SPECIAL ISSUE RESEARCH PAPER Construction of a tobacco master line to improve Rubisco engineering in chloroplasts

The inability to assemble Rubisco from any photosyn-thetic eukaryote within Escherichia coli has hampered structure–function studies of higher plant Rubisco. Precise genetic manipulation of the tobacco chloro-plast genome (plastome) by homologous recombina-tion has facilitated the successful production of transplastomic lines that have either mutated the Rubisco large subunit (L) gene, rbcL, or replaced it with foreign variants. Here the capacity of a new tobacco transplastomic line, cmtrL, to augment future Rubisco engineering studies is demonstrated. Initially the rbcL was replaced with the selectable marker gene, aadA, and an artificial codon-modified cmrbcM gene that codes for the structurally novel Rubisco dimer (L2,;100 kDa) from Rhodosprillum rubrum. To obtain cmtrL, the aadA was excised by transiently introduc-ing a T-DNA encoding CRE recombinase biolistically. Selection using aadA enabled transplantation of mu-tated and wild-type tobacco Rubisco genes into the cmtrL plastome with an efficiency that was 3- to 10-fold higher than comparable transformations into wild-type tobacco. Transformants producing the re-introduced form I tobacco Rubisco variants (hexadecamers com-prising eight L and eight small subunits,;520 kDa) were identified by non-denaturing PAGE with fully segregated homoplasmic lines (where no L2 Rubisco was produced) obtained within 6–9 weeks after trans-formation which enabled their Rubisco kinetics to be quickly examined. Here the usefulness of cmtrL in more readily examining the production, folding, and assem-bly capabilities of both mutated tobacco and foreign form I Rubisco subunits in tobacco plastids is discussed, and the feasibility of quickly assessing the kinetic properties of those that functionally assemble is demonstrated