tgd4-1 - MGDG - PtdGro - DGDG - SQDG - PtdEtn - TriGDG - PtdCho - TetraGDG

Transkript

1 Supplemental Data Xu et al. (2008). Lipid trafficking between the endoplasmic reticulum and the plastid in Arabidopsis requires the extraplastidic TGD4 protein A tgd4-1 tgd4-1/tgd4cgfp tgd4-1/tgd4cgfp - MGDG - PtdGro - DGDG - SQDG - PtdEtn - TriGDG - PtdCho - TetraGDG B Supplemental Figure 1. Complementation analysis using a TGD4cGFP fusion construct. (A) Rescue of the tgd4-1 lipid phenotype by stable expression of a cdna TGDcGFP fusion construct in Arabidopsis wild type an () and tgd4-1 mutant under the control of the 35S-CAMV promoter. Two independent transgenic lines are shown. Lipids from top to bottom: MDGD, monogalactosyldiacylglycerol; PtdGro, phosphatidylglycerol; DGDG, digalactosyldiacylglycerol; SQDG, sulfoquinovosyldiacylglycerol; PtdEtn, phosphatidylethanolamine; TriGDG, trigalactosyldiacylglycerol; PtdCho, phosphatidylcholine; TetraGDG, tetragalactosyldiacylglycerol. (B) genotyping of lines using a tgd4-1 specific cut amplified polymorphism DNA marker. A C/T mutation disrupts a HPaII site.

2 A Soy oil DAG tgd4-2 B tgd4-2 - TAG TAG - - Oleic acid - DAG DAG - Supplemental Figure 2. Accumulation of triglycerides in leaves of wild type (), the previously characterized mutant and the tgd4-2 and mutants. (A) A thin-layer chromatogram of lipid extracts is shown. Lipids were visualized by iodine staining. Soy oil was included as a standard. (B) In vivo pulse-chase labeling with labeled oleic acid. Leaves of 3-week-old plants were incubated with labeled oleic acid for 30min. Chase time was 4h following change to unlabeled medium. An autoradiograph of a thin-layer chromatogram is shown. Lipids: TAG, triacylglycerol; DAG, diacylglycerol.

3 V. vinifera 929 A. thaliana 889 B. vulgaris 1000 O. sativa 1000 P. patens 0.1 O. tauri 1000 O. lucimarinus Supplemental Figure 3. Unrooted tree showing the sequence relationships between presumed TGD4 orthologs from plants (Arabidopsis thaliana, NP_566296; Beta vulgaris, ABD83319; Oryza sativa, NP_ ; Vitis vinifera,cao70935, a moss (Physcomitrella patens, XP_ ), and two green algae (Ostreococcus lucimarinus, XP_ ; Ostreococcus tauri, CAL54660). Bootstrap values are indicated. Phylogenetic Analysis To generate the phylogenetic tree reported in Supplemental Figure 3, the predicted full length protein sequences were collected from GenBank through NCBI and aligned with ClustalX (version 1.81) (Thompson et al. 1997). The alignment is shown below. A distance matrix for the alignment was calculated, and an unrooted tree was constructed using the Protdist and Neighbor programs of the PHYLIP package (version 3.67), respectively (Felsenstein,2007). The reliability of the trees was assessed by 1000 bootstrap replicates. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. and Higgins, D.G. (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24: Felsenstein, J. (2007). PHYLIP (Phylogeny Inference Package), version 3.67 (Seattle: Department of Genome Sciences and Department of Biology, University of Washington)

4 Protein alignment used for phylogenetic analysis shown in supplemental Figure V.vinifera MK KLRWAMDGGF WELDMSTATT B.vulgaris AT3g MN RMRWVGEGDI WDLDMSTPVT O.sativa O.lucimari MGA RDGLRHAQFK PMKRAMDGAF GDREHGAAAN O.tauri MTSGRTNDRG GERSTRRARA SNGLRHSAFA PMRAVGSRIA DGGEIASSGA P.patens MKPVLSSQF WNESVSSSRS LDGVARAVPD DPLP LGLSRGTRLS RPMQIDFFQR FMSMPFVPSS MSK LEGTARAVPD DPLP LGLSRGTRLS RPKQVEFFHR FMASPLIPSF MEGAARPVPG DPLP LGLSRGPRVT RTKQLDFLHR FMASPLVPSF VEGTTRAHAS DGSS FVMTQPSGLA RSQQVFLAQK FLNLPAMPSY IDGSARAMGG METSGDGVGH FSMRVPSGLS RSQQVFLAQK LFNAPAVPSF IDGISHGLPA EPPP LGIVTGTRLS RSQQLLMLQR FLGIPCTPSY SISTH GFSLQRG FTFPFTENWF ASLLGQFNFQ KFVS KHRF TTLLGQFNLQ KFVS SPIRPNTGDG GGGGFSLQRV LTLPFSNNWL VSLLGQFDVQ RFVT SASGAG LSVHHA HLVHLAHNLS FTILEQLHVQ KLVA VESEGG MVLDRV VLNAGRGNWW TTLTARARAQ RAWN ARDEGG MVLDRV IANVGSGRWW TTVTARVRAQ RAWN VVDADNK--- --GGLVLDRV LGSAGGENWW ATLTGRVRPQ RLFRRKCSTG ---SVKEGRL LQPSES---- SWLQGIGRRF SDKSLYALGL SSELLITPDD ---AVRKAKS EELSDS---- SWMQVLGRHL SEKSFYAFGF CSEFLVTSED ---EIDKTKA FGRGSSSTVA SRLNTIGKHL KDKSLYALGF CSEFLLSPDD ---VVKEKLS NRQEVS---- -WSNDIKRHL HD--VMSLGV GTELLITPDT ---TEQVQEG KAHRALLDPS MYALGGRFRA NALENVAVKA IGELGSVKGV ---TEQVQEG KAHRALLDAS MYALGGRFRM NLLENVALKA VGELGSARAI ESSSSSSSRT GSDSAAQHTS RNLVESAHRF LDNSLYALCF RSRLQLGSRT TLLVSLEAY GDK-KVP RKKAVFLHK TLLFSYEAS GDT-KTA RKKAVFHHKA STKRYEGTLY TLLLSYDAYK GDLDKNP RAKAIFNHE TLLL--ELY NLKKGD RGKAIFRHK TRAFAKKFKL SDEKDDD AEAALNP RGRVSVQAK TRAITKKFVK KNETKSDAIE VNAGEAREDA RARVLLQAK TLSTTSELDS LSCQAFADQQ STGEQTRPPW RGRVSLRHK FPNHNLMVE AVWPGLFVDK FGTYWDVPLS MAIDLASVAS DSGASYHLSV QFPHLNLTVE RVWPGLFIDK HGNYWDVPAT LAADLASVTS DSGARYHLSL -FPLHNLTAE AVWPGLFVDK HGEYWDVPLS MAIDLASLPA ESGPSYHLCL -LPHQNITLE ASWPGLFIDK NGVYWDVPLS LSADLASVGS ESGLSYHVLL -LPSHLLSVD LSHNERYQTD D-RYVDGPTS ASVSVSSRG- RRAINYRVSA -IPRHVINAE LSHNERFETK K-SHVDGPTS ASVSLASRG- RRMINYRVMA -LDQQKIVAE AAYHERYVDR ESRYWDVPHT VSFDFASLGA PGGFRYRLGV HHNTGTPKQF DG----NQTH EVPATLLPGL CAKGAFALKK NIDLWRSKAQ HHISGAAKQV EG----NKSS DVPATLLPGL YVKSAFSFKK NIDFWRSQAK HHNSGSPKKL HS----DTME VPPPSLLPGL SLKSAVSYRT NMDLWRGTTP QQNSGEPKCF GGG---EETS DVPVALLPGL CAKAAVSIKK SIDVWRKKED RKWLG---DL VPF---DRKH GGGMRTPPRK ELQAGVSYEQ QAVLWRGRRR RKWLGT--EL KPF---ESAP DGEMQKPPRN EVLGGVSVEQ QVILWHGRRR HHSAGSPLEC GGTRPEVNAG EPPVKALPGL RLQAAASVEK SLNLWKADLE

5 Protein alignment used for phylogenetic analysis shown in supplemental Figure 3 K----LKMVQ PFDIFLSNPH ISFSGIIGAA GTACLG K----LKFVQ PFDLFLSSPH VSGAGLIGVV ATALVG K----LETCK PYDVFLSSPH VAVSGIIGSV MTAAFG K----LKNVQ PYDVFLSEPH VSFTGIIGAV ASGSFG RKPKSSSAVS GYTALPQVPT ITVGGIYGAV VRKSLD RKRASTSAVN GYTALPQVPT IAVGGIFGTV ARKSLD S----IISQK SYSLFDARPC ISLSGVLGGL LSSRLDPLYG ITQSLDPTGA DNSVRVQVED ESHGFKGFKL HLPRVKSALV ADIFASVAFT AQHGNFQRLF DNSLRSPLDD QPKDYRRLDL YASRGKSAFL ADLFATLSFS AQYGNFQRKF ENSIRSKFEN DSEGVGGFSL HFPSVNSGFM ADALGRASLT AQYGNFQKFF DCSKRMSMQN EILKSNAFKY FDERNKFAAF ADLFASVNFT AQHGNFQRLL DDYK-DGEQT ELQNFGSLSV NAQVGSFARP LLDFTSINLR LDAGGMGTPN EDYT-DGDEV RLQNFASIAL HGQIGSFSRP LFDFTSVNLR LDAGSVGNPY DNSPPIGRRH SADLFASVGL NAQFGRFKRN FWDFTKMGIR LDIGAVSALK LDLTRFYAR LDFPS GSKFLAGTTR LTQDLYNSQQ PSLEA----- FDLTRIHGR LDVPS GSKFCSAAAC LAKDIYKSQQ LSLEA----- FDLTRFHAR LDFPH GLRFLTGATS VAQDLLNSRQ PSLEA----- LDLTRVSAR LDISS GSLFLRGASQ LAQDFFFSRR PDLET----- KPLDAAAQTP LPLADRLITM GGRLKANPVS VTLSLGQQLL GPLRF----- KPLDASEQKP MSLGDRLITA DGRLKASPTS VTISFAQQLL GPLRF----- LHKMQDVSPS SNGESGDIGD ERGHPTLALE LQQQVIGPLR ARVHSRVSFN F QAICPTATLS LQQQIVGPFS FRIDSGVAVN V QAVCPNPMVS FQQQIVGPFS FRVDSKIAVN F QKICPEVLVS LQQQIVGPFS FKVESGIEID F CDVCPDVIVS LQQQIVGPFS FRVESTVTID R AEVRASGAEA LSATRAGLSA LKERKTMTEV R TEVRTSGAEA LAATCAGLSA IKQRKPMAQV PLNLNQQPGS QCVCEKAPDR RCKFVVGPLL RVRSALLYQR YSYARQKNTT LKNREWHIDV DEPVFAIEYA LQ-VLGSAKA IAW YSPKH LENQGCKVSL DNPVFAIEYA LQ-VLFSAKA VAW DHDSP LRNGANPVTV DKTVFAIEYA LQ-VLLSAKA VVS YSPKQ PKKQDHFVRV DDSVFAIDWA LK-VLGSAKA TAW YSPKH RESIQKQIVK PEVVYGLDCA LPPTIGSARV VAW YNATR RDVFKKEVAS PEVLYGLDCP LPPTLGSARA VVW YNATR RHSNREKIPC SQTWIASDLT TKCLIAEPSC EYFNFLCILV HKTRDLQPVR EEFMVELRFF EI PGSNCIHHLY SSMRGK-- NEFMVELRFF ET QEAMVELRFF ET QEAMAELRLF DL QDAFAEIRLF DL NMKNVCSMIF EPTDLAAD

6 MGDG - PtdGro - SQDG - Ptdcho - 60 Radiolabel (%) MGDG PtdGro DGDG SQDG PtdEtn PtdCho PtdIns 14 Supplemental Figure 4. In vivo labeling of polar lipids with [ C]-acetate. Wild type (), the previously studied mutant and mutant were compared. Excised leaves of three-week-old plants were exposed for 10 min to [C14]-acetate prior to analysis. (A) Representative autoradiograph of labeled lipids separated by thin-layer chromatography. (B) Quantitative analysis of polar lipid labeling. Three independent replicates were averaged. Lipids were identified by iodine staining, scraped into vials and radioactivity was quantified by scintillation counting. Standard deviation is indicted. Lipids: MGDG, monogalactosyldiacylglycerol; PtdGro, phosphatidylglycerol; DGDG, digalactosyldiacylglycerol; SQDG, sulfoquinovosyldiacylglycerol; PtdIns, phosphatidylinositol; PtdEtn, phosphatidylethanolamine; PtdCho, phosphatidylcholine.

7 Suppl. Table 1. Complete fatty acid composition data sets for wild type (), tgd4-2 and mutants. Abbreviations of lipids and fatty acids are as provided in the legends of Tables 1, 2, and 3 in the main text. MGDG (sn-2) and DGDG (sn-2) refer to the fatty acids in the sn-2 position only of the respective lipid (rf. Tab. 3 main text). In addition, representative data for TGDG (trigalactosyldiacylglycerol) and TAG (triacylglycerol) accumulating in the tgd4 mutants are provided for the mutant. Sample repeats were as indicated in the Tables in the main body of the text. Three repeats were averaged for the TGDG and TAG data sets. Standard deviation is indicated. Fatty Acid (mol%) 16:0 16:1 16:2 16:3 18:0 18:1 18:2 18:3 MGDG 3.9 ± ± ± ± ± ± ± ± 2.7 MGDG (sn-2) 1.8 ± ± ± ± ± ± ± ± 0.2 PtdGro 25.1 ± ± ± ± ± ± ± ± 0.1 DGDG 12.6 ± ± ± ± ± ± ± ± 1.1 DGDG (sn-2) 19.0 ± ± ± ± ± ± ± ± 3.6 SQDG 31.0 ± ± ± ± ± ± ± ± 3.1 PtdIns 27.2 ± ± ± ± ± ± ± ± 4.2 PtdEtn 27.5 ± ± ± ± ± ± ± ± 0.0 PtdCho 18.5 ± ± ± ± ± ± ± ± 0.1 tgd4-2 MGDG 7.2 ± ± ± ± ± ± ± ± 0.3 MGDG (sn-2) 18.3 ± ± ± ± ± ± ± ± 0.9 PtdGro 23.7 ± ± ± ± ± ± ± ± 0.6 DGDG 37.2 ± ± ± ± ± ± ± ± 0.2 DGDG (sn-2) 65.4 ± ± ± ± ± ± ± ± 1.3 SQDG 50.4 ± ± ± ± ± ± ± ± 1.1 PtdIns 27.8 ± ± ± ± ± ± ± ± 0.5 PtdEtn 26.2 ± ± ± ± ± ± ± ± 0.1 PtdCho 14.1 ± ± ± ± ± ± ± ± 0.2 MGDG 8.0 ± ± ± ± ± ± ± ± 0.1 MGDG (sn-2) 16.8 ± ± ± ± ± ± ± ± 0.7 PtdGro 23.9 ± ± ± ± ± ± ± ± 0.8 DGDG 37.7 ± ± ± ± ± ± ± ± 1.0 DGDG (sn-2) 74.0 ± ± ± ± ± ± ± ± 0.0 SQDG 50.6 ± ± ± ± ± ± ± ± 0.3 PtdIns 30.0 ± ± ± ± ± ± ± ± 2.7 PtdEtn 25.6 ± ± ± ± ± ± ± ± 0.2 Ptdcho 12.7 ± ± ± ± ± ± ± ± 0.1 TGDG 12.6 ± ± ± ± ± ± ± ± 2.8 TAG 41.7 ± ± ± ± ± ± ± ± 5.5