Cloning and expression analysis of DNA sequences for the major latex protein of opium poppy

Opium-poppy (Papaver somniferum L.) latex contains a group of very abundant, laticifer-specific peptides called the major latex proteins (MLPs). We determined a partial amino-acid sequence of an MLP cyanogen bromide peptide fragment that was used to design an MLP oligonucleotide primer. An MLP-specific DNA probe was then generated by polymerase chain reaction (PCR) of first-strand complementary DNA (cDNA) templates primed with the MLP oligonucleotide and oligodeoxythymidylic acid. This DNA fragment, called MLP-PCR, and two partial MLP cDNAs isolated with it, all contain open reading frames matching the known MLP amino-acid sequence. RNA gel blots of latex, tissue cultures, and the major organs of mature plants of opium poppy show that MLP is coded for by an 860-nucleotide mRNA and that this accumulates exclusively in laticifers.     

Glycoproteins of the opium poppy

Two carbohydrate-protein fractions were isolated from the water-soluble biopolymer from opium poppy capsules by chromatography on SP-Sephadex. The carbohydrate chains are composed of arabinose, rhamnose, xylose, mannose, glucose, galactose, galacturonic acid, glucuronic acid and 4-O-methyl glucuronic acid. Methylation analysis indicated a high degree of branching suggesting a very complex structure. Treatment of the glycoprotein with NaOH in the presence of NaBH₄ resulted in a significant decrease in the serine and threonine content. The carbohydrate side chains released contained the sugar alcohol, galactitol. These results indicate that polysaccharide chains are linked to protein via serine-O-galactoside linkages.     

The latex capacity of opium poppy capsules is fixed early in capsule development and is not a major determinant in morphine yield

This study found that the latex capacity (mg latex mg⁻¹ dry weight capsule) of opium poppy capsules is fixed early in capsule development. Latex capacity, which represents the proportion of the capsule wall allocated to laticifers (specialised cells for latex storage), had peaked in the capsule at 1 week after flowering. In contrast, the morphine content of capsules continued to increase with capsule development until commercial harvest. Morphine content was correlated with capsule mass and total latex mass, but there was no correlation between latex capacity and morphine yield. The most important morphological characteristic in terms of morphine end yield (commercial harvest stage) was capsule mass. The findings of this study demonstrate that although latex yield per plant is a highly heritable morphological characteristic, it may have limited potential for use in a breeding strategy aimed at increasing the morphine yield from capsules.     

Benzylisoquinoline alkaloid biosynthesis in opium poppy

Opium poppy (Papaver somniferum) is one of the world’s oldest medicinal plants and remains the only commercial source for the narcotic analgesics morphine, codeine and semi-synthetic derivatives such as oxycodone and naltrexone. The plant also produces several other benzylisoquinoline alkaloids with potent pharmacological properties including the vasodilator papaverine, the cough suppressant and potential anticancer drug noscapine and the antimicrobial agent sanguinarine. Opium poppy has served as a model system to investigate the biosynthesis of benzylisoquinoline alkaloids in plants. The application of biochemical and functional genomics has resulted in a recent surge in the discovery of biosynthetic genes involved in the formation of major benzylisoquinoline alkaloids in opium poppy. The availability of extensive biochemical genetic tools and information pertaining to benzylisoquinoline alkaloid metabolism is facilitating the study of a wide range of phenomena including the structural biology of novel catalysts, the genomic organization of biosynthetic genes, the cellular and sub-cellular localization of biosynthetic enzymes and a variety of biotechnological applications. In this review, we highlight recent developments and summarize the frontiers of knowledge regarding the biochemistry, cellular biology and biotechnology of benzylisoquinoline alkaloid biosynthesis in opium poppy.     

Somatic embryogenesis in the opium poppy, Papaver somniferum

A procedure is described for the induction of somatic embryos in the opium poppy. Papaver somniferum L. Callus was obtained from seedling hypocotyls on an agar solidified medium [Murashige and Skoog (1962) Physiol. Plant. 15: 473–497] containing 0.25 mg/l (1.2 μ M ) kinetin and 2.0 mg/l (10.7 μ M ) naphtalene acetic acid (NAA). Suspension cultures were initiated from callus using a liquid medium in which 2.0 mg/l (9.0 μ M ) 2,4-dichlorophenoxyacetic acid was substituted for NAA. Meristemoids, spheres of closely packed cells, developed in suspensions and on the surface of a few callus cultures. Differentiation of meristemoids into somatic embryos was accomplished by removing growth regulators from the liquid medium. Embryoids appeared morphologically normal and similar to torpedo stage embryos, however, they possessed mature tracheary elements and laticifers in areas that should have contained only procambium. Whole plants have been obtained by placing embryos in the light on solid medium that also lacked growth regulators.     

Honeybees foraging response in genetically diversified opium poppy

Studies were carried out on honeybees foraging on plant flowers. Results showed significantly higher foraging response of honeybees (Apis mellifera) in genetically divergent narcotic plant opium poppy (Papaver somniferum). Of the 18 mutants and two locally adapted cultivars of diverse genotypes screened, eight revealed significantly greater foraging response manifesting honeybee's preference towards specific plant morphotypes. The number of flower bloom did not correspond to number of foraging bees in both mutant and cultivar plant types of opium poppy. The genotype specific foraging response of honeybees could be attributed to physico-chemical properties of opium poppy flowers. This could have implications for the development of opium alkaloid fortified honeys for novel pharmaceuticals and isolation of natural spray compounds to attract honeybee pollinators for promoting crossing and sustainable hybridity in crops.     

Biochemical properties of lipoxygenase from opium poppy chloroplasts

Lipoxygenase (LOX) from opium poppy (Papaver somniferum L.) chloroplasts was isolated and 126.1-fold purified to electrophoretic homogeneity by combination of ion-exchange chromatography on HA-Ultragel column and affinity chromatography on a linoleyl-aminopropyl agarose column. The relative molecular mass of the LOX determined by SDS-PAGE was 92 kDa. Kinetic properties of purified LOX were determined in spectrophotometric assay by using of linoleic acid (K_M = 1.78 mM and V_max = 11.4 μmol mg⁻¹ min⁻¹) and linolenic acid (K_M = 1.27 mM and V_max = 10.2 μmol mg⁻¹ min⁻¹). The optimum pH was 6.0 for both linoleic and linolenic acid dioxygenation catalyzed by LOX. HPLC analysis of the products revealed a dual positional specificity of linoleic acid dioxygenation at pH 6.0 with ratio of 9- and 13-hydroperoxide products being about 1:1. The activity of purified LOX was stimulated by Mg²⁺ and Ca²⁺.     

Identification and characterization of latex-specific proteins in opium poppy

Latex from the opium poppy, Papaver somniferum L., was analyzed by polyacrylamide gel electrophoresis (PAGE). Two latex-specific bands were identified in protein samples of poppy latex using one-dimensional native PAGE. Second dimension analysis with SDS-PAGE indicates that these proteins have a relative molecular weight of approximately 20 kilodaltons. We have termed these polypeptides the major latex proteins (MLPs). Polyclonal antibodies prepared against the MLPs were used to probe protein gel blots of latex and poppy tissues known to lack laticifers. Laticifer-free tissues showed no reaction with anti-MLP immunoglobulin G indicating that MLPs are found only in poppy latex. MLP distribution was also examined in mature opium poppy tissues by immunocytochemistry. Laticifers were differentially labeled by fluorescein isothiocyanate secondary labeling of anti-MLP immunoglobulin G and could easily be identified in both transverse and longitudinal section. Fractionation studies of isolated latex showed that MLPs are concentrated in the latex cytosol and not in alkaloidal vesicles. Analysis of latex proteins by conventional two-dimensional electrophoresis indicates that the two MLP bands are composed of several distinct polypeptides with similar relative molecular weights. The pIs of these molecules range from 6.0 to 3.5. The role(s) of MLPs in laticifer metabolism has not been determined.