Isoleucine 309 acts as a C4 catalytic switch that increases ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) carboxylation rate in Flaveria

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 C₄ photosynthesis has evolved multiple times, enabling plants to evade the catalytic inadequacies of the CO₂-fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco). Compared with their C₃ ancestors, C₄ plants combine a faster rubisco with a biochemical CO₂-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 C₄-rubisco that can be transplanted into C₃-rubisco to improve carboxylation rate (V_C). Using transplastomic tobacco lines expressing native and mutated rubisco large subunits (L-subunits) from Flaveria pringlei (C₃), Flaveria floridana (C₃-C₄), and Flaveria bidentis (C₄), we reveal that Met-309-Ile substitutions in the L-subunit act as a catalytic switch between C₄ (³⁰⁹Ile; faster V_C, lower CO₂ affinity) and C₃ (³⁰⁹Met; slower V_C, higher CO₂ affinity) catalysis. Application of this transplastomic system permits further identification of other structural solutions selected by nature that can increase rubisco V_C in C₃ crops. Coengineering a catalytically faster C₃ rubisco and a CO₂-concentrating mechanism within C₃ crop species could enhance their efficiency in resource use and yield.