Cell-Specific Expression of Tetrahydrobenzylisoquinoline Alkaloid Biosynthetic Genes

The opium poppy contains specialized internal secretory cells called laticifers. In the aerial parts of the plant, the laticifer cells are anastomosed, forming a reticulated network. Laticifers are found associated with the vascular bundle in all plant parts. Morphine is found both in roots and in aerial plant parts and specifically accumulates in vesicles in laticifers. The benzo[c]phenathridine sanguinarine is found in root tissue. In plant cell cultures of P. somniferum, accumulation of sanguinarine can be elicited by addition of methyl jasmonate (Huang and Kutchan, 2000), but conditions have not been found under which morphine accumulates. The reason for the absence of morphine in cell culture is not completely clear, since all of the enzymes for which in vitro assays have been developed are also found in cell culture extracts. With availability of several biosynthetic cDNAs from P. somniferum, information as to the localization of selected biosynthetic enzymes and, therefore, the spatial distribution of alkaloid biosynthesis becomes clearer.

Tyrosine/dopa decarboxylase participates in the very early stages of tetrahydrobenzylisoquinoline alkaloid biosynthesis. In P. somniferum, this enzyme is encoded by a multigene family, which is classified into two groups tydc1 and tydc2 (Facchini and De Luca, 1994). From in situ hybridization experiments, transcript of tydc1 was more abundant than tydc2 in roots, while tydc2 transcript was more abundant than tydc1 transcript in stem (Facchini and De Luca, 1995). tydc transcript was detected in the metaphloem and protoxylem of vascular bundles in aerial plant parts (Fig. 10.9A and C). This localization is consistent with latex as the site of morphinan alkaloid accumulation.

FIGURE 10.9 Localization of tydc (Facchini and De Luca, 1995), SalAT, COR1, and MLP (Weid et al., 2004) in stem of P. somniferum. Panel (A) root cross-section stained with aniline safranine and astra blue; (B) in situ hybridization of tydc1; (C) in situ hybridization of tydc2; (D) immunolocalization of MLP to laticifers (red fluorescence) and SalAT to phloem parenchyma (green fluorescence); (E) immunolocalization of MLP to laticifers (red fluorescence); and (F) coimmunolocalization of MLP and COR1 to laticifers (yellow fluorescence). Green fluorescence indicates COR1 is present also in phloem parenchyma. xy, xylem; ph, phloem; vc, vascular cambium; la, laticifers; tydc, tyrosine/dopa decarboxylase; SalAT, salutaridinol 7-O-acetyltransferase; COR1, codeinone reductase; MLP, major latex protein.

Immunolocalization of five enzymes of alkaloid biosynthesis, two ofwhich occur late in the morphine-specific pathway, has been carried out with P. somniferum. Morphine biosynthesis is localized to the vascular bundle in capsule, stem, and root, and involves two different cell types—paranchyma associated with phloem and laticifer cells (Weid et al., 2004). Whereas 4'-omt and SalAT were detected in phloem parenchyma cells, COR1 was abundantly colocalized with major latex proteins to laticifers (Fig. 10.9D and F). It appears that at a late stage in morphine formation, at the level of salutaridinol-7-O-acetate or thebaine, biosynthesis moves out of the phloem parenchyma into the laticifers, the ultimate site of thebaine, codeine, and morphine accumulation. As for vindoline biosynthesis in C. roseus (St-Pierre et al., 1999), more than one cell type is implied in morphine biosynthesis in P. somniferum. This spatial distribution of the biosynthetic pathway infers that transport processes are integral to alkaloid formation. Again, cellular localization and intermediate transport could be one level of regulation of alkaloid biosynthesis in P. somniferum.

Due to commercial importance, P. somniferum is an alkaloid-producing plant of choice for metabolic engineering. Promoters will need to be chosen, however, that will direct transgene expression to the cell types in P. somniferum in which the appropriate biosynthetic gene transcripts are expected to occur. As model systems, plant cell cultures of a multitude of isoquinoline alkaloid-producing species can be used for metabolic engineering experiments, bypassing in some instances the complications that arise from multicellular compartmentation in differentiated plants.