Subcellular localization of tryptophan decarboxylase, strictosidine synthase and strictosidine glucosidase in suspension cultured cells of Catharanthus roseus and Tabernaemontana divaricata

Stevens, Lucas H.; Blom, Theo J.; Verpoorte, Robert

doi:10.1007/bf00233063

Cited by 88 publications

(58 citation statements)

References 23 publications

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“…Substrate specificity studies have suggested that only loganin can serve as a substrate for SLS (Yamamoto et al, 2000). The association of this cytochrome P450 with endoplasmic reticulum membranes or related vesicles could rapidly convert loganin into secologanin followed by its transfer to the vacuole (Contin et al, 1999) together with tryptamine for conversion into strictosidine by vacuole-associated STR (Stevens et al, 1993). In this way, the SLS-mediated conversion of loganin into secologanin, together with its subcellular compartmentalization for MIA biosynthesis, could prevent loganin from inhibiting LAMT.…”

Section: Kinetic Analyses and Substrate Specificity Studies With Rlammentioning

confidence: 99%

The Leaf Epidermome ofCatharanthus roseusReveals Its Biochemical Specialization

et al. 2008

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Catharanthus roseus is the sole commercial source of the monoterpenoid indole alkaloids (MIAs), vindoline and catharanthine, components of the commercially important anticancer dimers, vinblastine and vincristine. Carborundum abrasion technique was used to extract leaf epidermis-enriched mRNA, thus sampling the epidermome, or complement, of proteins expressed in the leaf epidermis. Random sequencing of the derived cDNA library established 3655 unique ESTs, composed of 1142 clusters and 2513 singletons. Virtually all known MIA pathway genes were found in this remarkable set of ESTs, while only four known genes were found in the publicly available Catharanthus EST data set. Several novel MIA pathway candidate genes were identified, as demonstrated by the cloning and functional characterization of loganic acid O-methyltransferase involved in secologanin biosynthesis. The pathways for triterpene biosynthesis were also identified, and metabolite analysis showed that oleanane-type triterpenes were localized exclusively to the cuticular wax layer. The pathways for flavonoid and very-longchain fatty acid biosynthesis were also located in this cell type. The results illuminate the biochemical specialization of Catharanthus leaf epidermis for the production of multiple classes of metabolites. The value and versatility of this EST data set for biochemical and biological analysis of leaf epidermal cells is also discussed.

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Section: Kinetic Analyses and Substrate Specificity Studies With Rlammentioning

confidence: 99%

The Leaf Epidermome ofCatharanthus roseusReveals Its Biochemical Specialization

et al. 2008

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“…For example, enzymes of the monoterpenoid indole alkaloid pathway in Catharanthus roseus have been localized to the cytosol (De Luca and Cutler, 1987), vacuole (McKnight et al, 1991), tonoplast (Stevens et al, 1993), thylakoid membranes (De Luca and Cutler, 1987), and endoplasmic reticulum (ER; St-Pierre and De Luca, 1995). The involvement of multiple subcellular compartments is also apparent in the biosynthesis of quinolizidine alkaloids.…”

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

et al. 2005

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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...

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“…In particular, we wanted to examine the effects of localizing TDC to the chloroplast, the site of biosynthesis of the enzyme's natural substrate, l-Trp (Radwanski and Last, 1995). We also studied the effects of targeting TDC to the endoplasmic reticulum (ER) because it has been demonstrated that targeting proteins to the ER (Iturriaga et al, 1989;Wandelt et al, 1992; Boevink et al, 1996) significantly enhances accumulation of the respective recombinant protein in plant cells (Fiedler et al, 1997; Gomord et al., 1997;Fischer et al, 1999).TDC is a cytosolic enzyme in TIA-producing plants such as C. roseus (De Luca and Cutler, 1987;Stevens et al, 1993), and ideally, studies on localization of TDC should be carried out using transgenic material from the plant expressing TDC as part of the pathway of interest; in our case, TIA biosynthesis in C. roseus. However, although transgenic undifferentiated C. roseus cell cultures can be recovered with relative ease, TIA biosynthesis is severely impeded in such cultures.…”

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confidence: 99%

“…TDC is a cytosolic enzyme in TIA-producing plants such as C. roseus (De Luca and Cutler, 1987;Stevens et al, 1993), and ideally, studies on localization of TDC should be carried out using transgenic material from the plant expressing TDC as part of the pathway of interest; in our case, TIA biosynthesis in C. roseus. However, although transgenic undifferentiated C. roseus cell cultures can be recovered with relative ease, TIA biosynthesis is severely impeded in such cultures.…”

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confidence: 99%

Targeting Tryptophan Decarboxylase to Selected Subcellular Compartments of Tobacco Plants Affects Enzyme Stability and in Vivo Function and Leads to a Lesion-Mimic Phenotype

Fiore

Leech

et al. 2002

Plant Physiology

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Tryptophan decarboxylase (TDC) is a cytosolic enzyme that catalyzes an early step of the terpenoid indole alkaloid biosynthetic pathway by decarboxylation of l-tryptophan to produce the protoalkaloid tryptamine. In the present study, recombinant TDC was targeted to the chloroplast, cytosol, and endoplasmic reticulum (ER) of tobacco (Nicotiana tabacum) plants to evaluate the effects of subcellular compartmentation on the accumulation of functional enzyme and its corresponding enzymatic product. TDC accumulation and in vivo function was significantly affected by the subcellular localization. Immunoblot analysis demonstrated that chloroplast-targeted TDC had improved accumulation and/or stability when compared with the cytosolic enzyme. Because ER-targeted TDC was not detectable by immunoblot analysis and tryptamine levels found in transient expression studies and in transgenic plants were low, it was concluded that the recombinant TDC was most likely unstable if ER retained. Targeting TDC to the chloroplast stroma resulted in the highest accumulation level of tryptamine so far reported in the literature for studies on heterologous TDC expression in tobacco. However, plants accumulating high levels of functional TDC in the chloroplast developed a lesion-mimic phenotype that was probably triggered by the relatively high accumulation of tryptamine in this compartment. We demonstrate that subcellular targeting may provide a useful strategy for enhancing accumulation and/or stability of enzymes involved in secondary metabolism and to divert metabolic flux toward desired end products. However, metabolic engineering of plants is a very demanding task because unexpected, and possibly unwanted, effects may be observed on plant metabolism and/or phenotype.Plants produce large arrays of chemicals, many of which are referred to as secondary metabolites. Despite the minor role initially assigned to these molecules, secondary metabolites are now considered crucial for the interaction of plants with their environment (Verpoorte, 1998). Many secondary metabolites are also important therapeutic agents or pharmaceuticals, and the generally low abundance to which they accumulate has prompted extensive research into their biosynthetic pathways. To date, few plant secondary metabolic pathways have been fully characterized, and some have been partially characterized, although investigation into many is at an early stage. Nevertheless, it is clear that the biosynthesis of secondary metabolites is under strict developmental, temporal, and spatial control in plants (St. Pierre et al., 1999; De Luca and St. Pierre, 2000). As a consequence of the strictly regulated biosynthesis, natural products accumulate to only trace amounts in plants, and their extraction and purification is often difficult and cost intensive. Attempts to use plant cell cultures as an alternative source to natural products have also been problematic, often due to the lack of fully functional pathways required for the production of the target molecules. With the exceptio...

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Subcellular localization of tryptophan decarboxylase, strictosidine synthase and strictosidine glucosidase in suspension cultured cells of Catharanthus roseus and Tabernaemontana divaricata

Cited by 88 publications

References 23 publications

The Leaf Epidermome ofCatharanthus roseusReveals Its Biochemical Specialization

The Leaf Epidermome ofCatharanthus roseusReveals Its Biochemical Specialization

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

Targeting Tryptophan Decarboxylase to Selected Subcellular Compartments of Tobacco Plants Affects Enzyme Stability and in Vivo Function and Leads to a Lesion-Mimic Phenotype

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