Papaver somniferum pathways

The benzylisoquinolines are formed from two molecules of fhe aromafic amino acid, tyrosine. In the past ten years, this pathway has been probed at the enzyme and gene level. The recent linking of the phloem-specific expression of tyrosine/Dopa decarboxylase (TYDC) genes with the bios)mthesis of the isoquinoline alkaloids in the opium poppy, Papaver somniferum (Facchini and De Luca, 1994, 1995, 2008 Liscombe and Facchini, 2008), and the association with alkaloid accumulation as part of the plant defence mechanism (Wink, 1993 Facchini et al, 1996) are of particular interest in furthering our knowledge of the location of alkaloid biosynthesis. 

Morishige T, Dubouzet E, Choi K-B, Yazaki K, Sato F. Molecular cloning of columbamine O- methyltransferase from cultured Coptis japonica cells. Eur. J. Biochem. 2002 269 5659-5667. Hirata K, Poeaknapo C, Schmidt J, Zenk MH. 1,2-Dehydroreti-culine synthase, the branch point enzyme opening the morphinan biosynthetic pathway. Phytochemistry 2004 65 1039-1046. De-Eknamktil W, Zenk MH. Purification and properties of 1,2-dehydroreticuUne reductase from Papaver somniferum seedlings. Phytochemistry 1992 31 813-821. 

Reticuline (67) is also a precursor for isoboldine (70) (in Papaver somniferum). Orientaline (62) has been isolated from P. somniferum for the first time, by adding inactive material as a carrier during isolation, after feeding ( + )-[3- C]nor-laudanosoline. However, it was shown not to be a precursor for isoboldine (70) in this plant, thus indicating a unique biosynthetic pathway for isoboldine from reticuline. 

Our recent research suggests organ-, tissue-, and cell-specific localization of constitutive and induced terpenoid defense pathways in conifers. For example, linalool synthase (PaTPS-Lin) seems to be preferentially expressed in needles of Norway spruce and Sitka spruce with little or no expression in sterns. ft is also likely that expression of PaTPS-Lin in spruce needles is not associated with resin ducts but could reside in other cells involved with induced terpenoid emission. In contrast, we can speculate that most other mono-TPS and di-TPS are associated with epithelial cells of constitutive and induced resin ducts. The possible localization of conifer sesqui-TPS is difficult to predict. Furthermore, the exact spatial and temporal patterns of terpenoid pathway gene expression associated with traumatic resin duct development in the cambium zone and outer xylem remain to be studied at the tissue and cell level. In situ hybridization and immuno-localization of TPS will address these open questions. These methods have worked well in identifying cell type specific gene and protein expression of alkaloid formation in opium poppy Papaver somniferum) As the biochemistry of induced terpene defenses and the development of traumatic resin ducts have been well described in spruce, this system is ideal for future studies of tissue- and cell-specific localization of transcripts and proteins associated with oleoresin defense and induced volatile emissions in conifers. In addition, the advent of laser dissection microscopy techniques presents a fascinating means by which to further address RNA and protein analysis in a tissue-and cell-specific manner. These techniques, when applied to the cambium zone, xylem mother cells, and the epithelial cells that surround traumatic resin ducts, and will allow a temporal and spatial analysis of cellular functions occurring in the traumatic resin response. 

Various types of cultures of Papaver somniferum and P. bracteatum have been found to be excellent producers of sanguinarine. The production of this alkaloid is dealt with separately (see Section VI,F). Pathways leading to the major alkaloids are summarized in Fig. 9. Several reviews on... 

Morphinan Alkaloids.—Extensive research on the biosynthesis of morphine (51) and related alkaloids in Papaver somniferum has allowed a detailed description of the pathway from the amino-acid tyrosine through reticuline (44), thebaine (46), and codeine (50) to morphine (51) (Scheme The incorporation of (R)-... 

Fig. 2. Alkaloid biosynthetic pathways are associated with a diverse variety of cell types. The tissue-specific localization (shaded) of enzymes and/or gene transcripts are depicted for the biosynthesis of tropane alkaloids in Atropa belladonna and Hyoscyamus niger roots (A), monoterpenoid indole alkaloids in Catharanthus roseus leaves (B), pyrrolizidine alkaloids in Senecio vernalis roots (C), pyrrolizidine alkaloids in Eupatorium cannabinum roots (D), benzyl-isoquinoline alkaloids in Papaver somniferum vascular bundles (E), and protoberberine alkaloids in Thalictrwn flamtm roots (F).

Several studies have reported the production in microorganisms of plant enzymes that are involved in alkaloid biosynthesis [29, 99-102], In this sense, Unterlinner et al. [100] have cloned and expressed in Escherichia coli four cDNAs encoding for different isoforms of the Codeinone reductase NADPH-dependent enzyme isolated from Papaver somniferum. In this report has been investigated the substrate specificity of the enzyme and the structural analysis, being the first report about the cloning and expression of genes of the biosynthetic pathway of morphine [90],... 

The results of numerous labeling and feeding studies and other experimental results have established that the steps of the pathway are as indicated (Fig. 32.20). 2- " C-Tyrosine produces morphine labeled at C-9 and C-16. l- " C-Dopa-mine gives morphine labeled at C-16.3,4-Dihydroxyphenyl-alanine is readily incorporated into morphine (58), thebaine (59), narcotine (63), and papaverine (31) (Santavy, 1979). (/ ,5)-Norcoclaurine (23) and (/ ,S )-coclaurine (24) (both labeled at C-1) were incorporated into thebaine and morphine (labeled at position C-9) when fed to Papaver somniferum plants (Loeffler et al., 1987). Norlaudanosoline (26) (Fig. 32.5) is an efficient precursor of morphine. Studies of the rates of incorporation of radioactivity demonstrated the sequence thebaine (59) to codeine (62) to morphine (58). 

The isoquinoline pathway s enzyme orthologs in other plants showed that genes of methyltransferase family such as (R,S)-reticuline 7-0-methyltransferase, coclaurine 4 -0-methyltransferase, (S)-norcoclaurine 6-0-methyltrasferase, columbamine 0-methyltransferase, coclaurine A-methyltransferase, putrescine A-methyltransferase responsible for the production of reticuline, coclaurine, norcoclaurine, columbaine have been reported from Papaver somniferum, Coptis japonica, Thalictrum flavum, Thalictrum tuberosum, Coffea liberica, Cojfea arabica, Cojfea canephora, Nicotiana tabacum. Solarium tuberosum. Datura stramonium, Hyoscyamus niger, and Atropa belladonna. Pathway contains information of about four alkaloids such as dopamine, colchicine, ephedrine, and methamphetamine and identified two missing links. 

Mishra S, Meena A, Singh S, Yadav DK, Khan F, Shukla RK (2010) Detection of substrate binding motifs for morphine biosynthetic pathway intermediates in novel wound inducible (R,S)-reticuline 7-O-methyltransferase of Papaver somniferum. In Proceedings of international symposium on current status and opportunities in Aromatic Medicinal Plants (AROMED), CIMAP (CSIR), Lucknow, India, 21-24 Feb 2010, Session I P-25, p 51... 

Brochmann-Hanssen E, Chen CH, Chen CR, Chiang HC (1975) Opium alkaloids, part XVI. The biosynthesis of 1-benzylisoquinolines in Papaver somniferum. Preferred and secondary pathways stereochemical aspects. J Chem Soc Perkin Trans 1 1931-1937 Brochmann-Hanssen E, Cheng CY, Chiang HC (1982) Biosynthesis of opium alkaloids The effects of structural modification of reticuline on racemization and bio transformation. J Nat Prod 45 629-634... 

Roberts MF, Antoun MD (1978) The relationship between L-DOPA decarboxylase in the latex of Papaver somniferum and alkaloid formation. Ph3rtochemistry 17 1083-1087 Roberts MF, McCarthy D, Kutchan TM, Coscia CJ (1983) Localization of enzyme and alkaloidal metabolites in Papaver latex. Arch Biochem Biophys 222 599-609 Rueffer M, El-Shagi H, Nagakura N, Zenk MH (1981) (S)-Norlaudanosoline synthetase the first enzyme in the benzylisoquinoline biosynthetic pathway. FEBS Lett 129 5-9 Sasse F, Backs-Hiisemann D, Barz W (1979) Isolation and characterization of vacuoles from cell suspension cultures of Daucus carota. Z Naturforsch 34 848-853 Schuchmann R, Wellmann E (1983) Somatic embryogenesis of tissue cultures of Papaver somniferum and Papaver orientate and its relationship to alkaloid and lipid metabolism. Plant Cell Rep 2 88-91... 

With a morphine biosynthetic gene in hand, we believed we could begin to address the question why only P. somniferum produces morphine, while other Papaver species such as P. rhoeas, P. orientale, and P. bracteatum do not. Unexpectedly, we found that the codeinone reductase transcript was present to some degree in all four species investigated. A review of the literature revealed no alkaloids reported in P. rhoeas for which codeinone reductase should participate in the synthesis. Similarly, P. orientale accumulates the alternate morphine biosynthetic precursor oripavine, but codeinone reductase is not involved in the biosynthesis of oripavine, acting instead after this alkaloid along the biosynthetic pathway to morphine.22 P. bracteatum produces the morphine precursor thebaine as a major alkaloid. As for oripavine in P. orientale, codeinone reductase would act in P. bracteatum after thebaine formation on the pathway to morphine. It appears, therefore, that the reason that P. rhoeas, P. orientale, and P. bracteatum do not produce morphine is not related to the absence of the transcript of the morphine biosynthesis-specific gene codeinone reductase. The expression of codeinone reductase may simply be an evolutionary remnant in these species. 

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