Overexpression of Rubisco subunits with RAF1 increases Rubisco content in maize

Rubisco catalyses a rate-limiting step in photosynthesis and has long been a target for improvement due to its slow turnover rate. An alternative to modifying catalytic properties of Rubisco is to increase its abundance within C₄ plant chloroplasts, which might increase activity and confer a higher carbon assimilation rate. Here, we overexpress the Rubisco large (LS) and small (SS) subunits with the Rubisco assembly chaperone RUBISCO ASSEMBLY FACTOR 1 (RAF1). While overexpression of LS and/or SS had no discernable impact on Rubisco content, addition of RAF1 overexpression resulted in a >30% increase in Rubisco content. Gas exchange showed a 15% increase in CO₂ assimilation (A_SAT) in UBI-LSSS-RAF1 transgenic plants, which correlated with increased fresh weight and in vitro V_cmax calculations. The divergence of Rubisco content and assimilation could be accounted for by the Rubisco activation state, which decreased up to 23%, suggesting that Rubisco activase may be limiting V_cmax, and impinging on the realization of photosynthetic potential from increased Rubisco content.