ABSTRACT C 4 grasses often outperform C 3 species under hot, arid conditions due to superior water and nitrogen use efficiencies and lower rates of photorespiration. A method of concentrating CO 2 around the site of carbon fixation in the bundle sheath (BS) is required to realize these gains. In NADP-malic enzyme (NADP-ME)-type C 4 grasses such as maize, suberin deposition in the BS cell wall is hypothesized to act as a diffusion barrier to CO 2 escape and O 2 entry from surrounding mesophyll cells. Suberin is a heteropolyester comprised of acyl-lipid-derived aliphatic and phenylpropanoid-derived aromatic components. To disrupt BS suberization, we mutated two paralogously duplicated, unlinked maize orthologues of Arabidopsis thaliana ALIPHATIC SUBERIN FERULOYL TRANSFERASE, ZmAsft1 and ZmAsft2 , using closely linked Dissociation transposons. Loss-of-function double mutants revealed a 97% reduction in suberin-specific omega-hydroxy fatty acids without a stoichiometric decrease in ferulic acid. However, BS suberin lamellae were deficient in electron opaque material, and cohesion between the suberin lamellae and polysaccharide cell walls was attenuated in double mutants. There were no other morphological phenotypes under ambient conditions. Furthermore, there was no significant effect on net CO 2 assimilation at any intercellular CO 2 concentration, and no effect on 13 C isotope discrimination relative to wild type. Thus, ZmAsft expression is not required to establish a functional CO 2 concentrating mechanism in in maize. Double mutant leaves exhibit elevated cell wall elasticity, transpirational, and stomatal conductance relative to WT. Thus, the ZmAsft genes are dispensable for gas exchange barrier function but may be involved in regulation of leaf water movement. One-sentence Summary Double mutants of two paralogously duplicated maize Aliphatic Suberin Feruloyl Transferase (ZmAsft) genes exhibit reduced aliphatic suberin content, cell wall cohesion defects, and elevated leaf transpiration, but no changes in CO2 assimilation relative to wild type.