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.

Figure 1.

Conversions catalyzed by CYP80B1, BBE, and COR in the biosynthesis of sanguinarine and morphine.

Figure 2.

Time course showing the induction of CYP80B1, BBE, and COR protein levels in cultured opium poppy cells treated with a fungal elicitor. Total cellular proteins were extracted, fractionated on by SDS-PAGE, and transferred to nitrocellulose membranes. Specific proteins were detected on western blots using antibodies raised against CYP80B1, BBE, COR, or calreticulin. Blots are representative of three independent experiments.

Figure 3.

Suc density gradient fractionation of cultured opium poppy cells treated with a fungal elicitor for 30 h. Every second fraction recovered from the gradient was used. Results are representative of three independent experiments.

Figure 4.

Suc density gradient fractionation, in the presence of magnesium or EDTA, of opium poppy cells treated with a fungal elicitor for 30 h. Every second fraction recovered from the gradient was used. Results are representative of three independent experiments.

Figure 5.

Time course of osmium tetroxide-fixed cultured opium poppy cells treated with a fungal elicitor. A, Control cell. B to F, Cells at different times after elicitor treatment: B, 2 h; C, 5 h; D, 10 h; E, 50 h; and F, 80 h. The actively growing control cells have a dense cytoplasm, and many plastids and mitiochondria (A). By 5 h posttreatment, some cells show the presence of large vesicles in the cytoplasm (C). After10 h, there is evidence of smaller vesicles fusing to form large vesicles (D), and by 50 h the movement of vesicles to the tonoplast is apparent (E). Eighty hours after treatment, many cells show disruption of the cytoplasm and the presence of multivesicular bodies (F). Asterisks show the location of large cytoplasmic vesicles. Abbreviations are as follows: a, amyloplasts; cw, cell wall; er, endoplasmic reticulum; m, mitochondrion; p, plastid; and v, vacuole. Bars = 500 nm.

Figure 6.

Permanganate-fixed (A–C), and high-pressure frozen and freeze-substituted (D–F) cultured opium poppy cells. A, Permanganate-fixed control cell showing extensive lamellar ER but lacking large vesicles. B, Permanganate-fixed cultured cell 50 h after elicitor treatment showing proliferation of large cytoplasmic vesicles containing a flocculent, electron-dense material, which also appears in the vacuole. C, Permanganate-fixed cultured cell 50 h after elicitor treatment showing dilations of lamellar ER, which develop into larger vesicles with flocculent material. D, High-pressure frozen and freeze-substituted control cell showing extensive lamellar ER but lacking large vesicles. E, High-pressure frozen and freeze-substituted cultured cell 50 h after elicitor treatment showing large cytoplasmic vesicles with an electron-dense membrane lining. Note the proximity of vesicle with tonoplast at bottom right of image. F, High-pressure frozen and freeze-substituted cultured cell 50 h after elicitor treatment showing the fusion of a large cytoplasmic vesicle with the central vacuole. Asterisks show the locations of large cytoplasmic vesicles. Abbreviations are as follows: cw, cell wall; m, mitochondrion; p, plastid; and v, vacuole. Bars = 500 nm.

Figure 7.

Immunogold localization of CYP80B1, BBE, and COR in cultured opium poppy cells treated with a fungal elicitor for 50 h. A, Anti-CYP80B1 serum. Labeling is associated with the ER (arrowheads). B, Anti-BBE. Labeling is associated with the ER. C, Anti-COR serum. Labeling is associated with the cytosol. D, Preimmune serum from the mouse used to produce anti-CYP80B1 serum. No labeling is visible. Ten-nanometer, gold-conjugated IgGs (anti-mouse or anti-rabbit, as appropriate) were used as the secondary antibodies in all cases. Abbreviations are as follows: v, vacuole; m, mitochondrion; and cw, cell wall. Bars = 500 nm.

Figure 8.

Autofluorescence of control and elicitor-treated opium poppy cell cultures. A, Cultured cell 50 h after elicitor treatment poppy cells showing distinct yellow-green fluorescence due to the accumulation of sanguinarine in the vacuoles. Only some of the cells show this fluorescence. B, Noninduced cells in which yellow-green fluorescence is not visible. Bar = 25 μm.