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C4 grasses often outperform C3 species under hot, arid conditions due to superior water and nitrogen use efficiencies and lower rates of photorespiration. A method of concentrating CO2 around the site of carbon fixation in the bundle sheath (BS) is required to realize these gains. In NADP-malic enzyme (NADP-ME)-type C4 grasses such as maize (Zea mays), suberin deposition in the BS cell wall is hypothesized to act as a diffusion barrier to CO2 escape and O2 entry from surrounding mesophyll cells. Suberin is a heteropolyester comprised of acyl-lipid-derived aliphatic and phenylpropanoidderived aromatic components. Suberin is synthesized by a large network of biosynthetic and regulatory genes, none of which have been characterized in C4 grasses to date. A set of candidate genes expressed concurrently with BS suberization were identified in maize, rice (Oryza sativa), and Setaria viridis and a putative biosynthetic pathway was assembled based on functional characterizations from model dicots. To disrupt aromatic suberin synthesis, two paralogously duplicated maize homologues of Arabidopsis ALIPHATIC SUBERIN FERULOYL TRANSFERASE, ZmAsft1 and ZmAsft2, were mutated using tightly linked Dissociation transposons. Loss-of-function double mutants exhibited a significant reduction in suberin-specific aliphatic monomers in both leaves and roots without a stoichiometric decrease in aromatic monomers. There was no     evidence that increased esterification to arabinoxylan, the major polymer sink for ferulic acid in maize leaves, masked the aromatic defect. Reduced staining of osmiophilic material and attenuated cohesion between the BS suberin lamellae and polysaccharides were observed by TEM, indirectly implicating aromatic monomers in the ultrastructural defect. Cell wall elasticity, transpiration, and stomatal conductance significantly increased without pleiotropic changes in cuticular permeability, suggesting that the mutation facilitated water movement through the BS apoplast. However, there were no morphological phenotypes under ambient conditions. Likewise, net CO2 assimilation and 13 C isotope discrimination were indistinguishable from wild type, indicating no difference in BS leakiness between genotypes. Thus, ZmAsft expression has a minor effect on leaf water movement but is not required for the CO2 concentrating mechanism. A more severe disruption to the suberin lamella is necessary to determine whether these structures are essential for CO2 concentration in NADP-ME-type C4 grasses.