What does it take to be C4? Lessons from the evolution of C4 photosynthesis

Twenty-five years ago research had already established a firm biochemical and physiological understanding of the CO2-concentrating mechanism that creates a high CO2 environment (1,000‐3,000 mbar) in bundle-sheath cells in leaves of C4 plants and accounts for most of their distinctive photosynthetic properties (5). It was then clear that the minimum requirements for this CO2 concentrating mechanism included: (a) cell-specific amplification of enzymes of C4 photosynthesis (i.e. phosphoenolpyruvate carboxylase [PEPC] in mesophyll, and C4 acid decarboxylases and Rubisco in bundle-sheath cells), with complementary adjustments of photosystem and electron transport activities; (b) novel cell-specific organelle metabolite translocators; (c) symplastic connections of the spatially separated sources and sinks of 4Cdicarboxylic acid transport metabolites; and (d) barriers to CO2 diffusion between the site of CO2 fixation by PEPCase in mesophyll cells and sites of CO2 release and refixation by Rubisco in bundle-sheath cells. These requirements have been met in a great variety of ways during the evolution of C4 plants, through diverse cooperative pathways of carbon metabolism and integrated photoreactions in adjacent,