consensus nucleotides for the symmetrical and · pdf filesee also figure 4 for conserved...

Supplemental Data. Träger et al. (2012). Plant Cell 10.1105/tpc.112.102996

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- Supplemental Figure 1 -

Plastid SRP RNA alignments

Consensus nucleotides for the symmetrical and asymmetrical internal loops, and secondary structure base pairings are marked as "<< >>" in the alignments.

See also Figure 4 for conserved nucleotides (circled). The conserved core region of SRP RNA is shown below the alignments.


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Gene order for all plastid SRP RNA regions

Depicted are the gene orders in the regions plastid SRP RNAs are located. “RNA” = ffs = plastid-encoded

SRP RNA; “()” = gene on other strand; ‘*’ = pseudo gene; number = unnamed genes. Organisms are listed

alphabetically with example organism on top (bold).

Streptophytes Monilophytes ----(trnE)--psbM------------(petN)-RNA-(trnC)-(trnG)-(psbZ)-trnS Pteridium aquilinum ----(trnE)--psbM------------(petN)-RNA-(trnC)-(trnG)-(psbZ)-trnS Adiantum capillus-veneris ----(trnE)--psbM-----*ycf66-(petN)-RNA-(trnC)-(trnG)-(psbZ)-trnS Alsophila spinulosa ----(trnD)--psbM------ycf66-(petN)-RNA-(trnC)-rpoB-rpoC1-rpoC2 Angiopteris evecta ----trnD-(trnY)-(trnE)-psbM-(petN)-RNA-(trnC)-(trnG)-(psbZ)-trnS Asplenium australasicum ----trnD-(trnY)-(trnE)-psbM-(petN)-RNA-(trnC)-(trnG)-(psbZ)-trnS Azolla caroliniana trnE-trnY-(trnD)-psbM-ycf66-(petN)-RNA-(trnC)-rpoB Botrychium strictum ----(trnE)--psbM------------(petN)-RNA-(trnC)-(trnG)-(psbZ)-trnS Cheilanthes lindheimeri trnE-trnY-(trnD)-psbM-ycf66-(petN)-RNA-(trnC)-rpoB Dicranopteris linearis trnE-trnY-(trnD)-psbM-ycf66-(petN)-RNA-(trnC)-rpoB Diplopterygium chinensis trnE-trnY-(trnD)-psbM-ycf66-(petN)-RNA-(trnC)-rpoB Dipteris chinensis ----(trnD)--psbM-----*ycf66-(petN)-RNA-(trnC)-rpoB-rpoC1-rpoC2 Equisetum arvense ----trnY-(trnD)-psbM-*ycf66-(petN)-RNA-(trnC)-rpoB Equisetum ramosissimum --------psbZ-trnG-trnE-trnY-(petN)-RNA-(trnC)-rpoB-chlB-rps16 Gleichenia japonica ----trnY-(trnD)-psbM-*ycf66-(petN)-RNA-(trnC)-rpoB Helminthostachys zeylanica ------------rps11-rpoA-(rbcL)-trnG-RNA-(trnC)-rpoB-matK-ndhB Lygodium japonicum trnE-trnY-(trnD)-psbM-ycf66-(petN)-RNA-(trnC)-rpoB Lygodium microphyllum ----(trnD)--psbM------------(petN)-RNA-(trnC)-rpoB-rpoC1-rpoC2 Mankyua chejuensis -----------(rpoB)-trnD-(trnY)-psbM-RNA-(trnG)-(rpl2)-(rpl23 Marsilea mutica ----trnD-(trnY)-(trnE)-psbM-(petN)-RNA-(trnC)-(trnG)-(psbZ)-trnS Marsilea quadrifolia -trnG-trnE-trnY-(trnD)-psbM-(petN)-RNA-(trnC)-rpoB Ophioglossum vulgatum trnE-trnY-(trnD)-psbM-ycf66-(petN)-RNA-(trnC)-rpoB Osmunda vachellii ---------(rrn5)-(rpl2)-(rbcL)-trnG-RNA-(trnC)-rpoB-chlB-rps16 Osmundastrum cinnamomeum trnE-trnY-(trnD)-psbM-*ycf66-(petN)-RNA-(trnC)-(trnG)-(psbZ)-trnS Plagiogyria japonica ----trnD-(trnY)-(trnE)-psbM-(petN)-RNA-(trnC)-(psbZ)-trnS-(psbC) Platycerium bifurcatum ----(trnD)--psbM------------(petN)-RNA-(trnC)-rpoB-rpoC1-rpoC2 Psilotum nudum ----trnD-(trnY)-(trnE)-psbM-(petN)-RNA-(trnC)-(trnG)-(psbZ)-trnS Salvinia molesta ----trnY-(trnD)-psbM-*ycf66-(petN)-RNA-(trnC)-rpoB Vandenboschia radicans Lycophytes -----trnL---psbM------ycf66-(petN)-RNA-(trnC)-rpoB-rpoC1-rpoC2 Huperzia lucidula -----trnL---psbM------ycf66-(petN)-RNA-(trnC)-rpoB-rpoC1-rpoC2 Isoetes flaccida Bryophytes Hornworts -----trnL---psbM------------(petN)-RNA-(trnC)-rpoB-rpoC1-rpoC2 Anthoceros formosae Mosses -----trnL---psbM------ycf66-(petN)-RNA-(trnC)-(petD)-(petB)-(psbH) Physcomitrella patens (petN)-RNA—-trnC--(petD) Aphanorrhegma serratum (petN)-RNA--trnC--(petD) Encalypta armata (petN)-RNA--trnC--(petD) Entosthodon bonplandii (petN)-RNA--trnC--(petD) Entosthodon laevis (petN)-RNA--trnC--(petD) Entosthodon serratus (petN)-RNA--trnC--(petD) Funariella curviseta (petN)-RNA--trnC--(petD) Funaria flavicans (petN)-RNA--trnC--(petD) Funaria hygrometrica (petN)-RNA--trnC--(petD) Physcomitrium pyriforme -----trnL---psbM------ycf66--------RNA-(trnC)-rpoB-rpoC1-rpoC2 Syntrichia ruralis Liverworts -----trnL---psbM------ycf66-(petN)-RNA-(trnC)-rpoB-rpoC1-rpoC2 Marchantia polymorpha -----trnL---psbM------------(petN)-RNA-(trnC)-rpoB-rpoC1-rpoC2 Aneura mirabilis -----trnL---psbM------ycf66-(petN)-RNA-(trnC)-rpoB-rpoC1-rpoC2 Ptilidium pulcherrimum Charophytes (psaM)-ycf12----------------(petN)-RNA-(trnC)-rpoB-rpoC1-rpoC2 Chlorokybus atmophyticus clpP-rpl20-------------------------RNA-(rps18)-(rpl33)-(psaJ) Chaetosphaeridium globosum ndhB-trnL---psbM-trnL-ycf66-(petN)-RNA-(trnC)-rpoB-rpoC1-rpoC2 Chara vulgaris (psaM)-ycf12----------------(petN)-RNA-(trnC)-rpoB-rpoC1-rpoC2 Mesostigma viride ndhB-trnL--(psbM)-----ycf66-(petN)-RNA-(trnC)-rpoB-(trnQ)-(psbK) Staurastrum punctulatum (trnR)-(trnF)-trnT----ycf66-(petN)-RNA--------rpoB-rpoC1-rpoC2 Zygnema circumcarinatum

partial genome sequences no flanks


7

Chlorophytes and secondary plastid-containing green algae Prasinophyceae rps12--rps7-tufA-----------RNA------(trnC)-rpoB-rpoC1-rpoC2 Ostreococcus tauri rps12--rps7-tufA-----------RNA------(trnC)-rpoB-rpoC1-rpoC2 Micromonas pusilla CCMP1545 rps12--rps7-tufA-----------RNA------(trnC)-rpoB-rpoC1-rpoC2 Micromonas sp. RCC299 rnpB-clpP-trnI-------------RNA-rpl36-------rpoB-rpoC2-rpoC1 Monomastix sp. OKE-1 chlI--trnG-ndbH------------RNA------(trnC)-rpoB-rpoC1-rpoC2 Nephroselmis olivacea # not available # Ostreococcus lucimarinus trnR-(trnT)-trnY-trnS-trnG-RNA-trnI-(ycf12)-(psbZ)-(trnL) Pycnococcus provasolii (psbZ)-(trnH)-trnMe-(rps2)-RNA------(trnC)-rpoB-rpoC1-rpoC2 Pyramimonas parkeae Trebouxiophyceae (psbN)-(trnR)-trnS-(trnL)-RNA------(trnC)-rpoB-rpoC1-rpoC2 Coccomyxa subellipsoidea clpP-petB-petD------------RNA------(trnC)-rpoB-rpoC1-rpoC2 Chlorella variabilis rpl32--cysT-ycf78---------RNA-p134-(trnC)-rpoB-rpoC1-rpoC2 Chlorella vulgaris petL-trnR-minD-tufA-rpl19-RNA-ycf4--ftsH--psbD-psbC Leptosira terrestris (atpH)-(atpI)-(rps2)------RNA------(trnC)-rpoB-rpoC1-rpoC2 Oocystis solitaria clpP-petB-petD------------RNA------(trnC)-rpoB-rpoC1-rpoC2 Parachlorella kessleri psbN-(psbT)-(psbB)-clpP---RNA------(trnC)-rpoB-rpoC1-rpoC2 Pedinomonas minor ycf20-RNA------(trnC) Trebouxia aggregata Ulvophyceae psaB--atpE-rps4-rpoB-rpoC1-rpoC2-trnR-(RNA)-(petG)-(petL) Oltmannsiellopsis viridis psbJ-rrn16-trnS-----------RNA-psaJ----------(rpoC1)-(rpoC2) Bryopsis hypnoides # ONLY RNA AVAILABLE Codium fragile (psbD)-rps12-rps7-RNA-(orf521)-(orf100)-(orf108)-(orf239) Pseudendoclonium akinetum Chlorophyceae tufA-trnP-atpB-rps18-petB--trnI--RNA-trnQ-rpoC1-(clpP) Scenedesmus obliquus (rrl)-(trnA)-(trnI)-(rrs)-(trnS)-RNA-psbF-psbZ-chlL-ycf12 Schizomeris leibleinii ** (rrl)-(trnA)-(trnI)-(rrs)--trnS--RNA-psbF-psbZ-chlL-ycf12 Stigeoclonium helveticum (rrs)--------RNA-psbF Uronema belkae ** ** = the SRP RNA in these organisms overlaps a putative replication origin on the opposite strand (Brouard et al., 2011) Secondary plastid-containing green algae (rps14)-trnS-psbK----------RNA------(trnC)-rpoB-rpoC1-rpoC2 Bigelowiella natans

Red lineage and Paulinella Paulinella (clpP)-(0079)-0080-RNA-(ftsZ)-(0082)-(ddl)-(0084) Paulinella chromatophora Rhodophytes psaJ--apcD--RNA------(trnL)-psbX-accD--------psbV Cyanidioschyzon merolae 10D // DBV201 psaJ--apcD--RNA------(trnL)-psbX-accD--------psbV Cyanidium caldarium psaJ--apcD--RNA-fabH--------psbX-accD--------psbV Gracilaria tenuistipitata psaJ--apcD--RNA-fabH-(trnL)-psbX-accD--------psbV Porphyra purpurea psaJ--apcD--RNA-fabH-(trnL)-psbX-accD-(trnA)-psbV Porphyra yezoensis Heterokontophytes dnaB-(trnF)-RNA-------------psbX------ycf66--psbV Phaeodactylum tricornutum dnaB-(trnF)-RNA-------------psbX------ycf66--psbV Fistulifera sp. strain JPCC DA0580 dnaB-(trnF)-RNA-------------psbX------ycf66--psbV Odontella sinensis dnaB-(trnF)-RNA-------------psbX------ycf66--psbV Synedra acus rbcL--rbcS--RNA-(ycf45)-(rpl20)-(rpl35) Thalassiosira oceanica dnaB-(trnF)-RNA-(ycf45)-(rpl20)-(rpl35) Thalassiosira pseudonana dnaB-(trnF)-RNA-------------psbX------ycf66--psbV Durinskia baltica dnaB-(trnF)-RNA-------------psbX------ycf66--psbV Kryptoperidinium foliaceum (trnG)-(ycf4)-(petL)-psaL-RNA-(trnS)-(trnM)-psbK Aureococcus anophagefferens Cryptophytes psaF--psaJ--RNA-------------psbX-------------psbV Rhodomonas salina psaF--psaJ--RNA-------------psbX-------------psbV Guillardia theta


8


Promoter sequences for all organisms with plastid SRP RNAs

Predicted TATA promoters marked as “-10 box” are shown together with spacer regions and the 5’ of the

plastid SRP RNA sequences. Organisms are listed alphabetically with example organism on top (bold).

-10 box spacer ffs XXX TATAAT XXXXXXXXXX XXXXXXXX... organism

Streptophytes

Monilophytes TGC TATACT TGATC GGGATTGT... Pteridium aquilinum TAG TAGTAT ACTTATTC GGGATTGT... Adiantum capillus-veneris TGA TATACT GTTATT GGGATTGC... Alsophila spinulosa CGA TATAAT ACTATTA GGGTTGTC... Angiopteris evecta TGA TATACT TGATCT GGATTGTT... Asplenium australasicum TGT TATACT AATATT GGGGTTGC... Azolla caroliniana CGA TATACC CTATAATA GGGTTGTC... Botrychium strictum TGG TATACT TAACC GGGGTTGT... Cheilanthes lindheimeri TGA TATACT ACAAAT GGGATTGT... Dicranopteris linearis TGA TATACT ACAAGT GGGATTGT... Diplopterygium chinensis TGA TACACT AAGATCA GGATTGCC... Dipteris chinensis CAT TAAAGT ATAAT GGGTTGCC... Equisetum arvense CAT TAAACT ATACT GGGTTGCC... Equisetum ramosissimum TGA TATACT ACAAGT GGGATTGT... Gleichenia japonica TGA TATAAC ATAAT GGGTTGTC... Helminthostachys zeylanica CGA TATACT GCAGATT GGGATTGA... Lygodium japonicum GGA TATACT GCAGATT GGGATTGA... Lygodium microphyllum TGG TATAAC CTATAATA GGGTTGTC... Mankyua chejuensis TGA TATACT AGTATC GGGATTGT... Marsilea mutica TGA TATACT AGTATC GGGATTGT... Marsilea quadrifolia TGG TATACC CTACAATA GGGTTGTC... Ophioglossum vulgatum CGA TACACT ATATTC GGGGTTGT... Osmunda vachellii # NOT POSSIBLE BECAUSE OF GAP at 5’ Osmundastrum cinnamomeum TGA TATACT ACTATT GGGATTGC... Plagiogyria japonica TGA TATACT AAATTA GGATTGTT... Platycerium bifurcatum ATA TACCCT CTAGATA GGGTTGTC... Psilotum nudum TGA TATACT GATACT GGGATTGC... Salvinia molesta TGA TATAAT ATGATT GGGATTGT... Vandenboschia radicans

Lycophytes AAT TATCCT TAATGTA GGGTTGTC... Huperzia lucidula ATT TATACT TAACATA GGGTTGTC... Isoetes flaccida

Bryophytes Hornworts TAT TACCAT TAATACA GGGTTGTT... Anthoceros formosae Mosses ATT TATTCT TAATGTTA GGATTATC... Physcomitrella patens ATT TATTCT TAATGTTA GGATTATC... Aphanorrhegma serratum ATT TATCCC CAATATTA GGATTATC... Encalypta armata ATT TATTCT TAATATTA GGATTATC... Entosthodon bonplandii ATT TATTCT TAATGTTA GGATTATC... Entosthodon laevis ATT TATTCT TAATATTA GGATTATC... Entosthodon serratus ATT TATTCT TAATGTTA GGATTATC... Funariella curviseta ATT TATTCT TAGTATTA GGATTATC... Funaria flavicans ATT TATTCT TAGTATTA GGATTATC... Funaria hygrometrica ATT TATTCT TAATGTTA GGATTATC... Physcomitrium pyriforme ATT TATCCT TTGCATTA GGATTATC... Syntrichia ruralis Liverworts TTT TAATCT TATGAT GGGTTGTC... Marchantia polymorpha TTT TATGCT GTTTAT GGGTTGAT... Aneura mirabilis TTT CAAAAT ATATAT GGGTTGTC... Ptilidium pulcherrimum

Charophytes TGT TAAAAT AGTAAGCATA ACCCTCAA... Chlorokybus atmophyticus TAA TAATTT ATTTTTATA GGAACTAG... Chaetosphaeridium globosum ATG TAATAT CTGATATA GGATTGTT... Chara vulgaris TTA TATCAT ATAAAGAA GCCCATTA... Mesostigma viride GAA TACAAT AGTAGCTAA GGCTGTAC... Staurastrum punctulatum GAA TATAAT TCGGATGT GATTATGT... Zygnema circumcarinatum


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Chlorophytes and secondary plastid-containing green algae Prasinophyceae AAA TATACT ATTTAGTA GTCCTACT... Ostreococcus tauri AAA TATACT ATAGAGTA GTCCTACT... Micromonas pusilla CCMP1545 AAA TATACT TAAAGTA GTCCTACT... Micromonas sp. RCC299 TGT TATATT AAAGTTA CCCCATTT... Monomastix sp. OKE-1 TGC TATAAT AAATACAA GCCCATAG... Nephroselmis olivacea # No plastid genome available # Ostreococcus lucimarinus GTA TATACT AAATATAA GCCTGGGT... Pycnococcus provasolii TTA AATAAT AATAA GCCCATAG... Pyramimonas parkeae Trebouxiophyceae TAT TATACT TGACAA GCTCACAA... Coccomyxa subellipsoidea TGA TATCAT AGCATTAT GAGATTCAT... Chlorella variabilis TGC TAAAAT ATTTGTAT GAGATTCAT... Chlorella vulgaris GAG TAAAAT AAAAGGT GTTCACTGT... Leptosira terrestris TGG TATAAT AATTAAA GGTTCATTA... Oocystis solitaria TGT TATTAT TTGAAACTA GGATTCATT... Parachlorella kessleri GTT TAATTT ATAG GTCTATAAC... Pedinomonas minor TAT TATAAT AAATAGA GTCCATTGC... Trebouxia aggregata Ulvophyceae AGG TAAACT ACTATTAA GGATTTTAA... Oltmannsiellopsis viridis TGT TATAAT ACGCAT ACCATGTTA... Bryopsis hypnoides # No plastid genome available # Codium fragile ATC GAAAAT TCTTA ACAAAGCGT... Pseudendoclonium akinetum Chlorophyceae TGA TATTTT ATTAAGTAA GCCCAGAAT... Scenedesmus obliquus TTT TATAAT TAAAAGTAA GCTCATGTC... Schizomeris leibleinii TAT TATAAT TATAATTAA GCTCATGGT... Stigeoclonium helveticum TAG TATAAT TATAATTAA GCTCATGGT... Uronema belkae Secondary green algae AAT TATAAT AAATGT GTAAACTGA... Bigelowiella natans

Red lineage and Paulinella Paulinella TGC TACAAT GGACAT GACTCGGA... Paulinella chromatophora Rhodophytes TGG TATAAT TTCGTTA GCCCGCAA... Cyanidioschyzon merolae 10D // DBV201 TTG TATAAT GAGTATT GCCCGGAT... Cyanidium caldarium TGC TATAAT TTGAATTAT ACTCGGGA... Gracilaria tenuistipitata TGT TATAGT CAGCTTAA GCCCGAAA... Porphyra purpurea TGT TATAAT TACAATAA GCCCGAGA... Porphyra yezoensis Heterokontophytes GCC TATAAT AAGCTTTG GGTATAGT... Phaeodactylum tricornutum CAT TAAAGT TCGGCCCA GGATATAG... Fistulifera sp. strain JPCC DA0580 TAA TATAAT ATGATTTG GGTATAGT... Odontella sinensis GTG TATAAT ACTATTT GGATATAG... Synedra acus TTG TATAAT GTTAATT GGATATAA... Thalassiosira oceanica TAG TATAAT ATATATT GGATATAG... Thalassiosira pseudonana TCG TATACT TATATT GGATATAG... Durinskia baltica TAG TATAGT AAGTTTA GGATATAG... Kryptoperidinium foliaceum TGA TACAAT AATTAT GTATCTAG... Aureococcus anophagefferens Cryptophytes CTA TATTAT ATTCTA ATCCTGGT... Rhodomonas salina TGT TATATT ATAATA GCCTTGAT... Guillardia theta


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Verification of plastid SRP RNA expression by RT-PCR in different lineages

RT-PCR of predicted plastid SRP RNAs for seven different phylogenetic groups. After total RNA isolation

and generation of cDNA reverse transcription PCRs were done and fragments of the predicted plastid SRP

RNAs were amplified: 100 bp (Psilotum nudum); 81 bp (Physcomitrella patens); 85 bp (Marchantia

polymorpha); 95 bp (Porphyra purpurea); 80 bp (Thalassiosira pseudonana); 87 bp (Aureococcus

anophagefferens); 90 bp (Rhodomonas salina). Fragments were successfully cloned into pGEM-T or

pDrive vector and verified by sequencing (data not shown). M: DNA ladder (bp). Green lineage: Psilotum

nudum (fern), Physcomitrella patens (moss) and Marchantia polymorpha (liverwort); Red lineage: Porphyra

purpurea (red alga), Thalassiosira pseudonana (diatom), Aureococcus anophagefferens (pelagophyte) and

Rhodomonas salina (cryptophyte).


11


Full alignment of plastid SRP54 sequences (1/4)

Plastid SRP54 sequences of the red and green lineage (marked in red and green, respectively) are aligned

together with the plastid SRP sequence of the glaucophyte Cyanophora (purple) and the plastid SRP54

sequence of Paulinella (cyan). The SRP RNA binding motif that corresponds to conserved regions is

indicated by “SM” and “GXG” in the alignment.


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15


Domain structure of cpSRP43 proteins

Shown are cpSRP43 sequences aligned according to identified ankyrin and chromo domains, which are

numbered. The length of each sequence is indicated at the C-terminus, and its ChloroP predicted transit

peptide (TP) length is shown below 'TP'. Dashed lines refer to partial sequence and dashed boxes are

included due to high sequence similarity to related species (see supplemental Figure 7 online) although no

domain was predicted by the used programs. No cpSRP43 sequence is available for ulvophyceae.


16

- Supplemental Figure 7 –

Full alignment of cpSRP43 sequences

Alignment of the available cpSRP43 sequences of the chlorophytes and streptophytes. The marked

domains ankyrin (ANK) and chromo (CD) are included according to a prediction for A. thaliana cpSRP43.

‘TP’ and the vertical line mark the transit peptide (61 amino acids) for cpSRP43 of P. patens.


17


Phylogenetic tree of cytosolic SRα (SR) and chloroplast - FtsY (cpFtsY) proteins

Shown is a radial-style consensus tree generated by Maximum Likelihood calculation as indicated in

Methods. Internal edge label values represent bootstrap support of 2000 replicates of the Maximum

Likelihood analysis. At: Arabidopsis thaliana; Vv: Vitis vinifera; Os: Oryza sativa; Zm: Zea mays; Pp:

Physcomitrella patens; Cr: Chlamydomonas reinhardtii; Ot: Ostreococcus tauri.


18


Alignment for phylogenetic tree construction (Supplemental Figure 8)

Sequences were trimmed beginning with L104 (LLVSD) within the N-domain of A. thaliana cpFtsY up to

amino acid G311 (NEVVG) within the G-domain and the equivalent residues in the other aligned

sequences - in total 208 residues for A. thaliana cpFtsY. Ath: Arabidopsis thaliana; Vv: Vitis vinifera; Os:

Oryza sativa; Zm: Zea mays; Pp: Physcomitrella patens; Cr: Chlamydomonas reinhardtii; Ot: Ostreococcus

tauri.


19

- Supplemental Table 1 -

Plastid SRP components identification summary – Red lineage and others

Species Group Plastid Acc. Genome size pSRP RNA Loop size

pSRP54 Mutations

pSRP54 accession

Paulinella chromatophora (recent endosymbiosis) Cercozoa; Euglyphida CP000815 1021616 bp Pred 4 No mut. (ffh) ACB42577 Cyanophora paradoxa Glaucocystophyceae U30821 135599 bp NO MUT(1) ES233562+EST111910*(EST) Porphyra purpurea Rhodophyta; Bangiophyceae U38804 191028 bp VER 4 Porphyra yezoensis Rhodophyta; Bangiophyceae AP006715 191952 bp Pred 4 Gracilaria tenuistipitata Rhodophyta; Florideophyceae AY673996 183883 bp Pred 4 Cyanidium caldarium Rhodophyta; Bangiophyceae AF022186 164921 bp Pred 4 Cyanidioschyzon merolae strain 10D Rhodophyta; Bangiophyceae AB002583 149987 bp Pred 4 No mut. CMC066C (JP) Cyanidioschyzon merolae DBV201 Rhodophyta; Bangiophyceae AY286123 149705 bp Pred 4 ••• Red algal secondary plastid Fucus vesiculosus Phaeophyceae, Heterokonta FM957154 124986 bp NO Ectocarpus siliculosus Phaeophyceae, Heterokonta FP102296 139954 bp NO MUT CBN76263;FP246789 (EST) Heterosigma akashiwo (CCMP452) Raphidophyceae, Heterokonta EU168191 160149 bp NO Heterosigma akashiwo (NIES 293) Raphidophyceae, Heterokonta EU168190 159370 bp NO Aureococcus anophagefferens (CCMP1984) Pelagophyceae, Heterokonta GQ231541 88471 bp VER + anno 4 No mut. EGB12501 Aureoumbra lagunensis (CCMP1507) Pelagophyceae, Heterokonta GQ231542 94346 bp NO Odontella sinensis diatom,Heterokonta Z67753 119704 bp Pred 4 Thalassiosira pseudonana diatom,Heterokonta EF067921 128814 bp Pred + anno 4 No mut. EED94755 Thalassiosira oceanica diatom,Heterokonta GU323224 141790 bp Pred + anno 4 Phaeodactylum tricornutum diatom,Heterokonta EF067920 117369 bp Pred + anno 4 No mut. EEC48599 Fistulifera sp. strain JPCC DA0580 diatom,Heterokonta AP011960 134918 bp Pred + annS 4 Fragilariopsis cylindrus diatom,Heterokonta JGI scaffold95 122028 bp NO MUT JGI: Fracy1|189595 Synedra acus diatom,Heterokonta JQ088178 116251 bp Pred + anno 4 Vaucheria litorea Xanthophyceae, Heterokonta EU912438 115341 bp NO Guillardia theta Cryptophyta AF041468 121524 bp Pred 4 Rhodomonas salina Cryptophyta EF508371 135854 bp VER 4 Emiliania huxleyi Haptophyceae AY741371 105309 bp NO MUT JGI: Emihu1|350646 Phaeocystis antarctica Haptophyceae JN117275 105651 bp NO Isocrysis galbana (ESTs only) Haptophyceae MUT EC142378 (EST) Uncultured prymnesiophyte C19847 Haptophyceae (environmental) HM565909 45567 bp NO Chromera velia strain CCMP2878 Alveolata; Chromerida HM222967 119798 bp NO Alveolata sp. CCMP3155 Alveolata; Chromerida HM222968 85535 bp NO ••• Haptophyte tertiary endosymbiont plastid Karlodinium veneficum Dinophyceae; Gymnodiniales JN039300 142981 bp NO ••• Diatom tertiary endosymbiont, nucleus retained Durinskia baltica CSIRO CS-38 Dinophyceae; Peridiniales GU591327 116470 bp Pred + anno 4 Kryptoperidinium foliaceum CCMP 1326 Dinophyceae; Peridiniales GU591328 140426 bp Pred + anno 4 Abbreviations: “NO”= No pSRP RNA found; “Pred”= pSRP RNA predicted; “anno” = pSRP RNA (ffs) annotated; “annS”= ffs annotation lacks 30 nt at 5 prime; “VER” = pSRP RNA expression verified by RT-PCR; “MUT” = the crucial SRP RNA binding positions are changed; “MUT(1)” = only one position mutated in pSRP54; “ffh” = ffh gene is retained in plastid genome; “EST” = the analyzed protein is from a translated EST sequence; “JGI” = the identifier is from the Joint Genome Institute genome project annotation; “JP” = http://merolae.biol.s.u-tokyo.ac.jp/; '*' = http://cyanophora.rutgers.edu/cyanophora/.


20

Plastid SRP components identification summary – Chlorophytes and secondary plastid-containing green algae

Species Group Plastid accession

Sequence size

cpSRP RNA

Loop size cpSRP54 mutations

cpSRP54 accession cpSRP43 accession

Nephroselmis olivacea Prasinophyceae AF137379 200799 bp Pred 4 Ostreococcus tauri Prasinophyceae CR954199 71666 bp Pred 4 No mut. CAL49991 CAL52252.2 Ostreococcus lucimarinus Prasinophyceae No mut. ABO94038 Pred. based on JGI:

(Ost9901_3|30191) Micromonas pusilla CCMP1545 Prasinophyceae FJ858269 41811 bp Pred 4 No mut. EEH59526 JGI : MicpuC3|120831 Micromonas sp. RCC299 Prasinophyceae FJ858267 72585 bp Pred 4 No mut. ACO68481 ACO68982 Pyramimonas parkeae Prasinophyceae FJ493499 101605 bp Pred 4 Monomastix sp. OKE-1 Prasinophyceae FJ493497 114528 bp Pred 4 Pycnococcus provasolii Prasinophyceae FJ493498 80211 bp Pred 4 Parachlorella kessleri Trebouxiophyceae; Chlorellaceae FJ968741 123994 bp Pred 4 Oocystis solitaria Trebouxiophyceae; Oocystaceae FJ968739 96287 bp Pred 4 Chlorella vulgaris C-27 Trebouxiophyceae; Chlorellaceae AB001684 150613 bp Pred 4 Chlorella variabilis NC64A Trebouxiophyceae; Chlorellaceae HQ914635 124579 bp Pred 4 No mut. JGI ChlNC64A_1| 48710 EFN52925 Leptosira_terrestris Trebouxiophyceae; Ctenocladales; EF506945 195081 bp Pred 4 Coccomyxa subellipsoidea C-169 Trebouxiophyceae; Coccomyxaceae HQ693844 175731 bp Pred 4 No mut JGI Coc_C169_1|26298 JGI:

Coc_C169_1|42286 Asterochloris sp. Cgr Trebouxiophyceae; Microthamniales No mut. JGI: Astpho1|78832 JGI: Astpho1|38153 Trebouxia aggregate Trebouxiophyceae; Microthamniales EU124000 1691 bp Pred 4 Pedinomonas minor Pedinophyceae; (Trebouxiophyceae) FJ968740 98340 bp Pred 4 Pseudendoclonium akinetum Ulvophyceae; Ulvales AY835431 195867 bp Pred 4 Codium fragile Ulvophyceae; Bryopsidales M35276 94 bp Pred + VER 6 Bryopsis hypnoides Ulvophyceae; Bryopsidales GQ892829 153429 bp Pred 5 Oltmannsiellopsis viridis Ulvophyceae DQ291132 151933 bp Pred 7 Scenedesmus obliquus Chlorophyceae; Sphaeropleales DQ396875 161452 bp Pred 4 Stigeoclonium helveticum Chlorophyceae; Chaetophorales DQ630521 223902 bp Pred 3 Schizomeris leibleinii Chlorophyceae; Chaetophorales HQ700713 182759 bp Pred 1 Uronema belkae Chlorophyceae; Chaetophorales HQ700714 4385 bp Pred 4 Floydiella terrestris Chlorophyceae; Chaetopeltidales GU196268 521168 bp NO Oedogonium cardiacum Chlorophyceae; Oedogoniales EU677193 196547 bp NO Chlamydomonas reinhardtii Chlorophyceae; Chlamydomonadales BK000554 203828 bp NO MUT EDP00260 Xs Dunaliella salina Chlorophyceae; Chlamydomonadales GQ250046 269044 bp NO MUT GHEXJPO01D5BDI * Volvox carterii Chlorophyceae; Chlamydomonadales GU084820 461064 bp NO MUT EFJ41797.1;

Volca1_98179,corrected

••• Green algal secondary plastid Bigelowiella natans Chlorarachniophyte DQ851108 69166 bp Pred No mut. JGI: Bigna1|80087 Euglena gracilis Euglenozoa X70810 143171 bp NO Eutreptia viridis Euglenozoa JN643723 65513 bp NO Eutreptiella gymnastica Euglenozoa HE605038 67622 bp NO * = Dunaliella cpSRP54 EST example shown, more ESTs from SRR070440 were used in prediction. “Loop size” = apical loop size, assuming 3 bp stem.


21

Plastid SRP components identification summary – Streptophytes (non vascular)


Sequence size

cpSRP RNA

Loop size

cpSRP54 mutations

cpSRP54 accession cpSRP43 accession

• Charophytes • Mesostigma viride Mesostigmatophyceae AF166114 118360 bp Pred 4 Chlorokybus atmophyticus Chlorokybophyceae DQ422812 152254 bp Pred 4 No mut. HO416123 (EST) HO424045 (EST) Chara vulgaris Charophyceae; Charales; DQ229107 184933 bp Pred 10 SRP005673 (ESTs) Nitella hyalina Charophyceae; Charales; No mut. HO551413,HO546410 (EST) Staurastrum punctulatum Zygnematophyceae AY958085 157089 bp Pred 9 Zygnema circumcarinatum Zygnematophyceae AY958086 165372 bp Pred 9 Spirogyra pratensis Zygnematophyceae No mut. GW599775 (EST) GW599392 (EST) Penium margaritaceum Zygnematophyceae No mut. HO617664 (EST) HO615562 (EST) Chaetosphaeridium globosum Coleochaetophyceae AF494278 131183 bp Pred 5 MUT HO382660 (EST) HO370201 (EST) Klebsormidium flaccidum Klebsormidiophyceae No mut. HO461325 (EST) • Marchantiophyta (liverworts) • Marchantia polymorpha Marchantiopsida X04465 121024 bp Pred + VER 7 Ptilidium pulcherrimum Jungermanniopsida HM222519 119007 bp Pred 7 Aneura mirabilis (parasitic) Jungermanniopsida EU043314 108007 bp Pred 7 • Bryophyta (mosses) • Physcomitrella patens Bryophytina (Funariidae) AP005672 122890 bp Pred + VER 10 No mut. EDQ81867 EDQ63802 Physcomitrium pyriforme Bryophytina (Funariidae) EF173155 742 bp Pred 10 Aphanorrhegma serratum Bryophytina (Funariidae) EF173158 740 bp Pred 10 Entosthodon bonplandii Bryophytina (Funariidae) EF173153 730 bp Pred 10 Entosthodon laevis Bryophytina (Funariidae) EF173151 742 bp Pred 10 Entosthodon serratus Bryophytina (Funariidae) EF173156 718 bp Pred 10 Funaria hygrometrica Bryophytina (Funariidae) EF173161 702 bp Pred 10 Funaria flavicans Bryophytina (Funariidae) EF173159 702 bp Pred 10 Funariella curviseta Bryophytina (Funariidae) EF173152 725 bp Pred 10 Encalypta armata Bryophytina (Funariidae) EF173150 749 bp Pred 9 Aulacomnium turgidum Bryophytina (Bryidae) No mut. FL685744 (EST) Syntrichia ruralis Bryophytina (Dicranidae) FJ546412 122630 bp Pred 10 • Anthocerotophyta (hornworts) Anthoceros formosae Anthocerotidae AB086179 161162 bp Pred 10 “Mut?” = two of the three RNA binding positions are mutated, but one of these does not have the usual higher plant amino acid.


22

Plastid SRP components identification summary – Streptophytes (vascular)


Sequence size

cpSRP RNA

Loop size

cpSRP54 mutations

cpSRP54 accession

cpSRP43 accession

• Lycophyta (spike/club mosses) •

Huperzia lucidula Lycopodiophyta; Lycopodiopsida AY660566 154373 bp Pred 5 No mut. SRR027134.38356 (EST, H. serrata)

SRR027134.70069 (EST, H. serrata)

Isoetes flaccida Lycopodiophyta; Isoetopsida GU191333 145303 bp Pred 5 Selaginella uncinata Lycopodiophyta; Isoetopsida AB197035.2 144170 bp NO Selaginella moellendorffii Lycopodiophyta; Isoetopsida FJ755183 143780 bp NO MUT EFJ30972 EFJ08987 Selaginella moellendorffii Lycopodiophyta; Isoetopsida HM173080 143775 bp NO • Pteridophyta (ferns) •

Psilotum nudum Moniliformopses; Psilotopsida; Psilotales

AP004638 138829 bp Pred + VER 10

Angiopteris evecta Moniliformopses; Marattiopsida

DQ821119 153901 bp Pred 10

Adiantum capillus-veneris Moniliformopses; Polypodiopsida; Pteridaceae

AY178864 150568 bp Pred 10

Alsophila spinulosa Moniliformopses; Polypodiopsida; Cyatheales

FJ556581 156661 bp Pred 10

Pteridium aquilinum Moniliformopses; Polypodiopsida; Pteridaceae

HM535629 152362 bp Pred 10 Mut? Contig13250, Pteridium_unigenes*

Contig5416 Pteridium_unigenes*

Cheilanthes lindheimeri Moniliformopses; Polypodiopsida; Pteridaceae

HM778032 155770 bp Pred 10

Equisetum arvense Moniliformopses; Equisetopsida; Equisetales

GU191334 133309 bp Pred 10

Mankyua chejuensis Moniliformopses; Ophioglossopsida; Ophioglossales

JF343520 146221 bp Pred 10

Lygodium japonicum Moniliformopses; Polypodiopsida; Schizaeales

HM021803 40947 bp Pred 11

Marsilea mutica Moniliformopses; Polypodiopsida; Salviniales

HM021801 26302 bp Pred 10

Gleichenia japonica Moniliformopses; Polypodiopsida; Gleicheniales

HM021798 29940 bp Pred 10

Osmundastrum cinnamomeum Moniliformopses; Polypodiopsida; Osmundales

HM021799 15148 bp Pred 10

Asplenium australasicum Moniliformopses; Polypodiopsida; Polypodiales

HQ658095 8462 bp Pred 10

Platycerium bifurcatum Moniliformopses; Polypodiopsida; Polypodiales

HQ658094 8325 bp Pred 10

Plagiogyria japonica Moniliformopses; Polypodiopsida; Cyatheales

HQ658099 12063 bp Pred 10

Azolla caroliniana Moniliformopses; Polypodiopsida; Salviniales

HQ658096 6766 bp Pred 10

Marsilea quadrifolia Moniliformopses; Polypodiopsida; HQ658098 9344 bp Pred 10


23

Salviniales

Salvinia molesta Moniliformopses; Polypodiopsida; Salviniales

HQ658097 7093 bp Pred 10

Lygodium microphyllum Moniliformopses; Polypodiopsida; Schizaeales

HQ658100 9904 bp Pred 11

Dicranopteris linearis Moniliformopses; Polypodiopsida; Gleicheniales

HQ658102 9299 bp Pred 10

Diplopterygium chinensis

Moniliformopses; Polypodiopsida; Gleicheniales

HQ658103

9262 bp

Pred

10

Dipteris chinensis Moniliformopses; Polypodiopsida; Gleicheniales

HQ658101 9945 bp Pred 10

Vandenboschia radicans Moniliformopses; Polypodiopsida; Hymenophyllales

HQ658104 9278 bp Pred 10

Osmunda vachellii Moniliformopses; Polypodiopsida; Osmundales

HQ658105 9276 bp Pred 10

Botrychium strictum Moniliformopses; Ophioglossopsida; Ophioglossales

HQ658108 8136 bp Pred 10

Helminthostachys zeylanica Moniliformopses; Ophioglossopsida; Ophioglossales

HQ658107 7846 bp Pred 10

Ophioglossum vulgatum Moniliformopses; Ophioglossopsida; Ophioglossales

HQ658106 6784 bp Pred 10

Equisetum ramosissimum Moniliformopses; Equisetopsida; Equisetales

HQ658109 11471 bp Pred 10

• Gymnosperms (selection) •

Gingko biloba Ginkgophyta; Ginkgoales AB684440 156945 bp NO Mut? SRR077423.52566 +36362 (EST)

Cycas taitungensis Cycadopsida AP009339 163403 bp NO

Pinus thunbergii Coniferopsida D17510 119707 bp NO MUT DR092755, (EST, P.taeda)

Gnetum parvifolium Gnetophyta; Gnetopsida AP009569 114914 bp NO MUT SRR064399.417818 (EST)

“Mut?” = one if the critical RNA binding positions not mutated. *(Brouard et al., 2011)


24


Summary of crystallographic data collection and structure refinement statistics

Pp-cpFtsY

data collection

space group P21212

unit cell a = 67.58 b = 123.46 c = 75.04

α = β = γ = 90

X-ray source SLS PXII

temperature [K] 100

resolution limits [Å]a 47.7 - 1.8 (1.85 - 1.8)

wavelength [Å] 1.000000

number of observations 1261569 (113985)

number of unique reflections 58928 (4282)

multiplicity 21.41 (26.62)

completeness [%] 100 (100)

mean I/σ 15.89 (4.28)

R(sym) [%] 14.4 (94.4)

R(meas)[%]b 14.8 (96.2)

R(mrgd-F)[%]b 5.8 (32.8)

structure refinement

PDB code 4AK9

R(work) [%] 21.53

R(free) [%] 24.68

number of protein atoms 4547

number of solvent molecule 485

mean isotropic B-value [Å2] 33.38

rmsd from ideal bond length [Å] 0.011

rmsd from ideal bond angle [°] 1.205

molprobity

Ramachandran outliers [%] 0

Ramachandran allowed [%] 1.20

Ramachandran favored [%] 98.80

all atoms clash score 11.71

molprobity score 1.85 avalues for highest resolution bin are given in parenthesis bfor details see (Diederichs and Karplus, 1997)


25


ITC data collection of all conducted experiments

Kd

(µM) ∆H0

(kcal mol-1) T∆S0

(kcal mol-1) ∆G0

(kcal) N

Interaction of Pp-cpFtsY with GDP and XDP

GDP

10.8 (± 1.1) -5.9 (± 0.5) 0.96 (± 0.3) -6.86 (± 0.7) 1.06 (± 0.2)

GDP

8.1 (± 0.8) -4.6 (± 0.5) 2.50 (± 0.3) -7.10 (± 0.7) 1.26 (± 0.2)

GDP

7.9 (± 0.8) -4.5 (± 0.5) 2.59 (± 0.3) -7.09 (± 0.7) 1.28 (± 0.2)

XDP

41 (± 4) -2.7 (± 0.3) 3.37 (± 0.3) -6.07 (± 0.6) *)

XDP

60 (± 6) -3.0 (± 0.3) 2.87 (± 0.3) -5.87 (± 0.6) *)

Interaction of Pp-cpFtsY(D338N) with GDP and XDP

GDP

529 (± 50) -7.7 (± 0.8) -3.18 (± 0.3) -4.52 (± 0.5) *)

GDP

538 (± 50) -8.0 (±0.8) -3.49 (± 0.3) -4.51 (± 0.5) *)

XDP

8.2 (± 0.8) -8.6 (± 0.8) -1.55 (± 0.3) -7.05 (± 0.7) 0.93 (± 0.2)

XDP

6.9 (± 0.7) -8.3 (± 0.68) -1.09 (± 0.3) -7.21 (± 0.7) 0.98 (± 0.2)

*) Due to the low affinity the N value (stoichiometry of the complex) could not be determined, rather this value was fixed to 1.0 for fitting of affinity constant (1/Kd) and change of enthalpy ∆H0. The errors given include the experimental errors of concentration measurements.


26

Supplemental Methods

Plant material

Seven organisms from different lineages were selected: Psilotum nudum (fern) from the Botanical Garden,

Gothenburg, Sweden; Physcomitrella patens (moss) gift from Stefan A. Rensing Lab, Freiburg i.B.,

Germany; Marchantia polymorpha (liverwort) from the Botanical Garden, Gothenburg, Sweden; Porphyra

purpurea (red alga) from Tjärnö, Sweden; Thalassiosira pseudonana (diatom) from the collection at Marin

Botany at Department of Biological and Environmental Science, University of Gothenburg, Sweden;

Aureococcus anophagefferens (pelagophyte) from the Center for Culture of Marine Phytoplankton (CCMP),

USA; Rhodomonas salina (cryptophyte) from CCMP, USA.

Wild type P. patens ssp. patens gametophytes (Gransden 2004 strain) were axenically grown on

cellophane overlaid KNOP Medium (Reski and Abel, 1985) added with 12 g/l phyto agar (Applichem) in a

light/dark regime of 16/8 h at 25°C and 50-70 µmol*s-1*m-2 in plant chambers. Cultures were maintained by

monthly sub-cultivating onto fresh media.

RNA isolation, cDNA synthesis and RT-PCR

RNA from all the organisms was obtained using a Viogene RNA Kit (Techtum) or by using a miRNeasy Mini

Kit (Qiagen) according to the manufacturer’s instructions. Purified RNA from using the Viogene RNA Kit

was divided into two samples: with or without reverse transcriptase treatment and PCRs were run for both

samples. In the non-reverse transcriptase no amplification of the sample were detected indicating no

contamination with chloroplastic DNA (data not shown). Purified RNA from the miRNeasy Mini-kit was

treated with DNaseI (Fermentas), integrity was checked using 10 % polyacrylamide gels and ethidium

bromide stain and a control PCR was done using this DNaseI treated RNA as template. In no case did the

DNaseI treated RNA lead to an amplification (data not shown). DNase treated RNA was then used for

generating cDNA by reverse transcription and it was divided into two samples with and without reverse

transcriptase. Samples without the addition of reverse transcriptase led to no amplification (data not

shown). cDNA corresponding to the RNA was obtained using a reverse transcriptase kit (Promega) with

genespecific primers or by using First Strand cDNA Synthesis Kit (Fermentas) with random hexamer or

genespecific primers according to the manufacturer’s instructions. For PCR amplification following plastid

SRP RNA specific primer pairs were used:

P. nudum

LP 5’-GGGTTGTCTATTCATGCCAGA-3’,

RP 5’-TTGACTATTCTGCAAAACACAAA-3’;

P. patens

LP 5’- TAATAATGCGTTTAAATAAAAAA-3’,

RP 5’- AGTTTTTATTTAAAACATGACAT-3’;

M. polymorpha


27

LP 5’-AAACATTTAAAAACGTATCAGGCTA-3’,

RP 5’-AAATTGTGGATTGTCTATTATGTCA-3’;

P. purpurea

LP 5’-GCCCGAAAGTATATAATTGTAGAAAC-3’,

RP 5’- AACACCCGAAAATCATATCTTCTA-3’;

T. pseudonana

LP 5’-TGATATAATATACTGAAAGAATTGGAACA-3’,

RP 5’- AAATACTAATGTACCTTTTGAATCG-3’;

A. anophagefferens

LP 5’-TAAGTTTTATAAGACCATACCAATGTG-3’,

RP 5’-TTTGTACCTAAAATCAAGCACTTACG-3’;

R. salina

LP 5’-TCCTGGTTACATATAGCAATTCTTC-3’,

RP 5’-CCTGGATTACATCATTAACAAAGTC-3’.

The PCR products were subsequently run on agarose gels for size determination. The PCR products were

collected from the gel and purified before ligated using a pGEM vector kit (Promega, USA). The ligated

products were sequenced using M13 forward and reverse primers (Macrogen, South Korea). In contrast the

amplified P. patens cpSRP RNA fragment was subcloned into pDrive with the PCR-Cloning Kit (Qiagen)

according to the manufacturer’s instructions and subsequently sequenced using T7 and SP6 primers. For

this purpose PCR was done with Go-Taq DNA Polymerase (Promega).

Phylogenetic analyses

Amino acid sequences of cytosolic signal recognition particle receptor α-subunit (SR) or chloroplast signal

recognition particle receptor FtsY (cpFtsY) were retrieved from public databases with the following

accessions: Arabidopsis thaliana [NP_194789.1 (SR); CAB40382.1 (cpFtsY)], Vitis vinifera

[XP_002278056.1 (SR); CBI38758.3 (cpFtsY)], Oryza sativa [BAD10268.1 (SR); BAD88155.1 (cpFtsY)],

Zea mays [ACF87986.1 (SR); CAD70570.1 (cpFtsY)], Physcomitrella patens [XP_001767352.1 (SR);

EDQ67200.1 (cpFtsY)], Chlamydomonas reinhardtii [XP_001692081.1 (SR); EDO99904.1 (cpFtsY)],

Ostreococcus tauri [XP_003074769.1 (SR); CAL54699.1 (cpFtsY)]. The sequences were aligned with

ClustalW implemented in the MEGA program version 4.0.2 (Tamura et al., 2007; Kumar et al., 2008) using

standard parameters. All sequences were trimmed beginning with L104 (LLVSD) within the N-domain up to

amino acid G311 (NEVVG) within the G-domain and the equivalent residues in the other aligned

sequences - in total 208 residues of A. thaliana cpFtsY. A maximum likelihood tree with 1000 and 2000

bootstrap variations was calculated by using the PHYLIP (Phylogeny Inference Package) programs –

SEQBOOT, PROML, CONSENSE – (Felsenstein, 1989, 2005) in version 3.69 with the multiple alignment

above. Phylogenetic inference was performed using Bayesian analysis of the same alignment as described

above, with the MrBayes software (Huelsenbeck et al., 2001; Ronquist and Huelsenbeck, 2003) – version

3.1.2. Analysis was carried out with a gamma-distributed rate model and allowed jumping between fixed-


28

rate amino acid models for one and two million generations with a sample frequency of 100 and four chains

(burn-in: 25% of saved trees). The second tree (data not shown) corroborated the results of the Maximum

Likelihood analysis tree with a clear separation in cytosolic and chloroplast proteins. All four trees were

displayed with TREEVIEW (Page, 1996) and showed the same overall phylogenetic distribution of the

sequences.

Accession Numbers

Additional accession numbers used for supplementary alignments are: Picea sitchensis (cpSRP54:

ABR16458 from NCBI) and Amborella trichopoda (partial cpSRP54: CK764517 from EST).


29

Supplemental References

Brouard JS, Otis C, Lemieux C, Turmel M (2011) The chloroplast genome of the green alga Schizomeris

leibleinii (Chlorophyceae) provides evidence for bidirectional DNA replication from a single origin in the chaetophorales. Genome Biol Evol 3: 505-515

Diederichs K, Karplus PA (1997) Improved R-factors for diffraction data analysis in macromolecular crystallography. Nat Struct Biol 4: 269-275

Felsenstein J (1989) PHYLIP - Phylogeny Inference Package (Version 3.2). Cladistics 5: 164-166 Felsenstein J (2005) PHYLIP (Phylogeny Inference Package) version 3.6. Distributed by the author.

Department of Genome Sciences, University of Washington, Seattle. Huelsenbeck JP, Ronquist F, Nielsen R, Bollback JP (2001) Bayesian inference of phylogeny and its

impact on evolutionary biology. Science 294: 2310-2314 Kumar S, Nei M, Dudley J, Tamura K (2008) MEGA: a biologist-centric software for evolutionary analysis

of DNA and protein sequences. Brief Bioinform 9: 299-306 Page RD (1996) TreeView: an application to display phylogenetic trees on personal computers. Comput

Appl Biosci 12: 357-358 Reski R, Abel OW (1985) Induction of budding on chloronemata and caulonemata of the moss,

Physcomitrella patens, using isopentenyladenine. Planta Springer-Verlag 1985 165: 354-358 Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models.

Bioinformatics 19: 1572-1574 Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA)

software version 4.0. Mol Biol Evol 24: 1596-1599

consensus nucleotides for the symmetrical and · pdf filesee also figure 4 for conserved...

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