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Chlorophyll f synthesis by a super-rogue photosystem II complex

Nature Plants volume 6, pages 238–244 (2020)Cite this article

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Abstract

Certain cyanobacteria synthesize chlorophyll molecules (Chl d and Chl f) that absorb in the far-red region of the solar spectrum, thereby extending the spectral range of photosynthetically active radiation1,2. The synthesis and introduction of these far-red chlorophylls into the photosynthetic apparatus of plants might improve the efficiency of oxygenic photosynthesis, especially in far-red enriched environments, such as in the lower regions of the canopy3. Production of Chl f requires the ChlF subunit, also known as PsbA4 (ref. 4) or super-rogue D1 (ref. 5), a paralogue of the D1 subunit of photosystem II (PSII) which, together with D2, bind cofactors involved in the light-driven oxidation of water. Current ideas suggest that ChlF oxidizes Chl a to Chl f in a homodimeric ChlF reaction centre (RC) complex and represents a missing link in the evolution of the heterodimeric D1/D2 RC of PSII (refs. 4,6). However, unambiguous biochemical support for this proposal is lacking. Here, we show that ChlF can substitute for D1 to form modified PSII complexes capable of producing Chl f. Remarkably, mutation of just two residues in D1 converts oxygen-evolving PSII into a Chl f synthase. Overall, we have identified a new class of PSII complex, which we term ‘super-rogue’ PSII, with an unexpected role in pigment biosynthesis rather than water oxidation.

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Fig. 1: Isolation and characterization of ChlF complexes.
Fig. 2: Expression of chimeric ChlF/D1 proteins in Syn6803.
Fig. 3: Characterization of PSII complexes isolated from the D1-QD mutant.

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All data generated or analysed during this study are included in the published article and its Supplementary Information.

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Acknowledgements

We thank B. Nwaobi for excellent laboratory management and R. Debus and J. Eaton-Rye for providing plasmids and strains. Financial support was provided by the Biotechnology and Biological Sciences Research Council (BB/P00931X/1 to J.W.M. and P.J.N.), the Grant Agency of the Czech Republic (project no. 19-29225X to M.B., R.S. and J.K.) and the Ministry of Education, Youth and Sports of the Czech Republic (National Program of Sustainability I, ID: LO1416 to M.B., R.S. and J.K.). J.P.T. received a PhD scholarship from the Indonesia Endowment Fund for Education (LPDP).

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Author notes

  1. These authors contributed equally: Joko P. Trinugroho, Martina Bečková, Shengxi Shao.

Authors and Affiliations

  1. Sir Ernst Chain Building—Wolfson Laboratories, Department of Life Sciences, Imperial College London, South Kensington Campus, London, UK

    Joko P. Trinugroho, Shengxi Shao, Jianfeng Yu, Ziyu Zhao, James W. Murray & Peter J. Nixon

  2. Laboratory of Photosynthesis, Centre Algatech, Institute of Microbiology, Czech Academy of Sciences, Třeboň, Czech Republic

    Martina Bečková, Roman Sobotka & Josef Komenda

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Contributions

P.J.N., J.K. and J.W.M. conceived the research. J.P.T., S.S and J.K. prepared the figures. P.J.N and J.K. wrote the manuscript with contributions from all the other authors. P.J.N. and J.K. coordinated the activities. J.P.T. constructed the mutants. J.P.T., M.B., S.S., J.Y., Z.Z. and R.S. performed the biochemical analyses. All authors approved the final version of the manuscript.

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Correspondence to Peter J. Nixon.

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Extended data

Extended Data Fig. 1 Analysis of FLAG-ChlF complexes immunopurified from the FLAG-ChlF/WT strain.

a, FLAG-ChlF complexes isolated from the FLAG-ChlF/WT strain were separated by clear-native polyacrylamide gel electrophoresis (CN-PAGE) in the first dimension (1D colour) and visualized by fluorescence (1D fluor) then denatured, separated by SDS-PAGE in the second dimension and protein subunits stained by Sypro Orange (2D SYPRO). Proteins were identified in the stained gel by immunoblotting and mass spectrometry. Abbreviations: F.ChlF, FLAG-tagged ChlF; PSII(1)’, monomeric PSII-like complex containing ChlF; RC47’, PSII(1)’ complex lacking CP43 and containing ChlF; PSI(1)-RC47’, complex composed of PSI monomer and RC47’ complex; CP43, CP43 module; U.P., unassembled proteins. b, Cartoon representations comparing the monomeric PSII complex containing D1 (PSII(1)) and the PSII-like complexes containing ChlF (PSII(1)’ and RC47’). Small subunits are not shown for clarity.

Extended Data Fig. 2 Characterization of FLAG-ChlF complexes immunopurified from the FLAG-ChlF/ΔD1/ΔCP43 strain.

a, Cartoon showing the psbD1C locus found in the FLAG-ChlF/ΔD1strain and the resulting FLAG-ChlF/ΔD1/ΔCP43 strain following deletion of psbD1C. Positions of the forward (F4) and reverse (R4) primers (Supplementary Table 3) used for PCR genotyping and predicted sizes are indicated. b, Agarose gel of PCR fragments confirming the genotype of the FLAG-ChlF/ΔD1/ΔCP43 strain and non-tagged strains. c, FLAG-ChlF complexes isolated from the FLAG-ChlF/ΔD1/ΔCP43 strain were separated by clear-native polyacrylamide gel electrophoresis (CN-PAGE) in the first dimension (1D colour) and visualized by fluorescence (1D fluor) then denatured, separated by SDS-PAGE in the second dimension and protein subunits stained by Sypro Orange (2D SYPRO). Proteins were identified in the stained gel by immunoblotting (FLAG and D2 blots shown) and mass spectrometry. Abbreviations: F.ChlF, FLAG-tagged ChlF; RC47’, PSII(1)’ complex lacking CP43; PSI(1)-RC47’, complex composed of PSI monomer and RC47’ complex; U.P., unassembled proteins. d, Cartoon showing the RC47’ complex composed of ChlF/D2 heterodimer with attached CP47. Small subunits are not shown for clarity.

Source data

Extended Data Fig. 3 Characterization of FLAG-ChlF complexes immunopurified from the FLAG-ChlF/ΔD1/ΔCP47 strain.

a, Cartoon showing the psbB locus found in the FLAG-ChlF/ΔD1strain and the resulting FLAG-ChlF/ΔD1/ΔCP47 strain following deletion of psbB. Positions of the forward (F6) and reverse (R6) primers (Supplementary Table 3) used for PCR genotyping and predicted sizes are indicated. b, Agarose gel of PCR fragments confirming the genotype of the FLAG-ChlF/ΔD1/ΔCP47 strain and untagged strains. c, FLAG-ChlF complexes isolated from the FLAG-ChlF/ΔD1/ΔCP47 strain were separated by clear-native polyacrylamide gel electrophoresis (CN-PAGE) in the first dimension (1D colour) and visualized by fluorescence (1D fluor) then denatured, separated by SDS-PAGE in the second dimension and protein subunits stained by Sypro Orange (2D SYPRO). Proteins were identified in the stained gel by immunoblotting (D2 and FLAG blots shown) and mass spectrometry. Abbreviations: F.ChlF, FLAG-tagged ChlF; PSI(3), trimeric PSI; PSI(1)-RCa’, complex composed of PSI monomer and ChlF/D2 complex; RC43’, complex composed of ChlF/D2 with CP43 attached; RCIIa’, assembly complex of ChlF/D2 with accessory proteins; RCIIb’, assembly complex of ChlF/D2 lacking assembly factors. U.P., unassembled proteins. d, Cartoon showing the modified RC43’ complex, composed of a ChlF/D2 heterodimer with attached CP43 and the RCIIb’ complex. Small subunits are not shown for clarity.

Source data

Extended Data Fig. 4 Detection of FLAG-ChlF and FLAG-D1.

Detergent-solubilized membrane proteins from the a, FLAG-ChlF/PSII−strain or b, FLAG-D1/PSII− strain were separated by clear-native (CN) gel electrophoresis in the first dimension (1D colour) and visualized by fluorescence (1D fluor) then denatured, separated by SDS-PAGE in the second dimension and protein subunits stained by Sypro Orange (2D SYPRO), then transferred to PVDF membranes for immunochemical detection of FLAG-tagged proteins using specific FLAG-tag antibodies (FLAG). Abbreviations: PSI(3), trimeric PSI; PSI(1), monomeric PSI complex; U.P., unassembled proteins.

Source data

Extended Data Fig. 5 Analysis of FLAG-ChlF complexes in the FLAG-ChlF(MG)/ΔD1 mutant (ChlF-MG) in which the QD pair (Q149/D150) of ChlF is replaced by the MG pair found in D1.

Detergent-solubilized membrane proteins from the FLAG-ChlF(MG)/ΔD1 strain were separated by clear-native polyacrylamide gel electrophoresis (CN-PAGE) in the first dimension (1D colour) and visualized by fluorescence (1D fluor) then denatured, separated by SDS-PAGE in the second dimension and protein subunits stained by Sypro Orange (2D SYPRO), then transferred to PVDF membrane for immunochemical detection of FLAG-ChlF (FLAG), D2, CP47 and CP43 proteins. Abbreviations: PSI(3), trimeric PSI; PSII(1), monomeric PSII; PSI(1), monomeric PSI complex; U.P., unassembled proteins.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–6 and Tables 1–3.

Reporting Summary

Supplementary Data

Unprocessed Western Blot Supplementary Fig. 4.

Source data

Source Data Extended Data Fig. 2

Unprocessed western blot Extended Data Fig. 2c.

Source Data Extended Data Fig. 3

Unprocessed western blot Extended Data Fig. 3c.

Source Data Extended Data Fig. 4

Unprocessed Wwestern blot Extended Data Fig. 4.

Source Data Extended Data Fig. 5

Unprocessed western blot Extended Data Fig. 5.

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Trinugroho, J.P., Bečková, M., Shao, S. et al. Chlorophyll f synthesis by a super-rogue photosystem II complex. Nat. Plants 6, 238–244 (2020). https://doi.org/10.1038/s41477-020-0616-4

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