The metabolite pathway between bundle sheath and mesophyll: Quantification of plasmodesmata in leaves of C₃ and C₄ monocots

C₄ photosynthesis is characterized by a CO₂-concentrating mechanism between mesophyll (M) and bundle sheath (BS) cells of leaves. This generates high metabolic fluxes between these cells, through interconnecting plasmodesmata (PD). Quantification of these symplastic fluxes for modeling studies requires accurate quantification of PD, which has proven difficult using transmission electron microscopy. Our new quantitative technique combines scanning electron microscopy and 3D immunolocalization in intact leaf tissues to compare PD density on cell interfaces in leaves of C₃ (rice [Oryza sativa] and wheat [Triticum aestivum]) and C₄ (maize [Zea mays] and Setaria viridis) monocot species. Scanning electron microscopy quantification of PD density revealed that C₄ species had approximately twice the number of PD per pitfield area compared with their C₃ counterparts. 3D immunolocalization of callose at pitfields using confocal microscopy showed that pitfield area per M-BS interface area was 5 times greater in C₄ species. Thus, the two C₄ species had up to nine times more PD per M-BS interface area (S. viridis, 9.3 PD µm^-2; maize, 7.5 PD µm^-2; rice 1.0 PD µm^-2; wheat, 2.6 PD µm^-2). Using these anatomical data and measured photosynthetic rates in these C₄ species, we have now calculated symplastic C₄ acid flux per PD across the M-BS interface. These quantitative data are essential for modeling studies and gene discovery strategies needed to introduce aspects of C₄ photosynthesis to C₃ crops.