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 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.