PROTON GRADIENT REGULATION 5 enables efficient C4 photosynthesis under fluctuating light
PROTON GRADIENT REGULATION 5 (PGR5) plays a critical role in generating proton motive force across thylakoid membranes and supporting photoprotection under fluctuating light in C3 plants. It is proposed that this function is achieved by regulating cyclic electron flow around Photosystem I. During the evolutionary transition from C3 to C4 photosynthesis, PGR5 abundance in leaves has increased, coinciding with an enhancement in cyclic electron flow rate. To investigate the role of PGR5 in C4 photosynthesis and photoprotection, we generated Setaria viridis (a model C4 monocot) lines with null PGR5 alleles. We demonstrate that loss of PGR5 severely impairs the establishment of proton motive force, photosynthetic control, and energy-dependent non-photochemical quenching at high irradiances, leading to a loss of Photosystem I activity under light stress. Furthermore, plants lacking PGR5 exhibit drastically reduced growth and photosynthesis when grown under fluctuating daylight; however, they are less severely affected than C3  pgr5 mutants. This relative tolerance arises from the ability of S. viridis lacking PGR5 to maintain significant levels of photosynthetic control, in contrast to C3 mutants. Additionally, in the absence of PGR5 and qE, a slower-relaxing, zeaxanthin-dependent form of non-photochemical quenching supports survival under fluctuating light, albeit at the cost of reduced photochemical efficiency and assimilation. Our findings highlight the essential role of PGR5 in enabling efficient C4 photosynthesis under fluctuating light by regulating photosynthetic control and energy-dependent non-photochemical quenching. This study also uncovers the interplay between multiple photoprotective mechanisms safeguarding C4 photosynthesis under light stress. Overall design: PolyA-enriched RNA-seq was performed on S. viridis leaf discs harvested from WT, null NdhO and null PGR5 plants. Plants were germinated in 0.5 L pots with seed raising mix (Debco, Tyabb, Australia) containing 5 g L-1 of Osmocote fertiliser (Scotts, Bella Vista, Australia). Seeds were covered with inverted plastic cups to maintain humidity, with the cups removed one week after germination. Plants were grown in a controlled-environment chamber with a 16 h light/8 h dark photoperiod, an irradiance of 380 µmol m-2 s-1, ambient CO2, 28°C day, 22°C night and 60% humidity. The position of WT and edited plants within the chamber was blocked and randomised to minimise positional effects. Plants were analysed 3-4 weeks after sowing, with the youngest fully expanded leaves used for analysis. WT S. viridis plants were used as a control for all experiments.