Plastome Engineering of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Tobacco to Form a Sunflower Large Subunit and Tobacco Small Subunit Hybrid

Targeted gene replacement in plastids was used to explore whether the rbcL gene that codes for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, the key enzyme of photosynthetic CO₂ fixation, might be replaced with altered forms of the gene. Tobacco (Nicotiana tabacum) plants were transformed with plastid DNA that contained the rbcL gene from either sunflower (Helianthus annuus) or the cyanobacterium Synechococcus PCC6301, along with a selectable marker. Three stable lines of transformants were regenerated that had altered rbcL genes. Those containing the rbcL gene for cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase produced mRNA but no large subunit protein or enzyme activity. Those tobacco plants expressing the sunflower large subunit synthesized a catalytically active hybrid form of the enzyme composed of sunflower large subunits and tobacco small subunits. A third line expressed a chimeric sunflower/tobacco large subunit arising from homologous recombination within the rbcL gene that had properties similar to the hybrid enzyme. This study demonstrated the feasibility of using a binary system in which different forms of the rbcL gene are constructed in a bacterial host and then introduced into a vector for homologous recombination in transformed chloroplasts to produce an active, chimeric enzyme in vivo.