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 CO2-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 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 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 ((309)Ile; faster V-C, lower CO2 affinity) and C-3 ((309)Met; 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 C-3 crops. Coengineering a catalytically faster C-3 rubisco and a CO2-concentrating mechanism within C-3 crop species could enhance their efficiency in resource use and yield.