Mind the gap: the evolutionary engagement of the C₄ metabolic cycle in support of net carbon assimilation

C₄ photosynthesis evolved dozens of times, with a critical step being the engagement of a C₄ metabolic cycle to concentrate CO₂ into a bundle sheath-like compartment. While C₃-C₄ intermediate species show a progressive increase in the activity of a C₄ metabolic cycle, the integration of the C₄ and C₃ biochemical cycles in enhancing photosynthetic carbon gain occurs in a punctuated manner, at an initial C₄ cycle activity near 60%. Punctuated integration of the C₄ cycle could result from the evolutionary acquisition of traits that coordinate the C₃ and C₄ biochemical cycles (for example, an enzymatic, regulatory or transport function) or from a sudden reduction in the mesophyll C₃ cycle. Alternatively, a punctuated pattern could be an artifact of low numbers of C₃-C₄ intermediates in the evolutionary space where C₄ cycle engagement occurs, due to incomplete sampling of natural diversity or evolutionary dynamics rendering such intermediates unstable. Understanding how the C₄ cycle becomes integrated with the C₃ cycle could reveal new avenues for engineering the C₄ pathway into C₃ plants. Such efforts would be facilitated by the generation of hybrids, or the discovery of additional intermediates, that span the transition from low to high C₄ cycle engagement.