Diffusion of CO2 across the Mesophyll-Bundle Sheath Cell Interface in a C4 Plant with Genetically Reduced PEP Carboxylase Activity

Phosphoenolpyruvate carboxylase (PEPC), localized to the cytosol of the mesophyll cell, catalyzes the first carboxylation step of the C-4 photosynthetic pathway. Here, we used RNA interference to target the cytosolic photosynthetic PEPC isoform in Setaria viridis and isolated independent transformants with very low PEPC activities. These plants required high ambient CO2 concentrations for growth, consistent with the essential role of PEPC in C-4 photosynthesis. The combination of estimating direct CO2 fixation by the bundle sheath using gas-exchange measurements and modeling C-4 photosynthesis with low PEPC activity allowed the calculation of bundle sheath conductance to CO2 diffusion (g(bs)) in the progeny of these plants. Measurements made at a range of temperatures suggested no or negligible effect of temperature on g(bs) depending on the technique used to calculate g(bs) Anatomical measurements revealed that plants with reduced PEPC activity had reduced cell wall thickness and increased plasmodesmata (PD) density at the mesophyll-bundle sheath (M-BS) cell interface, whereas we observed little difference in these parameters at the mesophyll-mesophyll cell interface. The increased PD density at the M-BS interface was largely driven by an increase in the number of PD pit fields (cluster of PDs) rather than an increase in PD per pit field or the size of pit fields. The correlation of g(bs) with bundle sheath surface area per leaf area and PD area per M-BS area showed that these parameters and cell wall thickness are important determinants of g(bs) It is intriguing to speculate that PD development is responsive to changes in C-4 photosynthetic flux.