Rubisco oligomers composed of linked small and large subunits assemble in tobacco plastids and have higher affinities for CO2 and O2.

Manipulation of Rubisco within higher plants is complicated by the different genomic locations of the large (L; rbcL) and small (S; RbcS) subunit genes. Although rbcL can be accurately modified by plastome transformation, directed genetic manipulation of the multiple nuclear-encoded RbcS genes is more challenging. Here we demonstrate the viability of linking the S and L subunits of tobacco (Nicotiana tabacum) Rubisco using a flexible 40-amino acid tether. By replacing the rbcL in tobacco plastids with an artificial gene coding for a S40L fusion peptide, we found that the fusions readily assemble into catalytic (S40L)8 and (S40L)16 oligomers that are devoid of unlinked S subunits. While there was little or no change in CO2/O2 specificity or carboxylation rate of the Rubisco oligomers, their Kms for CO2 and O2 were reduced 10% to 20% and 45%, respectively. In young maturing leaves of the plastome transformants (called ANtS40L), the S40L-Rubisco levels were approximately 20% that of wild-type controls despite turnover of the S40L-Rubisco oligomers being only slightly enhanced relative to wild type. The reduced Rubisco content in ANtS40L leaves is partly attributed to problems with folding and assembly of the S40L peptides in tobacco plastids that relegate approximately 30% to 50% of the S40L pool to the insoluble protein fraction. Leaf CO2-assimilation rates in ANtS40L at varying pCO2 corresponded with the kinetics and reduced content of the Rubisco oligomers. This fusion strategy provides a novel platform to begin simultaneously engineering Rubisco L and S subunits in tobacco plastids.