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. 2003 Nov;15(11):2626-35.
doi: 10.1105/tpc.015396. Epub 2003 Sep 24.

A tale of three cell types: alkaloid biosynthesis is localized to sieve elements in opium poppy

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A tale of three cell types: alkaloid biosynthesis is localized to sieve elements in opium poppy

David A Bird et al. Plant Cell. 2003 Nov.

Abstract

Opium poppy produces a diverse array of pharmaceutical alkaloids, including the narcotic analgesics morphine and codeine. The benzylisoquinoline alkaloids of opium poppy accumulate in the cytoplasm, or latex, of specialized laticifers that accompany vascular tissues throughout the plant. However, immunofluorescence labeling using affinity-purified antibodies showed that three key enzymes, (S)-N-methylcoclaurine 3'-hydroxylase (CYP80B1), berberine bridge enzyme (BBE), and codeinone reductase (COR), involved in the biosynthesis of morphine and the related antimicrobial alkaloid sanguinarine, are restricted to the parietal region of sieve elements adjacent or proximal to laticifers. The localization of laticifers was demonstrated using antibodies specific to the major latex protein (MLP), which is characteristic of the cell type. In situ hybridization showed that CYP80B1, BBE, and COR gene transcripts were found in the companion cell paired with each sieve element, whereas MLP transcripts were restricted to laticifers. The biosynthesis and accumulation of alkaloids in opium poppy involves cell types not implicated previously in plant secondary metabolism and dramatically extends the function of sieve elements beyond the transport of solutes and information macromolecules in plants.

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Figures

Figure 1.
Figure 1.
Sites of Action of (S)-N-Methylcoclaurine-3′-Hydroxylase (CYP80B1), Berberine Bridge Enzyme (BBE), and Codeinone Reductase (COR) in the Biosynthesis of Morphine and Sanguinarine. Dashed arrows indicate multiple enzymatic steps.
Figure 2.
Figure 2.
Immunospecificity of Affinity-Purified Antibodies. Immunoblot showing the levels of CYP80B1, BBE, and COR in crude protein extracts of various opium poppy organs. Protein blots were probed with affinity-purified polyclonal antibodies. Data are representative of three independent experiments. R, root; S, stem; L, leaf; C, carpel.
Figure 3.
Figure 3.
Alkaloid Biosynthetic Enzymes Are Localized to a Specific Cell Type Adjacent or Proximal to Laticifers in Opium Poppy. (A) to (D) Anatomical staining and immunofluorescence localization of CYP80B1 (red), BBE (green), COR (blue), and MLP (yellow) in the phloem of serial root cross-sections. (E) to (H) Phloem of serial stem cross-sections. (I) to (L) Phloem of serial leaf cross-sections. (M) to (P) Phloem of serial carpel cross-sections. Asterisks show the locations of several laticifers in sections (A), (E), (I), and (M) stained with toluidine blue O. Bars = 25 μm.
Figure 4.
Figure 4.
Alkaloid Biosynthetic Gene Transcripts Are Localized to the Companion Cells Paired with Sieve Elements in Opium Poppy. (A) to (D) In situ hybridization using DIG-labeled antisense probes for CYP80B1 (A), BBE (B), COR (C), and MLP (D) performed on stem ([A] and [B]) and carpel ([C] and [D]) sections. (E) and (F) In situ hybridization using DIG-labeled sense probes for CYP80B1 (E) and MLP (F) performed on stem (E) and carpel (F) sections. Asterisks and arrowheads show the locations of several laticifers and labeled companion cells, respectively. Bars = 25 μm.
Figure 5.
Figure 5.
Colocalization of MLP, Biosynthetic Enzymes or Gene Transcripts, and Callose Confirms the Role of Sieve Elements and Companion Cells in Alkaloid Biosynthesis. (A) Immunofluorescence localization of COR (blue), MLP (yellow), and callose (red) in a serial overlay of LR White–embedded, longitudinal root sections (0.3 μm thick) of opium poppy. Callose was localized using a β-1,3-linked glucan monoclonal antibody. (B) and (C) In situ hybridization using a DIG-labeled antisense probe for CYP80B1 (B) and localization of callose using aniline blue (C) in a root longitudinal section. Closed arrowheads point to two sieve plates, and the open arrowhead shows a DIG-labeled companion cell. (D) and (E) In situ hybridization using a DIG-labeled antisense probe for BBE (D) and localization of callose using aniline blue (E) in a root cross-section. Arrowheads show the locations of a sieve plate and pit fields. Bars = 15 μm.
Figure 6.
Figure 6.
Immunofluorescence Localization of Alkaloid Biosynthetic Enzymes to the Parietal Layer of Sieve Elements. Root cross-sections were counterstained with calcofluor white. (A) CYP80B1 (red) and MLP (yellow). (B) BBE (green) and MLP (yellow). (C) COR (blue) and MLP (yellow). Bar = 25 μm.

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