The evolution of C4 photosynthesis is thought to occur from structural and biochemical modifications of C3 plants through C3–C4 intermediates (Brown & Hattersley 1989; Monson & Moore 1989). Intermediate C3–C4 species typically have C4-like Kranz anatomy and glycine decarboxylase (GDC) localized within bundle sheath (BS) mitochondria. However, instead of a fully operational CO2 concentrating mechanism, they typically use a ‘C2 cycle’ where glycolate is shuttled from the mesophyll to the BS cells and decarboxylated via GDC to increase the refixation of photorespired CO2 (Rawsthorne et al. 1988; Rawsthorne 1992; Sage et al. 2012). This biochemical compartmentalization is generally associated with numerous mitochondria, peroxisomes and chloroplasts arranged in a centripetal position within the BS cells (Edwards & Ku 1987; Brown & Hattersley 1989). Additionally, compared with C3 plants, the C3–C4 species have reduced rates of photorespiration and lower CO2 compensation points (Γ), particularly under high temperatures (Monson & Rawsthorne 2000).