Does the Arabidopsis proton gradient regulation 5 mutant leak protons from the thylakoid membrane?

Short title: 1 2 Proton conductivity of the pgr5 mutant 3 4 Article title: 5 6 Does the Arabidopsis proton gradient regulation 5 mutant leak protons from the thylakoid 7 membrane? 1 8 9 Hiroshi Yamamoto 2 and Toshiharu Shikanai 3 10 Department of Botany, Graduate School of Science, Kyoto University, Kyoto, 606-8502 Japan 11 ORCID IDs: 0000-0002-1739-1226 (H.Y); 0000-0002-6154-4728 (T.S.) 12 13 One sentence summary: 14 High gH + of the pgr5 mutant is compensated to the WT level by introduction of flavodiiron 15 protein and strong down-regulation of the cytochrome b6f complex, objecting to the proton 16 leakage from the thylakoid membrane. 17 18 Footnotes 19 20 Funding information: 21 This work was supported by the Japanese Society for the Promotion of Science KAKENHI 22 (16H06553 and 19H00992). 23 24 Responsibilities of the Author for Contact: 25 Senior author. 26 Author for contact: shikanai@pmg.bot.kyoto-u.ac.jp 27 The author responsible for distribution of materials integral to the findings presented in this 28 article in accordance with the policy described in the Instructions for Authors 29 (www.plantphysiol.org) is: Toshiharu Shikanai (shikanai@pmg.bot.kyoto-u.ac.jp). 30 31 Author contributions: 32 H.Y. and T.S. designed the research; H.Y. performed the experiments; H.Y. analyzed the data; 33 H.Y. and T.S. wrote the article. 34 Both authors contributed equally to this work. 35 36 Plant Physiology Preview. Published on July 7, 2020, as DOI:10.1104/pp.20.00850

photosystem I it is a topic of debate. Based on a parameter of electrochromic shift analysis (g H + ), 48 the proton conductivity of the thylakoid membrane in the pgr5 mutant is enhanced at high light 49 intensity. Given this observation, PGR5 was proposed to regulate ATP synthase activity rather 50 than mediating CET. The originally reported pgr5 phenotype reflects a smaller proton motive 51 force (pmf) and could be explained by this H + leakage model. In this study, we genetically 52 reexamined the high g H + phenotype of the pgr5 mutant. Transgenic lines in which flavodiiron 53 protein-dependent pseudo-CET replaced PGR5-dependent CET had wild-type levels of g H + ,

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suggesting that the high g H + phenotype in pgr5 plants is caused secondarily by the low pmf. The 55 pgr1 mutant shows a similar reduction in pmf because of enhanced sensitivity of its cytochrome 56 b 6 f complex to lumenal acidification. In contrast to the pgr5 mutant, g H + was lower in the pgr1 57 mutant than in the WT. In the pgr1 pgr5 double mutants, g H + was intermediate to those of the 58 respective single mutants. It is unlikely that the g H + is upregulated simply in response to a low 59 pmf, as we did not observe uncoupling of the thylakoid membrane in the pgr5 mutant upon 60 monitoring the quenching of 9-aminoacridine fluorescence. We conclude that the g H + parameter

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The light reactions of photosynthesis are driven by two photosystems, photosystem II and I 72 (PSII and PSI), functioning in that order. They mediate electron transport from water to NADP + .

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This linear electron transport does not satisfy the ATP/NADPH production ratio required by the 74 Calvin-Benson cycle (Allen, 2002). Additional ATP requirement is satisfied by cyclic electron 75 transport (CET) around PSI (Yamori and Shikanai, 2016). In angiosperms, PSI CET consists of 76 at least two pathways (Fig. 1). The main route of electron transport is sensitive to antimycin A 77 and depends on PROTON GRADIENT REGULATION 5 (PGR5) and PGR5-LIKE 78 PHOTOSYNTHETIC PHENOTYPE (PGRL1) (Munekage et al., 2002;DalCorso et al., 2008).

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The minor pathway is mediated by the NADH dehydrogenase-like (NDH) complex (Peltier et   (Avenson et al., 2005;Wang et al., 2015). It was monitored as the g H + 115 parameter of electrochromic shift (ECS) analysis (Kanazawa and Kramer, 2002 illumination (Bailleul et al., 2010). The pmf consists of ∆pH and membrane potential (∆ψ) If the ATP level limited the rate of the Calvin-Benson cycle in the pgr5 mutant, it would be 167 reasonable to conclude that ATP synthase activity was enhanced to supplement ATP production.

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PGR5 is unlikely to regulate ATP synthase directly, but the pgr5 mutant phenotype observed in 180 Figure 3A shows the light intensity-dependence of pmf. In this analysis, we also 181 characterized a weak allele of pgr5-2, in which PGR5-dependent PSI CET was partially

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The high g H + phenotype of the pgr5 mutants may reflect the general response of ATP 238 synthase to the reduced size of pmf. However, the size of g H + was lower in the pgr1 mutant than 239 in the WT (Fig. 3B) contaminated with an absorbance at 505 nm caused by the synthesis of zeaxanthin and also by a 251 qE-related 535-nm change (Johnson and Ruban, 2014). We have to be careful on the evaluation 252 of the steady-state ECS parameters. We focused on the rapid decay kinetics of the ECS signal 253 and observed the opposite response of g H + between the pgr1 and pgr5 mutants, both of which 254 are defective in the qE induction (Fig. 3). Most probably, g H + mainly reflects the H + 255 conductivity of ATP synthase during steady-state photosynthesis (Kanazawa and Kramer, 2002).

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The idea is supported by an early study (Schönfeld and Neumann, 1977). But we observed that

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The measurement of 9-AA fluorescence was performed using the Dual-ENADPH and          In angiosperms, CET consists of PGR5/PGRL1-and NDH-dependent pathways. Both protein complexes mediate the backflow of electrons from PSI to the plastoquinone (PQ) pool via ferredoxin (Fd) as an electron donor. CET contributes to the ΔpH formation across the thylakoid membrane via the Q-cycle in the Cyt b 6 f complex. In Flv-dependent pseudo-CET, Flv directly reduces O 2 to water using photoreductant X (NADPH or Fd) from PSI. Pseudo-CET contributes to ΔpH formation via water oxidation in PSII and the Q-cycle. Lumenal acidification slows down plastoquinol oxidation at the Cyt b 6 f complex to prevent excess electron flow toward PSI (photosynthetic control). Lumenal acidification also induces qE quenching in the PSII antennae to discard excess photon energy as heat. The pmf composed of ΔpH and ΔΨ drives ATP synthesis via ATP synthase. The pgr5 mutants are defective in PGR5/PGRL1-dependent ΔpH formation. In the pgr1 mutant, photosynthetic control is more sensitive to lumenal acidification. In the H + leakage model, PGR5 functions to down-regulate ATP synthase instead of PSI CET. PC represents plastocyanin.  Figure 2. Enhanced pseudo-CET by Flv suppresses the high g H + phenotype in the pgr5 mutants. The light intensity-dependence of pmf formation (A) and g H + (B) was monitored in the WT, pgr5-1, WT+35S;PpFlv no. 13, and pgr5-1+35S;PpFlv no. 13 (biological replicates n = 6 ± sd). Symbols with the same letters are not significantly different between genotypes at 252 and 663 µmol photons m -2 s -1 (Tukey-Kramer test, P < 0.05).  Figure 3. Enhanced photosynthetic control induced by the pgr1 mutation suppresses the high g H + phenotype of the pgr5 mutants. The light intensity-dependence of pmf formation (A) and g H + (B) was monitored in the WT, pgr1, pgr5 alleles, and pgr1 pgr5 alleles (biological replicates n = 8 -10 ± sd). Symbols with the same letters are not significantly different between genotypes at 252 and 663 µmol photons m -2 s -1 (Tukey-Kramer test, P < 0.05). . Kinetics of 9-aminoacridine (9-AA) quenching in ruptured chloroplasts during the four consecutive cycles of illumination (2 min) followed by the dark recovery (2 min). Ruptured chloroplasts were illuminated with an actinic light using the Dual-ENADPH and Dual-DNADPH modules in the presence of 2 mM ADP and 100 μM methyl viologen as a terminal electron acceptor. (A) Representative 9-AA quenching traces measured on WT and pgr5-1 ruptured chloroplasts were normalized to the initial dark levels. Arrows indicate actinic light on/off cycles. The intensity of actinic light was indicated in white boxes in the top bar. (B) The light intensity-dependence of 9-AA quenching upon illumination. (C) Half-time (t 1/2 ) of the 9-AA fluorescence recovery upon transition from illumination to darkness (C) were monitored in the WT and pgr5-1 ruptured chloroplasts (biological replicates n = 4 ± sd). In (B) and (C), there are no significant differences between WT and pgr5-1 plants at each light intensity (Welch's t-test, P < 0.05).