Metabolite profiles reveal interspecific variation in operation of the Calvin–Benson cycle in both C(4) and C(3) plants

Low atmospheric CO(2) in recent geological time led to the evolution of carbon-concentrating mechanisms (CCMs) such as C(4) photosynthesis in >65 terrestrial plant lineages. We know little about the impact of low CO(2) on the Calvin–Benson cycle (CBC) in C(3) species that did not evolve CCMs, representing >90% of terrestrial plant species. Metabolite profiling provides a top-down strategy to investigate the operational balance in a pathway. We profiled CBC intermediates in a panel of C(4) (Zea mays, Setaria viridis, Flaveria bidentis, and F. trinervia) and C(3) species (Oryza sativa, Triticium aestivum, Arabidopsis thaliana, Nicotiana tabacum, and Manihot esculenta). Principal component analysis revealed differences between C(4) and C(3) species that were driven by many metabolites, including lower ribulose 1,5-bisphosphate in C(4) species. Strikingly, there was also considerable variation between C(3) species. This was partly due to different chlorophyll and protein contents, but mainly to differences in relative levels of metabolites. Correlation analysis indicated that one contributory factor was the balance between fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, phosphoribulokinase, and Rubisco. Our results point to the CBC having experienced different evolutionary trajectories in C(3) species since the ancestors of modern plant lineages diverged. They underline the need to understand CBC operation in a wide range of species.