Abstract
Mitochondrial functions depend on the proper maintenance and expression of the mitochondrial genome (mtDNA). Therefore, understanding mtDNA replication and repair requires methods to assess its integrity. Mutations or chemical treatments that affect processes involved in the maintenance or stability of the mtDNA can affect its global copy number, but also the relative abundance of different genomic regions or the frequency of illegitimate recombination across repeated sequences. These can be conveniently tested by quantitative PCR (qPCR). Arabidopsis thaliana offers several advantages for studying these processes, because of the extensive collections of mutants, natural accessions and other genetic resources available from stock centers. Here we describe protocols we routinely use to explore changes in mtDNA copy number and relative stoichiometry in Arabidopsis mutants of genes involved in the replication, repair and recombination of the mtDNA.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Gray MW (2012) Mitochondrial evolution. Cold Spring Harb Perspect Biol 4:a011403. https://doi.org/10.1101/cshperspect.a011403
Gualberto JM, Newton KJ (2017) Plant mitochondrial genomes: dynamics and mechanisms of mutation. Annu Rev Plant Biol 68:225–252. https://doi.org/10.1146/annurev-arplant-043015-112232
Arrieta-Montiel MP, Shedge V, Davila J, Christensen AC, Mackenzie SA (2009) Diversity of the Arabidopsis mitochondrial genome occurs via nuclear-controlled recombination activity. Genetics 183:1261–1268. https://doi.org/10.1534/genetics.109.108514
Christensen AC (2018) Mitochondrial DNA repair and genome evolution. Annual Plant Reviews online, pp 11–32. https://doi.org/10.1002/9781119312994.apr0544
Wu Z, Waneka G, Broz AK, King CR, Sloan DB (2020) MSH1 is required for maintenance of the low mutation rates in plant mitochondrial and plastid genomes. Proc Natl Acad Sci U S A 117:16448–16455. https://doi.org/10.1073/pnas.2001998117
Wallet C, Le Ret M, Bergdoll M, Bichara M, Dietrich A, Gualberto JM (2015) The RECG1 DNA translocase is a key factor in recombination surveillance, repair, and segregation of the mitochondrial DNA in Arabidopsis. Plant Cell 27:2907–2925. https://doi.org/10.1105/tpc.15.00680
Miller-Messmer M, Kuhn K, Bichara M, Le Ret M, Imbault P, Gualberto JM (2012) RecA-dependent DNA repair results in increased heteroplasmy of the Arabidopsis mitochondrial genome. Plant Physiol 159:211–226. https://doi.org/10.1104/pp.112.194720
Shedge V, Arrieta-Montiel M, Christensen AC, Mackenzie SA (2007) Plant mitochondrial recombination surveillance requires unusual RecA and MutS homologs. Plant Cell 19:1251–1264. https://doi.org/10.1105/tpc.106.048355
Preuten T, Cincu E, Fuchs J, Zoschke R, Liere K, Borner T (2010) Fewer genes than organelles: extremely low and variable gene copy numbers in mitochondria of somatic plant cells. Plant J 64:948–959. https://doi.org/10.1111/j.1365-313X.2010.04389.x
Moraes CT (2001) What regulates mitochondrial DNA copy number in animal cells? Trends Genet 17:199–205. https://doi.org/10.1016/s0168-9525(01)02238-7
Paszkiewicz G, Gualberto JM, Benamar A, Macherel D, Logan DC (2017) Arabidopsis seed mitochondria are bioenergetically active immediately upon imbibition and specialize via biogenesis in preparation for autotrophic growth. Plant Cell 29:109–128. https://doi.org/10.1105/tpc.16.00700
Liu YJ, Nunes-Nesi A, Wallström SV, Lager I, Michalecka AM, Norberg FE, Widell S, Fredlund KM, Fernie AR, Rasmusson AG (2009) A redox-mediated modulation of stem bolting in transgenic Nicotiana sylvestris differentially expressing the external mitochondrial NADPH dehydrogenase. Plant Physiol 150:1248–1259. https://doi.org/10.1104/pp.109.136242
Parent JS, Lepage E, Brisson N (2011) Divergent roles for the two PolI-like organelle DNA polymerases of Arabidopsis. Plant Physiol 156:254–262. https://doi.org/10.1104/pp.111.173849
Cupp JD, Nielsen BL (2012) Arabidopsis thaliana organellar DNA polymerase IB mutants exhibit reduced mtDNA levels with a decrease in mitochondrial area density. Physiol Plant 149:91–103. https://doi.org/10.1111/ppl.12009
Small I, Suffolk R, Leaver CJ (1989) Evolution of plant mitochondrial genomes via substoichiometric intermediates. Cell 58:69–76. https://doi.org/10.1016/0092-8674(89)90403-0
Davila JI, Arrieta-Montiel MP, Wamboldt Y, Cao J, Hagmann J, Shedge V, Xu YZ, Weigel D, Mackenzie SA (2011) Double-strand break repair processes drive evolution of the mitochondrial genome in Arabidopsis. BMC Biol 9:64. https://doi.org/10.1186/1741-7007-9-64
Odahara M, Kuroiwa H, Kuroiwa T, Sekine Y (2009) Suppression of repeat-mediated gross mitochondrial genome rearrangements by RecA in the moss Physcomitrella patens. Plant Cell 21:1182–1194. https://doi.org/10.1105/tpc.108.064709
Evans-Roberts KM, Mitchenall LA, Wall MK, Leroux J, Mylne JS, Maxwell A (2016) DNA gyrase is the target for the quinolone drug ciprofloxacin in Arabidopsis thaliana. J Biol Chem 291:3136–3144. https://doi.org/10.1074/jbc.M115.689554
Springer NM (2010) Isolation of plant DNA for PCR and genotyping using organic extraction and CTAB. Cold Spring Harb Protoc 2010:pdb.prot5515. https://doi.org/10.1101/pdb.prot5515
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
Stupar RM, Lilly JW, Town CD, Cheng Z, Kaul S, Buell CR, Jiang J (2001) Complex mtDNA constitutes an approximate 620-kb insertion on Arabidopsis thaliana chromosome 2: implication of potential sequencing errors caused by large-unit repeats. Proc Natl Acad Sci U S A 98:5099–5103. https://doi.org/10.1073/pnas.091110398
Galbraith DW, Harkins KR, Knapp S (1991) Systemic endopolyploidy in Arabidopsis thaliana. Plant Physiol 96:985–989. https://doi.org/10.1104/pp.96.3.985
Acknowledgments
This work was supported by the LABEX MitoCross [ANR-11-LABX-0057_MITOCROSS] and benefited from a funding managed by the French National Research Agency as part of the “Investments for the future” program. We thank Rokas Kubilinskas for critical reading of the manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Schatz-Daas, D., Fertet, A., Lotfi, F., Gualberto, J.M. (2022). Assessment of Mitochondrial DNA Copy Number, Stability, and Repair in Arabidopsis . In: Van Aken, O., Rasmusson, A.G. (eds) Plant Mitochondria. Methods in Molecular Biology, vol 2363. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1653-6_20
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1653-6_20
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1652-9
Online ISBN: 978-1-0716-1653-6
eBook Packages: Springer Protocols