De novo transcriptome assemblies of C 3 and C 4 non-model grass species reveal key differences in leaf development

Background C 4 photosynthesis is a mechanism that plants have evolved to reduce the rate of photorespiration during the carbon fixation process. The C 4 pathway allows plants to adapt to high temperatures and light while more efficiently using resources, such as water and nitrogen. Despite decades of studies, the evolution of the C 4 pathway from a C 3 ancestor remains a biological enigma. Interestingly, species with C 3 -C 4 intermediates photosynthesis are usually found closely related to the C 4 lineages. Indeed, current models indicate that the assembly of C 4 photosynthesis was a gradual process that included the relocalization of photorespiratory enzymes, and the establishment of intermediate photosynthesis subtypes. More than a third of the C 4 origins occurred within the grass family (Poaceae). In particular, the Otachyriinae subtribe (Paspaleae tribe) includes 35 American species from C 3 , C 4 , and intermediates taxa making it an interesting lineage to answer questions about the evolution of photosynthesis. Results To explore the molecular mechanisms that underpin the evolution of C 4 photosynthesis, the transcriptomic dynamics along four different leaf segments, that capture different stages of development, were compared among Otachyriinae non-model species. For this, leaf transcriptomes were sequenced, de novo assembled, and annotated. Gene expression patterns of key pathways along the leaf segments showed distinct differences between photosynthetic subtypes. In addition, genes associated with photorespiration and the C 4 cycle were differentially expressed between C 4 and C 3 species, but their expression patterns were well preserved throughout leaf development. Conclusions New, high-confidence, protein-coding leaf transcriptomes were generated using high-throughput short-read sequencing. These transcriptomes expand what is currently known about gene expression in leaves of non-model grass species. We found conserved expression patterns of C 4 cycle and photorespiratory genes among C 3 , intermediate, and C 4 species, suggesting a prerequisite for the evolution of C 4 photosynthesis. This dataset represents a valuable contribution to the existing genomic resources and provides new tools for future investigation of photosynthesis evolution.