{"paper_id":"3fa6fe14-dd79-430b-b490-5294c65434c8","body_text":"1 \n \nFaster relaxation of nonphotochemical quenching (NPQ) in C4 than in 1 \nC3 species 2 \n 3 \nLucía Arce Cubas 1,2, Asuka Nakamura 3, Richard L. Vath 1, Julia Walter 1, Cristina Rodrigues 4 \nGabriel Sales1, Emmanuel L. Bernardo1,4, Yuri Nakajima Munekage3, Johannes Kromdijk1,2* 5 \n1 Department of Plant Sciences, University of Cambridge, Cambridge, CB2 3EA United Kingdom 6 \n2 Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 7 \nUrbana, 61801 Illinois, USA 8 \n3 School of Science and Technology, Kwansei Gakuin University, Sanda, 669-1337 Japan 9 \n4 Institute of Crop Science, College of Agriculture and Food Science, University of the Philippines 10 \nLos Baños, Laguna, 4031 Philippines 11 \n*Corresponding author:  12 \nJohannes Kromdijk, jk417@cam.ac.uk 13 \nClassification:  14 \nBiological Sciences, Plant Biology 15 \nKeywords:  16 \nPhotosynthesis, C4, NPQ, photoprotection 17 \nThis PDF file includes: 18 \nMain Text 19 \nReferences 20 \nFigures 1 to 5 21 \n  22 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 1, 2025. ; https://doi.org/10.1101/2025.04.01.646649doi: bioRxiv preprint \n\n \n \n2 \n \nAbstract 23 \nAcceleration of photoprotective non-photochemical quenching (NPQ) responses to changes in 24 \nlight intensity has been suggested as a strategy to enhance crop yield. Despite many key crops 25 \nutilising C4 photosynthesis, our current understanding of NPQ overwhelmingly comes from C3 26 \nspecies. Using a series of experiments on three phylogenetically controlled C3 and C4 27 \ncomparisons, we show that NPQ relaxation is faster in C4 species. Temporal analysis of NPQ 28 \nrelaxation in leaves infiltrated with inhibitors to block proton motive force formation or xanthophyll 29 \nde-epoxidation showed that the faster relaxation observed in C4 species is driven by a greater 30 \ncontribution of energy-dependent quenching (qE) to overall NPQ. We show that the C4-31 \nassociated enhancement of qE is linked to altered regulation of lumen pH in C4 species, 32 \nreflecting increases in cyclic electron flow and membrane proton conductivity to meet the 33 \nincreased ATP demands of the C4 pathway. Indeed, in two of the three tested C4 species, NPQ 34 \nrelaxation became significantly slower and statistically indistinguishable from paired C3 species 35 \nwhen ATP and NADPH consumption was suppressed by performing measurements in CO 2-free 36 \nair. Altogether, our results suggest that NPQ responses in C4 species may already be optimised 37 \nto maintain high photosynthetic efficiency in the fluctuating light conditions typically found within 38 \nC4 canopies. Given the intrinsically faster NPQ in C4 photosynthesis, further acceleration of NPQ 39 \nmay have limited scope to enhance crop photosynthetic efficiency. 40 \n 41 \nSignificance Statement 42 \nAcceleration of non-photochemical quenching has been proposed as a means to enhance crop 43 \nphotosynthetic efficiency in C3 species but whether this strategy has potential in C4 species, 44 \nwhich include several major crops, remains unclear. We use three phylogenetically paired C3 and 45 \nC4 species to show that NPQ relaxation is significantly faster in species with the C4 pathway, 46 \npossibly aiding the maintenance of photosynthetic efficiency in fluctuating light environments. As 47 \na result, accelerating the rate of NPQ relaxation in C4 crops may have a more limited scope to 48 \nenhance photosynthesis.  49 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 1, 2025. ; https://doi.org/10.1101/2025.04.01.646649doi: bioRxiv preprint \n\n \n \n3 \n \nIntroduction 50 \n 51 \nNonphotochemical quenching (NPQ) refers to a collection of photoprotective mechanisms 52 \nwherein excess light energy in photosystem II (PSII) is dissipated as heat, preventing 53 \noverexcitation and the formation of reactive oxygen species that would otherwise damage the 54 \nphotosynthetic machinery (1). NPQ components operate at different timescales and likely involve 55 \nconformational changes in PSII-associated antennae that trigger the quenched state (2). Energy-56 \ndependent quenching (qE) is activated within seconds to minutes by lumen acidification (3), 57 \nwhich also triggers xanthophyll cycle enzyme violaxanthin de-epoxidase (VDE) to convert 58 \nviolaxanthin to zeaxanthin, enhancing qE (4). Zeaxanthin accumulation further supports a qE-59 \nindependent quenching component (qZ) that activates and recovers over  minutes to hours (5). 60 \nEven more sustained quenching (qH) relies on the plastid lipocalin LCNP and is negatively 61 \nregulated by suppressor of quenching SOQ1 (6). Finally, photoinhibitory quenching (qI) is 62 \nassociated with photodamage and requires de novo synthesis of the D1 protein for recovery (7). 63 \n 64 \nOur molecular understanding of NPQ as detailed above overwhelmingly comes from C3 species, 65 \nwhere NPQ relaxation has been found to significantly lag behind changes in irradiance, 66 \ntemporarily lowering photosynthetic efficiency and leading to substantial losses in ‘foregone’ 67 \ncanopy carbon assimilation (8). Accelerating recovery from photoprotection represents a 68 \npromising strategy for improving crop yield in C3 species (9, 10), yet whilst many key crops are 69 \nC4 (11) the specifics of the C4 NPQ response remain largely unknown (12). 70 \n 71 \nIn C3 photosynthesis, CO 2 is directly fixed in mesophyll  (M) chloroplasts by ribulose-1,5-72 \nbiphosphate carboxylase/oxygenase (Rubisco) into 3-carbon compound 3-phosphoglycerate (3-73 \nPGA). Higher photosynthetic rates are generally found in C4 species, where a carbon 74 \nconcentrating mechanism (CCM) enhances photosynthesis by suppressing RuBP oxygenation 75 \nand concomitant photorespiration (13). The C4 pathway operates between morphologically 76 \ndistinct M and bundle sheath (BS) cells, typically arranged in ‘Kranz’ anatomy: CO 2 is initially 77 \nconverted to bicarbonate in the M and fixed into 4-carbon oxaloacetate that is further reduced or 78 \ntransaminated into malate or aspartate before diffusion into the BS, where decarboxylating 79 \nenzymes release CO 2 around Rubisco and into the C3 cycle (14). Different C4 “subtypes” use 80 \ndifferent decarboxylases, often in combination (15), and have additional cell and subtype-specific 81 \nATP requirements for the regeneration of CCM biochemical intermediates (16-18). The 82 \nATP:NADPH ratio generated by linear electron flow (LEF) from PSII to PSI is insufficient to satisfy 83 \nthe demands of the C3 cycle, and the additional ATP demands by C4 metabolism further the 84 \nimbalance (19). Cyclic electron flow (CEF) helps balance energy budgets by recycling electrons 85 \naround PSI back to plastoquinone (PQ), contributing to the proton motive force (pmf) that powers 86 \nATP synthesis without concurrent production of NADPH (20). Considerably higher ratios of 87 \nPSI:PSII (21, 22) and CEF:LEF (23) are found in C4 versus C3 species, reflecting the ATP cost of 88 \nthe C4 pathway. 89 \n 90 \nThe functional differences between C3 and C4 photosynthesis are likely to affect NPQ. In C3 91 \nspecies, CEF plays a major photoprotective role by contributing to ∆ pH and thus qE activation, 92 \nwith CEF-defective mutants having severely reduced NPQ (24, 25). This could suggest an 93 \nenhanced qE component in C4 species, given their higher CEF:LEF ratios. In C3 species, CEF is 94 \npredominantly mediated by proton gradient regulation 5 (PGR5) and PGR5-like photosynthetic 95 \nphenotype 1 (PGRL1), with a minor contribution by chloroplast NADH dehydrogenase-like 96 \ncomplex (NDH) in low and fluctuating light (26-28). In C4 species, although PGR5/PGRL1 is still 97 \nimportant, recent studies show CEF occurs primarily via NDH (29-31). Notably, NPQ amplitude 98 \nwas lower in C4 PGR5 and PGRL1-deficient mutants, but higher in NDH mutants (30, 31). Based 99 \non these results, it was suggested that PGR5/PGRL1 could play a similar photoprotective role in 100 \nC4 photosynthesis as in C3 species, with NDH mainly contributing to supplementing ATP 101 \nproduction (31), but the effect of CEF pathway interplay on C4 NPQ remains unclear. Finally, 102 \nNPQ modulation is also regulated by the proton conductivity of the thylakoid membrane (gH +, 103 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 1, 2025. ; https://doi.org/10.1101/2025.04.01.646649doi: bioRxiv preprint \n\n \n \n4 \n \nindicative of  ATP synthase activity), as changes to gH+ affect pmf formation and dissipation (32). 104 \nHigher ATP consumption in C4 species results in faster turnover of inorganic phosphate and 105 \nsubstrate availability for ATP synthase (33), potentially resulting in altered control of ∆ pH-106 \ndependent NPQ than in C3 photosynthesis. 107 \n 108 \nThe present work aimed to characterise differences in NPQ relaxation between C3 and C4 109 \nspecies using a combination of spectroscopic, chemical, and molecular approaches, including C4 110 \nCEF mutants. To account for the strong confounding effect of phylogenetic distance (34), we 111 \ncompared phylogenetically linked pairs of C3 and C4 species from three evolutionarily distinct 112 \ngenera operating three different C4 metabolic cycles. 113 \n 114 \n 115 \n 116 \nResults 117 \n 118 \nDifferences in NPQ relaxation between C3 and C4 species 119 \nTo compare NPQ between photosynthetic pathways, we selected C3 and C4 species from 120 \nAlloteropsis (C3 A. semialata KWT, C4 A. semialata MDG), Flaveria  (C3 F. cronquistii , C4 F. 121 \nbidentis), and Cleome (C3 T. hassleriana, C4 G. gynandra). This experimental design minimises 122 \nphylogenetic variation between each C3 and C4 pair, whilst maintaining substantial evolutionary 123 \ndistance between the three genera. The selected species represent both monocots (Alloteropsis) 124 \nand dicots ( Flaveria, Cleome), three independent C4 origins (35, 36), and the three major 125 \ndecarboxylating enzymes found across C4 photosynthetic species: NADP-ME/PEPCK in C4 A. 126 \nsemialata MDG (37), NADP-ME in C4 F. bidentis (38), and NAD-ME in C4 G. gynandra (39). 127 \n 128 \nNPQ induction and relaxation responses were measured during a 1 hour photoperiod (600 µmol 129 \nm-2 s-1 PFD) followed by 25 minutes of darkness. Since NPQ components can be resolved based 130 \non their relaxation kinetics (1, 2) and given current interest in NPQ relaxation for improving 131 \nphotosynthesis (40), we focused on NPQ following the light-to-dark transition ( Fig. 1A, full traces 132 \nin Fig. S1). NPQ relaxation was faster in all C4 species relative to their C3 counterparts, resulting 133 \nin significantly lower NPQ across the dark period. Integrated NPQ across the dark recovery 134 \nperiod was 40% lower in C4 Alloteropsis semialata MDG than in C3 Alloteropsis semialata KWT, 135 \n33% lower in C4 Flaveria bidentis  than in C3 Flaveria cronquistii , and 22% lower in C4 136 \nGynandropsis gynandra than in C3 Tarenaya hassleriana (values and statistics in Table S1). 137 \n 138 \nThe NPQ relaxation kinetics of C3 and C4 species were underpinned by clear differences in NPQ 139 \ncomposition (Fig. 1B, values and statistics in Table S2). NPQ components were analysed by 140 \nseparating NPQ relaxation into different timescales of deactivation expressed as a function of 141 \ntotal NPQ. Fast-relaxing components (0-2 min) constituted a significantly greater proportion of 142 \nNPQ in all C4 species than in their C3 pairs and conversely, slower-relaxing NPQ components (2-143 \n15 min) were a larger part of NPQ in C3 species, contributing to a more exponential decay. 144 \n 145 \nEffects of chemical inhibition of Δ pH and xanthophyll-dependent components 146 \nFast-relaxing NPQ components include Δ pH-sensitive and xanthophyll-dependent qE in the first 147 \ntwo minutes, and qZ dissipation in 2- 15 minutes (2). To identify the elements responsible for 148 \nNPQ differences between C3 and C4 species, leaves were infiltrated with nigericin to collapse the 149 \nproton gradient or dithiothreitol (DTT) to inhibit the xanthophyll cycle, and integrated NPQ of 150 \ntreated leaves during the light period was compared to a control to assess NPQ dependence on 151 \nboth mechanisms ( Fig. 2 , full traces in Fig. S2 ). Nigericin infiltration resulted in significantly 152 \ngreater suppression of NPQ in C4 than in C3 species (Fig. 2A, values and statistics in Table S3), 153 \nindicating higher reliance on Δ pH for C4 NPQ responses: 75±3% reduction in C4 A. semialata 154 \nMDG vs. 58±9% in C3 A. semialata KWT, 88±2% in C4 F. bidentis vs. 62±4% in C3 F. cronquistii, 155 \nand 94±3% in C4 G. gynandra vs. 68±3% in C3 T. hassleriana. Although both qE and xanthophyll 156 \nde-epoxidation depend on Δ pH, the lack of significant differences in NPQ suppression by DTT in 157 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 1, 2025. ; https://doi.org/10.1101/2025.04.01.646649doi: bioRxiv preprint \n\n \n \n5 \n \nany C3 and C4 pairs ( Fig. 2B ) suggests that differences in C4 NPQ mostly stem from Δ pH-158 \nsensitive qE rather than qZ or xanthophyll-dependent qE. 159 \n 160 \nEffects of the removal of photosynthetic and photorespiratory electron sinks 161 \nThe higher rates of CEF to satisfy ATP:NADPH requirements (23) and larger electron sinks found 162 \nin C4 photosynthesis (33) may contribute to the formation and collapse of Δ pH. To test whether 163 \nfast NPQ relaxation in C4 species is linked to C4 metabolism, we repeated our NPQ 164 \nmeasurements in 2% O 2 + 0 ppm CO 2\n air ( Fig. 3 ). The suppression of photosynthesis and 165 \nphotorespiration slowed down NPQ relaxation and removed differences between C3 and C4  166 \nAlloteropsis and Cleome pairs. Unlike the step-like decay observed in ambient air (Fig. 1A), NPQ 167 \nin C4 A. semialata MDG and C4 G. gynandra followed the approximately exponential decay 168 \npattern of their C3 counterparts in 2% O 2 + 0 ppm CO 2 (Fig. 3A, values and statistics in Table 169 \nS1), and had similar relative contributions of the 0-2 min and 2-15 min components to total NPQ 170 \n(Fig. 3B, Table S2). In contrast, differences in NPQ kinetics between C3 and C4 species seemed 171 \nenhanced in Flaveria in 2% O2 + 0 ppm CO2\n air. Integrated NPQ was lower in C4 F. bidentis than 172 \nin C3 F. cronquistii; and the 0-2 min component still represented a significantly larger proportion 173 \nof NPQ in C4 F. bidentis  than in C3 F. cronquistii , whilst the 2-15 min component showed the 174 \nopposite trend. 175 \n 176 \nBased on the pH requirement of qE, we speculated that C4 F. bidentis may retain H + efflux 177 \ncapacity in 2% O2 + 0 ppm CO2 air. We estimated gH+ from decay kinetics of electrochromic shift 178 \n(ECS) signal measurements during brief dark intervals across 20 minutes of light to encompass 179 \nthe 0-2 and 2-15 min NPQ components (Fig. 4). Under ambient air, all C4 species tended to have 180 \nhigher gH + than their C3 pairs ( Fig. 4A, C & E). However, C4 F. bidentis  retained H + efflux 181 \ncapacity in 2% O 2 + 0 ppm CO 2 (Fig. 4D), albeit diminished compared to 21% O2 + 410 ppm 182 \nCO2, whereas suppressing photosynthesis and photorespiration resulted in gH + values 183 \napproaching zero in all other species ( Fig. 4B, D & F ). These results show that fast NPQ 184 \nrelaxation in C4 species is strongly linked to proton efflux capacity, and that an alternative 185 \nelectron sink in C4 F. bidentis likely sustains proton efflux and qE even when photorespiration 186 \nand photosynthesis are suppressed. 187 \n 188 \nEvaluating CEF in C3 and C4 species 189 \nChlorophyll fluorescence reflects the PSII quinone redox state. In darkness, with no LEF-driven 190 \nreduction, a post-illumination chlorophyll fluorescence rise (PIFR) is attributed to residual CEF 191 \ntransferring electrons from stromal donors to the PQ pool that then equilibrate with PSII-192 \nassociated quinones (41). We measured PIFR as a proxy for CEF after 1 h light treatment, 193 \nperiodically flashing far-red light to preferentially excite PSI and temporarily enhance PQ 194 \noxidation (full PIFR trace in Fig. S3 ). After the far-red pulse, a larger PIFR response was 195 \nobserved in C3 A. semialata KWT, C4 F. bidentis, and C4 G. gynandra than in their phylogenetic 196 \npairs, indicating higher CEF activity in those species ( Fig. 5A, solid lines). This is consistent with 197 \nATP:NADPH ratio requirements being higher in NADP-ME (C4 F. bidentis) and NAD-ME (C4 G. 198 \ngynandra) subtypes than in mixed PEPCK pathways (C4 A. semialata MDG), which have lower 199 \nATP requirements (18). Leaves were also treated with PGR5/PGRL1 pathway inhibitor Antimycin 200 \nA (dashed lines), which resulted in significantly lower PIFR in C3 F. cronquistii  and C3 T. 201 \nhassleriana, indicating a substantial contribution of PGR5/PGRL1 to PQ reduction. The addition 202 \nof Antimycin A also resulted in higher PIFR values in some cases – a secondary effect of 203 \nAntimycin A that has been previously attributed to additional inhibition of electron transport 204 \ndownstream of PQ (31). Even with this effect, these results validate existing knowledge of C3 and 205 \nC4 CEF pathways– C3 species primarily use the PGR5/PGRL1 route while C4 species use both 206 \nthe PGR5/PGRL1 and the NDH pathways (26, 30, 31). C3 A. semialata KWT appears to be an 207 \nexception, but given that the C3 subspecies may have reversed from a C3-C4 intermediate, CEF 208 \noperation might differ from other C3 plants (42).  209 \n 210 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 1, 2025. ; https://doi.org/10.1101/2025.04.01.646649doi: bioRxiv preprint \n\n \n \n6 \n \nThe role of CEF in C4 NPQ relaxation was further studied in C4 F. bidentis  FbPGRL1-RNAi and 211 \nFbNdhO-RNAi knockdown lines (31) – as previously observed, carbon assimilation was notably 212 \nlower only in FbNdhO-RNAi (Fig. S4), suggesting an impaired C4 cycle. NPQ in FbNdhO-RNAi 213 \ndecayed more slowly than the step-wise drop observed in WT and FbPGRL1-RNAi (Fig. 5B, 214 \nvalues and statistics in Table S4). Whilst the 0–2 min component constituting most of WT NPQ 215 \nwas significantly reduced in both FbPGRL1-RNAi and FbNdhO-RNAi, the slower NPQ relaxation 216 \nof FbNdhO-RNAi is evidenced by a more significant 2-15 min component than in WT. At the end 217 \nof illumination, NPQ was lower in FbPGRL1-RNAi than in WT and FbNdhO-RNAi, suggesting that 218 \ndifferences in NPQ composition stem from impaired relaxation in FbNdhO-RNAi, but from overall 219 \nNPQ suppression in FbPGRL1-RNAi. 220 \n 221 \n 222 \n 223 \nDiscussion  224 \n 225 \nC4 species have faster NPQ relaxation 226 \nThis study sought to characterise differences in NPQ relaxation between C3 and C4 227 \nphotosynthesis, by comparing phylogenetically linked Alloteropsis, Flaveria, and Cleome C3 and 228 \nC4 species. Despite considerable evolutionary distance between the three tested genera, all C4 229 \nspecies had significantly faster and overall greater NPQ relaxation than their C3 pairs ( Fig. 1), 230 \nshowing that this is likely linked to the C4 pathway. The rapid return of PSII to the unquenched 231 \nstate in C4 species would support higher photosynthetic quantum yields following decreases in 232 \nirradiance, and could be contributing to the more sustained rates of CO 2 assimilation that have 233 \nbeen observed in C4 versus C3 species during light-shade transitions (43, 44). Whilst increasing 234 \nthe rate of photosynthetic efficiency has been remarkably successful at improving photosynthetic 235 \nefficiency in C3 species (9, 10), our results suggest that this approach may result in more limited 236 \ngains in carbon assimilation in C4 species given their intrinsically faster NPQ relaxation rate. 237 \n 238 \nA greater proportion of C4 NPQ is qE 239 \nThe comparatively faster NPQ relaxation found in C4 plants stemmed from differences in NPQ 240 \ncomposition between C3 and C4 species. Separation of NPQ into components based on decay 241 \ntimescales revealed that C4 species had a significantly higher proportion of a fast-relaxing (0-2 242 \nmin) component compared to their C3 phylogenetic pairs, with C3 species exhibiting a 243 \ncomparatively greater proportion of NPQ relaxation within the 2-15 min timeframe (Fig. 1B). NPQ 244 \ncomponent qE operates within the 0-2 min timescale, responding to lumen acidification and 245 \nenhanced by zeaxanthin accumulation (2, 45). When infiltrated with ∆ pH-inhibitor nigericin, NPQ 246 \nin C4 species was significantly more depressed than in C3 species, whereas inhibiting 247 \nxanthophyll cycle activity with DTT did not result in significant differences in the extent of NPQ 248 \nreduction between C3 and C4 species ( Fig. 2). These results demonstrate that the acceleration 249 \nof NPQ identified in all three C4 species involved ∆ pH-dependent qE. Activation of qE by lumen 250 \npH relies on the presence of photosystem II subunit S (PsbS), which initiates the quenched LHCII 251 \nstate via a conformational switch upon protonation of lumen-exposed protonatable residues (46, 252 \n47). Thus, one hypothesis could be that C4 species may show slight alterations in PsbS relative 253 \nabundance, structure, or interactions with LHCs to explain the enhancement of qE. 254 \n 255 \nThe energetic requirements of the C4 pathway affect NPQ 256 \nWhen photosynthesis and photorespiration were suppressed, NPQ relaxation in C4 A. semialata 257 \nMDG and G. gynandra conformed to the slower exponential decay and composition of C3 A. 258 \nsemialata KWT and T. hassleriana ( Fig. 3). In ambient air, C4 metabolism largely suppresses 259 \nphotorespiration so these results suggest that the fast NPQ relaxation observed in Alloteropsis 260 \nand Cleome C4 species is specifically related to the ATP and NADPH demand by the C4 261 \nphotosynthetic pathway. qE is regulated by lumen pH, which is in turn dependent on membrane 262 \nproton conductivity, gH+ (32). The higher gH+ found in all three C4 species in ambient conditions 263 \n(Fig. 4A, C & E) could contribute to accelerated relaxation of pH-dependent NPQ, primarily qE. 264 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 1, 2025. ; https://doi.org/10.1101/2025.04.01.646649doi: bioRxiv preprint \n\n \n \n7 \n \nDifferent C4 pathways have distinct ATP:NADPH requirements: in NADP-ME/PEPCK subtypes 265 \n(C4 A. semialata MDG), the energetic balance is comparable to C3 species, whereas NADP-ME 266 \n(F. bidentis) and NAD-ME ( G. gynandra) pathways have elevated ATP:NADPH demands. The 267 \nincreased ATP demand is localised in BS cells in NADP-ME subtypes but in M cells in NAD-ME 268 \nsubtypes (16-18). M cells are positioned in the outer layer of Kranz anatomy, and therefore 269 \nrepresent the majority of the chlorophyll fluorescence signals used to determine NPQ. However, 270 \nin all three C4 species, the large metabolic pools of C4 cycle intermediates required to sustain 271 \ndiffusion gradients between M and BS cells may underpin the observed increases in gH + relative 272 \nto C3 species. The high gH+ indicates upregulated proton efflux, be it ATP synthase dependent or 273 \nindependent as photoprotective Δ pH has been found to be linked both to ATP synthase 274 \nregulation (32, 48) as well as to thylakoid antiporters like KEA3 (49), both mechanisms that could 275 \ndiffer in C4 photosynthesis compared to C3. Uniquely, in C4 F. bidentis NPQ relaxation was not 276 \nslowed by the suppression of photosynthesis and photorespiration ( Fig. 3), and gH + was much 277 \nhigher than in the other C4 species ( Fig. 4D), indicating the presence of an alternative electron 278 \nsink. The identity of this electron sink remains unclear but it seems plausible that it relates to 279 \nspecific attributes of the canonical NADP-ME C4 pathway, such as the significantly larger malate 280 \npool sizes that could go towards ATP-consuming metabolic reactions (50), although sustained 281 \nleakiness of the membrane via ATP synthase-dependent and independent mechanisms is also 282 \npossible.  283 \n 284 \nContribution of CEF pathways to faster NPQ in C4 species 285 \nIn C3 species, CEF via the PGR5/PGRL1 pathway is essential for photoprotection by contributing 286 \nto ∆ pH formation and qE (24, 25). Relative to the ancestral C3 pathway,  PGR5/PGRL1 287 \nabundance increases in C4 species in both M and BS cells (17, 51), whilst NDH differentially 288 \naccumulates per ATP requirements: in M cells for NAD-ME pathways and in BS for NADP-ME 289 \n(16, 18). Our results point to both NDH and PGR5/PGRL1 pathways contributing to fast relaxation 290 \nin C4 NPQ. Both C4 FbNdhO-RNAi and FbPGRL1-RNAi lines showed altered NPQ relaxation 291 \nkinetics relative to WT ( Fig. 5B ). Consistent with our findings, previous work found the 292 \nPGR5/PGRL1 pathway to significantly contribute to NPQ induction in C4 F. bidentis  (31). Here, 293 \nwe also show that knocking down PGRL1 results in a specific decrease in qE activation (Fig. 5C), 294 \nprobably due to deficient pmf formation. In contrast, reduction of NDH expression primarily 295 \nslowed down NPQ responses.  C4 F. bidentis  NDH is primarily found in the BS, accounting for 296 \nmost of BS CEF (30). Although the chlorophyll fluorescence signal primarily comes from the M, 297 \nan impaired 0-2 min NPQ component was still observed in FbNdhO-RNAi mutants (Fig 5C). The 298 \ntissue-specific expression of NDH in C4 species and the deleterious effect of its suppression on 299 \ngrowth and carbon assimilation suggests NDH is the major route for ATP provision to C4 300 \nmetabolism (30, 31). Accordingly, the rate of carbon assimilation in FbNdhO-RNAi  was greatly 301 \nreduced relative to wild type ( Fig S4 ). An impaired C4 cycle in FbNdhO-RNAi would alter 302 \ninorganic phosphate availability and LEF:CEF energy balance, potentially diminishing CEF-303 \nrelated qE and leading to parallel decreases in gH+ and ∆ pH in M cells (32, 33).  Since the role of 304 \nNDH in C4 photosynthesis has thus far only been studied in NADP-ME plants with NDH-enriched 305 \nBS, it remains unclear if NDH-mediated CEF generally also plays a photoprotective role similar to 306 \nPGR5/PGRL1. Future research could test this in NAD-ME species with NDH-enriched M (22), 307 \nsuch as C4 G. gynandra. 308 \n 309 \nThe complexity of NPQ molecular mechanisms, evolution of CEF pathways, and biochemical 310 \ndiversity within C4 species leave many outstanding questions with relation to C4 NPQ. 311 \nNevertheless, the results presented here demonstrate that the higher rates of CEF and larger 312 \nelectron sinks of C4 metabolism result in enhancement of the qE component, leading to faster 313 \nrelaxation kinetics. The increased contribution of qE to total NPQ in C4 species mimics the 314 \nengineering attempts to accelerate NPQ responses in C3 crops via single overexpression of 315 \nPsbS (52, 53) or in combination with violaxanthin de-epoxidase and zeaxanthin epoxidase (9, 316 \n10), engineering attempts which may have less scope to further accelerate NPQ responses and 317 \nphotosynthetic efficiency in C4 species. 318 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 1, 2025. ; https://doi.org/10.1101/2025.04.01.646649doi: bioRxiv preprint \n\n \n \n8 \n \n 319 \n 320 \nMaterials and Methods 321 \n 322 \nPlant material and growth conditions 323 \nC4 F. bidentis, C3 F. cronquistii, C4 G. gynandra and C3 T. hassleriana were grown in soil under 324 \ngrowth chamber conditions at 20ºC and 150 µmol m -2 s-1 PFD over a 16-hour photoperiod. A. 325 \nsemialata MDG and A. semialata KWT  were grown in a 4:1 mix of soil and vermiculite under 326 \nsemi-controlled glasshouse conditions at 18-25 ºC with supplemental lightning provided to a 327 \nminimum of 140-160 µmol m -2 s-1 PFD over a 16-hour photoperiod (further detail in (54)). 328 \nMeasurements were conducted on fully expanded leaves during vegetative stage: at 8-10 weeks 329 \nfor both Flaveria  species and G. gynandra, 4-6 weeks for T. hassleriana , and 2 weeks after 330 \nvegetative propagation for both Alloteropsis  species. C4 F. bidentis WT, FbPGRL1-RNAi and 331 \nFbNdhO-RNAi RNAi plants with expression ~10% of WT (31), were grown in a 3:2 mix of soil and 332 \nvermiculate, in a growth chamber at 24ºC and 250 µmol m -2 s-1 PFD over a 12-hour photoperiod. 333 \nYoung, fully-expanded FbNdhO-RNAi leaves were measured after 12-16 weeks and of all other 334 \nplants after 8-10 weeks.  335 \n 336 \nChlorophyll fluorescence measurements 337 \nChlorophyll fluorescence was measured with a gas exchange system (LI-6400XT, LI-COR, 338 \nLincoln, NE, USA) equipped with a leaf chamber fluorometer (6400-40 LCF, LI-COR). Chamber 339 \nconditions were controlled at 410 or 0 ppm sample CO 2 concentration (the latter with 2% O 2 air), 340 \n40-60% relative humidity, 25ºC block temperature, 300 µmol s-1 flow rate, and 10% blue (470 nm) 341 \nand 90% red actinic light (630 nm). The LCF used a 0.25 Hz modulated measuring light and a 342 \nmultiphase flash (55) to measure chlorophyll fluorescence parameters. 343 \n 344 \nLeaves were dark-adapted until stomatal conductance and net CO2 exchange rate stabilised (30-345 \n60 min), illuminated with 600 µmol m -2 s-1 PFD for 1 hour, and returned to darkness for 25 346 \nminutes. Multiphase saturating flashes at 4000 µmol m -2 s-1 PFD were used to measure steady 347 \nand maximal fluorescence after dark-adaption ( F and F m), and during photoperiod and dark 348 \nrecovery (F’ and Fm’), occurring five minutes before illumination (for F v/Fm); after 3, 5, 10, 15, 25, 349 \n35, 45, and 60 minutes of light exposure; and 30s after dark transition, then every 90s thereafter. 350 \nNPQ was derived from fluorescence measurements (56). NPQ relaxation was compared by 351 \ncalculating integrated NPQ from area under the curve (AUC); and 0–2 min and 2–15 min 352 \ncomponent contributions were expressed as the integrated NPQ within each phase relative to 353 \ntotal post-illumination NPQ. 354 \n 355 \nChlorophyll fluorescence rise during dark recovery (following (41)) was monitored for 2.5 minutes 356 \nin darkness after the photoperiod, interspersed by 5s of far-red (FR) light to oxidise the PQ pool 357 \n(740 nm, ~50 μ mol of photons m−2 s−1) every 20-25s from instrument variation. We compared the 358 \nPIFR of the dark period after FR, normalised to the final fluorescence value post-oxidation ( F0*, 359 \nsee Fig. S3 ). Representative data from five biological replicates of the subsequent rise in 360 \nfluorescence is presented. 361 \n 362 \nNigericin, DTT, and Antimycin A leaf infiltration 363 \nDark adapted leaves were vacuum infiltrated with NPQ inhibitors in a syringe with a buffer (20 364 \nmM HEPES/KOH pH 7.0) supplemented with either 100 µM nigericin, 5 mM DTT, or 250 µM 365 \nAntimycin A. Controls were infiltrated with buffer and equivalent volume of solvent without 366 \ninhibitor. The effect of NPQ inhibitors was estimated by calculating NPQ AUC during the light 367 \nperiod and expressing as a proportion of control NPQ AUC (Fig S2 for full NPQ traces). 368 \n 369 \nElectrochromic Shift measurements 370 \nThe ECS signal was measured as absorptance changes at 515 nm using 535 nm as an 371 \nisosbestic waveband (Dual-KLAS fitted with a P515/535 module, Heinz Walz GmbH, Effeltrich, 372 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 1, 2025. ; https://doi.org/10.1101/2025.04.01.646649doi: bioRxiv preprint \n\n \n \n9 \n \nDE) (57). The fore-optics were integrated in a custom measuring chamber (GFS-3000 measuring 373 \nchamber for DUAL-KLAS, Walz) with temperature controlled at 25 °C. Chamber conditions were 374 \ncontrolled via the console of a LI-6800 gas exchange system (LI-COR, USA) at 410 or 0 ppm 375 \nsample CO2 concentration (the latter with 2% O2 air), 60% relative humidity, and 200 µmol s-1 flow 376 \nrate. Leaves were dark-adapted, and illuminated with 600 µmol m -2 s-1 PFD for 20 minutes. Dark 377 \nInterval Relaxation Kinetics (DIRK) measurements of the ECS signal (58) were taken at 1, 3, 5, 378 \n10, and 20 minutes of illumination. Estimates of thylakoid membrane proton conductivity (gH +) 379 \nwere obtained from the inverse of the decay time constant ( τ ECS) of a single exponential decay 380 \nfitted to the first 300 milliseconds of the dark interval (59). 381 \n 382 \nGas concentration manipulation 383 \nTo suppress photosynthesis and photorespiration, a pre-mixed 2% O 2 and 98% N 2 gas mixture 384 \n(BOC Ltd., Woking, UK) was supplied to the LI-6400XT or LI-6800 using a mass flow controller 385 \n(EL-FLOW, Bronkhorst Hight-tech BV, Ruurlo, NL), and CO2 controlled at 0 ppm. 386 \n 387 \nStatistical analysis 388 \nOne-way ANOVA was conducted on independent experiments comparing each C3 and C4 389 \nphylogenetic pair, and comparing FbPGRL1-RNAi and FbNdhO-RNAi mutants with WT. 390 \nAssumptions of normality and homogeneity of variance were tested for, respectively with a 391 \nShapiro-Wilk and Bartlett’s test. 392 \n 393 \n 394 \n  395 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 1, 2025. ; https://doi.org/10.1101/2025.04.01.646649doi: bioRxiv preprint \n\n \n \n10 \n \nAcknowledgments and funding sources 396 \n 397 \nFor providing the original plant material, we thank Dr. Luke Dunning ( Alloteropsis cuttings), Dr. 398 \nMarjorie Lundgren ( F. cronquistii cuttings), Prof. Peter Westhoff ( F. bidentis seeds) and Prof. 399 \nJulian Hibberd ( T. hassleriana  seeds). Additional thanks to Prof. Hibberd and Prof. David M. 400 \nKramer for kindly consulting on our results, and to Dr. Gustaf Degen for his helpful guidance on 401 \nECS. 402 \n 403 \nLAC was jointly funded by the Cambridge Trust; and by Consejo Nacional de Ciencia y 404 \nTecnología (CONACyT). This work was supported by the Biotechnology and Biological Sciences 405 \nResearch Council (BBSRC) via grant BB/T007583/1 awarded to JK. For the purpose of open 406 \naccess, the authors have applied a Creative Commons Attribution (CC BY) licence to any Author 407 \nAccepted Manuscript version arising from this submission. 408 \n 409 \nAuthor Contributions 410 \n 411 \nJK and LAC conceived the study and designed the experiments. AN measured the CEF F. 412 \nbidentis mutants developed by YM. LAC carried out all other experiments, data analysis and 413 \ninterpretation, and wrote the manuscript. RLV helped with the 2% O 2 experimental setup and 414 \nprovided support with gas exchange experiments. JW and CRGS helped with developing ECS 415 \nprotocols, and JW with chemical infiltration protocols. ELB procured the initial plant material. All 416 \nauthors contributed to and reviewed the final manuscript. Descriptive statistics and figures were 417 \ncreated using R 4.1.1 on RStudio 2023.03.1+446. 418 \n 419 \nCompeting Interest Statement 420 \n 421 \nThe authors declare no conflict of interest. 422 \n 423 \n 424 \nSupporting Information 425 \n 426 \nFig. S1: NPQ induction and relaxation in phylogenetic pairs 427 \nFig. S2: NPQ induction and relaxation with chemical inhibitors 428 \nFig. S3: Sample full trace of Post-Illumination Fluorescence Rise 429 \nFig. S4: Carbon assimilation of CEF mutants 430 \nTable S1: Values and statistics of integrated NPQ across dark recovery in phylogenetic pairs 431 \nTable S2: Values and statistics of NPQ composition in phylogenetic pairs 432 \nTable S3: Values and statistics of NPQ with chemical inhibitors in phylogenetic pairs 433 \nTable S4: Values and statistics of NPQ composition in CEF mutants 434 \n  435 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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It is made \nThe copyright holder for this preprint (whichthis version posted April 1, 2025. ; https://doi.org/10.1101/2025.04.01.646649doi: bioRxiv preprint \n\nFigures \n \nFig. 1: Differences in NPQ relaxation in phylogenetically linked C3 and C4 species (n=5).  A) NPQ \nrelaxation after 1h of illumination at 600 µmol m -2 s-2 PFD preceded by 5m of darkness for Fv/Fm. \nRibbons represent standard error of the mean. B) NPQ composition based on time relaxation \nkinetics as a percentage of total NPQ. Asterisks indicate significant differences between C3 and C4 \nspecies found by one-way ANOVA (* P ≤  0.05, ** P ≤  0.01, *** P ≤  0.001). \n \n \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 1, 2025. ; https://doi.org/10.1101/2025.04.01.646649doi: bioRxiv preprint \n\n \nFig. 2: Effect of Δ pH disruption and xanthophyll cycle inhibition on C3 and C4 NPQ. AUC of NPQ \ninduction of leaves infiltrated with A) 100 µM nigericin to collapse the proton gradient and B) 5 mM \ndithiothreitol to inhibit the xanthophyll cycle, as a percentage of a control (n=5). Asterisks indicate \nsignificant differences between C3 and C4 species found by one-way ANOVA (* P ≤  0.05, ** P ≤  \n0.01, *** P ≤  0.001). \n \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 1, 2025. ; https://doi.org/10.1101/2025.04.01.646649doi: bioRxiv preprint \n\n \nFig. 3: Differences in NPQ relaxation in phylogenetically linked C3 and C4 species when \nsuppressing photosynthesis and photorespiration with 2% O 2 and 0 ppm CO 2 air (n=5). A) NPQ \nrelaxation after 1h of illumination at 600 µmol m -2 s-2 PFD preceded by 5m of darkness for Fv/Fm. \nRibbons represent standard error of the mean. B) NPQ composition based on time relaxation \nkinetics as a percentage of total NPQ. Asterisks indicate significant differences between C3 and C4 \nspecies found by one-way ANOVA (* P ≤  0.05, ** P ≤  0.01, *** P ≤  0.001). \n \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 1, 2025. ; https://doi.org/10.1101/2025.04.01.646649doi: bioRxiv preprint \n\n \nFig. 4: Proton conductivity of the thylakoid membrane (gH +) in C3 and C4 phylogenetically linked \nspecies, under ambient and 2% O 2 air and 0 ppm CO 2 (n=5), during 20 minutes of illumination at \n600 µmol m -2 s-2 PFD. Asterisks indicate significant differences between species at a given \ntimepoint found by one-way ANOVA (n=5, * P ≤  0.05, ** P ≤  0.01, *** P ≤  0.001). \n \n \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 1, 2025. ; https://doi.org/10.1101/2025.04.01.646649doi: bioRxiv preprint \n\n \nFig. 5: CEF in C3 and C4 phylogenetically linked species. A) Representative trace (n=5) of post-\nillumination fluorescence rise (PIFR) in leaves infiltrated with a control buffer (solid lines) or with \nAntimycin A (dashed lines), an inhibitor of the CEF PGR5 pathway. B) NPQ relaxation in C4 F. \nbidentis WT and FbNdhO and FbPGRL1 RNAi knockdown mutants. Ribbons represent standard \nerror of the mean (n=3). C) NPQ composition based on time relaxation kinetics as a percentage of \ntotal NPQ. Asterisks indicate significant differences between each mutant and the WT found by \none-way ANOVA (n=3, * P ≤  0.05, ** P ≤  0.01, *** P ≤  0.001).  \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 1, 2025. ; https://doi.org/10.1101/2025.04.01.646649doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}