Rubisco oligomers composed of linked small and large subunits assemble in tobacco plastids and have higher affinities for CO₂ and O ₂^1[C][W][OA]

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 ismore challenging. Here wedemonstrate the viabilityoflinking the S and L subunits of tobacco (Nicotiana tabacum) Rubisco using aflexible 40-amino acid tether. Byreplacing the rbcLintobacco plastids withanartificial genecoding foraS40L 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 K _ms for CO₂ and O₂ were reduced 10% to 20% and 45%, respectively. In young maturing leaves of the plastome transformants (called A^NtS40L), 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 A ^NtS40L 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 CO ₂-assimilation rates in A^NtS40L 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.