Improving global yields of important agricultural crops is a complex challenge. Enhancing yield and resource use by engineering improvements to photosynthetic carbon assimilation is one potential solution. During the last 40 million years C ₄ photosynthesis has evolved multiple times, enabling plants to evade the catalytic inadequacies of the CO ₂-fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco). Compared with their C ₃ ancestors, C ₄ plants combine a faster rubisco with a biochemical CO ₂- concentrating mechanism, enabling more efficient use of water and nitrogen and enhanced yield. Here we show the versatility of plastome manipulation in tobacco for identifying sequences in C ₄-rubisco that can be transplanted into C ₃-rubisco to improve carboxylation rate (V _C). Using transplastomic tobacco lines expressing native and mutated rubisco large subunits (L-subunits) from Flaveria pringlei (C ₃), Flaveria floridana (C ₃-C ₄), and Flaveria bidentis (C ₄), we reveal that Met-309-Ile substitutions in the L-subunit act as a catalytic switch between C ₄ ( ³⁰⁹Ile; faster V _C, lower CO ₂ affinity) and C ₃ ( ³⁰⁹Met; slower VC, higher CO ₂ affinity) catalysis. Application of this transplastomic system permits further identification of other structural solutions selected by nature that can increase rubisco V _C in C ₃ crops. Coengineering a catalytically faster C ₃ rubisco and a CO ₂-concentrating mechanism within C ₃ crop species could enhance their efficiency in resource use and yield.