Control of the proton motive force (pmf) via regulation of ATP synthase constitutes a key mechanism for photosynthetic organisms to maintain redox balance and induce photoprotective mechanisms under light fluctuations. Using time-resolved electrochromic shift measurements in various photosynthetic organisms, we found that ATP synthase is dynamically regulated during light fluctuations. While light-induced reduction of the CF1γ subunit is known to activate chloroplast ATP synthase, it did not account for the regulation in fluctuating light in Arabidopsis thaliana, suggesting alternative mechanisms. The PROTON GRADIENT REGULATION 5 (PGR5) protein is important for photoprotection in algal and plant chloroplasts. PGR5 has been proposed to facilitate cyclic electron transport around PSI (CET), but it also affects ATP synthase activity. The physiological role of cyanobacterial Pgr5 has remained elusive. We characterised a ∆pgr5 mutant of Synechocystis sp. PCC 6803 and investigated pmf dynamics in pgr5 mutants of Chlamydomonas reinhardtii, Arabidopsis, and the C4 grass Setaria viridis. While PGR5 was not required for CET in Synechocystis, it was needed for downregulating ATP synthase under high irradiance in all tested organisms via a thiol redox state dependent mechanism. As AtPGR5 interacted with AtCF1γ, PGR5 may have a conserved function as an inhibitor of ATP synthase. ### Competing Interest Statement The authors have declared no competing interest.

The superior productivity of C4 plants is achieved via a metabolic C4 cycle which acts as a CO2 pump across mesophyll and bundle sheath (BS) cells and requires an additional input of energy in the form of ATP. The importance of chloroplast NADH dehydrogenase-like complex (NDH) operating cyclic electron flow (CEF) around Photosystem I (PSI) for C4 photosynthesis has been shown in reverse genetics studies but the contribution of CEF and NDH to cell-level electron fluxes remained unknown. We have created gene-edited Setaria viridis with null ndhO alleles lacking functional NDH and developed methods for quantification of electron flow through NDH in BS and mesophyll cells. We show that CEF accounts for 84% of electrons reducing PSI in BS cells and most of those electrons are delivered through NDH while the contribution of the complex to electron transport in mesophyll cells is minimal. A decreased leaf CO2 assimilation rate and growth of plants lacking NDH cannot be rescued by supplying additional CO2. Our results indicate that NDH-mediated CEF is the primary electron transport route in BS chloroplasts highlighting the essential role of NDH in generating ATP required for CO2 fixation by the C3 cycle in BS cells.

PROTON GRADIENT REGULATION 5 (PGR5) is essential for generating proton motive force across thylakoid membranes in C3 plants and supporting photoprotection under fluctuating light conditions. 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, the leaf abundance of PGR5 has increased, coinciding with a rise in the cyclic electron flow rate. To investigate the contribution of PGR5 to photoprotection in C4 photosynthesis, we generated model C4 monocot Setaria viridis with null pgr5 alleles. We show that plants lacking PGR5 struggle to establish proton motive force and energy-dependent non-photochemical quenching (qE) at higher irradiances during instantaneous measurements. This leads to a progressive decline in maximum Photosystem I activity when leaves are exposed to repeated cycles of high irradiance. Additionally, plants without PGR5 exhibit severely reduced growth and photosynthesis compared to wild type plants when grown under fluctuating daylight but not under constant daylight. In the absence of PGR5, a slower-relaxing, zeaxanthin-dependent form of non-photochemical quenching supports growth under fluctuating light, albeit at the cost of reduced photochemical efficiency and assimilation rate. Our findings underscore the role of PGR5 in enabling efficient C4 photosynthesis under fluctuating light by establishing proton motive force for the rapid upregulation of qE and preventing photodamage to the electron transport machinery. This study highlights the importance of various non-photochemical quenching mechanisms for C4 photosynthesis and emphasises the role of PGR5 in the evolution of C4 plants. ### Competing Interest Statement The authors have declared no competing interest.

Summary Sorghum is one of the most important crops providing food and feed in many of the world's harsher environments. Sorghum utilizes the C 4 pathway of photosynthesis in which a biochemical carbon‐concentrating mechanism results in high CO 2 assimilation rates. Overexpressing the Rieske FeS subunit of the Cytochrome b 6 f complex was previously shown to increase the rate of photosynthetic electron transport and stimulate CO 2 assimilation in the model C 4 plant Setaria viridis . To test whether productivity of C 4 crops could be improved by Rieske overexpression, we created transgenic Sorghum bicolor Tx430 plants with increased Rieske content. The transgenic plants showed no marked changes in abundances of other photosynthetic proteins or chlorophyll content. The steady‐state rates of electron transport and CO 2 assimilation did not differ between the plants with increased Rieske abundance and control plants, suggesting that Cytochrome b 6 f is not the only factor limiting electron transport in sorghum at high light and high CO 2 . However, faster responses of non‐photochemical quenching as well as an elevated quantum yield of Photosystem II and an increased CO 2 assimilation rate were observed from the plants overexpressing Rieske during the photosynthetic induction, a process of activation of photosynthesis upon the dark–light transition. As a consequence, sorghum with increased Rieske content produced more biomass and grain when grown in glasshouse conditions. Our results indicate that increasing Rieske content has potential to boost productivity of sorghum and other C 4 crops by improving the efficiency of light utilization and conversion to biomass through the faster induction of photosynthesis.

The superior productivity of C 4 plants is achieved via a metabolic C 4 cycle which acts as a CO 2 pump across mesophyll and bundle sheath (BS) cells and requires an additional input of energy in the form of ATP. Chloroplast NADH dehydrogenase-like complex (NDH) increases ATP production in C 3 plants by operating cyclic electron flow (CEF) around Photosystem I (PSI), and its importance for C 4 photosynthesis has been proposed from evolutionary and reverse genetics studies. We used the gene-edited C 4 species Setaria viridis with null ndhO alleles lacking NDH to study a contribution of the complex to the cell-level electron transport. Our results indicate that NDH is the primary PSI electron acceptor mediating the majority of CEF in BS cells whilst the contribution of the complex to CEF in mesophyll cells is minimal. Moreover, the reduced leaf CO 2 assimilation rate and growth of plants lacking the complex cannot be rescued by supplying additional CO 2 , indicating that NDH is essential for generating ATP required for CO 2 fixation by the C 3 cycle. Hereby we resolve a cell-level mechanism for the contribution of NDH to supporting high CO 2 assimilation rates in C 4 photosynthesis.

Photosynthesis is fundamental for plant growth and yield. The Cytochrome b 6 f complex catalyses a rate-limiting step in thylakoid electron transport and therefore represents an important point of regulation of photosynthesis. Here we show that overexpression of a single core subunit of Cytochrome b 6 f , the Rieske FeS protein, led to up to a 40% increase in the abundance of the complex in Nicotiana tabacum (tobacco) and was accompanied by an enhanced in vitro Cytochrome f activity, indicating a full functionality of the complex. Analysis of transgenic plants overexpressing Rieske FeS by the light-induced fluorescence transients technique revealed a more oxidised primary quinone acceptor of Photosystem II (QA) and plastoquinone pool and a faster electron transport from the plastoquinone pool to Photosystem I upon changes in irradiance, compared to control plants. A faster establishing of qE, the energy-dependent component of non-photochemical quenching, in transgenic plants suggested a more rapid build-up of the transmembrane proton gradient, also supporting the increased in vivo Cytochrome b 6 f activity. However, there was no consistent increase in steady-state rates of electron transport or CO2 assimilation in plants overexpressing Rieske FeS grown in either laboratory conditions or in field trials, suggesting that the in vivo activity of the complex was only transiently increased upon changes in irradiance. Our results show that overexpression of Rieske FeS in tobacco enhances abundance of functional Cytochrome b 6 f and electron transport capacity and may have a potential to increase plant productivity if combined with other traits. One-sentence summary Increased abundance of Cytochrome b 6 f complex leads to transient increases in photosynthetic electron transport rate in tobacco.

ATP, produced by the light reactions of photosynthesis, acts as the universal cellular energy cofactor fuelling all life processes. Chloroplast ATP synthase produces ATP using the proton motive force created by solar energy-driven thylakoid electron transport reactions. Here we investigate how increasing abundance of ATP synthase affects leaf photosynthesis and growth of rice, Oryza sativa variety Kitaake. We show that overexpression of AtpD, the nuclear-encoded subunit of the chloroplast ATP synthase, stimulates both abundance of the complex, confirmed by immunodetection of thylakoid complexes separated by Blue Native-PAGE, and ATP synthase activity, detected as higher proton conductivity of the thylakoid membrane. Plants with increased AtpD content had higher CO2 assimilation rates when a stepwise increase in CO2 partial pressure was imposed on leaves at high irradiance. Fitting of the CO2 response curves of assimilation revealed that plants overexpressing AtpD had a higher electron transport rate (J) at high CO2, despite having wild-type-like abundance of the cytochrome b6f complex. A higher maximum carboxylation rate (Vcmax) and lower cyclic electron flow detected in transgenic plants both pointed to an increased ATP production compared with wild-type plants. Our results present evidence that the activity of ATP synthase modulates the rate of electron transport at high CO2 and high irradiance.

Sorghum is one of the most important crops providing food and feed in many of the world’s harsher environments. Sorghum utilises the C 4 pathway of photosynthesis in which a biochemical carbon concentrating mechanism results in high CO 2 assimilation rates. Overexpressing the Rieske subunit of the Cytochrome b 6 f complex was previously shown to increase the rate of photosynthetic electron transport and stimulate CO 2 assimilation in the model C 4 plant Setaria viridis . To test whether productivity of C 4 crops could be improved by Rieske overexpression, we created transgenic Sorghum bicolor plants with increased Rieske content. The transgenic plants showed no marked changes in abundance of other photosynthetic proteins or chlorophyll content. Increases in yield of Photosystem II and CO 2 assimilation rate as well as faster responses of non-photochemical quenching during transient photosynthetic responses were observed as a result of an elevated in vivo Cytochrome b 6 f activity in plants overexpressing Rieske. The steady-state rates of electron transport and CO 2 assimilation did not differ between transgenic and control plants, suggesting that Cytochrome b 6 f is not the only factor limiting electron transport in sorghum at high light and high CO 2 . Nevertheless, more agile responses of photosynthesis to light transitions led to increases in biomass and grain yield in plants overexpressing Rieske. Our results indicate that increasing Rieske content could boost productivity of C 4 crops by improving the efficiency of light utilisation and conversion to biomass.