Supplemental Figure 1: Characterization of Toc159 co-suppression lines nts

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Supplemental Data. ischof et al. Plant Cell (211)..1/tpc.111.92882 Supplemental Figure 1: Characterization of Toc19 co-suppression lines nts () Phenotype of rabiopsis plants co-suppressing attoc19.

1 Supplemental Data. ischof et al. Plant Cell (211)..1/tpc Supplemental Figure 1: Characterization of Toc19 co-suppression lines nts () Phenotype of rabiopsis plants co-suppressing attoc19. Images from wil-type (wt) an from plants transforme with the pcmi-attoc19gm vector encoing an truncate form of attoc19 uner the control of the 3S promoter (a-g). Transformants showe various extent of leaf epigmentation. () PCR analysis showing the he presence of the attoc86 transgene in the plants a-g image in. (C) Domain structure of attoc19. ttoc19 GM M correspons to the GTPase (re) an membrane (green) omains of attoc19. mino aci positions are inicate. e. (D) TIL-PCR characterization of T-DN L flanking sequences with lines whose segragation pattern suggeste te single insertions. Insertions in lines 27 an 71 were not in a precite coing seqeunce. (E) Western blot analysis of attoc86 transformant lines 27 an 71. Total leaf protein extracts (2 µg) were probe using antiboies specific to attoc19, attoc an atc as inicate. The leaves analyze (images shown at the bottom) showe e epigmentation to ifferent egrees. 2

2 Supplemental Data. ischof et al. Plant Cell (211)..1/tpc Protein abunance Transcript TOC components wt wts Toc19cs ppi2 ppi2 / wt T4G2 TOC T1G228 TOC TG TOC T3G4674 TOC-III TG1962 OEP T3G1797 TOC64-III TIC components T2G4784 TIC2-II TG7 TIC2-V T2G129 TIC T4G333 TIC T3G237 TIC T4G2343 TIC32-IV TG1662 TIC T2G2482 TIC T3G1889 TIC T1G69 TIC Heat shock proteins an stromal processing peptiase T3G4887 HSP93-III TG92 HSP93-V TG4239 SPP TOC an TIC components not ientifie by MS/MS T2G1664 TOC T3G1662 TOC TG23 TOC9 1. T1G386 TOC-I T4G98 TOC-IV -1.6 TG942 TOC64-V 3.4 T1G898 TOC64-I -1.4 T1G892 TOC T4G114 TIC32-IVb T1G494 TIC2-I T4G332 TIC2-IV 7.87 Supplemental Figure 2: Quantification of components of the plasti import machinery. Provie are the PEX proteome quantification ata an the transcript ratios between wt an ppi2 (see Materials an Methos).

3 Supplemental Data. ischof et al. Plant Cell (211)..1/tpc wt: 1297 ppi2: 292 % only in ppi2 9% % up in ppi ll: 3231 wt: 78 ppi2: 737 % of proteins 8% 7% 6% % 4% 3% common 6 % 39 % no change up in wt Plastis: 6 2% % % N = 4 only in wt N = 788 C % of proteins plasti wt ppi2 D wt ppi2 c c El El peptie minimal position Supplemental Figure 3: Proteome profiling of isolate wt an ppi2 chloroplasts. () Proteins ientifie by mass spectrometry in isolate wt an ppi2 plastis. Plasti localization is base on a chloroplast proteome reference table of 113 proteins. () Comparison between nuclear-encoe plasti proteins ientifie in wt an ppi2 plastis. Down- an upregulate plasti proteins have a two times lower or higher mean abunance in ppi2 compare to wt. (C) Peptie minimal position of nuclear-encoe plasti proteins. The position of the most N-terminal etecte pepties of each ientifie protein were groupe into bins. The reuce etection of pepties at the N-terminus of plasti proteins supports the removal of transit pepties after import in wt an in ppi2 plants. (D) Sequence context aroun N-acetylation sites of plasti proteins. The similar sequence context in wt an in ppi2 plants inicates that precursor processing of importe proteins is also functional in ppi2. N-acetylation sites ranging between positions 2 an 9 were taken for alignment.

4 Supplemental Data. ischof et al. Plant Cell (211)..1/tpc XIC MS an MS 2 ata L * _1_Toc86_C4 #228 RT: V: 1 NL: 1.4E3 F: FTMS + p NSI Full ms [3.-16.] MS *1 6 * z=? z=? _1_Toc86_C4 #223 RT: V: 1 NL: 2.24E2 T: ITMS + c NSI Full ms2 9.49@ci27. [ ] P MS _23_ppi2_ #269 RT:.32 V: 1 NL : 1.29E F: FTMS + p NSI Full ms [3.-16.] MS * z= Supplemental Figure 4: Extracte ion chromatogram quantification of the peptie ientifie in the transit peptie region of LHC3 (T1G612). () Extracte exact mass ion chromatogram for the spectra matching to the transit peptie region from leaves (upper panel, marke with an *1) an the corresponing samples from isolate plastis (marke with *2). In one of the samples from isolate plastis (three wt an three ppi2), an ion matching the exact mass of the LHC3 transit peptie (*2) was etecte at a shifte retention time of approx. 11 minutes (panels below). L: Leaves, P: Plastis. () MS/MS (MS 2 ) information on this ion compare to the transit peptie peak (*1) reveale their ifference. The peptie ientifie from isolate plastis represents the +1 isotope of an unrelate parent ion, as oppose to the monoisotopic ( peak) mass of the transit peptie peak (*1). XIC: extracte ion chromatogram.

5 Supplemental Data. ischof et al. Plant Cell (211)..1/tpc Toc19 -inepenent (N=38) Toc19 -epenent (N=44) Supplemental Figure : Toc19-inepenent an Toc19-epenent substrates. () Transit peptie alignment of the first amino acis of 38 Toc19-inepenent proteins. () Transit peptie alignment of the first amino acis of 4 Toc19-epenent proteins. e

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