The roles of latex and the vascular bundle in morphine biosynthesis in the opium poppy, Papaver somniferum

Abstract

The opium poppy, Papaver somniferum, is one of mankind's oldest medicinal plants. Opium poppy today is the commercial source of the narcotic analgesics morphine and codeine. Along with these two morphinans, opium poppy produces approximately eighty alkaloids belonging to various tetrahydrobenzylisoquinoline-derived classes. It has been known for over a century that morphinan alkaloids accumulate in the latex of opium poppy. With identification of many of the enzymes of alkaloid biosynthesis in this plant, biochemical data suggested involvement of multiple cell types in alkaloid biosynthesis in poppy. Herein the immunolocalization of five enzymes of alkaloid formation in opium poppy is reported: (R,S)-3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase central to the biosynthesis of tetrahydroisoquinoline-derived alkaloids, the berberine bridge enzyme of the sanguinarine pathway, (R,S)-reticuline 7-O-methyltransferase specific to laudanosine formation, and salutaridinol 7-O-acetyltransferase and codeinone reductase, which lead to morphine. In capsule and stem, both O-methyltransferases and the O-acetyltransferase are found predominantly in parenchyma cells within the vascular bundle, and codeinone reductase is localized to laticifers, the site of morphinan alkaloid accumulation. In developing root tip, both O-methyltransferases and the O-acetyltransferase are found in the pericycle of the stele, and the berberine bridge enzyme is localized to parenchyma cells of the root cortex. Laticifers are not found in developing root tip, and, likewise, codeinone reductase was not detected. These results provide cell-specific localization that gives a coherent picture of the spatial distribution of alkaloid biosynthesis in opium poppy.

Fig. 1.

Schematic presentation of the biosynthetic pathway from (S)-norcoclaurine to codeine, laudanine, and (S)-scoulerine in P. somniferum. The positions of the enzymes localized in this study are indicated in bold print.

Fig. 2.

Western blot of P. somniferum protein probed with anti-4′OMT, anti-SalAT, anti-COR, anti-7OMT, anti-BBE, or anti-MLP 15. Crude protein extracts were prepared from capsule, stem, leaf, and root tissue of mature plants. Sites of secondary antibody binding were visualized with nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate.

Fig. 3.

Immunolabeling of alkaloid biosynthetic enzymes in cross sections of capsule of P. somniferum. Shown are cross sections through the large vascular bundle as indicated by the fusion of several vascular bundles (A, D, G, and J) and through a small vascular bundle of the capsule wall (B, E, H, and K); also shown are the preimmune serum controls (C, F, I, and L). The green arrows indicate the position of anti-biosynthetic enzyme antibody; the red arrows indicate the position of anti-MLP 15 antibody and is indicative of laticifer cells; the yellow arrows indicate colocalization of a biosynthetic enzyme with MLP 15. (A–C) Localization of 4′OMT. xy, xylem; la, laticifer. (D–F) Localization of SalAT. (G–I) Localization of COR (yellow is indicative of colocalization of COR and MLP 15). (J–L) Localization of 7OMT. Single sections were probed with two primary antibodies (anti-MLP and anti-biosynthetic enzyme) and two secondary antibodies. Red (excitation, 568 nm; emission long-pass filter, 590 nm) and green (excitation, 488 nm; emission long-pass filter, 520 nm) fluorescence images were overlaid in all micrographs.

Fig. 4.

Immunolabeling of alkaloid biosynthetic enzymes, callose, and MLP 15 in longitudinal sections of stem of P. somniferum. Each row of micrographs represents a single section irradiated with a different excitation wavelength to visualize alkaloid biosynthetic enzyme (A, D, G, and J), callose of sieve plates (B, E, H, and K), or MLP 15 of laticifer cells (C, F, I, and L). The green arrows indicate the position of anti-biosynthetic enzyme antibody; the red arrows indicate the position of anti-MLP 15 antibody and is indicative of laticifer cells; the blue arrows indicate the position of anti-callose antibody and is indicative of sieve plates. (A) Localization of 4′OMT. (D) Localization of SalAT. (G) Localization of COR. (J) Localization of 7OMT. (B, E, H, and K) Callose of sieve elements. (C, F, I, and L) MLP 15 in laticifers. Single sections were probed with three primary antibodies (anti-biosynthetic enzyme, anti-MLP, and anti-callose) and three secondary antibodies. Green (excitation, 488 nm; emission long-pass filter, 520 nm), blue (excitation, 568 nm; emission long-pass filter, 590 nm), and red (excitation, 365 nm; emission long-pass filter, 420 nm) fluorescence images are presented individually. la, laticifer; sp, sieve plate.