Shared characteristics underpinning C$_4$ leaf maturation derived from analysis of multiple C$_3$ and C$_4$ species of $\textit{Flaveria}$

Most terrestrial plants use C$_3$ photosynthesis to fix carbon. In multiple plant lineages a modified system known as C$_4$ photosynthesis has evolved. To better understand the molecular patterns associated with induction of C$_4$ photosynthesis, the genus $\textit{Flaveria}$ that contains C$_3$ and C$_4$ species was used. A base to tip maturation gradient of leaf anatomy was defined, and RNA sequencing was undertaken along this gradient for two C$_3$ and two C$_4$ $\textit{Flaveria}$ species. Key C$_4$ traits including vein density, mesophyll and bundle sheath cross-sectional area, chloroplast ultrastructure, and abundance of transcripts encoding proteins of C$_4$ photosynthesis were quantified. Candidate genes underlying each of these C$_4$ characteristics were identified. Principal components analysis indicated that leaf maturation and the photosynthetic pathway were responsible for the greatest amount of variation in transcript abundance. Photosynthesis genes were over-represented for a prolonged period in the C$_4$ species. Through comparison with publicly available data sets, we identify a small number of transcriptional regulators that have been up-regulated in diverse C$_4$ species. The analysis identifies similar patterns of expression in independent C$_4$ lineages and so indicates that the complex C$_4$ pathway is associated with parallel as well as convergent evolution.Seeds for all Flaveria species used were kindly provided by Udo Gowik (Universität Düsseldorf). The European Union 3to4 project and the Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/J011754/1 funded the project