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An atypical short-chain dehydrogenase–reductase functions in the relaxation of photoprotective qH in Arabidopsis

Nature Plants volume 6, pages 154–166 (2020)Cite this article

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Abstract

Photosynthetic organisms experience wide fluctuations in light intensity and regulate light harvesting accordingly to prevent damage from excess energy. The antenna quenching component qH is a sustained form of energy dissipation that protects the photosynthetic apparatus under stress conditions. This photoprotective mechanism requires the plastid lipocalin LCNP and is prevented by SUPPRESSOR OF QUENCHING1 (SOQ1) under non-stress conditions. However, the molecular mechanism of qH relaxation has yet to be resolved. Here, we isolated and characterized RELAXATION OF QH1 (ROQH1), an atypical short-chain dehydrogenase–reductase that functions as a qH-relaxation factor in Arabidopsis. The ROQH1 gene belongs to the GreenCut2 inventory specific to photosynthetic organisms, and the ROQH1 protein localizes to the chloroplast stroma lamellae membrane. After a cold and high-light treatment, qH does not relax in roqh1 mutants and qH does not occur in leaves overexpressing ROQH1. When the soq1 and roqh1 mutations are combined, qH can neither be prevented nor relaxed and soq1 roqh1 displays constitutive qH and light-limited growth. We propose that LCNP and ROQH1 perform dosage-dependent, antagonistic functions to protect the photosynthetic apparatus and maintain light-harvesting efficiency in plants.

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Fig. 1: Genetic screen uncovered mutants with constitutively quenched fluorescence.
Fig. 2: Schematic representation of ROQH1 protein and accumulation in roqh1 mutants.
Fig. 3: Constitutive quenching is due to the combination of soq1 and roqh1 mutations.
Fig. 4: Constitutively quenched mutants are light-limited.
Fig. 5: Constitutive quenching requires the peripheral antenna of PSII and LCNP.
Fig. 6: Overexpression of ROQH1 prevents qH from occurring.
Fig. 7: ROQH1 is required for relaxation of qH.
Fig. 8: ROQH1 functions in a complex after cold and high light.

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Data availability

Source data for Figs. 2, 6 and 8 and Extended Data Figs. 1 and 2 are provided with the paper. Sequence data from this article can be found in the Arabidopsis Genome Initiative under accession numbers At1g44446 (CAO; https://www.arabidopsis.org/servlets/TairObject?id=226583&type=locus), At1g44575 (PsbS; https://www.arabidopsis.org/servlets/TairObject?id=226577&type=locus), At1g56500 (SOQ1; https://www.arabidopsis.org/servlets/TairObject?id=27293&type=locus), At3g47860 (LCNP; https://www.arabidopsis.org/servlets/TairObject?id=40152&type=locus) and At4g31530 (ROQH1; https://www.arabidopsis.org/servlets/TairObject?type=locus&id=128060).

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Acknowledgements

We thank S. Ruzin and D. Schichnes from the Biological Imaging Facility and K. McDonald and R. Zalpuri from the Transmission Electron Microscopy Facility at University of California, Berkeley for technical advice and assistance; S. Shahrasbi and S. Lee for assistance with mutant screening and crosses; C. Marshall for advice regarding DNA sequencing; F. Ouellet for providing antibodies against LCNP; M. Iwai for advice regarding BN–PAGE and critical reading of the manuscript; and R. Croce and C. Gee for critical discussions. C.L.A. would like to thank Daniel L. Amstutz, who passed away during the writing of this manuscript, for guidance and support. This research was supported by the Division of Chemical Sciences, Geosciences and Biosciences, the Office of Basic Energy Sciences and the Office of Science, US Department of Energy (Field Work Proposal 449B). This work used the Vincent J. Coates Genomics Sequencing Laboratory at University of California, Berkeley, supported by National Institutes of Health S10 Instrumentation Grants S10RR029668 and S10RR027303. R.F. was supported by the Dutch Organization for Scientific Research via an ECHO grant to R. Croce and by the US Department of Energy Office of Science, Office of Biological and Environmental Research programme under award no. DE-FC02-02ER63421. A.S. was supported by the National Institutes of Health National Research Service Award Trainee appointment (grant no. GM007127). K.K.N. is an investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Howard Hughes Medical Institute, University of California, Berkeley, CA, USA

    Cynthia L. Amstutz, Krishna K. Niyogi & Alizée Malnoë

  2. Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA

    Cynthia L. Amstutz, Alex Schultink, Sabeeha S. Merchant, Krishna K. Niyogi & Alizée Malnoë

  3. Department of Physics and Astronomy, Vrije University of Amsterdam, Amsterdam, The Netherlands

    Rikard Fristedt

  4. Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, USA

    Rikard Fristedt & Sabeeha S. Merchant

  5. Institute for Genomics and Proteomics, University of California, Los Angeles, CA, USA

    Sabeeha S. Merchant

  6. Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA

    Sabeeha S. Merchant

  7. Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Krishna K. Niyogi & Alizée Malnoë

  8. Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, Umeå, Sweden

    Alizée Malnoë

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Contributions

C.L.A., K.K.N. and A.M. designed research; C.L.A. performed research; A.M. performed the genetic screen, crosses and preliminary biophysical and biochemical characterizations; A.S. performed bioinformatics analysis to identify mutated genes; R.F. designed the ROQH1 antibody and performed biochemical fractionation and salt washes experiments. All of the authors analysed and discussed the data, and C.L.A. and A.M. wrote the paper with input from K.K.N., A.S., R.F. and S.S.M.

Corresponding authors

Correspondence to Krishna K. Niyogi or Alizée Malnoë.

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Competing interests

A.M. and K.K.N. have filed a US patent application (no. 2019 292 556 A1) describing how the overexpression of ROQH1 accelerates relaxation of NPQ (qH). The remaining authors declare no competing interests.

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Peer review information Nature Plants thanks Peter Jahns and the other, anonymous, reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 ROQH1 is enriched at the chloroplast stroma lamellae.

Proteins were separated by SDS-PAGE and analyzed by immunodetection with antibodies against ROQH1, Rubisco, Lhca1, Lhcb2. Ponceau is shown as loading control. Molecular masses (kD) are indicated according to the migration of Precision Plus Protein Standards markers from Bio-Rad. Total leaf extract (Leaf) from plants grown under 120 µmol photons m−2 s−1, 21 °C were fractionated into chloroplasts, thylakoids, grana (appressed membranes), grana margins, stroma, and stroma lamellae (non-appressed membranes). Proteins were separated by SDS-PAGE and analyzed by immunodetection with antibodies against ROQH1, Rubisco, Lhca1 or Lhcb2. Ponceau is shown as loading control. Molecular masses (kD) are indicated according to the migration of Precision Plus Protein Standards markers from Bio-Rad. Samples were loaded by equal total chlorophyll content (3 μg). Immunoblot is representative of 5 biologically independent experiments.

Source data

Extended Data Fig. 2 ROQH1 functions in a complex after cold and high light.

Two-dimensional BN/SDS-PAGE analysis from wild-type thylakoids isolated before (-) and after (+) a 5 h cold and high light treatment (6 °C and 1,600 µmol photons m-2 s-1), solubilized with 1% β-DM and immunoblotted with antibodies for Flag, PsaA, D1, and Lhcb2. For an internal loading control, 1 μg total chlorophyll of solubilized soq1 roqh1-1: ROQH1 OE thylakoids was loaded in the control lane. Immunoblots are representative of 2 biologically independent experiments.

Source data

Extended Data Fig. 3 ROQH1 is required to turn off qH.

Under non-stress conditions, SOQ1 inhibits LCNP activity. Under stress conditions, such as cold and high light, SOQ1 inhibition is relieved (grey dashed line) and LCNP is active. Quenching sites indicated by purple color are produced in the peripheral antenna directly mediated by LCNP (solid arrow) or indirectly (dashed arrow) through LCNP modification of LHCII hydrophobic environment. ROQH1 recycle these quenching sites back to light harvesting sites either directly by acting at the antenna (solid line) or indirectly through modification of LHCII hydrophobic environment (dashed line). Adapted from ref. 17. Copyright American Society of Plant Biologists.

Supplementary information

Supplementary Information

Supplementary Figs. 1–16, Tables 1–4 and Source Data for Supplementary Figs. 2, 5, 6, 7, 10, 12, 14 and 15.

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Source Data Fig. 2

Unprocessed western blots.

Source Data Fig. 6

Unprocessed western blots.

Source Data Fig. 8

Unprocessed western blots.

Source Data Extended Data Fig. 1

Unprocessed western blots.

Source Data Extended Data Fig. 2

Unprocessed western blots.

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Amstutz, C.L., Fristedt, R., Schultink, A. et al. An atypical short-chain dehydrogenase–reductase functions in the relaxation of photoprotective qH in Arabidopsis. Nat. Plants 6, 154–166 (2020). https://doi.org/10.1038/s41477-020-0591-9

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  • DOI: https://doi.org/10.1038/s41477-020-0591-9

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