4.8 Article

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

期刊

PLANT JOURNAL
卷 106, 期 3, 页码 876-887

出版社

WILEY
DOI: 10.1111/tpj.15196

关键词

chloroplast transformation; food security; homologous recombination; Rubisco; photosynthesis; site‐ directed mutagenesis; Nicotiana tabacum; technical advance

资金

  1. UK Biotechnology and Biological Sciences Research Council (BBSRC) [BB/I024488/1]
  2. Chemical Sciences, Geosciences, and Biosciences Division, Office of Basic Energy Sciences, Office of Science, US Department of Energy [DE-SC0014339, DE-SC0020142]
  3. US National Science Foundation (NSF) [MCB-1642386]
  4. U.S. Department of Energy (DOE) [DE-SC0014339, DE-SC0020142] Funding Source: U.S. Department of Energy (DOE)
  5. BBSRC [BB/I024488/1, BB/N016009/1] Funding Source: UKRI

向作者/读者索取更多资源

Photosynthetic inefficiency hinders crop productivity and sustainability, but improving Rubisco, a key enzyme in carbon fixation during photosynthesis, is challenging due to its location in the plastome and the plastid's repair system. A study introduced silent mutations into rbcL in tobacco plants to simplify screening, resulting in the successful generation of transplastomic lines with stable point mutations in 40% of transformants. This approach shows promise for mutagenic testing of Rubisco function in tobacco and other crops where chloroplast transformation is possible.
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 for improving 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 modification of 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.

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