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We identify transcription factors that show conserved patterns of expression in multiple C4 species, both within the Flaveria genus and also in more distantly related C4 plants., 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 C4Flaveria 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.

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 C4Flaveria 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 then 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 publically available datasets 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.HighlightWe identify transcription factors that show conserved patterns of expression in multiple 29 C4 species, both within the Flaveria genus, but also in more distantly related C4 plants.

C4 photosynthesis acts as a carbon concentrating mechanism that leads to large increases in photosynthetic efficiency. The C4 pathway is found in more than 60 plant lineages1 but the molecular enablers of this evolution are poorly understood. In particular, it is unclear how non-photosynthetic proteins in the ancestral C3 system have repeatedly become strongly expressed and integrated into photosynthesis gene regulatory networks in C4 leaves. Here, we provide clear evidence that in C3 leaves, genes encoding key enzymes of the C4 pathway are already co-regulated with photosynthesis genes and are controlled by both light and chloroplast-to-nucleus signalling. In C4 leaves this regulation becomes increasingly dependent on the chloroplast. We propose that regulation of C4 cycle genes by light and the chloroplast in the ancestral C3 state has facilitated the repeated evolution of the complex and convergent C4 trait.

Leaves of angiosperms are made up of multiple distinct cell types. While the function of mesophyll cells, guard cells, phloem companion cells and sieve elements are clearly described, this is not the case for the bundle sheath (BS). To provide insight into the role of the BS in the C3 species Arabidopsis thaliana, we labelled ribosomes in this cell type with a FLAG tag. We then used immunocapture to isolate these ribosomes, followed by sequencing of resident mRNAs. This showed that 5% of genes showed specific splice forms in the BS, and that 15% of genes were preferentially expressed in these cells. The BS translatome strongly implies that the BS plays specific roles in sulfur transport and metabolism, glucosinolate biosynthesis and trehalose metabolism. Much of the C4 cycle is differentially expressed between the C3 BS and the rest of the leaf. Furthermore, the global patterns of transcript residency on BS ribosomes overlap to a greater extent with cells of the root pericycle than any other cell type. This analysis provides the first insight into the molecular function of this cell type in C3 species, and also identifies characteristics of BS cells that are probably ancestral to both C3 and C4 plants.