Man 1-P. GMPP (VTC1), KJCs. GDP-Man. GDP-Man 3,5-epimerase. GDP-L-Gal GDP-L-Gal phosphorylase (VTC2, VTC5)

Transkript

1 Man 1-P GMPP (VTC1), KJCs GDP-Man GDP-Man 3,5-epimerase GDP-L-Gal GDP-L-Gal phosphorylase (VTC2, VTC5) L-Gal 1-P L-Gal 1-P phosphatase (VTC4) L-Gal L-Gal dehydrogenase L-Galactono-1,4-lactone L-Galactono-1,4-lactone dehydrogenase AsA Supplemental Figure 1. The Man/L-Gal pathway for the generation of AsA in plants. The synthesis of GDP-Man by VTC1 is one of the rate-limiting reactions affecting AsA level at least in many vascular plants including Arabidopsis.

2 VTC MKALILVGG--FGTRLRPLTLSFPKPLVDFANKPMILHQIEALKAVGVDEVV 5 Mm-GMPPB MKALILVGG--YGTRLRPLTLSTPKPLVDFCNKPILLHQVEALAAAGVDHVI 5 KJC1 MGSSMEEKVVAVIMVGGPTKGTRFRPLSLNIPKPLFPIAGQPMVHHPISACKRIPNLAQI 6 KJC2 ---MSEEKVVAVIMVGGPTKGTRFRPLSFNTPKPLIPLAGQPMIHHPISACKKISNLAQI 57 Mm-GMPPA MLKAVILIGGPQKGTRFRPLSFEVPKPLFPVAGVPMIQHHIEACAQVPGMQEI 53 VTC1 LAINYQP--EVMLNFLKDFETKLEIKITCSQETEPLGTAGPLALARDKLLDGSGEPFFVL 18 Mm-GMPPB LAVSYMS--QMLEKEMKAQEQRLGIRISMSHEEEPLGTAGPLALARD-LLSETADPFFVL 17 KJC1 YLVGFYE-EREFALYVSAISNELKVPVRYLREDKPHGSAGGLYHFRNLIMEDSPSHIFLL 119 KJC2 FLIGFYE-EREFALYVSSISNELKIPVRYLKEDKPHGSAGALYYFRDRIMEEKPSHVFLL 116 Mm-GMPPa LLIGFYQPDEALTQFLEAAQQEFNLPVRYLQEFAPLGTGGGLYHFRDQILAGAPEAFFVL 113 VTC1 NSDVISEYPLKEMLEFHKSHGGEASIMVTKVD--EPSKYGVVVMEESTGRVEKFVEKPKL 166 Mm-GMPPB NSDVICDFPFQAMVQFHRHHGQEGSILVTKVE--EPSKYGVVVCEADTGRIHRFVEKPQV 165 KJC1 NCDVCCSFPLPKMLEAHRGYGGIGTLLVIKVSPESASQFGELVADPVTNELLHYTEKPET 179 KJC2 NCDVCCSFPLQGILDAHRRYGGIGTMLVIKVSAEAASQFGELIADPDTKELLHYTEKPET 176 Mm-GMPPA NADVCSDFPLSAMLEAHRRQRHPFLLLGTTANRTQSLNYGCIVENPQTHEVLHYVEKPST 173 VTC1 YVGNKINAGIYLLNPSVLDKIELRPTS IEKE 197 Mm-GMPPB FVSNKINAGMYILSPAVLQRIQLKPTS IEKE 196 KJC1 FVSDRINCGVYVFTPEIFNAIGDVSTQRKDRATLKRVSSFEALQPATR-IPTDFVRLDQD 238 KJC2 FVSDLINCGVYVFTSDIFNAIEEVYSQIRDTSSN YQSATRSVPADFVRLDQD 228 Mm-GMPPA FISDIINCGIYLFSPEALKPLRDVFQRNQQDGQLE ESPGSWPGAGTIRLEQD 225 VTC1 TFPKIAAAQGLYAMVLPGFWMDIGQPRDYITGLRLYLDSLRKKSPAKLT------SGPHI 251 Mm-GMPP IFPVMAKEGQLYAMELQGFWMDIGQPKDFLTGMCLFLQSLRQKHPERLY------SGPGI 25 KJC1 ILSPLAGKKRLYTYETMDFWEQIKSPGMSLRCSGLYLSQFRLTSPQLLASGDGT-RSAIV 297 KJC2 ILSPLAGKKQLYTYENKDFWEQIKTPGKSLKCSALYLSQFRETSPHILASGDGTNRKPTI 288 Mm-GMPPA VFSALAGQGQIYVHLTDGIWSQIKSAGSALYASRLYLGRYQITHPERLARHTPG--GPRI 283 VTC1 VGNVLVDETATIGEGCLIGPDVAIGPGCIVESGVRLSRCTVMRGVRIKKHACISSSIIGW 311 Mm-GMPPB VGNVLVDPSARIGQNCSIGPNVSLGPGVVVEDGVCIRRCTVLRDAHIRSHSWLESCIVGW 31 KJC1 IGDVYIHPSAKVHPTAKIGPNVSISANARVGPGVRLMSCIILDDVEIMENAVVTNAIVGW 357 KJC2 IGDVYIHPSVKLHPTAKIGPNVSISANVRVGPGVRLISCIILDDVEIKENAVVINSIIGW 348 Mm-GMPPA RGNVYIHPTAKVAPSAVLGPNVSIGKGVTIGEGVRLRESIVLHGATLQEHTCVLHSIVGW 343 VTC1 HSTVGQWARIENMT ILGEDVHVSDEIYSNGGV 343 Mm-GMPPB RCRVGQWVRMENVT VLGEDVIVNDELYLNGAS 342 KJC1 KSSIGRWSRVQAEG------VYNSKLG VTILGDSVAVEDEVVVTSSI 398 KJC2 KSSIGRWSRVQASG------DYNDRLG ITILGEAVTVEDEVAVIGSI 389 Mm-GMPPA GSTVGRWARVEGTPNDPNPNDPRARMDSESLFKDGKLLPAITILGCRVRIPAEVLILNSI 43 VTC1 VLPHKEIKSNILKPEIVM 361 Mm-GMPPB VLPHKSIGESVPEPRIIM 36 KJC1 VLPNKTLNVSVQDEIIL- 415 KJC2 VLQNKTLNVSVQDDIIL- 46 Mm-GMPPA VLPHKELSRSFTNQIIL- 42 Supplemental Figure 2. Alignment of amino acid sequences of GMPPs and KJC proteins. The amino acid sequences of VTC1, KJC1, KJC2, and mouse GMPP subunits (Mm-GMPPA) and (Mm-GMPPB) were aligned by the pairwise method using the ClustalW program (Thompson et al. 1994). The amino acid residues are numbered from the first Met. Gaps (-) were introduced to achieve maximum similarity. Residues conserved in all five proteins are highlighted in black, and those conserved in three or four proteins are in grey. The bar indicates a pyrophosphorylaseconsensus motif. Supplemental Reference Thompson J.D., Higgins D.G., Gibson T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:

3 A kjc1-3 kjc1-2 (GK_175D6) (SALK_44963) (SALK_11173) o849 LBb1 LBb1 F-3 R-3 F-1 R-1 F-2 R-2 (SALK_23876) LBb1 3 bp F-1 R-1 B kjc1-2 KJC1 1.4 kbp KJC2 1.4 kbp Supplemental Figure 3. Arabidopsis T-DNA insertion lines for KJC genes. (A) The schematic diagram of T-DNA insertion sites in kjc mutants. Arrows indicate primers used for the determination of genotype that are listed in Supplemental Table 1. (B) T-DNA insertion sites in kjc mutants. Knock-out of kjc genes was confirmed by the amplification of the full-length coding sequence of KJC genes.

4 GMPP activity (mu/g fresh weight) VTC1 OX #4 VTC1 OX #3 VTC1 OX #2 VTC1 OX #1 KJC1 OX #11 KJC1 OX #9 KJC1 OX #7 KJC1 OX #3 KJC1 OX #2 KJC1 OX #1 KJC1 OX #4 KJC1 OX #1 KJC2 OX #5 KJC2 OX #2 KJC2 OX #1 KJC2 OX #4 VTC1KJC1 OX #1 VTC1KJC1 OX #2 VTC1KJC1 OX #3 VTC1KJC1 OX #4 Supplemental Figure 4. GMPP activity in kjc mutants and KJC over-expressing plants. GMPP activity per g fresh weight is shown here.data are mean values with standard deviation (n = 3 biological replicates).the asterisk indicates significant difference from (Student s t-test, p <.5).

5 14 GMPP activity (mu/g fresh weight) #2 #3 with genomic KJC1 Supplemental Figure 5. Complementation of mutant with genomic KJC1 gene. Genomic fragment of KJC1 gene was introduced into mutant. GMPP activity in the complemented lines #2 and 3 was measured. The activity in the mutant was properly recovered by genomic KJC1 gene. Data are mean values with standard deviation (n = 3 biological replicates).

6 A KJC1 KJC2 VTC1 High Medium Low Stage of development B KJC (h) (h) (h) KJC2 VTC1 Supplemental Figure 6. Expression patterns of KJC1, KJC2, and VTC1. (A) Expression of KJC1, KJC2, and VTC1 at specific stage of development. The data were acquired from Genevestigator. Based on the expression level, KJC1 is expected to be the major isoform and KJC2 the minor isoform. (B) The regulation of the expression of KJC1, KJC2, and VTC1. The expression data for 7-days-old Arabidopsis (Ecotype Landsberg erecta) seedlings grown under long-day condition (upper, 16 h light, 9 microe, 22 C, Michael et al., 28) and short-day condition (lower, 8 h light, 18 microe, 22 C) were acquired from Arabidopsis efp browser. The expression patterns suggest that the expression of KJC1 is under diel regulation. Supplemental Reference Michael T.P., Mockler T.C., Breton G., McEntee C., Byer A., Trout J.D., Hazen S.P., Shen R., Priest H.D., Sullivan C.M., Givan S.A., Yanovsky M., Hong F., Kay S.A., Chory J. (28) Network discovery pipeline elucidates conserved time-of-day-specific cis-regulatory modules. PLoS Genet. 4: e14.

7 A Relative expression level (%) KJC1 * * Relative expression level (%) KJC2 * * B Relative expression level (%) VTC1 KJC1 OX #11 KJC1 OX #9 * * * * KJC2 OX #2 KJC2 OX #1 * VTC1 OX #2 KJC1 OX #11 KJC1 OX #9 KJC2 OX #2 KJC2 OX #1 VTC1KJC1 OX #2 VTC1KJC1 OX #3 Supplemental Figure 7. Expression of KJC1, KJC2, and VTC1 in kjc mutants and KJC over-expressing plants. (A) Increased expression levels of KJC1 and KJC2 in the over-expressing plants. (B) The expression level of VTC1 in kjc mutants and those over-expressing KJC1 (KJC1 OX #9 and 11), KJC2 (KJC2 OX #1 and 2), VTC1 (VTC1 OX #2), and both of KJC1 and VTC1 (VTC1KJC1 OX #2 and 3). The expression levels relative to that of ACTIN2 in 2 week-old seedlings were measured by quantitative RT-PCR. Data are mean values with standard deviation (n = 3 biological replicates). The asterisk indicates significant difference from (Student s t test, p <.5).

8 1 Green seeds (%) vtc1-1 vtc2-1 Supplemental Figure 8. Seed development in kjc mutants. The proportions of green seeds in immature silique in wild-type plant, vtc1-1, vtc2-1,, and mutants were determined. Data are mean values with standard deviation (n = 3 biological replicates). No significant difference in the proportion between kjc mutants and wildtype plant was observed.

9 A Manα1 Manα1 3 Manβ GlcNAcβ1 4GlcNAcβ1 3 Xylβ1 L-Fucα1 B kda S S C kda S S Supplemental Figure 9. Protein gel blot analysis of N-glycans of glycoproteins. (A) The structure of plant N-glycan. GlcNAc, N-acetylglucosamine; L-Fuc, L-fucose; Xyl, xylose. (B) Xylosyl residues attaching to the mannnosyl resiude through β-1,2-linkage in kjc mutants. (C) L-Fucosyl reusidues attaching to proximal N-acetylglucosaminosyl residue through α-1,3-linkage. The residues were detected with antibodies against the Xyl and L-Fuc, respectively. These antibodies were a kind gift from Veronique Gomord, University of Rouen, France. The left panels show the gels stained with Commassie Brilliant Blue R-25. Although and mutants had drastically decreased GMPP activity, neither reduced N-glycosylation nor structural change was observed. The arrow indicates an unidentified glycoprotein accumulated in mutant. S, standard proteins.

10 1 8 * * Composition (%) * * Hex Hex 1 Hex 2 Supplemental Figure 1. Sugar composition of GIPC in calli of kjc mutants. GIPCs were extracted from calli of plant, and mutants, and analyzed by LC-MS/MS. The GIPCs were categorized according to the number of hexoses in the sugar moiety (Hex n ). Data are mean values with standard deviation (n = 3 biological replicates). The asterisk indicates significant difference from (Student s t test, p <.5)

11 A kda S S S S B K A Pyrophosphorylaseconsensus motif Supplemental Figure 11. Preparation of native and point-mutated recombinant proteins. (A) Recombinant enzymes expressed in E. coli. The purity of recombinant proteins purified by chelating chromatography was examined on SDS-PAGE. Lane 1, crude extracte of native rvtc1; 2, purified native rvtc1; 3, crude extracte of native rkjc1; 4, purified native rkjc1; 5, crude extracte of native rkjc2; 6, purified native rkjc2; 7, crude extracte of rvtc1 K23A; 8, purified rvtc1 K23A; 9, crude extracte of rkjc1 K33A; 1, purified rkjc1 K33A; 11, crude extracte of rkjc2 K3A; 12, purified rkjc2 K3A; S, standard proteins. The arrowheads indicate purified recombinant proteins. The proteins in the gel were stained with Coomassie Brilliant Blue R-25. (B) Conserved Lys residue in the pyrophosphorylase-consensus motif. The Lys residue indicated with an arrow was replaced with Ala residue in the point-mutated proteins. Mm-GMPPA, mouse GMPP subunit α; Mm-GMPPB, mouse GMPP subunit β.

12 Supplemental Figure 12. KJC/GMPP subunit and VTC1/GMPP subunit subfamilies. The phylogenetic relationships were analyzed using MEGA software (version 6.). The bar indicates substitutions per site. Numbers in nodes represent bootstrap values. Accession numbers for the sequences are listed in Supplemental Table 3.

13 A Bait B Bait VTC1 KJC1 KJC2 USP Empty vector VTC1 KJC1 KJC2 USP Empty vector VTC1 Prey KJC1 USP -Leu, -Trp, -His -Leu, -Trp Supplemental Figure 13. Interaction of KJC1 with USP in yeast cells. (A), Interaction between KJC1, VTC1, and USP. The yeast cells were grown on SD medium without Leu, Trp, and His. Neither VTC1 nor KJC1 showed interaction with USP. (B) Confirmation of the transformation. The yeast cells were grown on SD medium without Leu and Trp.

14 Supplemental Table 1. Sequences of primers used in the present study Name of primer Sequence Primers for genotype determination KJC1--F-1 5 -GATCAACAGAGGCTCACAGGGG-3 KJC1--R-1 5 -CTGAGTAGAAACATCTCCTATGGC-3 KJC1--F-2 5 -CCTTTACAATCGTGAACCTG-3 KJC1--R-2 5 -CTTTTAATCGAGTTCTGAACGGGC-3 KJC1--F-3 5 -TTCTTGCGTGCTTCAGGGGAC-3 KJC1--R-3 5 -CAGGACCGAACTAGACAGAC-3 KJC2--F-1 5 -ATGTCGGAGGAGAAAGTCGTCGC-3 KJC2--R-1 5 -CGATAACGGGCGAAATCGAGTTCC-3 LBb1 5 -GCGTGGACCGCTTGCTGCAACT-3 o ATATTGACCATCATACTCATTGC-3 Primers for full length RT-PCR KJC1-RT-PCR-F 5 -TGGAGGGACCCCATCACCGC-3 KJC1-RT-PCR-R 5 -GCTGCTCCCCACTGGGTTGG-3 KJC2-RT-PCR-F 5 -GAGAATATCCGACTACAAAGATACGACC-3 KJC2-RT-PCR-R 5 -CACAAAGTACAGTGAGAGTTTACAAATCTTCC-3 Primers for quantitative RT-PCR KJC1-Q-RT-PCR-F 5 -TGGGAGCAAATCAAATCTCC-3 KJC1-Q-RT-PCR-R 5 -TGCAGAGATTGAGACGTTGG-3 KJC2-Q-RT-PCR-F 5 -GCGAAACATCGCCTCATATT-3 KJC2-Q-RT-PCR-R 5 -TACACCAGGTCCAACACGAA-3 VTC1-Q-RT-PCR-F 5 -CACTCGCTTGAGACCATTGA-3 VTC1-Q-RT-PCR-R 5 -CCTAGTGGCTCGGTCTCTTG-3 ACTIN2-Q-RT-PCR-F 5 -ACCTTGCTGGACGTGACCT-3 ACTIN2-Q-RT-PCR-R 5 -CACCAATCGTGATGACTTGC-3 Primers for heterologous expression in E.coli, co-purification assay, and Y2H experiment KJC1-cDNA-NdeI-F 5 -GACATATGGGGAGCTCAATGGAGGAG-3 KJC1-cDNA-BglII-R 5 -GCAGATCTTTACAAAATGATCTCGTCTTGAACAC-3 KJC1-PM-F 5 -ATTCCAGCGCCTCTGTTTCC-3 KJC1-PM-R 5 -CAGAGGCGCTGGAATATTCA-3 KJC2-cDNA-NdeI-F 5 -GACATATGTCGGAGGAGAAAGTCGTC-3 KJC2-cDNA-EcoRI-F 5 -GAAGAATTCATGTCGGAGGAGAAAGTCG-3 KJC2-cDNA-BamHI-R 5 -GTGGATCCTCACAAGATGATATCATCTTGAACAC-3 KJC2-PM-F 5 -ACACCCGCACCATTGATTCC-3 KJC2-PM-R 5 -CAATGGTGCCGGTGTATTGA-3 USP-cDNA-EcoRI-F 5 -ATCGAATTCATGGCTTCTACGGTTGAT-3 USP-cDNA-BanHI-R 5 -CGAGGATCCTCAATCTTCAACAGAAAA-3 VTC1-cDNA-NdeI-F 5 -GACATATGAAGGCACTCATTCTTGTTG-3 VTC1-cDNA-EcoRI-F 5 -GAAGAATTCATGAAGGCACTCATTCTTG-3 VTC1-cDNA-BamHI-R 5 -GTGGATCCTCACATCACTATCTCTGGCTTC-3 VTC1-PM-F 5 -TTCCCAGCGCCCCTTGTTGATTTTG-3 VTC1-PM-R 5 -AAGGGGCGCTGGGAAACTGAGAGTC-3 FLAG-F 5 -CATGGATTACAAGGATGACGACGATAAGCA-3 FLAG-R 5 -TATGCTTATCGTCGTCATCCTTGTAATCC-3 Primers for over-expression in Arabidopsis KJC1-cDNA-BglII-F 5 -GAAGATCTGTGGTGAATGGTGAGTGTG-3 KJC1-cDNA-SacI-R 5 -GAGCTCTTACAAAATGATCTCGTC-3 KJC2-cDNA-BamHI-F 5 -GAGGATCCGACTACAAAGATACGACC-3 KJC2-cDNA-SacI-R 5 -CGCGAGCTCAGTGAGAGTTTACAAATCTTCC-3 VTC1-cDNA-BamHI-F 5 -GAGGATCCGGTGAGATCTCTCTCAAGG-3 VTC1-cDNA-SacI-R 5 -CACGAGCTCGACCCTAACAAGATATACAGCG-3 Primers for complementation of gkjc1-ecori-f 5 -GAGGAATTCTTGCGTGCTTCAGGGGAC-3 gkjc1-hindiii-r 5 -CAGGGAATCAAGCTTTTGCAAAGG-3 The restriction sites used for subclonoing are underlined.

15 Supplemental Table 2. Substrate specificity of rvtc1 in the absence and presence of rkjcs. Data is shown as the average of 3 reactions, ± standard deviation. Substrate rvtc1 rvtc1+rkjc1 a rvtc1+rkjc2 a GDP-sugar pyrophospholysis GTP-Man + PPi 1 ± 1.1 b 1 ±.6 b 1 ± 1.4 b GDP-Glc+ PPi 2.3 ± ± ±.13 UDP-sugar pyrophospholysis UDP-Glc + PPi UDP-Gal + PPi UDP-GlcA + PPi a Mixture of VTC1 and KJC protein at equal molar ratio was used. b Activity is expressed in % of that toward GMPP activity.

16 Supplemental Table 3. Accession numbers for amino acid sequences Name Organism Abbreviation Accession number At-1g122.1 Arabidopsis (A. thaliana ) At1g122.1 At-1g561.1 Arabidopsis (A. thaliana ) At1g561.1 At-1g Arabidopsis (A. thaliana ) At1g At-1g317.2 Arabidopsis (A. thaliana ) At1g317.2 At-1g Arabidopsis (A. thaliana ) KJC1 At1g At-2g465.1 Arabidopsis (A. thaliana ) KJC2 At2g465.1 At-2g Arabidopsis (A. thaliana ) At2g At-2g352.1 Arabidopsis (A. thaliana ) At2g352.1 At-2g Arabidopsis (A. thaliana ) VTC1 At2g At-3g325.1 Arabidopsis (A. thaliana ) At3g325.1 At-3g Arabidopsis (A. thaliana ) At3G At-3g564.1 Arabidopsis (A. thaliana ) At3g564.1 At-4g357.1 Arabidopsis (A. thaliana ) At4g357.1 At-4g Arabidopsis (A. thaliana ) At4g At-5g Arabidopsis (A. thaliana ) At5g At-5g Arabidopsis (A. thaliana ) At5g At-5g483.1 Arabidopsis (A. thaliana ) At5g483.1 At-5g Arabidopsis (A. thaliana ) At5g Os-1g6331 Rice (O. sativa ) Os1g6331 Os-1g8472 Rice (O. sativa ) Os1g8472 Os-2g1177 Rice (O. sativa ) Os2g1177 Os-3g289 Rice (O. sativa ) Os3g289 Os-3g1151 Rice (O. sativa ) Os3g1151 Os-3g2684 Rice (O. sativa ) Os3g2684 Os-3g735 Rice (O. sativa ) Os3g735 Os-4g6137 Rice (O. sativa ) Os4g6137 Os-5g4686 Rice (O. sativa ) Os5g4686 Os-5g58 Rice (O. sativa ) Os5g58 Os-6g712 Rice (O. sativa ) Os6g712 Os-7g2432 Rice (O. sativa ) Os7g2432 Os-8g269 Rice (O. sativa ) Os8g269 Os-8g3458 Rice (O. sativa ) Os8g3458 Os-9g2982 Rice (O. sativa ) Os9g2982 Os-9g5532 Rice (O. sativa ) Os9g5532 Aa-GMPPa Aedes aegypti ABF18356 Aa-GMPPb A. aegypti ABF18442 Bt-GMPPA Bos Taurus NP_ Bt-GMPPB B. Taurus NP_ Cd-GMPP Corynebacterium diphtheriae WP_ Dm-GMPPa Drosophila melanogaster NP_ Dm-GMPPb D. melanogaster NP_ Dr-GMPPAa Danio rerio NP_ Dr-GMPPAb D. rerio NP_ Dr-GMPPB D. rerio NP_ Hs-GMPPA Human (Homo sapiens ) NP_ Hs-GMPPB Human (H. sapiens ) AAD Mm-GMPPA Mouse (Mus musculus ) NP_ Mm-GMPPB Mouse (M. musculus ) NP_ Mt-mgtA Mycobacterium tuberculosis WP_ Mu-ManB Mycobacterium ulcerans WP_ Pa-cpsB Pyrococcus abyssi WP_ Ph-cpsB Pyrococcus horikoshii WP_ Rj-GMPP Rhodococcus jostii WP_ Rn-GMPPa Rattus norvegicus NP_ Rn-GMPPb R. norvegicus NP_ Xl-GMPPa-A Xenopus laeviss NP_ Xl-GMPPa-B X. laeviss NP_ Xl-GMPPb-A X. laeviss NP_ Xl-GMPPb-B X. laeviss NP_