Chapter 19 Engineering the Sunflower Rubisco Subunits into Tobacco Chloroplasts: New Considerations

Abstract

Many challenges confront Rubisco engineering in tobacco plastids. Here we use the aadA selectable marker gene flanked with loxP sites to select for transplastomic tobacco lines that replace the plastomic Rubisco large subunit gene (rbcL) with the comparable gene from sunflower (rbcLS) or both rbcLS and a synthetic gene (cm rbcS) coding a sunflower Rubisco small subunit. Eight rbcLS transformants (called tRstLA) and three rbcLS-cm rbcS transformants were obtained. During regeneration on selective medium recombination between the repeated 221-bp psbA 3’-sequences flanking cm rbcS elicited its excision in all transformants producing lines identical to tRstLA. Two marker-free (ΔaadA) transplastomic lines (tRstL) were produced by transiently introducing a CRE expressing T-DNA plasmid biolistically into tRstLA. The rbcLS abundance in the tRstL increased five-fold relative to the tRstLA lines. However the level of hybrid LsSt Rubisco (comprising eight sunflower large and eight tobacco small subunits) only increased approximately 50% which improved the growth and phenotype of tRstL. The catalytic properties of LsSt were determined using whole leaf gas exchange measurements and verified previous findings that the hybrid enzyme is kinetically comparable to the native tobacco and sunflower Rubisco. Our results show LsSt production in tRstL is impeded post-translationally, either due to problems with the translational processing of rbcLS or (and) from chaperone incompatibility problems that hinder productive folding of the sunflower large subunits or their assembly with tobacco small subunits. In the context of these findings we propose engineering strategies for addressing issues related to effectively engineering Rubisco subunits in tobacco plastids. We highlight how the development of a highly transformable aadA-free transplastomic tobacco line that only produces the smaller Rhodospirillum rubrum Rubisco might simplify some of these future engineering hurdles.