Plant Defense Responses in Opium Poppy Cell Cultures Revealed by Liquid Chromatography-Tandem Mass Spectrometry Proteomics

Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids, including the narcotic analgesic morphine and the antimicrobial agent sanguinarine. In contrast to the plant, cell cultures of opium poppy do not accumulate alkaloids constitutively but produce sanguinarine in response to treatment with certain fungal-derived elicitors. The induction of sanguinarine biosynthesis provides a model platform to characterize the regulation of benzylisoquinoline alkaloid pathways and other defense responses. Proteome analysis of elicitor-treated opium poppy cell cultures by two-dimensional denaturing-polyacrylamide gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry facilitated the identification of 219 of 340 protein spots based on peptide fragment fingerprint searches of a combination of databases. Of the 219 hits, 129 were identified through pre-existing plant proteome databases, 63 were identified by matching predicted translation products in opium poppy-expressed sequence tag databases, and the remainder shared evidence from both databases. Metabolic enzymes represented the largest category of proteins and included S-adenosylmethionine synthetase, several glycolytic, and a nearly complete set of tricarboxylic acid cycle enzymes, one alkaloid, and several other secondary metabolic enzymes. The abundance of chaperones, heat shock proteins, protein degradation factors, and pathogenesis-related proteins provided a comprehensive proteomics view on the coordination of plant defense responses. Qualitative comparison of protein abundance in control and elicitor-treated cell cultures allowed the separation of induced and constitutive or suppressed proteins. DNA microarrays were used to corroborate increases in protein abundance with a corresponding induction in cognate transcript levels. Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids, including the narcotic analgesic morphine and the antimicrobial agent sanguinarine. In contrast to the plant, cell cultures of opium poppy do not accumulate alkaloids constitutively but produce sanguinarine in response to treatment with certain fungal-derived elicitors. The induction of sanguinarine biosynthesis provides a model platform to characterize the regulation of benzylisoquinoline alkaloid pathways and other defense responses. Proteome analysis of elicitor-treated opium poppy cell cultures by two-dimensional denaturing-polyacrylamide gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry facilitated the identification of 219 of 340 protein spots based on peptide fragment fingerprint searches of a combination of databases. Of the 219 hits, 129 were identified through pre-existing plant proteome databases, 63 were identified by matching predicted translation products in opium poppy-expressed sequence tag databases, and the remainder shared evidence from both databases. Metabolic enzymes represented the largest category of proteins and included S-adenosylmethionine synthetase, several glycolytic, and a nearly complete set of tricarboxylic acid cycle enzymes, one alkaloid, and several other secondary metabolic enzymes. The abundance of chaperones, heat shock proteins, protein degradation factors, and pathogenesis-related proteins provided a comprehensive proteomics view on the coordination of plant defense responses. Qualitative comparison of protein abundance in control and elicitor-treated cell cultures allowed the separation of induced and constitutive or suppressed proteins. DNA microarrays were used to corroborate increases in protein abundance with a corresponding induction in cognate transcript levels. Opium poppy (Papaver somniferum) is one of our most important medicinal plants as the source of several pharmacologically active benzylisoquinoline alkaloids, including the analgesic morphine and codeine, the muscle relaxant and vasodilator papaverine, the antineoplastic drug noscapine, and the antimicrobial agent sanguinarine. In opium poppy plants, several benzylisoquinoline alkaloids are abundant in cytoplasmic vesicles of specialized cells known as laticifers, which are proximal to sieve elements of the nutrient-conductive phloem. Alkaloid biosynthetic enzymes have been localized to sieve elements (1Bird D.A. Franceschi V.R. Facchini P.J. A tale of three cell types: alkaloid biosynthesis is localized to sieve elements in opium poppy.Plant Cell. 2003; 15: 2626-2635Crossref PubMed Scopus (148) Google Scholar, 2Samanani N. Alcantara J. Bourgault R. Zulak K.G. Facchini P.J. The role of phloem sieve elements and laticifers in the biosynthesis and accumulation of alkaloids in opium poppy.Plant J. 2006; 47: 547-563Crossref PubMed Scopus (73) Google Scholar). In contrast, de-differentiated cell cultures of opium poppy do not constitutively accumulate alkaloids. However, the biosynthesis of sanguinarine is rapidly induced in response to treatment with specific fungal-derived elicitors (3Facchini P.J. Johnson A.G. Poupart J. De Luca V. Uncoupled defense gene expression and antimicrobial alkaloid accumulation in elicited opium poppy cell cultures.Plant Physiol. 1996; 111: 687-697Crossref PubMed Scopus (66) Google Scholar). Elicitor-induced sanguinarine biosynthesis in opium poppy cell cultures provides a platform to characterize the induction of antimicrobial alkaloid and other plant defense pathways under controlled conditions.Benzylisoquinoline alkaloid biosynthesis in opium poppy begins with the condensation of dopamine and 4-hydroxyphenylacetaldehyde by norcoclaurine synthase (4Liscombe D.K. Facchini P.J. Evolutionary and cellular webs in benzylisoquinoline alkaloid biosynthesis.Curr. Opin. Biotechnol. 2008; 19: 1-8Crossref PubMed Scopus (58) Google Scholar) to yield (S)-norcoclaurine (Fig. 1). Dopamine formation involves the decarboxylation of tyrosine and/or dihydroxyphenylalanine by tyrosine/dopa decarboxylase, whereas norcoclaurine 6-O-methyltransferase (6OMT) 1The abbreviations used are: 6OMT, norcoclaurine 6-O-methyltransferase; CNMT, coclaurine N-methyltransferase; CYP80B3, (S)-N-methylcoclaurine-3′-hydroxylase; 4′OMT, 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase; BBE, berberine bridge enzyme; CYP719A2, stylopine synthase; HPLC, high pressure liquid chromatography; EST, expressed sequence tag; 2-DE, two-dimensional electrophoresis; DTT, dithiotheitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; COR, codeinone reductase; SAM, S-adenosylmethionine; TCA, tricarboxylic acid; PR, pathogenesis-related; DIR, dirigent protein; PAL, phenylalanine ammonia lyase; NADP-ME, NADP-malic enzyme; 3β-HSD, 3β-hydroxysteroid dehydrogenase/isomerase; HSP70, heat-shock protein 70; BiP, luminal-binding protein; IFR, isoflavone reductase; GDH, glutamate dehydrogenase; GST, glutathione S-transferase; LC-MS/MS, liquid chromatography-tandem mass spectrometry. 1The abbreviations used are: 6OMT, norcoclaurine 6-O-methyltransferase; CNMT, coclaurine N-methyltransferase; CYP80B3, (S)-N-methylcoclaurine-3′-hydroxylase; 4′OMT, 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase; BBE, berberine bridge enzyme; CYP719A2, stylopine synthase; HPLC, high pressure liquid chromatography; EST, expressed sequence tag; 2-DE, two-dimensional electrophoresis; DTT, dithiotheitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; COR, codeinone reductase; SAM, S-adenosylmethionine; TCA, tricarboxylic acid; PR, pathogenesis-related; DIR, dirigent protein; PAL, phenylalanine ammonia lyase; NADP-ME, NADP-malic enzyme; 3β-HSD, 3β-hydroxysteroid dehydrogenase/isomerase; HSP70, heat-shock protein 70; BiP, luminal-binding protein; IFR, isoflavone reductase; GDH, glutamate dehydrogenase; GST, glutathione S-transferase; LC-MS/MS, liquid chromatography-tandem mass spectrometry. and coclaurine N-methyltransferase (CNMT) convert (S)-norcoclaurine to (S)-N-methylcoclaurine. The P450-dependent monooxygenase (S)-N-methylcoclaurine-3′-hydroxylase (CYP80B3) catalyzes the 3′-hydroxylation of (S)-N-methylcoclaurine prior to the formation of (S)-reticuline by 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′OMT). (S)-Reticuline represents the last common intermediate in the biosynthesis of morphine and sanguinarine. Epimerization of (S)-reticuline to (R)-reticuline is the first step in the formation of morphine. Alternatively, berberine bridge enzyme (BBE) converts (S)-reticuline to (S)-scoulerine as the first committed step in the sanguinarine pathway (Fig. 1). (S)-Scoulerine is converted to (S)-stylopine via the formation of two methylenedioxy bridges by the P450-dependent monooxygenases cheilanthifoline synthase and stylopine synthase (CYP719A2). Tetrahydroprotoberberine cis-N-methyltransferase converts (S)-stylopine to (S)-cis-N-methylstylopine, which is hydroxylated by the P450-dependent N-methylstylopine 14-hydroxylase. The initial reaction product tautomerizes to protopine, which is hydroxylated by protopine 6-hydroxylase to yield dihydrosanguinarine. Subsequent oxidation by dihydrobenzophenanthridine oxidase yields sanguinarine.Elicitor-induced sanguinarine accumulation in opium poppy cell cultures provides a responsive model system to profile modulations in gene transcripts (5Zulak K.G. Cornish A. Daskalchuk T.E. Deyholos M.K. Goodenowe D.B. Gordon P.M. Klassen D. Pelcher L.E. Sensen C.W. Facchini P.J. Gene transcript and metabolite profiling of elicitor-induced opium poppy cell cultures reveals the coordinate regulation of primary and secondary metabolism.Planta. 2007; 225: 1085-1106Crossref PubMed Scopus (85) Google Scholar), proteins, and metabolites (5Zulak K.G. Cornish A. Daskalchuk T.E. Deyholos M.K. Goodenowe D.B. Gordon P.M. Klassen D. Pelcher L.E. Sensen C.W. Facchini P.J. Gene transcript and metabolite profiling of elicitor-induced opium poppy cell cultures reveals the coordinate regulation of primary and secondary metabolism.Planta. 2007; 225: 1085-1106Crossref PubMed Scopus (85) Google Scholar, 6Zulak K.G. Weljie A.M. Vogel H.J. Facchini P.J. Quantitative 1H NMR metabolomics reveals extensive metabolic reprogramming of primary and secondary metabolism in elicitor-treated opium poppy cell cultures.BMC Plant Biol. 2008; 8: 5Crossref PubMed Scopus (95) Google Scholar) related to alkaloid biosynthesis and other defense responses. An annotated expressed sequence tag (EST) database was assembled from 10,224 random clones isolated from an elicitor-treated opium poppy cell culture cDNA library. ESTs corresponding to 40 enzymes involved in the conversion of sucrose to sanguinarine were identified. A corresponding DNA microarray probed with RNA from cell cultures collected at various time points after elicitor treatment showed the coordinate induction of diverse transcript populations, with alkaloid biosynthetic enzyme and defense protein transcripts displaying the most rapid and substantial modulations. In addition to all known sanguinarine biosynthetic gene transcripts, transcripts encoding several upstream primary metabolic enzymes were also induced. A combination of Fourier transform-ion cyclotron resonance-mass spectrometry and proton nuclear magnetic resonance (1H NMR) were used to monitor corresponding changes in metabolite profiles (5Zulak K.G. Cornish A. Daskalchuk T.E. Deyholos M.K. Goodenowe D.B. Gordon P.M. Klassen D. Pelcher L.E. Sensen C.W. Facchini P.J. Gene transcript and metabolite profiling of elicitor-induced opium poppy cell cultures reveals the coordinate regulation of primary and secondary metabolism.Planta. 2007; 225: 1085-1106Crossref PubMed Scopus (85) Google Scholar, 6Zulak K.G. Weljie A.M. Vogel H.J. Facchini P.J. Quantitative 1H NMR metabolomics reveals extensive metabolic reprogramming of primary and secondary metabolism in elicitor-treated opium poppy cell cultures.BMC Plant Biol. 2008; 8: 5Crossref PubMed Scopus (95) Google Scholar). Extensive and rapid changes in pool sizes of primary and secondary metabolites were observed in elicitor-treated cell cultures, but not in controls. A dynamic separation was revealed in the metabolome in response to elicitor treatment. Several alkaloids and other metabolites showed temporal changes in abundance consistent with modulations in the profiles of relevant biosynthetic gene transcripts. A corresponding proteomics analysis has not been performed, but would provide information highly complementary to available transcriptomics and metabolomics datasets.The use of proteomics to study biological processes in plants has gained momentum, although most applications are focused on model systems that typically do not produce specialized metabolites such as alkaloids (7Rose J.K. Bashir S. Giovannoni J.J. Jahn M.M. Saravanan R.S. Tackling the plant proteome: practical approaches, hurdles and experimental tools.Plant J. 2004; 39: 715-733Crossref PubMed Scopus (255) Google Scholar, 8Bertone P. Snyder M. Prospects and challenges in proteomics.Plant Physiol. 2005; 138: 560-562Crossref PubMed Scopus (17) Google Scholar, 9Chen S. Harmon A.C. Advances in plant proteomics.Proteomics. 2006; 6: 5504-5516Crossref PubMed Scopus (196) Google Scholar, 10Rossignol M. Peltier J.-B. Mock H.-P. Matros A. Maldonado A.M. Jorrin J.V. Plant proteome analysis: a 2004–2006 update.Proteomics. 2006; 6: 5529-5548Crossref PubMed Scopus (142) Google Scholar, 11Jorrin J.V. Maldonado A.M. Castillego M.S. Plant proteome analysis: a 2006 update.Proteomics. 2007; 7: 1-16Crossref Scopus (144) Google Scholar). Proteome reference maps have been produced for a number of plants systems including cell cultures (12Lei Z. Elmer A.M. Watson B.S. Dixon R.A. Mendes P.J. Sumner L.W. A two-dimensional electrophoresis proteomic reference map and systematic identification of 1,367 proteins from a cell suspension culture of the model legume Medicago truncatula..Mol. Cell. Proteomics. 2005; 4: 1812-1825Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) and root (13Mathesius U. Keijzers G. Natera S.H. Weinman J.J. Djordjevic M.A. Rolfe B.G. Establishment of a root proteome reference map for the model legume Medicago truncatula using the expressed sequence tag database for peptide mass fingerprinting.Proteomics. 2001; 1: 1424-1440Crossref PubMed Scopus (195) Google Scholar) of the model legume Medicago suspension cultures of P. of a two-dimensional gel electrophoresis protein database for the cell suspension 2004; 4: PubMed Scopus Google Scholar), of R. S. Proteome of of 2005; PubMed Scopus Google Scholar), in S. M. N. J. Proteome reference maps of in An of from Physiol. 2004; PubMed Scopus Google Scholar), and V. S. M. M. A two-dimensional proteome map of 2004; PubMed Scopus (95) Google Scholar). plant proteomics has been used to and and the protein M. Peltier J.-B. Mock H.-P. Matros A. Maldonado A.M. Jorrin J.V. Plant proteome analysis: a 2004–2006 update.Proteomics. 2006; 6: 5529-5548Crossref PubMed Scopus (142) Google Scholar). proteomics have been used to study the of proteins to specific S. Z. M. M. Snyder M. of proteins to revealed through protein U. S. A. 2007; PubMed Scopus Google Scholar) and to the proteome in A. cell cultures R. A. protein identification analysis of proteins in Cell. Proteomics. 2007; 6: Full Text Full Text PDF PubMed Scopus Google on plants the of to proteins in the and of S. R. proteomics of plant 2004; 15: Google Scholar). spots were by two-dimensional gel electrophoresis protein were to the of specific for proteins that in abundance with the accumulation of alkaloids in cell cultures were to M. J. R. R. Proteome analysis of the medicinal plant 2005; PubMed Scopus Google Scholar). the of sequence the identification of proteomics have plants that produce benzylisoquinoline alkaloids. electrophoresis coupled with liquid chromatography-tandem mass spectrometry identified proteins in the of which is a of the related to opium involved in plant the tricarboxylic acid and acid R. A. A. analysis of using two-dimensional gel electrophoresis and mass 2007; PubMed Scopus Google Scholar). proteins in the and of opium poppy were to and were identified by and G. G. of proteins in of the opium poppy (Papaver somniferum) using two-dimensional gel electrophoresis and PubMed Scopus Google Scholar). and proteins were from the and metabolic enzymes were in both whereas and proteins were abundant in the However, codeinone G. G. of proteins in of the opium poppy (Papaver somniferum) using two-dimensional gel electrophoresis and PubMed Scopus Google Scholar) and A. G. J. and 6-O-methyltransferase of and of enzymes of alkaloid biosynthesis in opium poppy.Plant J. 2003; PubMed Scopus Google Scholar), which the step in morphine biosynthesis and the formation of were the alkaloid biosynthetic enzymes identified in opium poppy using proteomic analysis of opium poppy cell cultures by coupled with identified several proteins involved in primary and secondary metabolism and defense response in response to elicitor treatment. a of the identified proteins from the of a and database for opium The induction of proteins was by an in the accumulation of corresponding gene transcripts by DNA microarray Opium poppy (Papaver somniferum) is one of our most important medicinal plants as the source of several pharmacologically active benzylisoquinoline alkaloids, including the analgesic morphine and codeine, the muscle relaxant and vasodilator papaverine, the antineoplastic drug noscapine, and the antimicrobial agent sanguinarine. In opium poppy plants, several benzylisoquinoline alkaloids are abundant in cytoplasmic vesicles of specialized cells known as laticifers, which are proximal to sieve elements of the nutrient-conductive phloem. Alkaloid biosynthetic enzymes have been localized to sieve elements (1Bird D.A. Franceschi V.R. Facchini P.J. A tale of three cell types: alkaloid biosynthesis is localized to sieve elements in opium poppy.Plant Cell. 2003; 15: 2626-2635Crossref PubMed Scopus (148) Google Scholar, 2Samanani N. Alcantara J. Bourgault R. Zulak K.G. Facchini P.J. The role of phloem sieve elements and laticifers in the biosynthesis and accumulation of alkaloids in opium poppy.Plant J. 2006; 47: 547-563Crossref PubMed Scopus (73) Google Scholar). In contrast, de-differentiated cell cultures of opium poppy do not constitutively accumulate alkaloids. However, the biosynthesis of sanguinarine is rapidly induced in response to treatment with specific fungal-derived elicitors (3Facchini P.J. Johnson A.G. Poupart J. De Luca V. Uncoupled defense gene expression and antimicrobial alkaloid accumulation in elicited opium poppy cell cultures.Plant Physiol. 1996; 111: 687-697Crossref PubMed Scopus (66) Google Scholar). Elicitor-induced sanguinarine biosynthesis in opium poppy cell cultures provides a platform to characterize the induction of antimicrobial alkaloid and other plant defense pathways under controlled alkaloid biosynthesis in opium poppy begins with the condensation of dopamine and 4-hydroxyphenylacetaldehyde by norcoclaurine synthase (4Liscombe D.K. Facchini P.J. Evolutionary and cellular webs in benzylisoquinoline alkaloid biosynthesis.Curr. Opin. Biotechnol. 2008; 19: 1-8Crossref PubMed Scopus (58) Google Scholar) to yield (S)-norcoclaurine (Fig. 1). Dopamine formation involves the decarboxylation of tyrosine and/or dihydroxyphenylalanine by tyrosine/dopa decarboxylase, whereas norcoclaurine 6-O-methyltransferase (6OMT) 1The abbreviations used are: 6OMT, norcoclaurine 6-O-methyltransferase; CNMT, coclaurine N-methyltransferase; CYP80B3, (S)-N-methylcoclaurine-3′-hydroxylase; 4′OMT, 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase; BBE, berberine bridge enzyme; CYP719A2, stylopine synthase; HPLC, high pressure liquid chromatography; EST, expressed sequence tag; 2-DE, two-dimensional electrophoresis; DTT, dithiotheitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; COR, codeinone reductase; SAM, S-adenosylmethionine; TCA, tricarboxylic acid; PR, pathogenesis-related; DIR, dirigent protein; PAL, phenylalanine ammonia lyase; NADP-ME, NADP-malic enzyme; 3β-HSD, 3β-hydroxysteroid dehydrogenase/isomerase; HSP70, heat-shock protein 70; BiP, luminal-binding protein; IFR, isoflavone reductase; GDH, glutamate dehydrogenase; GST, glutathione S-transferase; LC-MS/MS, liquid chromatography-tandem mass spectrometry. 1The abbreviations used are: 6OMT, norcoclaurine 6-O-methyltransferase; CNMT, coclaurine N-methyltransferase; CYP80B3, (S)-N-methylcoclaurine-3′-hydroxylase; 4′OMT, 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase; BBE, berberine bridge enzyme; CYP719A2, stylopine synthase; HPLC, high pressure liquid chromatography; EST, expressed sequence tag; 2-DE, two-dimensional electrophoresis; DTT, dithiotheitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; COR, codeinone reductase; SAM, S-adenosylmethionine; TCA, tricarboxylic acid; PR, pathogenesis-related; DIR, dirigent protein; PAL, phenylalanine ammonia lyase; NADP-ME, NADP-malic enzyme; 3β-HSD, 3β-hydroxysteroid dehydrogenase/isomerase; HSP70, heat-shock protein 70; BiP, luminal-binding protein; IFR, isoflavone reductase; GDH, glutamate dehydrogenase; GST, glutathione S-transferase; LC-MS/MS, liquid chromatography-tandem mass spectrometry. and coclaurine N-methyltransferase (CNMT) convert (S)-norcoclaurine to (S)-N-methylcoclaurine. The P450-dependent monooxygenase (S)-N-methylcoclaurine-3′-hydroxylase (CYP80B3) catalyzes the 3′-hydroxylation of (S)-N-methylcoclaurine prior to the formation of (S)-reticuline by 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′OMT). (S)-Reticuline represents the last common intermediate in the biosynthesis of morphine and sanguinarine. Epimerization of (S)-reticuline to (R)-reticuline is the first step in the formation of morphine. Alternatively, berberine bridge enzyme (BBE) converts (S)-reticuline to (S)-scoulerine as the first committed step in the sanguinarine pathway (Fig. 1). (S)-Scoulerine is converted to (S)-stylopine via the formation of two methylenedioxy bridges by the P450-dependent monooxygenases cheilanthifoline synthase and stylopine synthase (CYP719A2). Tetrahydroprotoberberine cis-N-methyltransferase converts (S)-stylopine to (S)-cis-N-methylstylopine, which is hydroxylated by the P450-dependent N-methylstylopine 14-hydroxylase. The initial reaction product tautomerizes to protopine, which is hydroxylated by protopine 6-hydroxylase to yield dihydrosanguinarine. Subsequent oxidation by dihydrobenzophenanthridine oxidase yields sanguinarine. Elicitor-induced sanguinarine accumulation in opium poppy cell cultures provides a responsive model system to profile modulations in gene transcripts (5Zulak K.G. Cornish A. Daskalchuk T.E. Deyholos M.K. Goodenowe D.B. Gordon P.M. Klassen D. Pelcher L.E. Sensen C.W. Facchini P.J. Gene transcript and metabolite profiling of elicitor-induced opium poppy cell cultures reveals the coordinate regulation of primary and secondary metabolism.Planta. 2007; 225: 1085-1106Crossref PubMed Scopus (85) Google Scholar), proteins, and metabolites (5Zulak K.G. Cornish A. Daskalchuk T.E. Deyholos M.K. Goodenowe D.B. Gordon P.M. Klassen D. Pelcher L.E. Sensen C.W. Facchini P.J. Gene transcript and metabolite profiling of elicitor-induced opium poppy cell cultures reveals the coordinate regulation of primary and secondary metabolism.Planta. 2007; 225: 1085-1106Crossref PubMed Scopus (85) Google Scholar, 6Zulak K.G. Weljie A.M. Vogel H.J. Facchini P.J. Quantitative 1H NMR metabolomics reveals extensive metabolic reprogramming of primary and secondary metabolism in elicitor-treated opium poppy cell cultures.BMC Plant Biol. 2008; 8: 5Crossref PubMed Scopus (95) Google Scholar) related to alkaloid biosynthesis and other defense responses. An annotated expressed sequence tag (EST) database was assembled from 10,224 random clones isolated from an elicitor-treated opium poppy cell culture cDNA library. ESTs corresponding to 40 enzymes involved in the conversion of sucrose to sanguinarine were identified. A corresponding DNA microarray probed with RNA from cell cultures collected at various time points after elicitor treatment showed the coordinate induction of diverse transcript populations, with alkaloid biosynthetic enzyme and defense protein transcripts displaying the most rapid and substantial modulations. In addition to all known sanguinarine biosynthetic gene transcripts, transcripts encoding several upstream primary metabolic enzymes were also induced. A combination of Fourier transform-ion cyclotron resonance-mass spectrometry and proton nuclear magnetic resonance (1H NMR) were used to monitor corresponding changes in metabolite profiles (5Zulak K.G. Cornish A. Daskalchuk T.E. Deyholos M.K. Goodenowe D.B. Gordon P.M. Klassen D. Pelcher L.E. Sensen C.W. Facchini P.J. Gene transcript and metabolite profiling of elicitor-induced opium poppy cell cultures reveals the coordinate regulation of primary and secondary metabolism.Planta. 2007; 225: 1085-1106Crossref PubMed Scopus (85) Google Scholar, 6Zulak K.G. Weljie A.M. Vogel H.J. Facchini P.J. Quantitative 1H NMR metabolomics reveals extensive metabolic reprogramming of primary and secondary metabolism in elicitor-treated opium poppy cell cultures.BMC Plant Biol. 2008; 8: 5Crossref PubMed Scopus (95) Google Scholar). Extensive and rapid changes in pool sizes of primary and secondary metabolites were observed in elicitor-treated cell cultures, but not in controls. A dynamic separation was revealed in the metabolome in response to elicitor treatment. Several alkaloids and other metabolites showed temporal changes in abundance consistent with modulations in the profiles of relevant biosynthetic gene transcripts. A corresponding proteomics analysis has not been performed, but would provide information highly complementary to available transcriptomics and metabolomics The use of proteomics to study biological processes in plants has gained momentum, although most applications are focused on model systems that typically do not produce specialized metabolites such as alkaloids (7Rose J.K. Bashir S. Giovannoni J.J. Jahn M.M. Saravanan R.S. Tackling the plant proteome: practical approaches, hurdles and experimental tools.Plant J. 2004; 39: 715-733Crossref PubMed Scopus (255) Google Scholar, 8Bertone P. Snyder M. Prospects and challenges in proteomics.Plant Physiol. 2005; 138: 560-562Crossref PubMed Scopus (17) Google Scholar, 9Chen S. Harmon A.C. Advances in plant proteomics.Proteomics. 2006; 6: 5504-5516Crossref PubMed Scopus (196) Google Scholar, 10Rossignol M. Peltier J.-B. Mock H.-P. Matros A. Maldonado A.M. Jorrin J.V. Plant proteome analysis: a 2004–2006 update.Proteomics. 2006; 6: 5529-5548Crossref PubMed Scopus (142) Google Scholar, 11Jorrin J.V. Maldonado A.M. Castillego M.S. Plant proteome analysis: a 2006 update.Proteomics. 2007; 7: 1-16Crossref Scopus (144) Google Scholar). Proteome reference maps have been produced for a number of plants systems including cell cultures (12Lei Z. Elmer A.M. Watson B.S. Dixon R.A. Mendes P.J. Sumner L.W. A two-dimensional electrophoresis proteomic reference map and systematic identification of 1,367 proteins from a cell suspension culture of the model legume Medicago truncatula..Mol. Cell. Proteomics. 2005; 4: 1812-1825Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) and root (13Mathesius U. Keijzers G. Natera S.H. Weinman J.J. Djordjevic M.A. Rolfe B.G. Establishment of a root proteome reference map for the model legume Medicago truncatula using the expressed sequence tag database for peptide mass fingerprinting.Proteomics. 2001; 1: 1424-1440Crossref PubMed Scopus (195) Google Scholar) of the model legume Medicago suspension cultures of P. of a two-dimensional gel electrophoresis protein database for the cell suspension 2004; 4: PubMed Scopus Google Scholar), of R. S. Proteome of of 2005; PubMed Scopus Google Scholar), in S. M. N. J. Proteome reference maps of in An of from Physiol. 2004; PubMed Scopus Google Scholar), and V. S. M. M. A two-dimensional proteome map of 2004; PubMed Scopus (95) Google Scholar). plant proteomics has been used to and and the protein M. Peltier J.-B. Mock H.-P. Matros A. Maldonado A.M. Jorrin J.V. Plant proteome analysis: a 2004–2006 update.Proteomics. 2006; 6: 5529-5548Crossref PubMed Scopus (142) Google Scholar). proteomics have been used to study the of proteins to specific S. Z. M. M. Snyder M. of proteins to revealed through protein U. S. A. 2007; PubMed Scopus Google Scholar) and to the proteome in A. cell cultures R. A. protein identification analysis of proteins in Cell. Proteomics. 2007; 6: Full Text Full Text PDF PubMed Scopus Google Scholar). on plants the of to proteins in the and of S. R. proteomics of plant 2004; 15: Google Scholar). spots were by two-dimensional gel electrophoresis protein were to the of specific for proteins that in abundance with the accumulation of alkaloids in cell cultures were to M. J. R. R. Proteome analysis of the medicinal plant 2005; PubMed Scopus Google Scholar). the of sequence the identification of proteins. proteomics have plants that produce benzylisoquinoline alkaloids. electrophoresis coupled with liquid chromatography-tandem mass spectrometry identified proteins in the of which is a of the related to opium involved in plant the tricarboxylic acid and acid R. A. A. analysis of using two-dimensional gel electrophoresis and mass 2007; PubMed Scopus Google Scholar). proteins in the and of opium poppy were to and were identified by and G. G. of proteins in of the opium poppy (Papaver somniferum) using two-dimensional gel electrophoresis and PubMed Scopus Google Scholar). and proteins were from the and metabolic enzymes were in both whereas and proteins were abundant in the However, codeinone G. G. of proteins in of the opium poppy (Papaver somniferum) using two-dimensional gel electrophoresis and PubMed Scopus Google Scholar) and A. G. J. and 6-O-methyltransferase of and of enzymes of alkaloid biosynthesis in opium poppy.Plant J. 2003; PubMed Scopus Google Scholar), which the step in morphine biosynthesis and the formation of were the alkaloid biosynthetic enzymes identified in opium poppy using In proteomic analysis of opium poppy cell cultures by coupled with identified several proteins involved in primary and secondary metabolism and defense response in response to elicitor treatment. a of the identified proteins from the of a and database for opium The induction of proteins was by an in the accumulation of corresponding gene transcripts by DNA microarray with with

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