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 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.
Highlights
The transformation of chloroplast or plastid genomes in higher plants represents a promising technology in multiple biotechnological applications such as introducing agronomically important traits, metabolic engineering, recombinant protein expression and production of high-value therapeutic compounds (Maliga and Bock 2011)
One of the most commonly studied plastid genes is rbcL, which encodes the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)
The Nt-rbcLm gene was seamlessly joined with the native rbcL promoter from tobacco and inserted into a chloroplast transformation plasmid, pCT-rbcL, described previously (Lin et al 2014)
Summary
The transformation of chloroplast or plastid genomes in higher plants represents a promising technology in multiple biotechnological applications such as introducing agronomically important traits, metabolic engineering, recombinant protein expression and production of high-value therapeutic compounds (Maliga and Bock 2011). In addition to the introduction and expression of foreign genes, chloroplast transformation can be used to delete or mutate plastid-encoded protein subunits for functional studies. Since many of these proteins are involved in photosynthesis, the technology has great potential to improve photosynthesis and productivity in crops (Hanson et al 2013, Bock 2015, Martin-Avila et al 2020). Rubisco catalyses two competing reactions in the stroma of chloroplasts: carboxylation or oxygenation of ribulose-1,5-bisphosphate (RuBP) (Ogren and Bowes 1971, Tcherkez et al 2006, Andersson and Backlund 2008). Two general goals of engineering Rubisco are to improve its carboxylation efficiency under ambient
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