The role of phloem sieve elements and laticifers in the biosynthesis and accumulation of alkaloids in opium poppy^†

Abstract

SummaryThe benzylisoquinoline alkaloids of opium poppy, including the narcotic analgesics morphine and codeine, accumulate in the multinucleate cytoplasm of specialized laticifers that accompany vascular tissues throughout the plant. In mature opium poppy plants, immunofluorescence labeling using specific antibodies showed that four alkaloid biosynthetic enzymes, (S)-norcoclaurine 6-O-methyltransferase (6OMT), (S)-coclaurine N-methyltransferase (CNMT), (S)-3¢-hydroxy-N-methylcoclaurine-4¢-O-methyltransferase (4¢OMT) and salutaridinol-7-O-acetyltransferase (SAT) were restricted to sieve elements of the phloem adjacent or proximal to laticifers. The identity of sieve elements was confirmed by (i) the specific immunogold labeling of the characteristic cytoplasm of this cell type, (ii) the co-localization of a sieve element-specific H þ -ATPase with all biosynthetic enzymes and (iii) the strict association of sieve plates with immunofluorescent cells. The localization of laticifers was demonstrated antibodies specific to major latex protein (MLP), which is characteristic of this cell type. In situ hybridization using antisense RNA probes for 6OMT, CNMT, 4¢OMT and SAT showed that the corresponding gene transcripts were found in the companion cell paired with each sieve element. Seven benzylisoquinoline alkaloid biosynthetic enzymes, (S)-N-methylcoclaurine 3¢-hydroxylase (CYP80B1), berberine bridge enzyme, codeinone reductase, 6OMT, CNMT, 4¢OMT and SAT were localized by immunofluorescence labeling to the sieve elements in the root and hypocotyl of opium poppy seedlings. The abundance of these enzymes increased rapidly between 1 and 3 days after seed germination. The localization of seven biosynthetic enzymes to the sieve elements provides strong support for the unique, cell type-specific biosynthesis of benzylisoquinoline alkaloids in the opium poppy.

Benzylisoquinoline alkaloid biosynthesis in opium poppy

“…The biosynthesis and storage of BIAs in opium poppy has been extensively investigated and shown to involve three distinct cell types (Facchini and De Luca 1995;Bird et al 2003;Samanani et al 2006;Lee and Facchini 2010). In situ hybridization and immunofluorescence labeling demonstrated the localization of biosynthetic gene transcripts and enzymes to companion cells and sieve elements, respectively.…”

“…However, SalAT and COR transcripts have also been reported in the cytoplasm, or latex, of laticifers (Decker et al 2000;Larkin et al 2007;Allen et al 2008), although in situ hybridization and immunofluorescence labeling failed to detect SalAT and COR mRNA and corresponding enzymes in laticifers (Bird et al 2003;weid et al 2004;Samanani et al 2006). Recently, the application of shotgun proteomics to determine the relative abundance of proteins in isolated latex, or in whole stem including latex, revealed that the final three steps in morphine biosynthesis, catalyzed by T6ODM, COR and CODM, occur primarily in laticifers (Onoyovwe et al 2013).…”

“…The importance of transcriptional control in BIA metabolism is particularly evident from (1) the cell typespecific localization of biosynthetic gene transcripts (Bird et al 2003;Samanani et al 2006;Lee and Facchini 2010;Onoyovwe et al 2013) and (2) the coordinated induction of all expressed biosynthetic gene transcripts (Zulak et al 2007) and corresponding proteins (Zulak et al 2009;Desgagné-Penix et al 2010) in opium poppy cell cultures after the addition of a fungal-derived elicitor.…”

Tyrosine Aminotransferase Contributes to Benzylisoquinoline Alkaloid Biosynthesis in Opium Poppy

“…The highest transcript levels for most biosynthetic genes involved in BIA metabolism occur in the stems and roots of opium poppy (Bird et al, 2003;Samanani et al, 2006). In particular, transcripts encoding Tyr/ DOPA decarboxylase and NCS, the other known enzymes involved in the early step of the BIA pathway, were abundant in stems and roots and were found only at low levels in leaves and developing seed capsules (Facchini and De Luca, 1995;Lee and Facchini, 2010).…”

“…6). NCS (Lee and Facchini, 2010) and other BIA biosynthetic enzymes are also specifically localized or at least most abundant in sieve elements of the phloem (Bird et al, 2003;Samanani et al, 2006). The occurrence of TyrAT in sieve elements would facilitate access to L-Tyr translocated in the phloem to ensure adequate precursor availability for BIA biosynthesis.…”