Summary: Introduction of a C₄ photosynthetic mechanism into C₃ crops offers an opportunity to improve photosynthetic efficiency, biomass and yield in addition to potentially improving nitrogen and water use efficiency. To create a two‐cell metabolic prototype for an NADP‐malic enzyme type C₄ rice, we transformed Oryza sativa spp. japonica cultivar Kitaake with a single construct containing the coding regions of carbonic anhydrase, phosphoenolpyruvate (PEP) carboxylase, NADP‐malate dehydrogenase, pyruvate orthophosphate dikinase and NADP‐malic enzyme from Zea mays, driven by cell‐preferential promoters. Gene expression, protein accumulation and enzyme activity were confirmed for all five transgenes, and intercellular localization of proteins was analysed. ¹³CO₂ labelling demonstrated a 10‐fold increase in flux though PEP carboxylase, exceeding the increase in measured in vitro enzyme activity, and estimated to be about 2% of the maize photosynthetic flux. Flux from malate via pyruvate to PEP remained low, commensurate with the low NADP‐malic enzyme activity observed in the transgenic lines. Physiological perturbations were minor and RNA sequencing revealed no substantive effects of transgene expression on other endogenous rice transcripts associated with photosynthesis. These results provide promise that, with enhanced levels of the C₄ proteins introduced thus far, a functional C₄ pathway is achievable in rice.

Worldwide efforts to engineer C₄ photosynthesis into C₃ crops require a deep understanding of how this complex pathway operates. CO₂ is incorporated into four-carbon metabolites in the mesophyll, which move to the bundle sheath where they are decarboxylated to concentrate CO₂ around RuBisCO. We performed dynamic ¹³CO₂ labeling in maize to analyze C flow in C₄ photosynthesis. The overall labeling kinetics reflected the topology of C₄ photosynthesis. Analyses of cell-specific labeling patterns after fractionation to enrich bundle sheath and mesophyll cells revealed concentration gradients to drive intercellular diffusion of malate, but not pyruvate, in the major CO₂-concentrating shuttle. They also revealed intercellular concentration gradients of aspartate, alanine, and phosphenolpyruvate to drive a second phosphoenolpyruvate carboxykinase (PEPCK)-type shuttle, which carries 10–14% of the carbon into the bundle sheath. Gradients also exist to drive intercellular exchange of 3-phosphoglycerate and triose-phosphate. There is rapid carbon exchange between the Calvin–Benson cycle and the CO₂-concentrating shuttle, equivalent to ~10% of carbon gain. In contrast, very little C leaks from the large pools of metabolites in the C concentration shuttle into respiratory metabolism. We postulate that the presence of multiple shuttles, alongside carbon transfer between them and the Calvin–Benson cycle, confers great flexibility in C₄ photosynthesis.

Low atmospheric CO₂ in recent geological time led to the evolution of carbon-concentrating mechanisms (CCMs) such as C₄ photosynthesis in >65 terrestrial plant lineages. We know little about the impact of low CO₂ on the Calvin–Benson cycle (CBC) in C₃ 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 C₄ (Zea mays, Setaria viridis, Flaveria bidentis, and F. trinervia) and C₃ species (Oryza sativa, Triticium aestivum, Arabidopsis thaliana, Nicotiana tabacum, and Manihot esculenta). Principal component analysis revealed differences between C₄ and C₃ species that were driven by many metabolites, including lower ribulose 1,5-bisphosphate in C₄ species. Strikingly, there was also considerable variation between C₃ 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 C₃ species since the ancestors of modern plant lineages diverged. They underline the need to understand CBC operation in a wide range of species.

Plants regulate the distribution of electrons to activate chloroplast metabolism at the onset of light.

C₄ photosynthesis concentrates CO₂ 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 C₃ and C₄ photosynthesis. Previous studies have indicated that the first major change in metabolism probably involved relocation of glycine decarboxylase and photorespiratory CO₂ release to the bundle sheath and establishment of intercellular shuttles to maintain nitrogen stoichiometry. This was followed by selection for a CO₂-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 ¹³CO₂ labelling patterns for four species. Our results point to operation of multiple shuttles, including movement of aspartate in C₃–C₄ intermediates and a switch towards a malate/pyruvate shuttle in C₄-like species. The malate/pyruvate shuttle increases from C₄-like to complete C₄ 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 C₄ photosynthesis in the genus Flaveria.