PIAL1 (At1g08910) ORF sequence

Átírás

1 PIAL1 (At1g08910) ORF sequence ATGGTTATTCCGGCGACTTCTAGGTTTGGGTTTCGTGCTGAATTCAACACCAAGGAGTTTCAAGCTTCCTGCATCTCTCT! CGCTAATGAAATCGATGCGGCTATTGGGAGAAATGAAGTTCCAGGGAATATTCAAGAGCTCGCTTTAATCCTCAATAATG! TGTGCCGACGTAAATGTGATGATTATCAAACCAGGGCGGTGGTAATGGCGCTGATGATCTCAGTCAAGAGTGCTTGTCAG! CTTGGATGGTTCCCGGAGAGAGAGACTCAAGAATTGTTGGCTATCATAGACTTGATGTGGAATGGTTTCAGCTGTCCTGA! AAATGTCACCTCTTGTGTAAATAGTCCCGTCACTCTAATATCGCAGGTCATTGAGAGGTTCTATCCATGTGTGAAGCTGG! GGCATATTCTTGTTTCTTTTGAAGCTAAGCCAGAATCAAAGATGATGATGAAGGACTTCCATATTTCAAAGAAGATGCCA! CATTCTCCCAAACAGAAAGTTGGATTATTTGTTGTTCGGACAGAGGACATAAGCAGATCTAATTGTATTGTACATCCACA! AGGAGTCAGCTTTTTGTTGAATGGAAAGGGCATTGACAAGAGAGTTAACATCTCAATGGAGTCTGGGCCGCAGCTTCCAA! CTAATGTTACTGCCCTGCTAAACCTTGGGGCAAATCTTCTACAAGCAATAGGCTGTTTTGGAGGTAGTTACTTGATTGCT! ATTGCTTTCATGGATGTCATACCACTGCCCAACAAACCGTTACTGAAAGATTATGTTCATCCTGAAGTCGTTGGATCAAA! CTCAGATTGTGACATAATTGAGGGGCCATCGAGGATATCTCTCAGTTGTCCTATCAGCCGAACGCGTATCAAACTTCCTG! TGAAGGGTCATGTCTGTAAACATCTTCAGTGTTTTGATTTCTGGAATTATGTCAACATGAACACAAGACGACATCATGGC! GCTGCCCGCATTATTCTAGAAGAAGTGGGACGTAATGCTGCAGATGTGGTTATCTCTGCTGATGGAACCTGGATGGTTGA! GACGGAAAATGATGAGGACGTGGAACTTGTGCCAGAAACCACTCATGACCATGGAGACCCAAATAGTTTCATCAACTTGG! GGCCTACTGTTAAGAATCCTGCCAGAGATGAGAATGAAATGGAAACATCCACCCAAGTCGAAGAACATAATCCTTGTTTA! TCTGAGATTCAGGGTCCGTCAAATGACACACATAGGCCTGCTTCAGATTATACTATGCTTAACCAGTCTCATACTTCAAC! CAACACACTACCACAGCTGCCACGAACTTTGAATGCCTTTGATGGTCAACAATTCGTGAACTTACCACAAGTAATAAACA! CCCGAGATTCACCAGCAAGCCAAGCCTTACCTATGACATTCTCACCAACCCCATCTCCACAAGATATATTAGCTACTAAT! GCGGCAAACTTTGGCACATCAATGCCGGCTGCTCAGTCTTCTCAGTTTCAAGGCTCACATGTTACGTCCCTTGGAAATTG! TGAAGGAAGAACTTCTGATTTGATGGCGAGATGGAACCACATCTATGGACGTGTTCAAACTCAATTTCCACCTGCACCTC! TTTCGCATCATCATTATTCAATGCAGAATCAGAGCCCGTCCCCTGCACAGCAAAGACCAGTGCCATCTTACATTGCACAT! CCTCAAACTTTTCATGTCAACTACGGAGAGAACGCTGACCAAAGATGGATGCCGTCTTCCATTGCACATCCTCAAACTTT! ACCTGTTAACTACGGAGGAAATACTAACCAAAGACCGATACCATCTTCCATTGCACATCCTCAAACTTTACCTGTCAACT! ACAGGGGGAATACTGACCATAGATCGACGCCATATTCCATTACACATCTCCAAACATTACTTAACTACGGAGGGAACGCC! GATCAAAGACCAATGCCATCTTCCATTACAAATCTCCAAACTTTACCTGCCACGTACGGAGGGTACGCCCACCAAAGACC! AATGTCATCTTCCATTACACATCCCCGAACTTCACCTGTCAACTACGGAGGGACCCCTGACCAAAGACCAATGCCATCTT! CCATTACACATCCCCAAACTTTACCTGTCAGCTACGGAGGGACCACAGACCAAATACTGAATCCTGGTGGTGCAATGGGA! CAGTTCTCATCACGAGAGTTCATGAATTTGACTCCTGCTAACACTGAGAATTGGCGCCCACAAAGCCGGATGCGAGGCAG! CGTAGCACCTGGTACGGGATATGACCATATGATCATTCATCCTACCCGTCCAGTTCATCCACAAGCTCAAACACCTCCTG! CGCCTCTTTCAACATCTTATGATGGAGCAGATGAAATTCAAGCATTTATAGGGCACCCAAGCTATCCCGTTAGTAATAAC! GAAACACAAGCGGGTACCAGTTCGTTGCCAGTGGCAGAGGGTCTTGGGTATTCAGGGTCGTTTTGGTCAATGCCTCCTGA! GACATGGTGA PIAL1 (At1g08910) protein sequence MVIPATSRFGFRAEFNTKEFQASCISLANEIDAAIGRNEVPGNIQELALILNNVCRRKCDDYQTRAVVMALMISVKSACQ! LGWFPERETQELLAIIDLMWNGFSCPENVTSCVNSPVTLISQVIERFYPCVKLGHILVSFEAKPESKMMMKDFHISKKMP! HSPKQKVGLFVVRTEDISRSNCIVHPQGVSFLLNGKGIDKRVNISMESGPQLPTNVTALLNLGANLLQAIGCFGGSYLIA! IAFMDVIPLPNKPLLKDYVHPEVVGSNSDCDIIEGPSRISLSCPISRTRIKLPVKGHVCKHLQCFDFWNYVNMNTRRHHG! AARIILEEVGRNAADVVISADGTWMVETENDEDVELVPETTHDHGDPNSFINLGPTVKNPARDENEMETSTQVEEHNPCL! SEIQGPSNDTHRPASDYTMLNQSHTSTNTLPQLPRTLNAFDGQQFVNLPQVINTRDSPASQALPMTFSPTPSPQDILATN! AANFGTSMPAAQSSQFQGSHVTSLGNCEGRTSDLMARWNHIYGRVQTQFPPAPLSHHHYSMQNQSPSPAQQRPVPSYIAH! PQTFHVNYGENADQRWMPSSIAHPQTLPVNYGGNTNQRPIPSSIAHPQTLPVNYRGNTDHRSTPYSITHLQTLLNYGGNA! DQRPMPSSITNLQTLPATYGGYAHQRPMSSSITHPRTSPVNYGGTPDQRPMPSSITHPQTLPVSYGGTTDQILNPGGAMG! QFSSREFMNLTPANTENWRPQSRMRGSVAPGTGYDHMIIHPTRPVHPQAQTPPAPLSTSYDGADEIQAFIGHPSYPVSNN! ETQAGTSSLPVAEGLGYSGSFWSMPPETW*! Supplemental Figure 1. Open reading frame and protein sequence of PIAL1 as determined by sequencing of cdnas. 1

2 PIAL2 (At5g41580) ORF sequence ATGTCTACGGCGGCAGCGGCTCGTCCGGTGGCTGGAACTGGCTTACGGGAGAAGACGGCAGCGTCACTGGTCAATTCCTT! CCGATTAGCGTCGGTGACTCAACGTTTACGTTACCACATTCAAGACGGAGCTAAGGTTGATCCTAAGGAGTTTCAAATCT! GTTGCATCTCTTTCGCTAAAGGCATTGATTTTGCTATAGCGAATAATGATATTCCTAAGAAGGTTGAGGAGTTTCCGTGG! TTACTCAAACAGTTGTGCAGACATGGAACTGATGTGTACACTAAGACGGCGCTTATGGTGCTGATGATCTCTGTTAAGCA! TGCTTGTCATTTGGGATGGTTTTCGGATAGCGAGAGTCAAGAACTTATCGCTCTAGCTGATGAGATAAGGACTTGTTTTG! GGAGTTCTGGAAGCACTAGCCCTGGTATCAAAAGTCCTGGCAGTACATTTTCGCAGATCATGGAGAGGTTCTATCCATTT! GTAAAGCTTGGGCATGTTCTTGTTTCTTTTGAAGTGAAGGCTGGCTATACAATGCTGGCGCATGATTTTTATATCTCAAA! GAATATGCCACATTCACTCCAAGAGAAAATTCGGTTATTTGTTGCCCAGACAGACAACATAGACACGTCTGCCTGTATTT! CAAATCCTCCAGAAGTCAGCTTCTTGCTAAATGGAAAGGGGGTTGAGAAGAGAGTCAATATCGCAATGGATACAGGGCCT! CAACTCCCTACAAATGTTACTGCACAGCTGAAATATGGAACTAATCTTCTCCAAGTGATGGGGAATTTTAAAGGAAATTA! CATCATCATAATTGCTTTTACGGGACTGGTAGTGCCACCTGAAAAACCGGTTCTTAAAGATTACCTTCAGTCCGGAGTCA! TTGAAGCAAGTCCAGATTCAGACATCATTGAGGGGCCATCACGAGTATCTCTCAGTTGCCCTATAAGTCGCAAGCGGATC! AAGCTACCAGTCAAGGGCCAGTTATGTAAACATCTTCAGTGTTTTGATTTCTCAAACTACGTTCACATAAATATGAGGAA! CCCAACCTGGCGCTGCCCGCATTGCAATCAACCTGTTTGCTACCCTGACATTCGTTTAGATCAAAACATGGCCAAGATAT! TAAAAGATGTGGAACACAATGCTGCTGATGTAATCATCGATGCTGGTGGTACATGGAAGGTTACAAAAAATACTGGCGAG! ACCCCGGAACCTGTGCGTGAGATCATTCATGATCTAGAAGACCCGATGAGCTTATTAAACTCTGGTCCTGTTGTTTTCGA! TCTTACGGGGGATGATGATGCTGAACTGGAAGTTTTTGGTGACAACAAGGTTGAGGACCGGAAGCCCTGTATGTCTGATG! CTCAGGGTCAATCTAATAATAACAACACAAATAAACATCCTTCAAACGATGATTACTCTTCGATATTTGATATCTCTGAT! GTGATCGCACTTGACCCGGAGATTTTATCTGCTTTGGGAAACACTGCGCCACAACCGCATCAAGCTTCGAATACTGGAAC! TGGTCAACAATATTCAAACTTATCCCAAATACCAATGTCCATAGATCCAATGCCGGTACCTGTACCATTCTCGCAGACAC! CATCTCCAAGAGATAGACCAGCAACTACTTCCACTGTCTTCACCATACCGAATCCCTCTCCACAATACTCTCAGGTTCAT! GCTTCACCTGTTACACCCACCGGAACATATCTTGGTAGAACCACTAGTCCGAGATGGAACCAGACTTATCAATCTCAAGC! GCCACCAATGACAACTCCATATACGAGCCGGAAGGTCTCAGTTCCAGTAACGAGCCAGTCGCCAGCGAATGTATCCTCTT! TTGTTCAGTCTCAGCATGTCCCTAGAGTACTCAGCCAGCCTAATAATTATGGCGTCAGAGGTTTAACCAGTAGCCATGCA! AGCACTTCAAGGCAGCACCCAAGTGGTCCCACTGTTCAATCTGTTTCTCGATTAAGTGACCTAGTGGATGTAGACTTGAC! GGTTCCTGATACATCCAACTGGCGCCCGAGGATGCGAGGAAGTCTTGTGCCCGGTTCTCATTCCACTGCTCTTGACCACA! TGATCATCCGACCTAGCCAACAGTCTCAAACTTCCACTAGGCTGAATAGTTCACAGCCGGTTCAGACACCATCGGTTCAA! ACGTCTCAAGCTCAGTCACCTTTTACAACGGCTGCCTATAGAACCGAGACAGTTTTAGGAAACCGAAACCATCCGGTGCC! CGCTCCTCCTGGCATTGTTAGGCCTACTGGACCGACATCTTGA PIAL2 (At5g41580) protein sequence MSTAAAARPVAGTGLREKTAASLVNSFRLASVTQRLRYHIQDGAKVDPKEFQICCISFAKGIDFAIANNDIPKKVEEFPW! LLKQLCRHGTDVYTKTALMVLMISVKHACHLGWFSDSESQELIALADEIRTCFGSSGSTSPGIKSPGSTFSQIMERFYPF! VKLGHVLVSFEVKAGYTMLAHDFYISKNMPHSLQEKIRLFVAQTDNIDTSACISNPPEVSFLLNGKGVEKRVNIAMDTGP! QLPTNVTAQLKYGTNLLQVMGNFKGNYIIIIAFTGLVVPPEKPVLKDYLQSGVIEASPDSDIIEGPSRVSLSCPISRKRI! KLPVKGQLCKHLQCFDFSNYVHINMRNPTWRCPHCNQPVCYPDIRLDQNMAKILKDVEHNAADVIIDAGGTWKVTKNTGE! TPEPVREIIHDLEDPMSLLNSGPVVFDLTGDDDAELEVFGDNKVEDRKPCMSDAQGQSNNNNTNKHPSNDDYSSIFDISD! VIALDPEILSALGNTAPQPHQASNTGTGQQYSNLSQIPMSIDPMPVPVPFSQTPSPRDRPATTSTVFTIPNPSPQYSQVH! ASPVTPTGTYLGRTTSPRWNQTYQSQAPPMTTPYTSRKVSVPVTSQSPANVSSFVQSQHVPRVLSQPNNYGVRGLTSSHA! STSRQHPSGPTVQSVSRLSDLVDVDLTVPDTSNWRPRMRGSLVPGSHSTALDHMIIRPSQQSQTSTRLNSSQPVQTPSVQ! TSQAQSPFTTAAYRTETVLGNRNHPVPAPPGIVRPTGPTS*! Supplemental Figure 2. Open reading frame and protein sequence of PIAL2 as determined by sequencing of cdnas. 2

3 A Supplemental Fig. 3 3

4 B C Supplemental Figure 3. Alignment and structure of SP-RING proteins. A, Alignment of SUMO ligase protein sequences. Arabidopsis proteins PIAL1, PIAL2 and SIZ1 were aligned with human SUMO ligase PIAS1 and yeast SUMO ligase SIZ1. All of these proteins contain the SP-RING (zf-miz) domain as region of highest similarity. B, SP-RING (zf-miz) domain structure as modeled for PIAL2. C, Compared to PIAL2, PIAL1 contains an insertion of seven 23 amino acid repeats, which were aligned to emphasize the repeat structure (numbers denote amino acids in PIAL1). Supplemental Fig. 3 (contd.) 4

5 Supplemental Figure 4. Alignment and phylogenetic tree of plant SUMO ligases. Top, the sequence of the SP-RING domain of different plant SUMO ligases was aligned for building a phylogenetic tree. Bottom, phylogenetic tree, equivalent to the one shown in Fig. 1C, but with bootstrap values listed at the branch points. See also text and legend to Fig. 1 for further explanations. Entries were from Novatchkova et al. (2012). 5

6 Supplemental Figure 5. Gene models of PIAL1. Comparison of the intron-exon structure proposed by two repositories, TAIR (purple, and ARTADE (green, omicspace.riken.jp/artade/), with the structure obtained experimentally in this work (red). The cdna is identical to the TAIR model except for position of intron 15, and differs from the ARTADE model in the position of intron 2 and in one exon, which is part of intron 14 in the TAIR model and in the cdna. 6

7 Supplemental Figure 6. Expression of PIAL1 and PIAL2 in different tissues. RNA from the indicated tissues was isolated and used for RT-PCR (three technical replicates). AtUBC9 served as positive control. Transcripts were detected in young rosette leaves, cauline leaves, flowers, and stems. Expression was low in siliques and old leaves. Thus, the genes are apparently broadly expressed, but mostly at low levels. 7

8 WT pial1 pial2 pial1 pial2 pial1 pial2 pial1 pial1 pial2 pial2 pial1 pial2 Supplemental Figure 7. Growth habit of SUMO ligase mutants. pial1, pial2 or double mutants grow like WT under standard greenhouse conditions. In contrast, combination of PIAL mutations with SIZ1 loss of function leads to progressively decreased growth compared to. Plants were ca. 6 weeks old except in the top row, which shows plants aged ca. 4 weeks. 8

9 Growth of seedlings on plates without additives WT pial2-1 pial2-2 pial2-2 pial2-1 pial2-1 pial2-2 pial2-2 pial2-1 Growth of seedlings on plates with 300 mm mannitol WT pial2-1 pial2-2 pial2-2 pial2-1 pial2-1 pial2-2 pial2-2 pial2-1 Supplemental Fig. 8 9

10 Growth of seedlings on plates with 2.5 µm abscisic acid WT pial2-1 pial2-2 pial2-2 pial2-1 pial2-1 pial2-2 pial2-2 pial2-1 Growth of seedlings on plates with 150 mm NaCl WT pial2-1 pial2-2 pial2-2 pial2-1 pial2-1 pial2-2 pial2-2 pial2-1 Supplemental Fig. 8 (contd.) 10

11 fresh weight (mg) Supplemental Figure 8. Seedling growth without additives, or in presence of mannitol, ABA, or NaCl. Seeds of the indicated genotype were germinated on MS plates, and seedling growth was monitored after two weeks. For quantitation of seedling size, see Fig. 2. The diagram combines four diagrams of Fig. 2A in one graph. Supplemental Fig. 8 (contd.) 11

12 Supplemental Figure 9. Protein blots and Coomassie stained gels used to assess global SUMO conjugate levels., extract from plants grown on normal plates; +, extract from plants grown on plates containing 300 mm mannitol. Data with mannitol did in general not differ from data without. Lanes empty in the top images contain marker proteins (visible in the bottom images). A pair of identical gels was run in parallel, one was subsequently stained with Coomassie brilliant blue, the other one used for protein transfer to PVDF membranes and subsequent incubation with primary and secondary antibody. Resultant images were quantified using ImageJ software. Values were first normalized to protein content as determined by Coomassie staining, and then compared to the value of Col-0 WT without stress treatment. Each gel contained Col-0 WT samples to correct for blot-to-blot variation. 12

13 E1 + E2 PIAL2M SIZ1 fr. PIAL2 FL An: SUMO Immunoblot (strep tag) Supplemental Figure 10. PIAL2, but not SIZ1 catalyzes SUMO chain formation. In vitro SUMO chain formation assay using full length PIAL2 (PIAL2 FL), fragment PIAL2M, or a fragment of Arabidopsis SUMO ligase SIZ1 (SIZ1 fr.; all three linked to maltose binding protein) indicates that PIAL2, but not SIZ1 enhances SUMO chain formation. PIAL2M is not influenced by SIZ1 in its activity. 13

14 A EKPVLKDYLQ SGVIEASPDS DIIEGPSRVS LSCPISRKRI KLPVKGQLCK HLQCFDFSNY VHINMRNPTW RCPHCNQPVC YPDIRLDQNM AKILKDVEHN AADVIIDAGG TWKVTKNTGE TPEPVREIIH DLEDPMSLLN SGPVVFDLTG DDDAELEVFG DNKVEDRKPC MSDAQGQSNN NNTNKHPSND DYSSIFDISD VIALDPEILS ALGNTA* B C Supplemental Fig

15 Supplemental Figure 11. In vitro auto-sumoylation of PIAL2. A, Amino acid sequence of the region of PIAL2 contained in the maltose binding protein fusion protein PIAL2M. SP-RING in green, two SUMO1 modified Lys residues in bold. Two potential SUMO interaction motifs (SIMs) are in red. Two Cys residues mutated to inactivate the SP-RING are in bold blue. B, After conversion of two Lys residues to Arg (boldface in panel A), PIAL2M is still modified in vitro by (auto)sumoylation, as shown by Western blot of reaction contents using anti-mbp antibody (lanes 1 and 3, protein before in vitro reaction; lanes 2, 4, after reaction). C, Western blotting of in vitro reaction contents using anti-strep tag antibody to detect SUMO1 chains. SUMO modification of PIAL2M at the indicated Lys residues lowers the (initial) reaction rate of SUMO chain formation. Lanes 5, 8, SUMO1 input; lanes 6, 9, reaction with WT PIAL2M; lanes 7, 10, reaction with Lys to Arg mutant of PIAL2M (both bold Lys residues of panel A changed to Arg). Lanes 5 7, one hour reactions; lanes 8 10, reactions were allowed to proceed for two hours. Supplemental Fig. 11 (contd.) 15

16 Supplemental Figure 12. Auto-sumoylation of PIAL2 M indicates interaction with SUMO1, SUM3, SUM5 and SUM7. Protein blots with anti Maltose binding protein antibody to detect PIAL2M was assayed in vitro using different Arabidopsis SUMO isoforms as indicated. All tested isoforms result in auto-sumoylation. 16

17 A B ligase input Supplemental Figure 13. Dele$on analysis of PIAL2M. (A), Dele$on constructs tested in panel B. Yellow bars indicate intact func$onal elements (long bar, SP- RING; short bar, puta$ve SUMO interac$on mo$f, SIM1; red bar indicates mutated SIM1). (B), Ac$vity of dele$on constructs as determined by an$- SUMO protein blot of in vitro SUMO conjuga$on reac$ons. BoOom insert shows sec$on of a Coomassie- stained gel to document the amount of PIAL2M variant protein added to the reac$on. 17

18 A B Supplemental Fig

19 C Supplemental Figure 14. Kine%cs of in vitro SUMO conjuga%on. Components for in vitro SUMO conjuga%on were mixed as described in Methods. SAE, SCE and SUMO1 were either allowed to react without addi%onal protein (lanes 2, 5, 8, 11, 15, 18, 21, 24), or PIAL2M was added (lanes 4, 7, 10, 13, 17, 20, 23, 26). The VFDL to AAAA mutant PIAL2Msim1 was added to the reac%ons of lanes 3, 6, 9, 12, 16, 19, 22, and 25. Aliquots were withdrawn aver 0.5 hr, 1 hr, 2 hr and 16 hr. Note that at the 16 hr %me point, most of monomeric SUMO (dot to the right) is converted into chains. PIAL1Msim1 is apparently more ac%ve than PIAL2M (lanes 9 vs. 10 and 16 vs. 17). Lanes 1 and 14 show SUMO input. (A), Reac%ons with 0.15 µm PIAL2M / PIAL2Msim1 (B), Reac%ons with 1.5 µm PIAL2M / PIAL2Msim1 (C), Quan%fica%on of panels (A) and (B). 2 hr reac%on without ligase was used as reference (rela%ve value set to 1). Supplemental Fig. 14 (contd.) 19

20 Supplemental Table 1. A Number of seedlings used for wet weight determination. Genotype WT pial1 pial2 pial1 pial2 pial1 pial2 pial1 pial2 Row sum no stress mm NaCl mm mannitol µm ABA Column sum B Dry weight of seedlings as per cent of wet weight Genotype WT pial1 pial2 pial1 pial2 150 mm NaCl 7.5±1% 6.9±0.4% 6.2±0.2% 9.1±0.4% 20

21 Supplemental Table 2. Metabolite concentrations. Metabolites pial2-2 pial2-1 WT asp ± ± ± ± 8.33 glu * ± * ± ± ± asn * ± ± ± ± 6.61 ser * ± * ± * ± ± gln * ± 9.05 * ± 9.51 *88.53 ± ± 3.95 gly * ± * ± *95.28 ± ± hsn ± ± ± ± 0.42 thr ± * ± * ± ± 4.01 his *14.34 ± 0.96 *13.43 ± 0.98 *17.14 ± ± 0.58 ala * ± ± * ± ± arg * ± * ± * ± ± 2.99 tyr *4.20 ± 0.34 *4.04 ± ± ± 0.16 val * ± * ± * ± ± 6.48 met *5.31 ± 0.45 *4.30 ± 0.40 *3.82 ± ± 0.30 trp *54.49 ± 4.98 *56.48 ± ± ± 1.82 phe *22.25 ± ± ± ± 1.33 ile ± ± ± ± 0.56 fructose *1.80 ± 0.22 *1.64 ± ± ± 0.06 glucose *5.78 ± 0.52 *5.59 ± 0.61 *2.06 ± ± 0.38 citric acid *7.50 ± ± ± ± 0.86 sucrose *5.96 ± 1.13 *5.84 ± ± ± 0.20 putrescine 0.82 ± ± ± ± 0.07 spermidine 7.57 ± ± ± ± 0.68 lactate *5.90 ± 0.99 *8.45 ± ± ± 0.83 succinate *4.60 ± ± 0.35 *5.05 ± ± 0.23 glycerate 3.75 ± 0.41 *4.68 ± 1.05 *8.39 ± ± 0.26 fumarate 2.97 ± ± ± ± 0.10 malate 3.00 ± ± 0.70 *7.99 ± ± 0.27 sinapine 6.09 ± 0.61 *7.24 ± 0.72* 3.36 ± ± 0.45 xylose 6.30 ± ± ± ± 0.35 sorbose 6.47 ± 0.79 *8.79 ± 1.76 *8.15 ± ± 1.02 altrose *5.42 ± ± ± ± 0.18 galactose *7.43 ± 1.16 *11.01 ± ± ± 1.08 maltose 0.77 ± ± ± ± 0.12 tagatose 6.87 ± 0.74 *8.64 ± ± ± 0.96 The level of free amino acids in the leaves of wild type and SUMO ligase mutants are given in nmol/g FW. Metabolites determined by GC-MS in extracts of Arabidopsis leaves are given as ratio of the average of the relative amount of the internal standards. The data are presented as the mean ± SE obtained from four independent measurements. *Statistically significant at the 95% level (P < 0.05). 21

22 Supplemental Table 3. Primer sequences used for RNA detection and for qrt-pcr. # Primer Abbrev. Fwd Rev 1 At2g25680 SULTR5;2 AAATGCTGCCATAGGCTTTGTTGC CCCGTAGTTCCGCATCCACAAAAC 2 At1g80310 SULTR5;1 AAGCCGCTCCTCGTGATGTCTAAG AGCTGCCCGCCATTTCGTCATAAG 3 At3g12520 SULTR4;2 ACCACAGTGTGCTTTAGCAGCAAT TCTCTTGTCCACACGCCACAGA 4 At5g13550 SULTR4;1 GAGATCGGTGTCCTTGTTGGTGTT CCCAAGACAGCAATGTGAGGGTT 5 At5g19600 SULTR3;5 CAACGGGGCCATTTTCAAAGACAG AGAAGCACAAGCATCATGCAAACG 6 At3g15990 SULTR3;4 TCTTCAGCTTGTGCTGGTGAATCC TCCACTCAGACCCAATGCCTCAAT 7 At1g23090 SULTR3;3 AATGTCAGCCGTGAGCGGTGTA ACACAAGCTCGATGTCCTTCTTGG 8 At4g02700 SULTR3;2 TCACTACCGGGCCATTTTCACGTT TGCAACCGCCATCACCACGTTT 9 At3g51895 SULTR3;1 TCGTCGTAGCGGTGGCGATATCTA ACCGCAGTTTTTGGCCTCGACA 10 At1g77990 SULTR2;2 CGGTTAGTCATCGCTAGTCCCAGA TCTCGTCCAATTTTGCTCGCTTCA 11 At1g22150 SULTR1;3 GGCGATTACCTTCTCAAGGGCTTC TCCTATCGCTACAGCTTCCGTCAA 12 At5g10180 SULTR2;1 ATTGTTGCTCTAACCGAGGCGATT TGTACCCTTTTATTCCGGCGAACG 13 At1g78000 SULTR1;2 TCACCCTGTGGACGGAAGTC GTTTCATCGGAACATGTCCACC 14 At4g08620 SULTR1;1 TTCTGTCATGCACTCCGTATTCAA TGCCAATTCCACCCATGC 15 At5g43780 ATPS4 AAGATGCGTGGGTTGGCGAA CAACCAGAGGGACACATGAAACCA 16 At4g14680 ATPS3 GGCACTAAGATGAGAGCATTGGCA ATCCACAAGGACTTTCCAGCCTCC 17 At1g19920 ATPS2 TGGTTCTCTCCGAAGTGTGGAGAT TCCTGGAGAAGTAGTTCCCCAAGT 18 At3g22890 ATPS1 TGTTCATCTCCGGCACTAAGATGC TCCACCAGAACTTTCCATCCACCT 19 At4g39940 APSK2 GCTGATTTTCCCGCCCTTTCAGAA GCATATCGAACTCTCGTGCCACAC 20 At2g14750 APSK1 GAGCCACCATTGAACTGCGAGAT ATCCGACGACCTTTTCCGCCAT 21 At3g03900 APSKput1 TATACGCAGGGTCGGAGAAGTAGC GCAGGCGTCACGGTCTTTTCTATA 22 At5g67520 APSKput2 CGGTCCGGTGAAACGAAGCAAAT AGTAACGGGACAATCATGCCAGAC 23 At4g21990 APR3 GGAAGAGATCCTCCGTGAAAGC CTGTAACCTCAGAAGCAACAATGGA 24 At1g62180 APR2 TGATCGAACCCATTTGTCTCAGAG TCAGGAGCAATTAGAGTTGAAGCA 25 At4g04610 APR1 TCATTGGAGCCAAAAGTTTCGCAA TCAGAGACACAGGAGCAACATGAA 26 At1g08090 NRT2-1 TGCCAGATGGAAATCGAGCTACCT CGGCATACCACAGAATCTTTCCGA 27 At2g15620 Nitrite red GAAACCCCGATTTCACCAACTTGC CATGAGTCCCCACCACACACACAT 28 At4g05320 UBQ10 GGCCTTGTATAATCCCTGATGAATAAG AAAGAGATAACAGGAACGGAAACATAGT 29 At5g60390 EF1a TGAGCACGCTCTTCTTGCTTTCA GGTGGTGGCATCCATCTTGTTACA 30 At1g GAPDH5' TCTCGATCTCAATTTCGCAAAA CGAAACCGTTGATTCCGATTC 31 At1g GAPDH3' TTGGTGACAACAGGTCAAGCA AAACTTGTCGCTCAATGCAATC 32 At5g65080 INTRON TTTTTTGCCCCCTTCGAATC ATCTTCCGCCACCACATTGTAC 33 At1g08910 PIAL1 CATTATCTCCCCCGGAAAACTTCT CATCTGCAGCATTACGTCCCACTT 34 At5g41580 PIAL2 GTCCGGTGGCTGGAACTGGCTTAC CCCTTGTTCTAGCCTCTCAAGTTCT 35 At4g27960 UBC9 GTTGCGGAAGACATGTTTCATTGGCAGG CCATGGCATACTTTTGGGTCCAGGTCC Primer sequences of investigated genes with abbreviations (Abbrev.) and ATG numbers. SULTR Sulfate transporter, ATPS ATP sulfurylase, APSK APS-kinase, APR APS-reductase, NRT Nitrate transporter, UBQ Ubiquitin, EF1a elongation factor. 22

23 Supplemental Table 4. Vectors used in this work. pet42c-nafa pet9d- SAE1c2corr pet9d-sce1 pet9d-sce1 K15R pet-tag3-sum1 pet-tag3-sum1 H89K pet-tag3-sum3 pet-tag3-sum5 pet-tag3-sum7 pmal-pial1 pmal-pial1m pmal-pial1s pmal-pial2 pmal-pial2m pmal-pial2m- K2R pmal-pial2msim1 pmal-pial2msim1/sim2 pmal-pial2msim2 pmal-pial2m-spring pmal-pial2m-spring/sim1 pmal-pial2m-spring/sim2 pmal-pial2m-δ1 pmal-pial2m- Δ1-sim1 pmal-pial2m-δ2 pmal-pial2m- Δ2-sim1 pmal-pial2m-δ3 pmal-pial2m- Δ3-sim1 pmal-pial2m-δ4 pmal-pial2s Nucleosome Assembly Factor, a known sumoylation substrate, Flagtagged (Budhiraja et al., 2009). Dicistronic construct for the expression of both subunits of SUMO E1 in E. coli, His-tag on SAE2 (Budhiraja et al., 2009). A construct for the expression of SUMO E2 SCE1, no tags (Budhiraja et al., 2009). A mutant of SCE1 where the sumoylation site, identified by mass spectrometry, was substituted with arginine. The SUMO1 construct predominantly used for in vitro SUMOylation assays, carries a Strep-3xHA-8xHis tag (Budhiraja et al., 2009). SUMO1 used for mass spectrometry analysis, His 89 substituted with Lys to give a QTGG fragment after tryptic digest (Miller et al., 2010). SUMO3, used for in vitro SUMOylation assays, carries a Strep-3xHA- 8xHis tag (Budhiraja et al., 2009). SUMO5, used for in vitro SUMOylation assays, carries a Strep-3xHA- 8xHis tag (Budhiraja et al., 2009). SUMO7, used for in vitro SUMOylation assays, carries a Strep-3xHA- 8xHis tag (Budhiraja et al., 2009). MBP fusion construct for expression of the full-length PIAL1. MBP fusion construct for expression of the N264-P445 fragment of PIAL1 MBP fusion construct for expression of the S290-A353 fragment of PIAL1 MBP fusion construct for expression of the full-length PIAL2 MBP fusion construct for expression of the E281-A496 fragment of PIAL2. A mutant of PIAL2M where the two SUMOylation sites identified by mass spectrometry (K372, K448) were substituted with arginines. A mutant of PIAL2M where the SIM interacting motif 1 (VFDL ) was substituted with alanines. A double mutant of PIAL2M, combining the VFDL AAAA and the IFDI AAAA A mutant of PIAL2M where the SIM interacting motif 2 (IFDI ) was substituted with alanines. A mutant of PIAL2 where two Zn 2+ coordinating cysteines (C329, C355) of the SP-RING were substituted with alanines. A double mutant of PIAL2M, combining the C329A, C355A and VFDL AAAA mutations A double mutant of PIAL2M, combining the C329A, C355A and IFDI AAAA mutations A truncation of PIAL2M,encompassing S307-A496. A truncation of PIAL2M, encompassing S307-A496with VFDL to AAAA replacement. A truncation of PIAL2M, encompassing E281-P466. A truncation of PIAL2M, encompassing E281-P466 with VFDL to AAAA replacement. A truncation of PIAL2M, encompassing E281-K448. A truncation of PIAL2M, encompassing E281-K448with VFDL to AAAA replacement. A truncation of PIAL2M, encompassing E281-D414 MBP fusion construct for expression and Western blot detection of the S307-A388 fragment of PIAL2 23