Transcriptome dynamics in developing leaves from C₃ and C₄<i>Flaveria</i> species

C₄ species have evolved more than 60 times independently from C₃ ancestors. This multiple and parallel evolution of the complex C₄ trait suggests common underlying evolutionary mechanisms, which could be identified by comparative analysis of closely related C₃ and C₄ species. Efficient C₄ function depends on a distinctive leaf anatomy that is characterised by enlarged, chloroplast-rich bundle sheath cells and narrow vein spacing. To elucidate the molecular mechanisms that generate the Kranz anatomy, we analysed a developmental series of leaves from the C₄ plant Flaveria bidentis and the closely related C₃ species Flaveria robusta by comparing anatomies and transcriptomes. Vascular density measurements of all nine leaf developmental stages identified three leaf anatomical zones whose proportions vary with respect to the developmental stage. We then deconvoluted the transcriptome datasets using non-negative matrix factorisation, which identified four distinct transcriptome patterns in the growing leaves of both species. By integrating the leaf anatomy and transcriptome data, we were able to correlate the different transcriptional profiles with different developmental zones in the leaves. These comparisons revealed an important role for auxin metabolism, in particular auxin homeostasis (conjugation and deconjugation), in establishing the high vein density typical of C₄ species.