmodesmata density in C-4 grasses is a result of larger pit fields and/or more abundant plasmodesmata per pit field area.Proliferation of plasmodesmata (PD) connections between bundle sheath (BS) and mesophyll (M) cells has been proposed as a key step in the evolution of two-cell C-4 photosynthesis; However, a lack of quantitative data has hampered further exploration and validation of this hypothesis. In this study, we quantified leaf anatomical traits associated with metabolite transport in 18 species of BEP and PACMAD grasses encompassing four origins of C-4 photosynthesis and all three C-4 subtypes (NADP-ME, NAD-ME, and PCK). We demonstrate that C-4 leaves have greater PD density between M and BS cells than C-3 leaves. We show that this greater PD density is achieved by increasing either the pit field (cluster of PD) area or the number of PD per pit field area. NAD-ME species had greater pit field area per M-BS interface than NADP-ME or PCK species. In contrast, NADP-ME and PCK species had lower pit field area with increased number of PD per pit field area than NAD-ME species. Overall, PD density per M-BS cell interface was greatest in NAD-ME species while PD density in PCK species exhibited the largest variability. Finally, the only other anatomical characteristic that clearly distinguished C-4 from C-3 species was their greater S-b value, the BS surface area to subtending leaf area ratio. In contrast, BS cell volume was comparable between the C-3 and C-4 grass species examined.