Sanguinarine Biosynthesis Is Associated with the Endoplasmic Reticulum in Cultured Opium Poppy Cells after Elicitor Treatment

Abstract

Three key benzylisoquinoline alkaloid biosynthetic enzymes, (S)-N-methylcoclaurine-3#-hydroxylase (CYP80B1), berberine bridge enzyme (BBE), and codeinone reductase (COR), were localized in cultured opium poppy (Papaver somniferum) cells by sucrose density gradient fractionation and immunogold labeling. CYP80B1 catalyzes the second to last step in the formation of (S)-reticuline, the last common intermediate in sanguinarine and morphine biosynthesis. BBE converts (S)-reticuline to (S)-scoulerine as the first committed step in sanguinarine biosynthesis, and COR catalyzes the penultimate step in the branch pathway leading to morphine. Sanguinarine is an antimicrobial alkaloid that accumulates in the vacuoles of cultured opium poppy cells in response to elicitor treatment, whereas the narcotic analgesic morphine, which is abundant in opium poppy plants, is not produced in cultured cells. CYP80B1 and BBE were rapidly induced to high levels in response to elicitor treatment. By contrast, COR levels were constitutive in the cell cultures, but remained low and were not induced by addition of the elicitor. Western blots performed on protein homogenates from elicitor-treated cells fractionated on a sucrose density gradient showed the cosedimentation of CYP80B1, BBE, and sanguinarine with calreticulin, and COR with glutathione S-transferase. Calreticulin and glutathione S-transferase are markers for the endoplasmic reticulum (ER) and the cytosol, respectively. In response to elicitor treatment, large dilated vesicles rapidly developed from the lamellar ER of control cells and fused with the central vacuole. Immunogold localization supported the association of CYP80B1 and BBE with ER vesicles, and COR with the cytosol in elicitor-treated cells. Our results show that benzylisoquinoline biosynthesis and transport to the vacuole are associated with the ER, which undergoes major ultrastructural modification in response to the elicitor treatment of cultured opium poppy cells.Alkaloids are a diverse group of approximately 12,000 low molecular weight, nitrogen-containing compounds found in about 20% of plant species. Morphine and sanguinarine are members of the large and diverse group of benzylisoquinoline alkaloids, of which more than 2,500 different structures have been identified in plants. Although morphine is a potent narcotic analgesic, it has been implicated in the stress-induced cross-linking of galacturonic-containing polysaccharides in the cell walls of opium poppy (Papaver somniferum) plants ( Morimoto et al., 2001). By contrast, sanguinarine, which shows specific toxic effects to herbivores and microbial pathogens, is proposed to function as an inducible defense compound (Schmeller et al., 1997). Benzylisoquinoline alkaloids share a common biosynthetic pathway, beginning with the condensation of two L-Tyr derivatives to produce the central precursor (S)-norcoclaurine ( Fig. 1; Samanani et al., 2004). Specific O-and N-methyltransferases convert (S)-norcoclaurine to (S)-N-methylcoclaurine (Facchini, 2001). The P450-depe...

Benzylisoquinoline alkaloid biosynthesis in opium poppy

“…The subcellular localization of most enzymes involved in the biosynthesis of sanguinarine has been investigated in cultured opium poppy cells using microprojectile bombardment followed by imaging of green fluorescent protein fusions . As shown previously by density gradient fractionation (Amann et al 1986;Rueffer and Zenk 1987;Tanahashi and Zenk 1990;Bauer and Zenk 1991), and using immunogold localization (Alcantara et al 2005), BBe and CYPs associate with the endoplasmic reticulum (eR). Opium poppy BBe was also shown to contain a 25-amino acid, N-terminal signal peptide directing the enzyme to the eR, and an adjacent vacuolar-sorting determinant, resulting in its final translocation to the vacuole (Bird and Facchini 2001).…”

“…Although not localized as a GFP fusion along with the other enzymes , DBOX contains a predicted N-terminal signal peptide ) and related STOX enzymes have been associated with the eR (Amann et al 1988); thus, a third reentry of a pathway intermediate into the eR lumen is predicted. Oxidation of dihydrosanguinarine in the eR would facilitate the vesicle-mediated transport of sanguinarine to the central vacuole of cultured opium poppy cells (Alcantara et al 2005). The proposed subcellular trafficking features of sanguinarine biosynthesis might also occur in other BIA branch pathways, although no empirical evidence is available.…”

A rice high-affinity potassium transporter (HKT) conceals a calcium-permeable cation channel

“…Specimens from 10-day-old Nipponbare seedlings were trimmed to 1 mm 2 . Osmium tetroxide fixation, thin sectioning, immunogold labeling, and electron microscopy were carried out as described (31). 45 Ca 2+ Uptake Assay in the Oocytes.…”

Immunohistochemical localization of caffeine in young Camellia sinensis (L.) O. Kuntze (tea) leaves

“…The alkaloid ancistroheynine A was found in the tip of the shoot and leaf midrib of Liana Ancistrocladus heyneanus and ancistrocladine was found in the branch root of the plant by FT-Raman investigations (Urlaub et al 1997). Many more articles are published describing the localization of different alkaloids with different techniques (Verzár -Petri 1975;Yoder and Mahlberg 1976;Furr and Mahlberg 1981;Neumann et al 1983;Platt and Thomson 1992;Bringmann et al 1996;Mösli Waldhauser and Baumann 1996;Meininger et al 1997;Cai et al 1999;Corsi and Bottega 1999;Bottega et al 2004;Pasqua et al 2004;Alcantara et al 2005;Frosch et al 2006Frosch et al , 2007aNikolakaki and Christodoulakis 2006;Mesjasz -Przybyiowicz et al 2007;Liang et al 2009;Moraes et al 2009;Argyropoulou et al 2010;Christodoulakis et al 2010). …”