Shared characteristics underpinning C₄ leaf maturation derived from analysis of multiple C₃ and C₄ species of Flaveria

Most terrestrial plants use C₃ photosynthesis to fix carbon. In multiple plant lineages a modified system known as C₄ photosynthesis has evolved. To better understand the molecular patterns associated with induction of C₄ photosynthesis, the genus Flaveria that contains C₃ and C₄ species was used. A base to tip maturation gradient of leaf anatomy was defined, and RNA sequencing was undertaken along this gradient for two C₃ and two C₄ Flaveria species. Key C₄ traits including vein density, mesophyll and bundle sheath cross-sectional area, chloroplast ultrastructure, and abundance of transcripts encoding proteins of C₄ photosynthesis were quantified. Candidate genes underlying each of these C₄ 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 C₄ species. Through comparison with publicly available data sets, we identify a small number of transcriptional regulators that have been up-regulated in diverse C₄ species. The analysis identifies similar patterns of expression in independent C₄ lineages and so indicates that the complex C₄ pathway is associated with parallel as well as convergent evolution.