et al. (2013). plant cell 10.1105/tpc.113.117192 · 2013-11-20 · mimulus guttatus popolus...

Supplemental Data. Hackenberg et al. (2013). Plant Cell 10.1105/tpc.113.117192

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Supplemental Figure 1. Complementation of the nca1 mutant phenotype.

(A) Two-week old seedlings grown on plates without hydroxyurea. The genotype is indicated

in the top left corner and the construct introduced is shown in the lower left corner where

applicable. The nca1 mutants are characterized by their small size and yellow cotelydons. Pp:

Physcomitrella patens. Scale bar is 1 mm. All images are to scale. (B) Catalase activities of

nca1-1 mutants carrying the indicated constructs. Three independent transgenic lines were

used as biological replicates and each was measured in technical triplicates. Error bars

indicate SEM. One-way ANOVA followed by Dunnett's post test was used to calculate p-

values. (*: P < 0.05, **: P < 0.01). (C) Ion leakage on challenge with Pto DC3000 (avrRpm1).

Two independent lines, O1 and O2, are shown. These two lines were assayed in the

experiment illustrated in Figure 2A and therefore the Col-0, nca1-1 and rpm1-3 data is

identical to that in Figure 2A.


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Species Accession number Arabidopsis thaliana Ath: AT3G54360 NCA1 Arabidopsis lyrata Aly:485837 Cucumis sativus Csa:Cucsa.281150.1 Glycine max 1 Gma:Glyma04g15130.1 Glycine max 2 Gma:Glyma06g47010.1 Mimulus guttatus Mgu:mgf005331m Popolus trichocarpa 1 Ptr:POPTR_0003s18860.1 Popolus trichocarpa 2 Ptr:POPTR_0001s07350.1 Manihot esculenta Mes:cassava1463.valid.m1 Ricinus communis Rco:29686.m000895 Zea mays Zma:GRMZM2G049672_P03 Sorghum bicolor 1 Sbi:Sb03g008890.1 Sorghum bicolor 2 Sbi:Sb07g001060.1 Brachypodium distachyon 1 Bdi:Bradi1g33800.1 Brachypodium distachyon 2 Bdi:Bradi1g09110.1 Oryza sativa 1 Osa:LOC_Os02g55200.2 Oryza sativa 2 Osa:LOC_Os01g01420.2 Physcomitrella patens Ppa:115595 Selaginella moellendorfii Smo:154638

Supplemental Figure 2. NCA1 phylogeny.

Phylogenetic tree of NCA1-related protein sequences. Numbers at nodes show bootstrap

confidence values with 1000 indicated maximum confidence. The bar shows an evolutionary

distance corresponding to 0.2 amino acid substitutions per site. Species used in this study are

shown in bold.

1000

572872

611

1000998

1000

995

1000978

1000

1000

1000

0,200

580

1000

1000

Magnoliophyta

eudicotyledons

monocotyledons

Arabidopsis thalianaArabidopsis lyrataCucumis sativusGlycine max 1Glycine max 2Mimulus guttatusPopolus trichocarpa 1Popolus trichocarpa 2Manihot esculentaRicinus communisZea maysSorghum bicolor 1Sorghum bicolor 2Brachypodium distachyon 1Brachypodium distachyon 2Oryza sativa 1Oryza sativa 2Physcomitrella patensSelaginella moellendorfii


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Ch

r

Po

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Ref b

ase

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base

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istance

Co

re sup

po

rt

Co

nco

rdan

ce

Base q

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SN

P typ

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rm

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Co

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wt A

A

mu

t AA

3 6642770 G A 23485 3 1.00 40 NEWSNP CDS AT3G19190 1 1878 3 Nonsyn W * 3 6691289 G A 25034 3 1.00 40 NEWSNP intronic/noncoding AT3G19300 1 3 6710942 G A 44687 4 1.00 40 NEWSNP intronic/noncoding AT3G19370 1 3 6896641 G A 230386 4 1.00 40 NEWSNP intronic/noncoding AT3G19840 1 3 6918098 G A 251843 6 1.00 40 NEWSNP intronic/noncoding AT3G19895 1 3 6308262 G A 357993 7 1.00 40 NEWSNP CDS AT3G18370 1 889 1 Nonsyn V I 3 7028540 T G 362285 3 1.00 40 NEWSNP CDS AT3G20130 1 1469 2 Nonsyn V G 3 6271374 G T 394881 3 1.00 40 NEWSNP intergenic 3 7081829 G A 415574 5 1.00 40 NEWSNP five_prime_UTR AT3G20300 1 3 6231373 G A 434882 5 1.00 40 NEWSNP CDS AT3G18180 1 902 2 Nonsyn R Q 3 6200949 G A 465306 5 1.00 40 NEWSNP CDS AT3G18100 1 255 3 Syn A A 3 7185705 G A 519450 7 1.00 40 NEWSNP CDS AT3G20560 1 1047 3 Syn K K 3 6106333 G A 559922 3 1.00 40 NEWSNP CDS AT3G17840 1 236 2 Nonsyn G D 3 6101106 G C 565149 4 0.80 40 NEWSNP intergenic 3 6101102 A T 565153 4 0.80 40 NEWSNP intergenic 3 6073730 T A 592525 3 1.00 40 NEWSNP intergenic 3 7306635 G A 640380 7 1.00 40 NEWSNP intronic/noncoding AT3G20860 1 3 7517355 G A 851100 4 1.00 40 NEWSNP CDS AT3G21350 1 244 1 Nonsyn D N 3 7739220 T G 1072965 4 0.80 40 NEWSNP intergenic 3 7739221 T C 1072966 4 0.80 40 NEWSNP intergenic

Supplemental Table 1. SHOREmap output for the atg2-3 mutant. Candidate polymorphisms

for the hydroxyurea resistant mutant with normal catalase activity levels (atg2-3) are shown

prioritized with respect to the allele frequency peak (Peak distance).


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Ch

r

Po

sition

Ref b

ase

Called

base

Peak d

istance

Co

re sup

po

rt

Co

nco

rdan

ce

Base q

uality

SN

P typ

e

Locatio

n

Gen

e ID

Isofo

rm

OR

F po

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Co

do

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n

Co

do

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wt A

A

mu

t AA

3 20140743 C T 93688 3 1.0 40 NEWSNP CDS AT3G54360 1 896 2 Nonsyn G E 3 19898935 C T 148120 3 1.0 40 NEWSNP CDS AT3G53640 1 979 1 Nonsyn A T 3 19681433 C T 365622 9 1.0 40 NEWSNP intergenic 3 19598989 C T 448066 10 1.0 40 NEWSNP CDS AT3G52850 1 13 1 Nonsyn L F 3 19589130 C G 457925 4 0.8 40 NEWSNP CDS AT3G52830 1 148 1 Nonsyn L V 3 19570913 G A 476142 5 1.0 40 NEWSNP intergenic 3 20573676 C T 526621 4 1.0 40 NEWSNP intronic/noncoding AT3G55460 1 3 20744362 A T 697307 4 1.0 40 REFERR intronic/noncoding AT3G55870 1 3 20765256 C T 718201 6 1.0 40 NEWSNP CDS AT3G55950 1 2071 1 Nonsyn D N 3 20805990 A T 758935 4 1.0 40 REFERR CDS AT3G56040 1 1590 3 Nonsyn D G 3 20805991 T C 758936 4 1.0 40 REFERR CDS AT3G56040 1 1589 2 Nonsyn D G 3 19260194 T G 786861 3 1.0 40 REFERR Intergenic 3 19219272 A C 827783 7 1.0 40 REFERR five_prime_UTR AT3G51780 1 3 19219271 A G 827784 7 1.0 40 REFERR five_prime_UTR AT3G51780 1 3 19217855 A T 829200 5 1.0 40 REFERR three_prime_UTR AT3G51780 1 3 19217852 A T 829203 6 1.0 40 REFERR three_prime_UTR AT3G51780 1 3 19217851 A T 829204 6 1.0 40 REFERR three_prime_UTR AT3G51780 1 3 19197345 C T 849710 6 1.0 40 NEWSNP CDS AT3G51720 1 1072 1 Nonsyn L F 3 19093682 G T 953373 4 1.0 40 NEWSNP intergenic 3 19003919 C G 1043136 4 0.8 40 REFERR intergenic

Supplemental Table 2. SHOREmap output for the nca1-1 mutant. The candidate mutations

are prioritized with respect to the allele frequency peak (Peak distance).


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 S. bicolor 1 1 90 85 77 79 72 74 52 53 53 51 51 53 53 51 53 51 46 39 Z. mays 2 90 90 81 77 76 77 54 55 55 53 53 54 54 53 54 52 48 40 S. bicolor 2 3 85 90 79 76 74 76 54 54 54 53 53 54 54 54 54 53 48 39 B. distachyon 2 4 77 81 79 91 74 76 55 56 56 54 53 56 55 53 54 52 46 40 B. distachyon 1 5 79 77 76 91 69 71 52 54 54 52 51 54 54 52 53 51 45 38 O. sativa 2 6 72 76 74 74 69 92 57 58 58 52 53 57 57 52 53 52 47 41 O. sativa 1 7 74 77 76 76 71 92 57 58 58 53 54 56 56 52 54 52 49 42 M. guttatus 8 52 54 54 55 52 57 57 70 69 60 67 70 69 57 56 55 44 42 G. max 1 9 53 55 54 56 54 58 58 70 96 65 68 70 71 60 60 59 47 43 G. max 2 10 53 55 54 56 54 58 58 69 96 65 67 70 71 58 60 58 46 43 R. communis 11 51 53 53 54 52 52 53 60 65 65 75 71 72 60 56 56 47 41 M. esculenta 12 51 53 53 53 51 53 54 67 68 67 75 75 76 60 59 58 48 42 P. trichocarpa 2 13 53 54 54 56 54 57 56 70 70 70 71 75 89 60 60 58 45 41 P. trichocarpa 1 14 53 54 54 55 54 57 56 69 71 71 72 76 89 60 59 58 44 42 C. sativus 15 51 53 54 53 52 52 52 57 60 58 60 60 60 60 64 65 46 39 A. thaliana 16 53 54 54 54 53 53 54 56 60 60 56 59 60 59 64 92 46 38 A. lyrata 17 51 52 53 52 51 52 52 55 59 58 56 58 58 58 65 92 46 38 P. patens 18 46 48 48 46 45 47 49 44 47 46 47 48 45 44 46 46 46 45 S. moellendorfii 19 39 40 39 40 38 41 42 42 43 43 41 42 41 42 39 38 38 45

Supplemental Table 3. Comparison of NCA1 protein sequences across species. Percent

amino acid identity is shown. Accession numbers for individual sequences are given in

Supplemental figure 2.


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Supplemental Table 4. Unique catalase peptides. Replicate indicates the biological replicate

number. *No unique CAT2 peptides were detected in the cat2 mutant.

Peptide'sequence cat2/wt ncat1/wt Replicate Peptide'sequence cat2/wt ncat1/wt Replicate

CAT1 %& %& %& %& CAT3 dGIQFPDVVHALkPNPk 13.1% 9.5% 1yRPSSAYNAPFYTTNGGAPVSNNISSLTIGER 50.3% 47.7% 1

CAT2 gPILLEDYHLVEk ND* 15.3% 2 yRPSSAYNAPFYTTnGGAPVSNNISSLTIGER 71.7% 65.2% 1gPILLEDYHLVEk ND* 5.3% 2 iFAYGDTQR 46.1% 30.9% 2fPDMVHALkPNPk ND* 1.8% 2 tNIQEYWR 48.5% 33.2% 2fPDMVHALkPNPk ND* 1.8% 3 tNIQEYWR 43.3% 34.5% 2

CAT2%average 6.1% aPGVQTPVIVR 56.5% 60.3% 2aPGVQTPVIVR 40.8% 30.2% 2

CAT3 iFAYGDTQR 16.6% 19.6% 1 aPGVQTPVIVR 34.1% 27.8% 2tNIQEYWR 28.5% 27.8% 1 vPTPTNSYTGIR 48.1% 23.0% 2aPGVQTPVIVR 32.5% 33.9% 1 gPVLLEDYHLIEk 43.5% 20.2% 2eGNFDLVGNNTPVFFIR 18.1% 10.6% 1 gPVLLEDYHLIEk 45.6% 18.4% 2iFAYGDTQR 50.4% 36.9% 1 gPVLLEDYHLIEk 48.3% 20.2% 2tNIQEYWR 23.6% 20.7% 1 vPTPTNSYTGIR 49.0% 26.6% 2aPGVQTPVIVR 18.7% 14.7% 1 vPTPTNSYTGIR 45.6% 21.7% 2vPTPTNSYTGIR 34.9% 22.8% 1 eGNFDLVGNNTPVFFIR 53.7% 55.8% 2eGNFDLVGNNTPVFFIR 9.5% 7.0% 1 dLHDAIASGNYPEWk 79.8% 88.7% 2gPVLLEDYHLIEk 14.1% 8.8% 1 dGIQFPDVVHALkPNPk 44.1% 22.5% 2iFAYGDTQR 5.1% 10.3% 1 aPGVQTPVIVR 25.9% 14.7% 3tNIQEYWR 26.8% 16.1% 1 aPGVQTPVIVR 29.7% 16.1% 3aPGVQTPVIVR 50.9% 52.5% 1 vPTPTNSYTGIR 35.5% 16.0% 3eGNFDLVGNNTPVFFIR 11.1% 6.7% 1 tNIQEYWR 46.8% 33.6% 3vPTPTNSYTGIR 23.4% 15.6% 1 aPGVQTPVIVR 27.2% 17.1% 3gPVLLEDYHLIEk 19.0% 10.6% 1 eGNFDLVGNNTPVFFIR 25.1% 10.7% 3dLHDAIASGNYPEWk 36.0% 38.2% 1 aPGVQTPVIVR 27.8% 14.4% 3vPTPTNSYTGIR 19.6% 9.0% 1 vPTPTNSYTGIR 33.5% 17.0% 3vPTPTNSYTGIR 27.7% 16.0% 1 tNIQEYWR 44.0% 27.8% 3hMEGFGVHTYTLIAk 18.5% 11.7% 1 aPGVQTPVIVR 38.9% 29.8% 3eGNFDLVGNNTPVFFIR 46.7% 37.4% 1 eGNFDLVGNNTPVFFIR 24.6% 7.6% 3sWAPDRQDR 6.1% 3.7% 1 aPGVQTPVIVR 25.1% 15.3% 3dLHDAIASGNYPEWk 50.7% 38.3% 1 vPTPTNSYTGIR 32.1% 14.2% 3dLHDAIASGNYPEWk 17.3% 11.0% 1 fHWkPTcGIk 31.7% 12.0% 3dGIQFPDVVHALkPNPk 11.1% 8.7% 1 dLHDAIASGNYPEWk 33.7% 19.4% 3

continues)next)column dLHDAIASGNYPEWk 44.1% 28.5% 3CAT3%average 34.0% 24.1%


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Biological replicate 114 115 116 117

1 wild type cat2 nca1 1/3 wild type, cat2, and nca1



Supplemental Table 5. iTRAQ proteomics experiment setup. Four technical replicates were

performed for each of the three biological replicates. The resulting data can be found in

Supplemental Data Set 2 online.


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Name Sequence Target gene Used with Purpose

PM-01910 TTACGAAGGCGGTGTTTTTC At5g25760 PM-01911 transcript quantification

PM-01911 CAAGCAGGACTCCAAGCATT At5g25760 PM-01910 transcript quantification

PM-01912 TGCGAAATACCGAACATCAA At1g13320 PM-01913 transcript quantification

PM-01913 GGCCAAAATGATGCAATCTC At1g13320 PM-01912 transcript quantification

PM-01794 TTCAAACCCGTGTCTTCTCC At3g35090 (CAT2 ) PM-02549 transcript quantification

PM-02549 TTCTCAGCATGACGAACCTG At3g35090 (CAT2 ) PM-01794 transcript quantification

PM-04120 ttaaGGTACCcttgcaacacgtttcatctacg At3g54360 (NCA1) PM-04284 amplify pNCA1

PM-04284 atatAAGCTTtcgtgaaggtagcaacaacc At3g54360 (NCA1) PM-04120 amplify pNCA1

PM-04453 tataAAGCTTAAAGCAGGCTTCATGGTGAG eYFP PM-04454 amplify eYFP

PM-04454 tataAAGCTTTgtggtggtggtggtggtgGTACAGCTCGTCCATGCC

eYFP PM-04453 amplify eYFP

PM-03812 GGGGACAAGTTTGTACAAAAAAGCAGGCTCTATGACGACGACTTCTGTTTGC

At3g54360 (NCA1) PM-03815 amplify NCA1 cDNA & gDNA

PM-03815 GGGGACCACTTTGTACAAGAAAGCTGGGTTttcacagctgtagccgctta

At3g54360 (NCA1) PM-03812 amplify NCA1 gDNA

PM-03813 GGGGACCACTTTGTACAAGAAAGCTGGGTTTTAGAGTGCAGTTTCAGCATCG

At3g54360 (NCA1) PM-03812 amplify NCA1 cDNA

PM-03858 GGGGACAAGTTTGTACAAAAAAGCAGGCTCTATGGAAGTTTGTGAGGCGAAAG

PP_12622_C1 PM-03859 amplify PpNCA1 cDNA

PM-03859 GGGGACCACTTTGTACAAGAAAGCTGGGTTCTATGATGCAGAGTCGGTGAGC

PP_12622_C1 PM-03858 amplify PpNCA1 cDNA

PM-03807 GGGGACAAGTTTGTACAAAAAAGCAGGCTCTATGGATCCTTACAAGTATCGTC

At3g35090 (CAT2 ) PM-03808 amplify CAT2 cDNA

PM-03808 GGGGACCACTTTGTACAAGAAAGCTGGGTTTTAGATGCTTGGTCTCACGTTC

At3g35090 (CAT2 ) PM-03807 amplify CAT2 cDNA

PM-04122 TTAAGGTACCAGAAAATTGTTAAGACCAAACTACCC

At3g35090 (CAT2 ) PM-04285 amplify CAT2 promoter

PM-04285 ATATAAGCTTGGTTTGATGAGAAGAGAGCTTG

At3g35090 (CAT2 ) PM-04122 amplify CAT2 promoter

Supplemental Table 6. Oligonucleotide sequences.


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Gene Mutant Background Gene ID References NCA1 nca1-‐1 Col-‐0 At3g54360 current study NCA1 nca1-‐2 Col-‐0 At3g54360 current study CAT2 cat2-‐2 Col-‐0 At4g35090 5,6 NDR1 ndr1-‐1 Col-‐0 At3g20600 7

EDS1 eds1-‐1 Ws At3g48090 8

RPM1 rpm1-‐3 Col-‐0 At3g07040 9

ATG2 atg2-‐1 Col-‐0 At3g19190 10

ATG2 atg2-‐3 Col-‐0 At3g19190 current study ATG4A and ATG4B atg4a4b-‐1 Ws At2g44140, At3g59950 11

ATG5 atg5-‐1 Col-‐0 At5g17290 11

ATG7 atg7-‐1 Ws At5g45900 12

CAT1 -‐ -‐ At1g20630 -‐ CAT3 -‐ -‐ At1g20620 -‐ PEX4 -‐ -‐ At5g25760 13 PP2A -‐ -‐ At1g13320 13 UGT74E2 -‐ -‐ At1g05680 14 FES1A -‐ -‐ At3g09350 15

Supplemental Table 7. Accession numbers.


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Supplemental experimental procedures

Phylogenetic analysis

The phytozome family 23259700 comprises twenty-one protein member sequences, which

were downloaded from phytozome.net. The sequence Cpa:evm.model.supercontig_33.143

was disregarded, as it appears to be a partial sequence. For the sequence

Mes:cassava1463.valid.m1, the N-terminal part of the sequences up until the consensus start

methionine was disregarded to include only coding sequence. The phylogenetic tree was

generated by aligning using the T-Coffee and UPGMA implementations in CLC workbench

v. 5.6 with standard settings and 1000-fold bootstrap testing.

Cloning NCA1 and CAT2 constructs

The NCA1 promoter was amplified from Col-0 gDNA and cloned into a pGreen00291 vector

carrying a Gateway (GW) cassette using restriction sites KpnI and HindIII followed by eYFP2

amplification and cloning into the pGreen0029-pNCA1-GW plasmid using the HindIII site to

construct a pGreen0029-pNCA1-eYFP-GW (pMC-00637) plasmid. The same strategy was

used for cloning the CAT2 promoter to construct the pGreen0029-pCAT2-eYFP-GW (PMC-

00642) plasmid. NCA1 gDNA excluding promoter was amplified from Col-0 gDNA and

recombined into the pMC-00637 plasmid to create the pGreen0029-pNCA1-eYFP-NCA1

(pMC-00638, pNCA1:NCA1) in-frame fusion expression construct. NCA1 cDNA was

amplified from the U13379 clone, obtained from the Arabidopsis Biological Resource Center,

Columbus, USA and then recombined into a pEarleyGate1043 vector to create the


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pEarleyGate-35S-YFP-NCA1 (pMC-00570, 35S:NCA1) construct. P. patens NCA1 cDNA

was amplified from RNA extracted as described4 and recombined into pEarleyGate104 to

create the pEarleyGate-35S-YFP-PpNCA1 (pMC-00588, 35S:PpNCA1) construct. CAT2

cDNA was amplified from RNA extracted as described4 and recombined into PMC-00642 to

create the pGreen0029-pCAT2-eYFP-CAT2 (PMC-00647) expression construct.

iTRAQ proteomics

Samples were kept on ice and centrifuged at 4°C throughout the protein extraction procedure.

For each of the three biological replicates, between 50 and 60 mg of leaves were transferred

to 400 µL of PBS and ground using the Tissuelyser (Qiagen) for 3 min. Then, the sample was

centrifuged for 5 min at 14,000g and the supernatant was transferred and centrifuged at

100,000g for 45 min. The supernatant was transferred and (2 % SDS and 0.1g/mL sucrose)

were added to a final volume of 500 µL, mixed 1:1 with phenol (AppliChem, pH 8.0),

vortexed, and centrifuged at 10,000g for 5 min. The lower phase was transferred and proteins

were precipitated using 5 volumes of pre-chilled (-20°C) 100 mM ammonium acetate in

methanol for 30 min at - 20°C and centrifuged at 10.000g for 5 min. The pellet was washed

twice in 100 mM ice-cold ammonium acetate in methanol and twice in 80% acetone. The

pellet was dried and re-dissolved in 50 µL sample buffer (7 M urea, 2 M thiourea, and 200

mM triethylammonium hydrogen bicarbonate buffer (TEAB)) for 3 hours and the protein

concentration was determined using Qubit Quantitation Kit (Invitrogen). The disulfide bonds

were reduced using 10 mM DTT for 1 hour at 25°C followed by alkylation using 40 mM

iodoacetamide for 40 min in the dark. Then, 50 µg protein was transferred and diluted to 1 M

urea and digested overnight at 37°C with 1:50 of trypsin (Promega). A final concentration of


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1 % FA was added and clean-up of peptides was performed using half of POROS 50 R2 and

POROS 50 R3 as previously described (Larsen et al., 2002) and dried. Peptides were

redissolved in 50 µL 200 mM TEAB (pH 8,5) and the peptide concentration was determined

using Qubit Quantitation Kit (Invitrogen). The labelling with the 4-plex iTRAQ reagents

(Applied Biosystems) was performed according to the manufacturer’s recommendations. To

each vial, 70 µL of ethanol was added, combined with peptides, and incubated 1 hour and

dried. Supplemental table S6 shows the experimental set-up together with the amount of

peptides used for each biological replicate. Prior to LC-MS, peptides were redissolved in (90

% acetonitrile and 0.1 % TFA) and cleaned up and fractionated using a TSK Amide-80 HILIC

column in a microHPLC system as previously described (Palmisano et al., 2010).

Then, the labelled peptides were redissolved in mobile phase A buffer (aqueous 0.1 % formic

acid) for the LC-MS/MS analysis. A nanoflow RP HPLC (EASY-nanoLC, Proxeon

Biosystems, Denmark) in line with a nano-electrospray LTQ-Orbitrap XL mass spectrometer

(Thermo Fisher Scientific, USA) was used. The labelled peptides were separated on a two

column system with a C18 pre- and analytical column (home-packed with either ReproSil-Pur

(5 µm material) or C18-AQ (3 µm material)). The labelled peptides were either analysed as

one total fraction (3 µg peptides) or separated into ten sub-fractions where each sub-fraction

was analysed individually and subsequently combined. For elution, a 150 min gradient for the

total fraction and a 100 min gradient for the sub-fraction from 0 % to 28 % of mobile phase B

buffer (0.1 % formic acid in acetonitrile) was used with a constant flow of 250 nl/min. Mass

spectra were generated in the positive ion mode with data dependent acquisition. Survey MS

scans in the m/z range 400–1800 were acquired in the Orbitrap at a resolution of 60000 at 400

m/z. Data-dependent collision induced dissociation (CID) MS/MS analysis of the three most


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intense ions was performed in the LTQ followed by higher energy collision dissociation

(HCD) MS/MS analysis of the corresponding ions with detection in the Orbitrap.

For MS/MS, the instrument was set at 30.000 at 400 m/z resolution, 15.000 signal threshold,

500-ms maximum ion injection time, 2.5 m/z isolation width, 30-ms activation time at 35

normalized collision energy and dynamic exclusion enabled for 30 s with a repeat count of 1

to do CID. MS/MS for acquiring HCD was set at 7,500 at 400 m/z resolution, 30.000 signal

threshold, 5-ms activation time at 48 normalized collision energy and dynamic exclusion

enabled for 30 s with a repeat count of 1.

Finally, the raw data were deconvoluted and converted into peak lists by ProteomeDiscoverer

software version 1.2 (Thermo Scientific), which were then used for searches against the

Arabidopsis proteome using Mascot version 2.3. The searches were performed with the

following parameters: MS accuracy 10 ppm, MS/MS accuracy 0.6 Da for CID and 0.1 Da for

HCD, trypsin digestion, two missed cleavage allowed and fixed carbamidomethyl

modification of cysteine. Variable modifications were oxidized methionine together with

iTRAQ 4plex (monoisotopic mass = 144.102) for N-terminus and lys amino acid residues.

iTRAQ quantification was performed using Proteome Discoverer based in reporter ion

integration within 50 ppm window.


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