NDH complex-mediated cyclic electron flow in bundle sheath cells enables C ₄ photosynthesis

The superior productivity of C ₄ plants is achieved via a metabolic C ₄ cycle which acts as a CO ₂ pump across mesophyll and bundle sheath (BS) cells and requires an additional input of energy in the form of ATP. Chloroplast NADH dehydrogenase-like complex (NDH) increases ATP production in C ₃ plants by operating cyclic electron flow (CEF) around Photosystem I (PSI), and its importance for C ₄ photosynthesis has been proposed from evolutionary and reverse genetics studies. We used the gene-edited C ₄ species Setaria viridis with null ndhO alleles lacking NDH to study a contribution of the complex to the cell-level electron transport. Our results indicate that NDH is the primary PSI electron acceptor mediating the majority of CEF in BS cells whilst the contribution of the complex to CEF in mesophyll cells is minimal. Moreover, the reduced leaf CO ₂ assimilation rate and growth of plants lacking the complex cannot be rescued by supplying additional CO ₂ , indicating that NDH is essential for generating ATP required for CO ₂ fixation by the C ₃ cycle. Hereby we resolve a cell-level mechanism for the contribution of NDH to supporting high CO ₂ assimilation rates in C ₄ photosynthesis.