Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Grafting Rhodobacter sphaeroides with red algae Rubisco to accelerate catalysis and plant growth

Nature Plants volume 9, pages 978–986 (2023)Cite this article

Subjects

Abstract

Improving the carboxylation properties of Rubisco has primarily arisen from unforeseen amino acid substitutions remote from the catalytic site. The unpredictability has frustrated rational design efforts to enhance plant Rubisco towards the prized growth-enhancing carboxylation properties of red algae Griffithsia monilis GmRubisco. To address this, we determined the crystal structure of GmRubisco to 1.7 Å. Three structurally divergent domains were identified relative to the red-type bacterial Rhodobacter sphaeroides RsRubisco that, unlike GmRubisco, are expressed in Escherichia coli and plants. Kinetic comparison of 11 RsRubisco chimaeras revealed that incorporating C329A and A332V substitutions from GmRubisco Loop 6 (corresponding to plant residues 328 and 331) into RsRubisco increased the carboxylation rate (kcatc) by 60%, the carboxylation efficiency in air by 22% and the CO2/O2 specificity (Sc/o) by 7%. Plastome transformation of this RsRubisco Loop 6 mutant into tobacco enhanced photosynthesis and growth up to twofold over tobacco producing wild-type RsRubisco. Our findings demonstrate the utility of RsRubisco for the identification and in planta testing of amino acid grafts from algal Rubisco that can enhance the enzyme’s carboxylase potential.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Three-dimensional crystal structure of G. monilis Rubisco.
Fig. 2: Structural comparison between Form IB SoRubisco, Form IC RsRubisco and Form ID GmRubisco.
Fig. 3: Phylogenetic grafting of GmRubisco into RsRubisco to enhance catalysis.
Fig. 4: The simulated improvement of tobacco photosynthesis by RsRubiscoChi11.
Fig. 5: The improved carboxylation properties of RsRubisco-chi11 translate to improvements in the photosynthesis and growth of tobacco.

Similar content being viewed by others

Data availability

Atomic coordinates and structure factors for G. monilis Rubisco are accessible via Protein Data Bank (http://www.wwpdb.org) accession number 8BDB. All other data are available online as source files and via https://doi.org/10.6084/m9.figshare.21547515. Source data are provided with this paper.

References

  1. Bailey-Serres, J., Parker, J. E., Ainsworth, E. A., Oldroyd, G. E. D. & Schroeder, J. I. Genetic strategies for improving crop yields. Nature 575, 109–118 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Simkin, A. J., López-Calcagno, P. E. & Raines, C. A. Feeding the world: improving photosynthetic efficiency for sustainable crop production. J. Exp. Bot. 70, 1119–1140 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Evans, J. R. & Lawson, T. From green to gold: agricultural revolution for food security. J. Exp. Bot. 71, 2211–2215 (2020).

    CAS  PubMed  Google Scholar 

  4. Sharwood, R. E. Engineering chloroplasts to improve Rubisco catalysis: prospects for translating improvements into food and fiber crops. New Phytol. 213, 494–510 (2017).

    CAS  PubMed  Google Scholar 

  5. von Caemmerer, S. Rubisco carboxylase/oxygenase: from the enzyme to the globe: a gas exchange perspective. J. Plant Physiol. 252, 153240 (2020).

    Google Scholar 

  6. Busch, F. A. Photorespiration in the context of Rubisco biochemistry, CO2 diffusion and metabolism. Plant J. 101, 919–939 (2020).

    CAS  PubMed  Google Scholar 

  7. Evans, J. & Seemann, J. in Photosynthesis (ed. Briggs, W. R.) 183–205 (Alan R Liss, 1989).

  8. Evans, J. R. & Clarke, V. C. The nitrogen cost of photosynthesis. J. Exp. Bot. https://doi.org/10.1093/jxb/ery366 (2018).

  9. Iñiguez, C. et al. Evolutionary trends in RuBisCO kinetics and their co-evolution with CO2 concentrating mechanisms. Plant J. 101, 897–918 (2020).

    PubMed  Google Scholar 

  10. Ermakova, M. et al. Installation of C4 photosynthetic pathway enzymes in rice using a single construct. Plant Biotech. J. 19, 575–588 (2021).

    CAS  Google Scholar 

  11. Long, B. M., Rae, B. D., Rolland, V., Förster, B. & Price, G. D. Cyanobacterial CO2-concentrating mechanism components: function and prospects for plant metabolic engineering. Curr. Opin. Plant Biol. 31, 1–8 (2016).

    CAS  PubMed  Google Scholar 

  12. Fei, C., Wilson, A. T., Mangan, N. M., Wingreen, N. S. & Jonikas, M. C. Modelling the pyrenoid-based CO2-concentrating mechanism provides insights into its operating principles and a roadmap for its engineering into crops. Nat. Plants 8, 583–595 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Andersson, I. & Backlund, A. Structure and function of Rubisco. Plant Physiol. Biochem. 46, 275–291 (2008).

    CAS  PubMed  Google Scholar 

  14. Iñiguez, C., Aguiló-Nicolau, P. & Galmés, J. Improving photosynthesis through the enhancement of Rubisco carboxylation capacity. Biochem. Soc. Trans. 49, 2007–2019 (2021).

    PubMed  Google Scholar 

  15. Bracher, A., Whitney, S. M., Hartl, F. U. & Hayer-Hartl, M. Biogenesis and metabolic maintenance of Rubisco. Annu. Rev. Plant Biol. 68, 29–60 (2017).

    CAS  PubMed  Google Scholar 

  16. Aigner, H. et al. Plant Rubisco assembly in E. coli with five chloroplast chaperones including BSD2. Science 358, 1272–1278 (2017).

    CAS  PubMed  Google Scholar 

  17. Whitney, S. M., Baldet, P., Hudson, G. S. & Andrews, T. J. Form I Rubiscos from non-green algae are expressed abundantly but not assembled in tobacco chloroplasts. Plant J. 26, 535–547 (2001).

    CAS  PubMed  Google Scholar 

  18. Zhu, X. G., Portis, A. R. & Long, S. P. Would transformation of C3 crop plants with foreign Rubisco increase productivity? A computational analysis extrapolating from kinetic properties to canopy photosynthesis. Plant Cell Env. 27, 155–165 (2004).

    CAS  Google Scholar 

  19. Lin, M. T. & Hanson, M. R. Red algal Rubisco fails to accumulate in transplastomic tobacco expressing Griffithsia monilis RbcL and RbcS genes. Plant Direct 2, e00045 (2018).

    PubMed  PubMed Central  Google Scholar 

  20. Gunn, L. H., Martin Avila, E., Birch, R. & Whitney, S. M. The dependency of red Rubisco on its cognate activase for enhancing plant photosynthesis and growth. Proc. Natl Acad. Sci. USA 117, 25890–25896 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Andersson, I. Catalysis and regulation in Rubisco. J. Exp. Bot. 59, 1555–1568 (2008).

    CAS  PubMed  Google Scholar 

  22. Hanson, D. T. Breaking the rules of Rubisco catalysis. J. Exp. Bot. 67, 3180–3182 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Martin-Avila, E. et al. Modifying plant photosynthesis and growth via simultaneous chloroplast transformation of Rubisco large and small subunits. Plant Cell 32, 2898–2916 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Lin, M. T., Salihovic, H., Clark, F. K. & Hanson, M. R. Improving the efficiency of Rubisco by resurrecting its ancestors in the family Solanaceae. Sci. Adv. 8, abm6871 (2022).

    Google Scholar 

  25. Young, J. N. et al. Large variation in the Rubisco kinetics of diatoms reveals diversity among their carbon-concentrating mechanisms. J. Exp. Bot. 67, 3445–3456 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Whitney, S. M. & Sharwood, R. E. Construction of a tobacco master line to improve Rubisco engineering in chloroplasts. J. Exp. Bot. 59, 1909–1921 (2008).

    CAS  PubMed  Google Scholar 

  27. Heureux, A. M. C. et al. The role of Rubisco kinetics and pyrenoid morphology in shaping the CCM of haptophyte microalgae. J. Exp. Bot. 68, 3959–3969 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Iñiguez, C., Galmés, J. & Gordillo, F. J. L. Rubisco carboxylation kinetics and inorganic carbon utilization in polar versus cold-temperate seaweeds. J. Exp. Bot. 70, 1283–1297 (2018).

    PubMed Central  Google Scholar 

  29. Chen, Z. X., Yu, W. Z., Lee, J. H., Diao, R. & Spreitzer, R. J. Complementing amino acid substitutions within loop 6 of the alpha/beta-barrel active site influence the CO2/O2 specificity of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase. Biochemistry 30, 8846–8850 (1991).

    CAS  PubMed  Google Scholar 

  30. Bouvier, J. W. et al. Rubisco adaptation is more limited by phylogenetic constraint than by catalytic trade-off. Mol. Biol. Evol. 30, 2880–2896 (2021).

    Google Scholar 

  31. Wilson, R. H., Alonso, H. & Whitney, S. M. Evolving Methanococcoides burtonii archaeal Rubisco for improved photosynthesis and plant growth. Sci. Rep. 6, 22284 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Wilson, R. H., Martin-Avila, E., Conlan, C. & Whitney, S. M. An improved Escherichia coli screen for Rubisco identifies a protein–protein interface that can enhance CO2-fixation kinetics. J. Biol. Chem. 293, 18–27 (2018).

    CAS  PubMed  Google Scholar 

  33. Zhou, Y. & Whitney, S. Directed evolution of an improved Rubisco; in vitro analyses to decipher fact from fiction. Int. J. Mol. Sci. 20, 5019 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Savir, Y., Noor, E., Milo, R. & Tlusty, T. Cross-species analysis traces adaptation of Rubisco toward optimality in a low-dimensional landscape. Proc. Natl Acad. Sci. USA 107, 3475–3480 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Tcherkez, G., Farquhar, G. & Andrews, T. Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized. Proc. Natl Acad. Sci. USA 103, 7246–7251 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Mueller-Cajar, O. et al. Structure and function of the AAA+ protein CbbX, a red-type Rubisco activase. Nature 479, 194–199 (2011).

    CAS  PubMed  Google Scholar 

  37. Taylor, T. C. & Andersson, I. Structural transitions during activation and ligand binding in hexadecameric Rubisco inferred from the crystal structure of the activated unliganded spinach enzyme. Nat. Struct. Biol. 3, 95–101 (1996).

    CAS  PubMed  Google Scholar 

  38. Chen, Z. X. & Spreitzer, R. J. Chloroplast intragenic suppression enhances the low CO2/O2 specificity of mutant ribulose-bisphosphate carboxylase/oxygenase. J. Biol. Chem. 264, 3051–3053 (1989).

    CAS  PubMed  Google Scholar 

  39. Karkehabadi, S., Satagopan, S., Taylor, T. C., Spreitzer, R. J. & Andersson, I. Structural analysis of altered large-subunit loop-6/carboxy-terminus interactions that influence catalytic efficiency and CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase. Biochemistry 46, 11080–11089 (2007).

    CAS  PubMed  Google Scholar 

  40. Gutteridge, S., Rhoades, D. F. & Herrmann, C. Site-specific mutations in a loop region of the C-terminal domain of the large subunit of ribulose bisphosphate carboxylase/oxygenase that influence substrate partitioning. J. Biol. Chem. 268, 7818–7824 (1993).

    CAS  PubMed  Google Scholar 

  41. Parry, M. A. J., Madgwick, P., Parmar, S., Cornelius, M. J. & Keys, A. J. Mutations in loop six of the large subunit of ribulose-1,5-bisphosphate carboxylase affect substrate specificity. Planta 187, 109–112 (1992).

    CAS  PubMed  Google Scholar 

  42. Galmés, J. et al. Environmentally driven evolution of Rubisco and improved photosynthesis and growth within the C3 genus Limonium (Plumbaginaceae). New Phytol. 203, 989–999 (2014).

    PubMed  Google Scholar 

  43. Zhu, G., Jensen, R. G., Bohnert, H., Wildner, G. & Schlitter, J. Dependence of catalysis and CO2/O2 specificity of Rubisco on the carboxy-terminus of the large subunit at different temperatures. Photosynth. Res. 57, 71–79 (1998).

    CAS  Google Scholar 

  44. Joshi, J., Mueller-Cajar, O., Tsai, Y. C., Hartl, F. U. & Hayer-Hartl, M. Role of small subunit in mediating assembly of red-type Form I Rubisco. J. Biol. Chem. 290, 1066–1074 (2015).

    CAS  PubMed  Google Scholar 

  45. Bhat, J. Y. et al. Mechanism of enzyme repair by the AAA+ chaperone Rubisco activase. Mol. Cell 67, 744–756.e6 (2017).

    CAS  PubMed  Google Scholar 

  46. Genkov, T. & Spreitzer, R. J. Highly conserved small subunit residues influence Rubisco large subunit catalysis. J. Biol. Chem. 284, 30105–30112 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Spreitzer, R. J., Peddi, S. R. & Satagopan, S. Phylogenetic engineering at an interface between large and small subunits imparts land-plant kinetic properties to algal Rubisco. Proc. Natl Acad. Sci. USA 102, 17225–17230 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Whitney, S. M., Birch, R., Kelso, C., Beck, J. L. & Kapralov, M. V. Improving recombinant Rubisco biogenesis, plant photosynthesis and growth by coexpressing its ancillary RAF1 chaperone. Proc. Natl Acad. Sci. USA 112, 3564–3569 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Whitney, S. M. et al. Isoleucine 309 acts as a C4 catalytic switch that increases ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) carboxylation rate in Flaveria. Proc. Natl Acad. Sci. USA 108, 14688–14693 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Buck, S. et al. Escherichia coli expressing chloroplast chaperones as a proxy to test heterologous Rubisco production in leaves. J. Exp. Bot. 74, 664–676 (2023).

    PubMed  Google Scholar 

  51. Sharwood, R. E., Ghannoum, O., Kapralov, M. V., Gunn, L. H. & Whitney, S. M. Temperature responses of Rubisco from Paniceae grasses provide opportunities for improving C3 photosynthesis. Nat. Plants 2, 16186 (2016).

    CAS  PubMed  Google Scholar 

  52. Whitney, S. M. & Sharwood, R. E. Linked Rubisco subunits can assemble into functional oligomers without impeding catalytic performance. J. Biol. Chem. 282, 3809–3818 (2007).

    CAS  PubMed  Google Scholar 

  53. Sharwood, R. E., Sonawane, B. V., Ghannoum, O. & Whitney, S. M. Improved analysis of C4 and C3 photosynthesis via refined in vitro assays of their carbon fixation biochemistry. J. Exp. Bot. 67, 3137–3148 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Whitney, S. M. & Sharwood, R. E. Rubisco engineering by plastid transformation and protocols for assessing expression. Methods Mol. Biol. 2317, 195–214 (2021).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This research was supported by the Australian Government through the Australian Research Council Centre of Excellence for Translational Photosynthesis CE140100015 and through the Formas Future Research Leaders grant 2017-00963. The work was also supported by the project ‘Structural dynamics of biomolecular systems (ELIBIO)’ (NO. CZ.02.1.01/0.0/0.0/15_003/0000447) from the European Regional Development Fund. We thank Diamond Light Source for access to beamline I02 (proposal MX11171-5) and B. Li for technical assistance.

Author information

Author notes

  1. These authors contributed equally: Yu Zhou, Laura H. Gunn.

Authors and Affiliations

  1. Plant Science Division, Research School of Biology, The Australian National University, Canberra, Australian Capital Territory, Australia

    Yu Zhou, Rosemary Birch & Spencer M. Whitney

  2. Laboratory of Molecular Biophysics, Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden

    Laura H. Gunn & Inger Andersson

  3. Norwegian College of Fisheries Sciences, UiT Arctic University of Norway, Tromsø, Norway

    Inger Andersson

  4. Institute of Biotechnology, Academy of Sciences of the Czech Republic, Biocev, Vestec, Czech Republic

    Inger Andersson

Authors
  1. Yu Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Laura H. Gunn
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rosemary Birch
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Inger Andersson
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Spencer M. Whitney
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y.Z., L.H.G. and S.M.W. conceived the project and all authors wrote the paper; L.H.G. and I.A. determined the crystal structure; L.H.G. and S.M.W. generated and analysed all enzymes; Y.Z. and R.B. generated the transgenic tobacco lines, performed the molecular and protein analyses, and measured photosynthesis and growth.

Corresponding author

Correspondence to Spencer M. Whitney.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Plants thanks Cong-Zhao Zhou and Thomas Sharkey for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Interactions of the GmRbcS βE-βF hairpin loop.

Interactions between residues within the βE-βF sheet (hairpin loop) of adjacent GmRbcS subunits (coloured teal, residue labeling in red and blue correspond to GmRbcS subunits B and C in PDB 8BDB, respectively) and residues in neighboring RbcL subunits C and A (coloured grey, labels in black). Residue contacts are indicated by dashed lines.

Extended Data Fig. 2 Quantifying Rubisco Ko.

(A) Linear response of Kc to increasing [O2] for E. coli produced RsRubisco, (B) RsRubisco-chi11 and (C) Rubisco from tobacco leaves. The Kc is derived from the y-axis (that is where [O2] = 0) and Ko calculated as Kc/slope of the linear fit. Fits were made to the mean Kc values (±S.E) derived from Michaelis-Menten fits to 14CO2 fixation assays measured using n = 3 or 4 independent biological samples per Rubisco isoform (each symbol representing a differing enzyme preparation). See Table 1 for Ko values.

Source data

Extended Data Fig. 3 Generation of the tobRr::X transforming master-line and tobRsL11S.

(A) Schematic summary of the Rubisco genetics and transformation process used by20 to generate the tobRsLS::X genotype from which a homozygous line coding a single cbbX allele was generated and used to pollinate the Rhodospirillum rubrum L2 RrRubisco tobRr genotype26 to produce (B) the heterozygous RsCbbX (RsRca) producing tobRr::X line with all the progeny (C) kanamycin resistant (see pBinTP-cbbX plasmid in panel A showing co-transformation of the nptII and cbbX alleles). (D) The RsRubisco bioengineering tobRr::X master-line was plastome transformed with pLEVRsL11S that contained plastome flanking sequence (see dashed lines with numbering relative to the corresponding tobacco plastome GenBank accession NC_001879.2 sequence) that directed the replacement of the rbcM gene with the Rs-rbcL-rbcS-aadA operon coding RsRubisco-chi11 that differs by 3 nucleotides (as shown) to the RsRbcL in the tobRsLS::X line produced by20. Shown are the location of the 5UTR-probe and the BglII sites and fragment size it hybridizes to. (E) 32P-5UTR-probe labelled genomic DNA-blot screen of 6 independent tobRsL11S::X lines following 2 rounds of spectinomycin selection, a tobRsLS::X line (positive control) and tobRr::X (negative control). Lines #2 and #5 were deemed homoplasmic as no rbcM containing plastome copies (5.5 kb fragment) were evident. (F) Subsequent native-PAGE screen of soluble protein from three tobacco (wt), three tobRsLS::X plants and three of the six T0 tobRsL11S::X lines grown to maturity for seed. The seed (T1 progeny) from tobRsL11S line #1::X was used in subsequent growth comparisons (Fig. 5).

Source data

Extended Data Fig. 4 Comparative Loop 6 interactions.

Comparative interactions around the GmRubisco Loop 6 site 1 (A331) and site 2 (V334) residues relative to (A, C) Rs and (B, D) SoRubisco. Residue contacts are indicated by dashed lines.

Supplementary information

Supplementary Information

Supplementary Protocols, Figs.1 and 2, Tables 1–3 and References.

Reporting Summary

Source data

Source Data Fig. 3

Unprocessed Coomassie-stained native PAGE.

Source Data for Figs. 3 and 5, and Extended Data Fig. 2

Spreadsheet-based data for Table 1, Figs. 3c and 5c–g, and Extended Data Fig. 2 combined into a workbook with multiple tabs.

Source Data Extended Fig. 3e

Unprocessed DNA blot (only low-resolution source image available) for Extended Data Fig. 3e and unprocessed Coomassie-stained native PAGE for Extended Data Fig. 3f.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhou, Y., Gunn, L.H., Birch, R. et al. Grafting Rhodobacter sphaeroides with red algae Rubisco to accelerate catalysis and plant growth. Nat. Plants 9, 978–986 (2023). https://doi.org/10.1038/s41477-023-01436-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41477-023-01436-7

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing