Leaf transcriptomes from C3, C3-C4 intermediate, and C4 Neurachne species give insights into C4 photosynthesis evolution

The C-4 photosynthetic pathway is hypothesized to have evolved from the ancestral C-3 pathway through progressive changes in leaf anatomy and biochemistry with extant C-3-C-4 photosynthetic intermediate species representing phenotypes between species demonstrating full C-3 and full C-4 states. The Australian endemic genus Neurachne is the only known grass group that contains distinct, closely related species that carry out C-3, C-3-C-4 intermediate, or C-4 photosynthesis. To explore and understand the molecular mechanisms underlying C-4 photosynthesis evolution in this genus, leaf transcriptomes were generated from two C-3, three photosynthetic intermediate (proto-Kranz, C-2-like, and C-2), and two C-4 Neurachne species. The data were used to reconstruct phylogenetic relationships in Neurachne, which confirmed two independent C-4 origins in the genus. Relative transcript abundances substantiated the photosynthetic phenotypes of individual species and highlighted transcriptional investment differences between species, including between the two C-4 species. The data also revealed proteins potentially involved in C-4 cycle intermediate transport and identified molecular mechanisms responsible for the evolution of C-4-associated proteins in the genus.