Elevated auxin biosynthesis and transport underlie high vein density in C₄ leaves

High vein density, a distinctive trait of C₄ leaves, is central to both C₃-to-C₄ evolution and conversion of C₃ to C₄-like crops. We tested the hypothesis that high vein density in C₄ leaves is due to elevated auxin biosynthesis and transport in developing leaves. Up-regulation of genes in auxin biosynthesis pathways and higher auxin content were found in developing C₄ leaves compared with developing C₃ leaves. The same observation held for maize foliar (C₄) and husk (C₃) leaf primordia. Moreover, auxin content and vein density were increased in loss-of-function mutants of Arabidopsis MYC2, a suppressor of auxin biosynthesis. Treatment with an auxin biosynthesis inhibitor or an auxin transport inhibitor led to much fewer veins in new leaves. Finally, both Arabidopsis thaliana auxin efflux transporter pin1 and influx transporter lax2 mutants showed reduced vein numbers. Thus, development of high leaf vein density requires elevated auxin biosynthesis and transport.