Characteristics of the C ₄ bundle sheath emerge in C ₃ rice after editing a plasma membrane proton ATPase

C ₄ photosynthesis improves light, water and nitrogen-use efficiencies and can raise yield by 50% compared with the ancestral C ₃ pathway. Engineering C ₄ traits into C ₃ crops could substantially boost food production but requires coordinated modifications to leaf anatomy and cell-specific photosynthetic function. For example, C ₄ leaves contain more numerous, shorter bundle sheath cells that are photosynthetically active. In searching for transcriptional regulators of bundle sheath development in C ₃ rice, we unexpectedly found OSA3, a plasma membrane H ⁺ -ATPase that is expressed in bundle sheath cells as they elongate, and when knocked out reduces their length due to reduced apoplastic acidification. Bundle sheath cell number and chloroplast occupancy are increased. Thus, switching between C ₃ and C ₄ bundle sheath identity is controlled by acid growth, and OSA3 represents a simple tool for C ₄ engineering.