Investigating the NAD-ME biochemical pathway within C₄ grasses using transcript and amino acid variation in C₄ photosynthetic genes

Expanding knowledge of the C₄ photosynthetic pathway can provide key information to aid biological improvements to crop photosynthesis and yield. While the C₄ NADP-ME pathway is well characterised, there is increasing agricultural and bioengineering interest in the comparably understudied NAD-ME and PEPCK pathways. Within this study, a systematic identification of key differences across species has allowed us to investigate the evolution of C₄-recruited genes in one C₃ and eleven C₄ grasses (Poaceae) spanning two independent origins of C₄ photosynthesis. We present evidence for C₄-specific paralogs of NAD-malic enzyme 2, MPC1 and MPC2 (mitochondrial pyruvate carriers) via increased transcript abundance and associated rates of evolution, implicating them as genes recruited to perform C₄ photosynthesis within NAD-ME and PEPCK subtypes. We then investigate the localisation of AspAT across subtypes, using novel and published evidence to place AspAT3 in both the cytosol and peroxisome. Finally, these findings are integrated with transcript abundance of previously identified C₄ genes to provide an updated model for C₄ grass NAD-ME and PEPCK photosynthesis. This updated model allows us to develop on the current understanding of NAD-ME and PEPCK photosynthesis in grasses, bolstering our efforts to understand the evolutionary ‘path to C₄’ and improve C₄ photosynthesis.