A T6ODM MEKAKLMKLGNGMEIPSVQELAKLTLAEIPSRYVCANENLLLPMGASVIN Contig36010 MEKAKLMKLGNGMEIPSVQELAKLTLAEIPSRYVCTDENLLLPMGASVIN Contig40340 T6ODM DHETIPVIDIENLLSPEPIIGKLELDRLHFACKEWGFFQVVNHGVDASLV Contig36010 DHETIPVIDIENLLSPEPIIGKLELDRLHSACKEWGFFQVVNHGVDASLV Contig40340 T6ODM DSVKSEIQGFFNLSMDEKTKYEQEDGDVEGFGQGFIESEDQTLDWADIFM Contig36010 DSVKSEIQGFFNLSMDEKTKYEQEDGDVEGFGQGFIASEDQTLDWADIFM Contig40340 T6ODM MFTLPLHLRKPHLFSKLPVPLRETIESYSSEMKKLSMVLFNKMEKALQVQ Contig36010 MFTLPLHLRKPHLFSKLPVPLRETIESYSSEMKKLSMVLFNKMEKALQVQ Contig40340 RETIESYSSEMKKLSMVLFNKMEKALQVQ T6ODM AAEIKGMSEVFIDGTQAMRMNYYPPCPQPNLAIGLTSHSDFGGLTILLQI Contig36010 AAEIKGMSEVFKDGTQAMRMNYYPPCPQPNLAIGLTS Contig40340 AAEIKGMSEVFKDGTQAMRMNYYPPCPQPNLAIGLTSHSDFGGLTILLQI T6ODM NEVEGLQIKREGTWISVKPLPNAFVVNVGDILEIMTNGIYHSVDHRAVVN Contig36010 EIMTNGIYHSVDHRAVLN Contig40340 NEVEGLQIKREGTWISVKPLPNAFVVNVGDILEIMTNGIYHSVDHRAVLN T6ODM STNERLSIATFHDPSLESVIGPISSLITPETPALFKSGSTYGDLVEECKT Contig36010 STNERLSIATFHDPSLESVIGPISSLITPETPALFKSGSTYG CKT Contig40340 STNERLSIATFHDPSLESVIGPISSLITPETPALFKSGSTYGDLVEECKK T6ODM RKLDGKSFLDSMRI Contig36010 RKLDGKSFLDSMRM Contig40340 RKLDGKSFLDSMRM

B COR MESNGVPMITLSSGIRMPALGMGTAETMVKGT-EREKLAFLKAIEVGYRH Singlet99907 MESNGVPMITLSSGIRMPALGMGTAETMVKGT-EREKLAFLKAIEVGYRH Singlet96716 MESNGVPMITLSSGIRMPALGMGTVETMEKGT-EREKLAFLKAIEVGYRH Contig44472 MESSGVPVITLGSGKVMPVLGMGTFEKVGKGS-ERERLAILKAIEVGYRY Contig34291 MENSGVPVITLSSSKGMPVLGLGTAENLTKGS-ERE--AILKAIEAGYRH Singlet89743 MESSGVPVITLGSGKVMPVLGMGTFEKVGKGS-ERERLAILKAIEVGYRY Contig22291 MESNGVPIITLSSGRGMPVLGMGTCETLGKGSHERERLAILKAIEVGYR- Contig32681 Contig21418 MENSGVPVITLSSGKGVPVLGLGTAE--LKGS-ERVKLAILKAIEVGYRH Contig1346 MESNGVPMITLSSGIRMPALGMGTVETMEKGT-EREKLAFLKAIEVGYRH Contig6762 COR F-DTAAAYQSEECLGEAIAEALQLGLIKSRDELFITSKLWCADAHADLVL Singlet99907 F-DTAAAYQSEECLGEAIAEALQLGLIKSRDELFITSKLWCADAHADLVL Singlet96716 F-DTAAAYQTEECLGEAIAEALQLGLIKSRDELFITSKLWCADAHADLVL Contig44472 F-DTAAAYETEEVLGEAIAEALQLGLVKSRDELFISSMLWCTDAHADRVL Contig34291 F-DTAFIYQTEESVGEAIAEALQLGFIKSRDELF-TSKLWGSDAHPDRVL Singlet89743 F-DTAAAYETEEVLGEAIAEALQLGLVKSRDELFISSMLWCTDAHADRVL Contig22291 FIDTAAAYGTEEVVGEVISEALQLGLIKSRDELFISSQLWCTDAHPDRVL Contig32681 Contig21418 F-DTAFVYQTEGSVGDAVAEALQLGLIKSRDELFITSKLWCADAYPDHVL Contig1346 F-DTAAAYQTEECLGEAIAEALQLGLIKSRDELFITSKLWCADAHADLVL Contig6762 COR PALQNSLRNLKLEYLDLYLIHHPVSLKPGKFVNEIP-KDHILPMDYKSVW Singlet99907 PALQNSLRNLKLEYLDLYLIHHPVSLKPGKFVNEIP-KDHILPMDYKTVW Singlet96716 PALQNSLRNLKLDYLDLYLIHHPVSLKPGKFVNEIP-KDHILPMDYKSVW Contig44472 LALQNSLRNLKLEYVDLYMLPFPASLKPGKI Contig34291 LALQNSLRNLKLQYLDLYLIHYPVSLKPGTTVKDLGNKDNFLPMDYKSVW Singlet89743 LALQNSLRNLKLEYVDLYMLPFPASLKPGKITMDIPEED-ICRMDYRSVW Contig22291 LALQNSLRNLRLEYLDLYLIHFPVSLNPGELVMDVPG-DEIFLMDYRSVW Contig32681 Contig21418 PALQNSLRNLKLEYLDLYLIHWPVSLKPGKFVHPIP-KDEIFPIDYKSVW Contig1346 PALQNSL SLKPGKFVNEIP-KDHILPMDYKSVW Contig6762 COR ALRAMEECQTLGFTRAIGVSNFSCKKLQELMAAAK-----IPPVVNQVEM Singlet99907 ALRAMEECQTLGFTRAIGVSNFSCKKLQELMAAAK-----IPPVVNQVEM Singlet96716 ALRAMEECQTLGFTRAIGVCNFSCKKLQELMATAN-----SPPVVNQVEM Contig44472 Contig34291 ALRAMEECQKLGLTKSIGVSNFSSKKIQELMSTAS-----IPPAVNQVEM Singlet89743 ALRAMEECQNLGFTKSIGVSNFSCKKLQELMATAN-----IPPAVNQVEM Contig22291 ALRAMEECQNLGFTKSIGVSNFSIKKLQELMAIAN-----IPPVVNQVEM Contig32681 KVEM Contig21418 ALRAMEKCQMLGLTKSIGVSNFSCKKLQYLMATAN-----IPPAVNQVEM Contig1346 ALRAMEECQTLGFTRAIGVCNFSCKKLQELMATANRGKSVSPPVVNQVEM Contig6762 NFSCKKLQELMATAK-----IPPVVNQVEM COR SPTLHQKNEYCKANNIMITAHSVLGAIGAPWGSNAVMDSKVLHQIAVARG Singlet99907 SPTLHQKNEYCKANNIMITAHSVLGAIGAPWGSNAVMDSKVLHQIAVARG Singlet96716 SPTLHQKNEY Contig44472 Contig34291 NPTWQQKKEYCQANNILVTAYSTLGAKGTGWGSNAVMGSEVLNQIALARG Singlet89743 SPAFQQKKEYCNANNILVSAISVLGSNGTPWGSNAVLGSEVLKKIAMAKG Contig22291 SPVFQQKNEYCKANNILVTAISVLGSKGTPWGSNAVLGSEVLNQIATARG Contig32681 SPTLHQKNEYCKANNIMITAHSVLGAVGAAWGTKAVMHSKVLHQIAVARG

Contig21418 NPIWQQQKDYCKSNNIMVTAYSPLGAKGTMWGSSGVMDSEVLNQISQVRG Contig1346 SPTLHQKNEYCKANNIMITAHSVLGAVGAAWGTNAVMHSKVLHQIAVA Contig6762 SPTLHQKNEYCKANNIMITAHSVLGAIGAPWGSNAVMDSKVLHQIAVARG COR KSVAQVSMRWVYQQGASLVVKSFNEGRMKENLKIFDWELTAEDMEKISEI Singlet99907 KSVAQVSMRWVYQQGASLVVKSFNEGR Singlet96716 RWVYQQGASLVVK Contig44472 Contig34291 KSIAQISLRWVYEQGVSLVVKSFNEERMRENLKIFDWELTAEDLKKIDEL Singlet89743 KSVAQVSMRWVYEQGASLVVKSFSEERLRENLNIFDWELTKEDHEKIGEI Contig22291 KSIAQVSLRWVYEQGASLVVKSFDEDRMRENLNIFDWELTKEDSEKIIVI Contig32681 KSVAQVSMRWVYQQGASLVVKSFNEGRMKENLKIFDWELTAED--KISEI Contig21418 KSVAQVSLRWVYEQGASLLVKSFNEERMKENLKIFDWELSPEDLKKISEL Contig1346 KISEI Contig6762 KSVAQVSMRWVYQQGASLVVKSFNEARIEENLKIFDWELTAEDMEKISEI COR PQSRTSS-AAFLLSPTGPFKTEEEFWDEKD Singlet99907 Singlet96716 Contig44472 Contig34291 PQSRVAT-AAFVVSETGPFKSLEEFWDDE Singlet89743 PQCRILS-AYFLVSPNGPFKSQEELWDDE Contig22291 PERKIII-ENFMVSVNGPFKSVEEFWD Contig32681 PQSRTSS-ADFLLSPTGPFKTEEEFWDEKD Contig21418 PQRRVSTGDPF-VSINGPFKSVEELWDDEV Contig1346 PQSRTSS-AAFLLSPTGPF Contig6762 PQSRTSS-AAFLLSPTGPFK

C CODM METPILIKLGNGLSIPSVQELAKLTLAEIPSRYTCTGESPLNNIGASVTD Contig28380 METPILIKLGNGLSIPSVQELAKLTLAEIPSRYTCTGESPLNNIGASVTD Contig45746 CODM DETVPVIDLQNLLSPEPVVGKLELDKLHSACKEWGFFQLVNHGVDALLMD Contig28380 DETVPVIDLQNLLSPEPVVGKLELDKLHSACKEWGFFQLVNHGVDALLMD Contig45746 CODM NIKSEIKGFFNLPMNEKTKYGQQDGDFEGFGQPYIESEDQRLDWTEVFSM Contig28380 NIKSEIKGFFNLPMNEKTKYGQQDGDFEGFGQPYIESEDQRLDWTEVFSM Contig45746 CODM LSLPLHLRKPHLFPELPLPFRETLESYLSKMKKLSTVVFEMLEKSLQLVE Contig28380 LSLPLHLRKPHLFPELPLPFRETLESYLSKMKKLSTVVFEMLEKSLQLVE Contig45746 SKMKKLSTVVFEMLEKSLQLVE CODM IKGMTDLFEDGLQTMRMNYYPPCPRPELVLGLTSHSDFSGLTILLQLNEV Contig28380 IKGMTDLFEDGLQTMRMNYYPPCPRPELVLGLTSHSDFSGLTILLQLNEV Contig45746 IKGMTDLFEDGLQTMRMNYYPPCPRPELVLGLTSHSDFSGLTILLQLNEV CODM EGLQIRKEERWISIKPLPDAFIVNVGDILEIMTNGIYRSVEHRAVVNSTK Contig28380 EGLQIRKEERWISIK EIMTNGIYRSVEHRAVVNSTK Contig45746 EGLQIRKEERWISIKPLPDALIVNVGDILEIMTNGIYRSVEHRAVVNSTK CODM ERLSIATFHDSKLESEIGPISSLVTPETPALFKRGRYEDILKENLSRKLD Contig28380 ERLSIATFHDSKLESEIGPI LSRKLD Contig45746 ERLSIATFHDSKLESEIGPISSLVTPETPALFKRGRYEDILKENLSRKLD CODM GKSFLDYMRM Contig28380 GKSFLDYMRM Contig45746 GKSFLDYMRM Supplemental Figure 6. Amino acid sequence alignment of contigs and singletons in the opium poppy transcriptome database encoding the following BIA biosynthetic enzymes: (A) thebaine 6-O-demethylase (T6ODM), (B) codeinone reductase (COR), and (C) codeine O-demethylase (CODM). Names in red represent sequences detected in both the whole stem and latex sub-proteomes, whereas names in black indicate sequences occurring only in the whole stem sub-proteome. Sequences with names in italics were not included in cumulative emPAI scores. Gaps in amino acid sequences resulted from incomplete coverage or errors in the assembly of the 454 pyrosequencing data.

Supplementary Table 1. Top 30 most abundant proteins based on emPAI score in the whole stem and latex sub-proteomes of opium poppy. BIA biosynthetic enzymes are highlighted in gray.

Whole stem

Number Accession Score Mass Number of significant matches

Number of significant sequences

emPAI wt % Description

1 gi|18477626 49096 47977 2216 24 17.7 4.1 Ribulose-1,5-biphosphate carboxylase 2 gi|125982 4009 17855 127 10 12.4 1.1 Major latex protein 15 3 gi|29726138 5725 52757 147 23 8.4 2.2 ATP synthase beta subunit 4 gi|58013197 20796 41792 800 15 7.4 1.5 Actin 5 gi|61814646 2621 25984 66 7 5.9 0.8 S-Norcoclaurine synthase 2 6 gi|15233891 2537 54271 77 21 5.6 1.5 V-type proton ATPase subunit B2 7 gi|14718210 4628 51973 116 21 5.3 1.4 ATP synthase beta subunit 8 gi|46906215 5973 91670 228 33 5.2 2.3 Phospholipase D1 9 gi|1703374 670 20690 26 8 5.1 0.5 ADP-ribosylation factor 1

10 gi|356569000 15851 70795 674 21 4.9 1.7 Heat shock protein 70 11 gi|355754836 5908 53150 140 18 4.7 1.2 PLP-dependent aminotransferase 12 gi|222101934 4966 35771 165 11 4.4 0.8 Codeinone reductase 13 gi|315113446 9110 34028 245 10 3.4 0.6 Salutaridine reductase 14 gi|168024396 12308 49594 535 16 3.4 0.8 Alpha tubulin 15 gi|255544516 4843 63200 131 20 3.4 1 ATP synthase alpha subunit

16 gi|33413894 6599 39665 240 15 3.2 0.6 3'-Hydroxy-N-methylcoclaurine 4'-O-methyltransferase 2

17 gi|194705850 7662 49192 482 15 3.2 0.8 Elongation factor 1 alpha 18 gi|377685892 10422 42591 383 14 3.1 0.7 Scoulerine 9-O-methyltransferase 1 19 gi|108709466 1840 22804 64 7 3.0 0.3 40S ribosomal protein S3 20 gi|255560725 4201 46812 167 14 2.9 0.7 Dead box RNA helicase 21 gi|33286376 3919 39750 177 11 2.9 0.6 Catechol O-methyltransferase 22 gi|166702 5839 37652 317 11 2.9 0.5 Glyceraldehyde 3-phosphate

dehydrogenase A subunit 23 gi|224293 335 11329 18 3 2.8 0.2 Histone H4 24 gi|93280042 4848 38856 182 8 2.7 0.5 Glutamine synthetase 25 gi|224117728 1389 21941 61 5 2.6 0.3 60S ribosomal protein L6 26 gi|355755168 10836 52931 264 16 2.6 0.7 PLP-dependent aminotransferase 27 gi|225438619 233 12302 13 4 2.4 0.1 60S ribosomal protein L30 28 gi|224100953 2039 39082 55 8 2.4 0.5 UDP-glucuronate decarboxylase 29 gi|3021483 434 15270 22 3 2.3 0.2 Histone H2B-2 30 gi|313471270 2565 40823 91 8 2.2 0.4 Codeine O-demethylase

Latex

Number Accession Score Mass Number of significant matches

Number of significant sequences

emPAI wt % Description

1 gi|125982 32713 17855 2013 13 253.1 35.4 Major latex protein 15 2 gi|6478208 8732 35774 535 18 21.3 6 Codeinone reductase 3 gi|313471270 30910 40823 1524 15 10.2 3.3 Codeine O-demethylase 4 gi|46906217 6067 91813 379 34 4.8 3.4 Phospholipase D2 5 gi|313471275 8718 40598 597 13 4.6 1.5 Thebaine 6-O-demethylase 6 gi|377685908 9440 38790 422 10 4.6 1.4 Short-chain dehydrogenase/reductase 7 gi|356569000 11299 70795 734 19 4.4 2.4 Heat shock protein 70 8 gi|33286372 2376 39816 121 10 4.3 1.4 (R,S)-Reticuline 7-O-methyltransferase 9 gi|32186898 4710 41761 264 12 3.6 1.2 Actin

10 gi|224981577 1461 28888 112 9 3.1 0.7 14-3-3 protein 11 gi|15229033 3393 42769 131 9 2.6 0.9 S-Adenosylmethionine synthase 4

12 gi|356568567 2409 31954 130 5 2.3 0.6 2-Dehydro-3-deoxyphosphooctonate aldolase

13 gi|388571232 763 38039 32 11 2.2 0.7 Pavine N-methyltransferase 14 gi|14594931 634 18598 22 6 2.2 0.3 Beta-5 proteasome subunit 15 gi|255560725 2479 46812 134 12 2.2 0.8 Dead box RNA helicase

16 gi|225470846 1105 22014 35 5 2.1 0.4 Nascent polypeptide-associated complex subunit alpha

17 gi|4680249 3127 49369 310 10 2.0 0.8 Elongation factor 1-alpha 1 18 gi|297831158 3942 53303 239 14 1.9 0.8 S-adenosyl-L-homocysteine hydrolase 2 19 gi|356529147 4564 65950 196 14 1.9 1 ATP-citrate synthase beta chain protein 1 20 gi|77999077 12045 35718 570 6 1.9 0.5 Malate dehydrogenase 21 gi|255648341 677 27393 40 7 1.8 0.4 Proteasome subunit 22 gi|157812633 3450 110726 246 23 1.8 1.5 Phosphoenolpyruvate carboxylase 23 gi|30687535 735 21778 24 4 1.7 0.3 Quinone reductase 24 gi|225466253 1070 46254 45 10 1.6 0.6 Isocitrate dehydrogenase 25 gi|33621084 1676 13472 185 2 1.5 0.2 Thioredoxin H3 26 gi|224100953 937 39082 36 7 1.5 0.4 UDP-glucuronate decarboxylase 27 gi|15233891 779 54271 47 13 1.4 0.6 V-type proton ATPase subunit B2 28 gi|585783 1300 25274 134 6 1.4 0.3 GTP-binding nuclear protein Ran/TC4 29 gi|388506768 1107 33417 57 5 1.4 0.4 Unknown 30 gi|351721357 428 25964 20 7 1.3 0.3 Proteasome subunit

Supplemental Table 2. Sequences of PCR primers used to assemble expression constructs and to perform quantitative RT-PCR.

Expression constructs

Quantitative RT-PCR

Vector name

Forward primer (5’-3’)

Reverse primer (5’-3’)

Insert size (bp)

pQE30-SalSyn

GCGGATCCATGATCGTCATAAGCGAT BamHI

GCAAGCTTATCTACTGTCGAAAAGGT HindIII 1243

pQE30-SalR

GCGGATCCATGCCTGAAACATGTCCA BamHI

GCCTGCAGAAATGCAGATAGTTCTGA PstI 933

pQE30-COR

GCGGATCCATGGAGAGTAATGGTGTA BamHI

GCCTGCAGTTTCAATCCTTCTCATCC PstI 968

Gene Forward primer (5’-3’)

Reverse primer (5’-3’)

Size (bp)

SalSyn CGGGATATGTACGCCACAAGAGACAGAG GAGCAAAAGCTACCATCTGCTTGGCT 628

SalR GGTAAACAATGCTGGGGTTGCAGGTT CAGAAGGTCCATCGTCGGGGAAAAGA 614

SalAT GAGTTCATGTCGCAACCGGATGTGCCAC TGCTGGTAAGAACGCCGAAACAGTTACAG 642

T6ODM GATGGAAAAAGCTCTACAAGTACAAGCAGCC CATTCCTCCACAAGATCCCCATATGTAGAT 468

COR TTCATCACTTCCAAGCTCTGGTGCGCTGAT ACCCATCTCATACTAACCTGGGCAACAG 515

CODM CACATGCACCGGTGAAAGCCCGTTGAATAA AATCCGAGTGTGACGTAAGACCAAGTACA 614

Supplemental Table 3. CID spectra of substrates and reaction products generated by native cell-free latex protein extracts.

Compound [M+H]+ Retention time (min)

ESI[+]-CID, m/z (Relative abundance) Reference

Thebaine 312 3.55 312 (2), 281 (2), 266(4), 255 (1), 251 (11), 249 (2), 237 (1), 234 (2), 223 (2), 221 (7), 218 (4), 195 (2), 177 (1), 58 (100)

Authentic standard

Oripavine 298 2.50 298 (1), 267 (3), 249 (8), 237(4), 221 (3), 223 (2), 218 (6), 58 (100)

Authentic standard

Codeinone 298 3.05 298 (1), 241 (4), 239 (100), 211 (5), 181(4), 153 (3), 58 (46), 44 (9)

Authentic standard

Morphinone 284 1.45 284 (92), 227 (67), 225 (32), 209 (43), 199 (41), 197 (50), 185 (62), 181 (79), 153 (32), 58 (100), 44 (26)

Raith et al., 2003

Codeine 300 1.75 300 (100), 282 (6), 243 (8), 241 (4), 225 (13), 215 (23), 199 (12), 183 (12), 165 (7), 155 (3), 58 (11)

Authentic standard

Morphine 386 0.50 286 (100), 229 (8), 227 (4), 211 (9), 201 (20), 185 (10), 183 (10) 173 (9), 155 (6), 58 (9)

Authentic standard

Supplemental References

Raith, K., Neubert, R., Poeaknapo, C., Boettcher, C., Zenk, M.H., and Schmidt, J. (2003). Electrospray tandem mass spectrometric investigations of morphinans. J. Am. Soc. Mass Spectrom., 14: 1262-1269.