Bundle sheath suberisation is required for C₄ photosynthesis in a Setaria viridis mutant

C₄ photosynthesis provides an effective solution for overcoming the catalytic inefficiency of Rubisco. The pathway is characterised by a biochemical CO₂ concentrating mechanism that operates across mesophyll and bundle sheath (BS) cells and relies on a gas tight BS compartment. A screen of a mutant population of Setaria viridis, an NADP-malic enzyme type C₄ monocot, generated using N-nitroso-N-methylurea identified a mutant with an amino acid change in the gene coding region of the ABCG transporter, a step in the suberin synthesis pathway. Here, Nile red staining, TEM, and GC/MS confirmed the alteration in suberin deposition in the BS cell wall of the mutant. We show that this has disrupted the suberin lamellae of BS cell wall and increased BS conductance to CO₂ diffusion more than two-fold in the mutant. Consequently, BS CO₂ partial pressure is reduced and CO₂ assimilation was impaired in the mutant. Our findings provide experimental evidence that a functional suberin lamellae is an essential anatomical feature for efficient C₄ photosynthesis in NADP-ME plants like S. viridis and have implications for engineering strategies to ensure future food security.