Abstract
The complex syndromes of C4 photosynthesis include several biochemical and anatomical modifications evolved from the ancestral C3 photosynthetic pathway. Although the biochemical modifications in C4 plants are well understood, the mechanisms responsible for C4 anatomical modifications are still largely unknown. High vein density, which is a distinctive anatomical trait of C4 leaves, is central to C3-to-C4 evolution and is considered a key step in the conversion of C3 to C4-like crops in the future. I tested the hypothesis that high vein density in C4 leaves is due to elevated auxin biosynthesis and transport in developing leaves. To test this hypothesis I conducted the following studies. First, I obtained transcriptomes of developing leaves from Tarenaya hassleriana and Gynandropsis gynandra, a C3 and a C4 species in Cleomaceae. I analyzed and compared expressed genes of the two Cleomaceae species by RNA-seq. In C4 G. gynandra many genes promoting auxin biosynthesis or transport were up-regulated while the gene for MYC2, a transcription factor that suppresses the biosynthesis of tryptophan, which is a precursor of auxin, was down-regulated. Second, I found higher auxin contents in the developing leaves of G. gynandra. The same observation held for maize foliar (C4) and husk (C3) leaf primordia. Third, I found increased auxin contents and vein densities in both of the two Arabidopsis thaliana loss-of-function myc2 mutants. Fourth, treating G. gynandra with yucasin, an auxin biosynthesis inhibitor, reduced leaf vein density. Fifth, treating plants with NPA, an auxin transport inhibitor, led to much fewer higher-order (minor) veins in new leaves. Finally, both A. thaliana auxin efflux transporter pin1 and influx transporter lax2 mutants showed reduced vein numbers. These observations point to the central role of auxin biosynthesis and transport in vein density control. I conclude that elevated biosynthesis and transport of auxin is the molecular basis underlying high vein density in C4 leaves.