Transcriptome dynamics in developing leaves from C3 and C4 Flaveria species reveal determinants of Kranz anatomy

C4 species have evolved more than 60 times independently from C3 ancestors. This multiple and parallel evolution of the complex C4 trait indicates common underlying evolutionary mechanisms that might be identified by comparative analysis of closely related C3 and C4 species. Efficient C4 function depends on a distinctive leaf anatomy that is characterized by enlarged, chloroplast rich bundle sheath cells and a narrow vein spacing. To elucidate molecular mechanisms generating this so called Kranz anatomy, we analyzed a developmental series of leaves from the C4 plant Flaveria bidentis and the closely related C3 species Flaveria robusta using leaf clearing and whole transcriptome sequencing. Applying non-negative matrix factorization on the data identified four different zones with distinct transcriptome patterns in growing leaves of both species. Comparing these transcriptome patterns revealed an important role of auxin metabolism and especially auxin homeostasis for establishing the high vein density typical for C4 leaves.