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Photosynthetic inefficiencies limit the productivity and sustainability of crop production and the resilience of agriculture to future societal and environmental challenges. Rubisco is a key target for improvement as it plays a central role in carbon fixation during photosynthesis and is remarkably inefficient. Introduction of mutations to the chloroplast‐encoded Rubisco large subunitrbcL is of particular interest for improving the catalytic activity and efficiency of the enzyme. However, manipulation ofrbcL is hampered by its location in the plastome, with many species recalcitrant to plastome transformation, and by the plastid's efficient repair system, which can prevent effective maintenance of mutations introduced with homologous recombination. Here we present a system where the introduction of a number of silent mutations intorbcL within the model plantNicotiana tabacumfacilitates simplified screening via additional restriction enzyme sites. This system was used to successfully generate a range of transplastomic lines from wild‐typeN. tabacumwith stable point mutations withinrbcL in 40% of the transformants, allowing assessment of the effect of these mutations on Rubisco assembly and activity. With further optimization the approach offers a viable way forward for mutagenic testing of Rubisco functionin plantawithin tobacco and modification ofrbcL in other crops where chloroplast transformation is feasible. The transformation strategy could also be applied to introduce point mutations in other chloroplast‐encoded genes.

Photosynthesis in C3 plants is limited by features of the carbon‐fixing enzyme Rubisco, which exhibits a low turnover rate and can react with O₂instead ofCO₂, leading to photorespiration. In cyanobacteria, bacterial microcompartments, known as carboxysomes, improve the efficiency of photosynthesis by concentratingCO₂near the enzyme Rubisco. Cyanobacterial Rubisco enzymes are faster than those of C3 plants, though they have lower specificity towardCO₂than the land plant enzyme. Replacement of land plant Rubisco by faster bacterial variants with lowerCO₂specificity will improve photosynthesis only if a microcompartment capable of concentratingCO₂can also be installed into the chloroplast. We review current information about cyanobacterial microcompartments and carbon‐concentrating mechanisms, plant transformation strategies, replacement of Rubisco in a model C3 plant with cyanobacterial Rubisco and progress toward synthesizing a carboxysome in chloroplasts.