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

Abstract

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.