Metabolic profiles in C(3), C(3)–C(4) intermediate, C(4)-like, and C(4) species in the genus Flaveria

C(4) photosynthesis concentrates CO(2) around Rubisco in the bundle sheath, favouring carboxylation over oxygenation and decreasing photorespiration. This complex trait evolved independently in >60 angiosperm lineages. Its evolution can be investigated in genera such as Flaveria (Asteraceae) that contain species representing intermediate stages between C(3) and C(4) photosynthesis. Previous studies have indicated that the first major change in metabolism probably involved relocation of glycine decarboxylase and photorespiratory CO(2) release to the bundle sheath and establishment of intercellular shuttles to maintain nitrogen stoichiometry. This was followed by selection for a CO(2)-concentrating cycle between phosphoenolpyruvate carboxylase in the mesophyll and decarboxylases in the bundle sheath, and relocation of Rubisco to the latter. We have profiled 52 metabolites in nine Flaveria species and analysed (13)CO(2) labelling patterns for four species. Our results point to operation of multiple shuttles, including movement of aspartate in C(3)–C(4) intermediates and a switch towards a malate/pyruvate shuttle in C(4)-like species. The malate/pyruvate shuttle increases from C(4)-like to complete C(4) species, accompanied by a rise in ancillary organic acid pools. Our findings support current models and uncover further modifications of metabolism along the evolutionary path to C(4) photosynthesis in the genus Flaveria.