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
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SUMMARY rbc L is of particular interest for improving the catalytic activity and efficiency of the enzyme. However, manipulation ofrbc L 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 intorbc L within the model plantNicotiana tabacum facilitates simplified screening via additional restriction enzyme sites. This system was used to successfully generate a range of transplastomic lines from wild‐typeN. tabacum with stable point mutations withinrbc L 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 functionin planta within tobacco and modification ofrbc L in other crops where chloroplast transformation is feasible. The transformation strategy could also be applied to introduce point mutations in other chloroplast‐encoded genes. -
Orr, Douglas J. ; Worrall, Dawn ; Lin, Myat T. ; Carmo-Silva, Elizabete ; Hanson, Maureen R. ; Parry, Martin A. J. ( , Plant Physiology)