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.