Targeted genome editing to replace nuclear-encoded Rubisco in higher plants

Improving the efficiency of the Rubisco enzyme (ribulose-1,5-bisphosphate carboxylase/oxygenase) is a key strategy to enhance photosynthesis and yields in crops. Rubisco catalyses net CO2 assimilation in all photosynthetic organisms but is slow and cannot fully discriminate between O2 and CO2. The small subunit of Rubisco (SSU) can play an important role in determining catalytic rates. However, SSUs are encoded by large, nuclear rbcS gene families. Nicotiana tabacum (tobacco) is a model often used for testing Rubisco engineering approaches. Nevertheless, the rbcS family in tetraploid tobacco remains poorly understood. First, this work characterised the rbcS gene family to identify the major isoforms that contribute to the tobacco Rubisco enzyme. The regulation of individual rbcS isoforms in response to light quantity and quality was also explored. Second, a strategy to knock-out the major rbcS isoforms using an RNA-guided endonuclease (RGEN) was established. A tobacco mutant with ca. 5% of wild-type Rubisco content was successfully generated as a tool for future studies to engineer Rubisco. Finally, an approach to examine the impact of heterologous SSUs on growth and photosynthesis in Arabidopsis and tobacco was also explored. These findings contribute to efforts to engineer Rubisco by providing a platform to express non native SSUs in a model crop species