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Reverse Genetics in Flowering Plant Plastids

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Genomics of Chloroplasts and Mitochondria

Part of the book series: Advances in Photosynthesis and Respiration ((AIPH,volume 35))

Summary

Plastid reverse genetics exploits the predominance of homologous DNA recombination in this organelle, which allows targeted mutations to be introduced into plastid genes. Most studies have used tobacco and involve replacement of wild-type plastid genes with mutant alleles. Mutant alleles are either disrupted by the marker gene or lie adjacent to the marker gene. Marker selection with antibiotics is required to remove wild-type plastid genomes and reveal the phenotype of homoplasmic mutant plants. Targeted knock-outs have shown that tobacco plastid genes are either dispensable or essential. Dispensable plastid genes include those encoding photosynthesis-related proteins, subunits of the plastid-encoded RNA polymerase, ribosomal protein rpl33, valyl transfer RNA(GAC), glycyl transfer RNA(GCC) and putative origins of DNA replication. Loss-of-photosynthesis is dispensable if mutant plants are propagated on sucrose-containing medium. Knock-outs were particularly useful for elucidating the roles of conserved but dispensable hypothetical reading frames (ycf genes) in photosynthesis. Site-directed mutations allow structure-function studies on the products of plastid genes. Marker-free plants containing deletions of dispensable plastid genes, e.g. the large subunit of RuBisCO gene, facilitate the rapid isolation of plants containing site-directed mutant alleles. Knock-outs of essential tobacco plastid genes (accD, clpP, ycf1, ycf2, rps2, rps4, rps18, rpl20, trnC-GCA, trnN-GUU, trnG-UCC) persist as heteroplasmic mixtures with the wild-type allele under antibiotic selection; removal of selection results in loss of the knock out allele. Homoplasmic cells containing knock out alleles of essential genes would not be viable and this explains the leaf-lamina-loss phenotype of mutant plants. Strong selection for the wild-type gene may hinder the isolation of partial-function alleles of essential plastid genes containing site-directed mutations. New methods are required to study essential plastid genes involving regulated expression or inducible excision mediated by site-specific recombinases. Progress may require the use of angiosperm species, in which homologues of essential tobacco plastid genes are dispensable.

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Abbreviations

LS RuBisCO:

Large subunit of Ribulose Bisphosphate Carboxylase/Oxygenase;

NEP:

Nucleus-encoded plastid RNA polymerase;

ORF:

Open reading frame;

ori :

Origin of DNA replication;

PEP:

Plastid-encoded ­plastid RNA polymerase;

pt DNA:

Plastid DNA;

WT:

Wild-type;

ycf –:

Hypothetical chloroplast open reading frame

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Acknowledgements

I thank: Dr Elisabeth Mudd and the editors for improving the manuscript, and Prof Ulrich Koop (München) for information on the ycf10 knockout. This work was supported by research grants BB/E020445 and BB/I011552 from the Biotechnology and Biological Sciences Research Council (UK).

Note Added in Proof

Articles published after going to press: Fleischmann et al. (2011) used aadA-knockouts to show that the plastid genes encoding ribosomal proteins rpl22, rpl23, rpl32, rps3 and rps16 were essential whereas ribosomal proteins rps15 and rpl36 were nonessential. Whitney et al. (2011) introduced rbcL genes from Flaveria C3 and C4 species into aadA-free rbcM tobacco plants (Fig. 18.12) to identify amino acids affecting the carboxylation rate and CO2 affinity of RuBisCO.

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  1. Faculty of Life Sciences, The University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK

    Anil Day

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  1. AM MUEHLENBERG 1, POTSDAM-GOLM 14476, Germany

    Ralph Bock

  2. Inst. Zelluläre und Molekulare, Botanik (IZMB), Universität Bonn, Kirschallee 1, Bonn, 53115, Germany

    Volker Knoop

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Day, A. (2012). Reverse Genetics in Flowering Plant Plastids. In: Bock, R., Knoop, V. (eds) Genomics of Chloroplasts and Mitochondria. Advances in Photosynthesis and Respiration, vol 35. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-2920-9_18

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