One sentence summary: Simultaneous stimulation of RuBP regeneration and electron transport results in improvements in biomass yield in glasshouse and field grown tobacco.

C 4 plants contribute 20% to the global primary productivity despite representing only 4% of higher plant species. Their CO 2 concentrating mechanism operating between mesophyll and bundle sheath cells increases CO 2 partial pressure at the site of Rubisco and hence photosynthetic efficiency. Electron transport chains in both cell types supply ATP and NADPH for C 4 photosynthesis. Since Cytochrome b 6 f is a key point of control of electron transport in C 3 plants, we constitutively overexpressed the Rieske FeS subunit in Setaria viridis to study the effects on C 4 photosynthesis. Rieske FeS overexpression resulted in a higher content of Cytochrome b 6 f in both mesophyll and bundle sheath cells without marked changes in abundances of other photosynthetic complexes and Rubisco. Plants with higher Cytochrome b 6 f abundance showed better light conversion efficiency in both Photosystems and could generate higher proton-motive force across the thylakoid membrane. Rieske FeS abundance correlated with CO 2 assimilation rate and plants with a 10% increase in Rieske FeS content showed a 10% increase in CO 2 assimilation rate at ambient and saturating CO 2 and high light. Our results demonstrate that Cytochrome b 6 f controls the rate of electron transport in C 4 plants and that removing electron transport limitations can increase the rate of C 4 photosynthesis.