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JXB Advance Access originally published online on February 3, 2008
Journal of Experimental Botany 2008 59(7):1909-1921; doi:10.1093/jxb/erm311
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© The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

RESEARCH PAPER

Construction of a tobacco master line to improve Rubisco engineering in chloroplasts

Spencer M. Whitney* and Robert E. Sharwood

Molecular Plant Physiology Group, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory 0200, Australia

* To whom correspondence should be addressed. E-mail: spencer.whitney{at}anu.edu.au

The inability to assemble Rubisco from any photosynthetic eukaryote within Escherichia coli has hampered structure–function studies of higher plant Rubisco. Precise genetic manipulation of the tobacco chloroplast genome (plastome) by homologous recombination 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 introducing a T-DNA encoding CRE recombinase biolistically. Selection using aadA enabled transplantation of mutated 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 comprising 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 transformation which enabled their Rubisco kinetics to be quickly examined. Here the usefulness of cmtrL in more readily examining the production, folding, and assembly 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.

Key words: CO2 fixation, mutagenesis, photosynthesis, plastome transformation, Rubisco

Received 21 September 2007; Revised 26 October 2007 Accepted 12 November 2007


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