Pigment Deficiency in Nightshade/Tobacco Cybrids Is Caused by the Failure to Edit the Plastid ATPase -Subunit mRNA

Supplemental Data

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• Supplemental Figure 1 - Supplemental Figure 1. Karyological and molecular analyses of somatic hybrids. A) Chromosomes of Ab27 symmetric nuclear hybrid stained with acetoorcein at metaphase. Chromosomes are fully condensed. B) Chromosomes of Ab27 symmetric nuclear hybrid stained with acetoorceine at late prophase. Chromosomes are not completely condensed, which helps to distinguish between small chromosomes of nightshade (2n=72) and significantly larger chromosomes of tobacco (2n=48). In here and in (A), (C) and (D) arrows indicate small chromosomes of nightshade. C) Chromosomes of the F1 progeny plant Ab27/2 (see also Supplemental Figure 3E). D) Chromosomes of the F1 progeny plant Ab27/3 (see also Supplemental Figure 3F). E)Analysis of isoforms of amylases. Protein extracts of leaf tissue were prepared and resolved by polyacrylamide gel electrophoresis (PAGE) under native conditions. The gel contained soluble starch. After PAGE separation, the gel was incubated in a reaction buffer at 37°C for 2 hours and starch was visualized by staining with iodine. The order of loaded protein samples from left to right: (1) Nt(Nt) tobacco SR1; (2) AbNt(Nt) line Ab7; (3) AbNt(Nt) line Ab11; (4) AbNt(Nt) line Ab12; (5) AbNt(Nt) line Ab13; (6) AbNt(Nt) line Ab15; (7) AbNt(Nt) line Ab27; (8) Ab(Ab) nightshade line Ab5; (9) Ab(Nt^m) cybrid line L3 (see also Table 1 in the text). F) Isoform analysis of peroxidases. Activity of electrophoretically separated peroxidase isoforms is shown. Protein extracts were loaded in the following order: (1) Ab(Ab) nightshade line Ab5; (2) Nt(Nt) tobacco SR1; (3) Ab(Nt^m) cybrid line L3. G) DNA-gel-blot analysis of DNA extracted from parental lines and symmetric somatic hybrids. Approximately 10 ??g of total genomic DNA were digested with HindIII, separated by agarose gel electrophoresis, transferred onto nylon filter and hybridized with radioactively-labeled probe derived from the rRNA genes of tobacco. The order of lanes was: (1) Nt(Nt) tobacco SR1; (2) Ab(Ab) nightshade line Ab5; (3 to 9) independent lines of AbNt(Nt) symmetric hybrids. H)DNA-gel-blot analysis of hybrids and cybrids. For this analysis, DNA was digested with BamHI and hybridized with an rRNA gene probe. Lanes: (1) Ab(Nt) albino cybrid Abw; (2) Ab(Nt) albino cybrid Abw3; (3) Ab(Nt^m) green cybrid L3; (4) Ab^Nt(Nt) green, asymmetric hybrid line Bar103; (5) Nt(Nt) green tobacco SR1; (6) Ab(Ab) green nightshade line Ab5; (7) Ab27/2, a plant from F1 progeny of Ab27; (8) AbNt(Nt) symmetric nuclear hybrid Ab27; (9) Ab27/3, a plant from F1 progeny of Ab27. I) Inheritance of the tobacco-specific Inp repeat in cybrids and hybrids. The same membrane as shown in (H) was hybridized with a radioactively labeled probe derived from the Inp-repeat (Boutry and Chua, 1985). Note that there is no cross-hybridization of the probe with genomic sequences in nightshade and nightshade cybrids. The asymmetric hybrid Bar103 contains much less of tobacco-specific Inp sequences as expected from its genomic composition being Ab^Nt(Nt).
• Supplemental Figure 2 - Supplemental Figure 2. RNA editing in plastids of cybrids and rescued lines. A) RNA editing in the albino Ab(Nt) cybrid in comparison to editing in wild type tobacco, Nt(Nt), the rescued lines Ab(Nt^m) and Ab^Nt(Nt) and the cybrid lines Ab(Nt:CCC) and Ab(Nt:CTC). As a control, albino tobacco mutants Nt(δrpo) were assayed as well. Note that site atpA-264 is mutated from C to T at the DNA level in line Ab(Nt^m) and Ab(Nt:CTC). RNA editing was assessed by sequencing PCR products derived from plastid cDNA. Asterisks denote editing sites. B) RNA editing of atpA-264 in line mA is partial, but not abolished. Transformation vector prepared analogous to pWAT in Figure 2. Open circles: point mutations introduced. Note that the coding context is not affected by point mutations. C) An RNA editing defect in the rpoB transcript of the green Nt(Ab) cybrid in comparison to wild type nightshade.
• Supplemental Figure 3 - Supplemental Figure 3. Phenotypic analysis of somatic hybrids. A) Flowers and fruit type of nightshade. B) Flowers and fruit type of tobacco. C) Intermediate morphology of symmetric nuclear hybrid AbNt(Nt). From left to right, side views of tobacco, Nt(Nt) line SR1; Ab27 hybrid, AbNt(Nt) and nightshade, Ab(Ab) flowers. D) Intermediate morphology of symmetric nuclear hybrid AbNt(Nt). Top view of flowers, arrangement is the same as in (F). E) Ab27/2, an F1 progeny plant after pollination of Ab27, AbNt(Nt) with tobacco pollen. Plant shown is grown in a field. F) Ab27/3, an F1 progeny plant after pollination of Ab27, AbNt(Nt) with tobacco pollen. This is a different line from the one shown in (H). Plants in (H) and (I) retain about 33-36 chromosomes of nightshade (Supplemental Figure 1), and illustrate that the symmetric hybrid Ab27 is partially fertile.
• Supplemental Figure 4 - Supplemental figure 4. DNA sequence analysis of cybrids. Genotyping of cybrid lines generated from fusion of various CAT transplastomic lines with tnightshade recipient cells was done by sequencing of PCR products derived from total cellular DNA. Excerpts of chromatograms are shown for the atpA region including editing site 264, and two sites in the ndhA gene that are polymorphic between nightshade and tobacco. Boxed base triplets indicate specified codon positions; ndhA codons 95 and 108 are different between tobacco's and nightshade's plastid chromosome. The ndhA sequence analysis confirms the presence of the tobacco plastome in all cybrids.
• Supplemental Figure 5 - Supplemental Figure 5. Phenotypes of A) Nt(Nt:NtC2), B) Nt(Nt:AbC2) and C) tobacco Petit Havana plants grown in soil.
• Supplemental Figure 6 - Supplemental Figure 6. Analysis of the presence of the rpoC2 mutation in C2N and C2A lines. The presence of the tobacco-specific three-codon insertion was tested by sequence analysis of PCR products in cybrid lines F1CLN-3 and F2CLN-5. Other polymorphisms analyzed in the genes atpA and ndhA are identical to wild type tobacco sequences as expected. Shown are DNA sequences in tobacco and nightshade (top) and excerpts of relevant chromatograms (bottom).
• Supplemental Figure 7 - Supplemental Figure 7. Analysis of all editing sites shared between nightshade and tobacco in Ab(Nt) cybrids. RNA editing was analysed by sequencing PCR products derived from plastid cDNA. Excerpts of chromatograms are limited to the respective codons. Editing sites are marked with an asterisk. Number and position of editing sites in tobacco are given as described earlier (Hirose et al., 1999; Schmitz-Linneweber et al., 2002). Note that sites atpA-265, ndhB-204, ndhD-1 are known to remain unprocessed in non-photosynthetic tissue, while other sites in ndhB, rpoB and rpoA are only partially processed in this tissue (Hirose et al., 1999; Karcher and Bock, 2002; Chateigner-Boutin and Hanson, 2003; and own unpublished observations).