Plastome engineering of ribulose-1,5-bisphosphate carboxylase/oxygenase intobacco 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 carboxyl ase/oxygenase, the key enzyme of photosynthetic CO, fixation, might be repl aced with altered forms of the gene. Tobacco (Nicotiana tabacum) plants wer e transformed with plastid DNA that contained the rbcL gene from either sun flower (Helianthus annuus) or the cyanobacterium Synechococcus PCC6301, alo ng with a selectable marker. Three stable lines of transformants were regen erated that had altered rbcL genes. Those containing the rbcL gene for cyan obacterial ribulose-1,5-bisphosphate carboxylase/oxygenase produced mRNA bu t no large subunit protein or enzyme activity. Those tobacco plants express ing the sunflower large subunit synthesized a catalytically active hybrid f orm of the enzyme composed of sunflower large subunits and tobacco small su bunits. A third line expressed a chimeric sunflower/tobacco large subunit a rising from homologous recombination within the rbcL gene that had properti es similar to the hybrid enzyme. This study demonstrated the feasibility of using a binary system in which different forms of the rbcL gene are constr ucted in a bacterial host and then introduced into a vector for homologous recombination in transformed chloroplasts to produce an active, chimeric en zyme in vivo.