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    Rights statement: This is the peer reviewed version of the following article: Lin, M.T., Orr, D.J., Worrall, D., Parry, M.A., Carmo‐Silva, E. and Hanson, M.R. (2021), A procedure to introduce point mutations into the Rubisco large subunit gene in wild‐type plants. The Plant Journal. Accepted Author Manuscript. https://doi.org/10.1111/tpj.15196 which has been published in final form at https://onlinelibrary.wiley.com/doi/10.1111/tpj.15196This article may be used for non-commercial purposes in accordance With Wiley Terms and Conditions for self-archiving.

    Accepted author manuscript, 588 KB, PDF document

    Embargo ends: 11/02/22

    Available under license: CC BY-NC: Creative Commons Attribution-NonCommercial 4.0 International License

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A procedure to introduce point mutations into the Rubisco large subunit gene in wild-type plants

Research output: Contribution to journalJournal articlepeer-review

E-pub ahead of print
<mark>Journal publication date</mark>11/02/2021
<mark>Journal</mark>The Plant Journal
Publication StatusE-pub ahead of print
Early online date11/02/21
<mark>Original language</mark>English

Abstract

Photosynthetic inefficiencies limit the productivity and sustainability of crop production, and the resilience of agriculture to future societal and environmental challenges. Rubisco is a key target for improvement as it plays a central role in carbon fixation during photosynthesis and is remarkably inefficient. Introduction of mutations to the chloroplast-encoded Rubisco large subunit rbcL is of particular interest to improve the catalytic activity and efficiency of the enzyme. However, manipulation of rbcL is hampered by its location in the plastome, with many species recalcitrant to plastome transformation, and by the plastid’s efficient repair system, which can prevent effective maintenance of mutations introduced with homologous recombination. Here we present a system where the introduction of a number of silent mutations into rbcL within the model plant Nicotiana tabacum facilitates simplified screening via additional restriction enzyme sites. This system was used to successfully generate a range of transplastomic lines from wild-type N. tabacum with stable point mutations within rbcL in 40% of the transformants, allowing assessment of the effect of these mutations on Rubisco assembly and activity. With further optimization, the approach offers a viable way forward for mutagenic testing of Rubisco function in planta within tobacco and modifying rbcL in other crops where chloroplast transformation is feasible. The transformation strategy could also be applied to introduce point mutations in other chloroplast-encoded genes.

Bibliographic note

This is the peer reviewed version of the following article: Lin, M.T., Orr, D.J., Worrall, D., Parry, M.A., Carmo‐Silva, E. and Hanson, M.R. (2021), A procedure to introduce point mutations into the Rubisco large subunit gene in wild‐type plants. The Plant Journal. Accepted Author Manuscript. https://doi.org/10.1111/tpj.15196 which has been published in final form at https://onlinelibrary.wiley.com/doi/10.1111/tpj.15196This article may be used for non-commercial purposes in accordance With Wiley Terms and Conditions for self-archiving.