Transcriptome dynamics in developing leaves from C3and C4Flaveriaspecies reveal determinants of Kranz anatomy

C 4 species have evolved more than 60 times independently from C 3 ancestors. This multiple and parallel evolution of the complex C 4 trait indicates common underlying evolutionary mechanisms that might be identified by comparative analysis of closely related C 3 and C 4 species. Efficient C 4 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 C 4 plant Flaveria bidentis and the closely related C 3 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 C 4 leaves.