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

High vein density, a distinctive trait of C-4 leaves, is central to both C-3-to-C-4 evolution and conversion of C-3 to C-4-like crops. We tested the hypothesis that high vein density in C-4 leaves is due to elevated auxin biosynthesis and transport in developing leaves. Upregulation of genes in auxin biosynthesis pathways and higher auxin content were found in developing C-4 leaves compared with developing C-3 leaves. The same observation held for maize foliar (C-4) and husk (C-3) 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.