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BIOENERGETICS AND PHOTOSYNTHESIS

Rubisco Oligomers Composed of Linked Small and Large Subunits Assemble in Tobacco Plastids and Have Higher Affinities for CO₂ and O₂^{1,[C],[W],[OA]}

Molecular Plant Physiology, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory 2601, Australia (S.M.W., H.J.K., R.E.S.); and Department of Horticulture, Plant Physiology/Biochemistry/Molecular Biology Program, University of Kentucky, Lexington, Kentucky 40546–0312 (R.L.H.)

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 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 CO₂-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.

² Present address: Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY 14850.

The author responsible for the distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Spencer Michael Whitney (spencer.whitney{at}anu.edu.au).

^[C] Some figures in this article are displayed in color online but in black and white in the print edition.

^[W] The online version of this article contains Web-only data.

^[OA] Open access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.109.135210

^* Corresponding author; e-mail spencer.whitney{at}anu.edu.au.

Received January 1, 2009; accepted February 15, 2009; published February 20, 2009.