Chloroplast NADH dehydrogenase-like complex-mediated cyclic electron flow is the main electron transport route in C bundle sheath cells.

The superior productivity of C plants is achieved via a metabolic C cycle which acts as a CO pump across mesophyll and bundle sheath (BS) cells and requires an additional input of energy in the form of ATP. The importance of chloroplast NADH dehydrogenase-like complex (NDH) operating cyclic electron flow (CEF) around Photosystem I (PSI) for C photosynthesis has been shown in reverse genetics studies but the contribution of CEF and NDH to cell-level electron fluxes remained unknown. We have created gene-edited Setaria viridis with null ndhO alleles lacking functional NDH and developed methods for quantification of electron flow through NDH in BS and mesophyll cells. We show that CEF accounts for 84% of electrons reducing PSI in BS cells and most of those electrons are delivered through NDH while the contribution of the complex to electron transport in mesophyll cells is minimal. A decreased leaf CO assimilation rate and growth of plants lacking NDH cannot be rescued by supplying additional CO. Our results indicate that NDH-mediated CEF is the primary electron transport route in BS chloroplasts highlighting the essential role of NDH in generating ATP required for CO fixation by the C cycle in BS cells.