PCR analysis of pulsed-field gel electrophoresis-purified plastid DNA, a sensitive tool to judge the hetero-/homoplastomic status of plastid transformants

Świątek, M.; Greiner, Stephan; Kemp, Sabine; Drescher, Axel; Koop, Hans‐Ulrich; Herrmann, Reinhold G.; Maier, Rainer M.

doi:10.1007/s00294-003-0369-4

Cited by 20 publications

(8 citation statements)

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“…also Oldenburg and Bendich, ) we assessed quality and integrity of ptDNA during leaf development in several higher plant species by three independent methods other than PCR: by visualizing unfractionated high‐molecular‐mass ptDNA released from gently embedded protoplasts by pulsed‐field gel electrophoresis (cf. Swiatek et al , ), by ultracentrifugation of single‐stranded and double‐stranded ptDNA in analytical CsCl equilibrium gradients, and by restriction of unfractionated DNA prepared from chloroplasts and gerontoplasts purified by combined differential and isopycnic centrifugation (Figure d,e; cf. Schmitt and Herrmann, ; Herrmann, ).…”

Section: Resultsmentioning

confidence: 99%

“…Protoplast suspensions (8 × 10 6 cells per ml) were gently mixed with three parts of 1.1% low‐melting‐point agarose. The embedded cells were then lysed and DNA was separated using a CHEF Mapper® XA System (Bio‐Rad, Munich, Germany) essentially as previously described (Swiatek et al , ). The DNA was then blotted by alkaline transfer onto a nitrocellulose membrane and hybridized to a radiolabelled Sal I restriction fragment library covering the entire plastid genome of Nicotiana tabacum in 11 ptDNA fragments inserted into vector pBR322 (Medgyesy et al , ).…”

Section: Methodsmentioning

confidence: 99%

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Chloroplast nucleoids are highly dynamic in ploidy, number, and structure during angiosperm leaf development

et al. 2020

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Chloroplast nucleoids are large, compact nucleoprotein structures containing multiple copies of the plastid genome. Studies on structural and quantitative changes of plastid DNA (ptDNA) during leaf development are scarce and have produced controversial data. We have systematically investigated nucleoid dynamics and ptDNA quantities in the mesophyll of Arabidopsis, tobacco, sugar beet, and maize from the early postmeristematic stage until necrosis. DNA of individual nucleoids was quantified by DAPI-based supersensitive epifluorescence microscopy. Nucleoids occurred in scattered, stacked, or ring-shaped arrangements and in recurring patterns during leaf development that was remarkably similar between the species studied. Nucleoids per organelle varied from a few in meristematic plastids to >30 in mature chloroplasts (corresponding to about 20-750 nucleoids per cell). Nucleoid ploidies ranged from haploid to >20-fold even within individual organelles, with average values between 2.6-fold and 6.7-fold and little changes during leaf development. DNA quantities per organelle increased gradually from about a dozen plastome copies in tiny plastids of apex cells to 70-130 copies in chloroplasts of about 7 lm diameter in mature mesophyll tissue, and from about 80 plastome copies in meristematic cells to 2600-3300 copies in mature diploid mesophyll cells without conspicuous decline during leaf development. Pulsed-field electrophoresis, restriction of highmolecular-weight DNA from chloroplasts and gerontoplasts, and CsCl equilibrium centrifugation of singlestranded and double-stranded ptDNA revealed no noticeable fragmentation of the organelle DNA during leaf development, implying that plastid genomes in mesophyll tissues are remarkably stable until senescence.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Chloroplast nucleoids are highly dynamic in ploidy, number, and structure during angiosperm leaf development

et al. 2020

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“…To estimate the number of wild-type plastid DNA copies in the transformants, we used PCR analysis with a mixture of wild-type plasmid (pCStrnR) and knock-out plasmid (pCSDtrnR) in different molar ratios as the DNA templates, according to the detection procedure described by Swiatek et al (2003). The detection limit for copies of wild-type plastid DNA was estimated to be 10 )4 by ethidium bromide staining of PCR products (Figure 3c) and 10 )5 by Southern blot hybridization (Figure 3b).…”

Section: Discussionmentioning

confidence: 99%

“…To estimate the number of wild‐type plastid DNA copies in the transformants, we used PCR analysis with a mixture of wild‐type plasmid (pCStrnR) and knock‐out plasmid (pCSΔtrnR) in different molar ratios as the DNA templates, according to the detection procedure described by Swiatek et al. (2003).…”

Section: Discussionmentioning

confidence: 99%

Plastid transformation reveals that moss tRNA^Arg‐CCG is not essential for plastid function

Sugiura

Sugita²

2004

The Plant Journal

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SummaryThree distinct arginine tRNA genes, trnR-CCG, trnR-ACG, and trnR-UCU, are present in the plastid genome of bryophytes, whereas only the latter two trnR genes are present in the major vascular plants, except for black pine. trnR-CCG is located between rbcL and accD in the moss Physcomitrella patens and it was previously believed to be functional in plastids. However, no trnR-CCG transcript has been detected by Northern hybridization, and the codon usage of CGG is quite low in plastid protein-coding sequences. This raises the possibility that trnR-CCG is non-functional. To investigate this possibility, we integrated a foreign gene into the trnR-CCG coding region via homologous recombination, and constructed stable plastid trnR-CCG knockout moss transformants. The trnR-CCG knock-out transformants grew normally, indicating that the P. patens trnR-CCG gene is not essential for plastid function.

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“…Petit Havanna were transformed by particle bombardment of leaves [21]. Selection and culture of transformed material as well as assessment of plastome segregation and the homoplastomic state were performed essentially as described by De Santis-Maciossek et al [36] and Swiatek et al [37]. Essentially, 10 leaves were used for particle bombardment, and 19 antibiotic resistant transformants were selected.…”

Section: Methodsmentioning

confidence: 99%

Knock‐out of the chloroplast‐encoded PSI‐J subunit of photosystem I in Nicotiana tabacum

et al. 2007

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The plastid‐encoded psaJ gene encodes a hydrophobic low‐molecular‐mass subunit of photosystem I (PSI) containing one transmembrane helix. Homoplastomic transformants with an inactivated psaJ gene were devoid of PSI‐J protein. The mutant plants were slightly smaller and paler than wild‐type because of a 13% reduction in chlorophyll content per leaf area caused by an ≈ 20% reduction in PSI. The amount of the peripheral antenna proteins, Lhca2 and Lhca3, was decreased to the same level as the core subunits, but Lhca1 and Lhca4 were present in relative excess. The functional size of the PSI antenna was not affected, suggesting that PSI‐J is not involved in binding of light‐harvesting complex I. The specific PSI activity, measured as NADP+ photoreduction in vitro, revealed a 55% reduction in electron transport through PSI in the mutant. No significant difference in the second‐order rate constant for electron transfer from reduced plastocyanin to oxidized P700 was observed in the absence of PSI‐J. Instead, a large fraction of PSI was found to be inactive. Immunoblotting analysis revealed a secondary loss of the luminal PSI‐N subunit in PSI particles devoid of PSI‐J. Presumably PSI‐J affects the conformation of PSI‐F, which in turn affects the binding of PSI‐N. This together renders a fraction of the PSI particles inactive. Thus, PSI‐J is an important subunit that, together with PSI‐F and PSI‐N, is required for formation of the plastocyanin‐binding domain of PSI. PSI‐J is furthermore important for stability or assembly of the PSI complex.

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PCR analysis of pulsed-field gel electrophoresis-purified plastid DNA, a sensitive tool to judge the hetero-/homoplastomic status of plastid transformants

Cited by 20 publications

References 50 publications

Chloroplast nucleoids are highly dynamic in ploidy, number, and structure during angiosperm leaf development

Chloroplast nucleoids are highly dynamic in ploidy, number, and structure during angiosperm leaf development

Plastid transformation reveals that moss tRNA^Arg‐CCG is not essential for plastid function

Knock‐out of the chloroplast‐encoded PSI‐J subunit of photosystem I in Nicotiana tabacum

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PCR analysis of pulsed-field gel electrophoresis-purified plastid DNA, a sensitive tool to judge the hetero-/homoplastomic status of plastid transformants

Cited by 20 publications

References 50 publications

Chloroplast nucleoids are highly dynamic in ploidy, number, and structure during angiosperm leaf development

Chloroplast nucleoids are highly dynamic in ploidy, number, and structure during angiosperm leaf development

Plastid transformation reveals that moss tRNAArg‐CCG is not essential for plastid function

Knock‐out of the chloroplast‐encoded PSI‐J subunit of photosystem I in Nicotiana tabacum

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Plastid transformation reveals that moss tRNA^Arg‐CCG is not essential for plastid function