Developmental regulation of benzylisoquinoline alkaloid biosynthesis in opium poppy plants and tissue cultures

Summary Opium poppy (Papaver somniferum L.) contains a number of pharmaceutically important alkaloids of the benzylisoquinoline type including morphine, codeine, papaverine, and sanguinarine. Although these alkaloids accumulate to high concentrations in various organs of the intact plant, only the phytoalexin sanguinarine has been found at significant levels in opium poppy cell cultures. Moreover, even sanguinarine biosynthesis is not constitutive in poppy cell suspension cultures, but is typically induced only after treatment with a funga-derived elicitor. The absence of appreciable quantities of alkaloids in dedifferentiated opium poppy cell cultures suggests that benzylisoquinoline alkaloid biosynthesis is developmentally regulated and requires the differentiation of specific tissues. In the 40 yr since opium poppy tissues were first culturedin vitro, a number of reports on the redifferentiation of roots and buds from callus have appeared. A requirement for the presence of specialized laticifer cells has been suggested before certain alkaloids, such as morphine and codeine, can accumulate. Laticifers represent a complex internal secretory system in about 15 plant families and appear to have multiple evolutionary origins. Opium poppy laticifers differentiate from procambial cells and undergo articulation and anastomosis to form a continuous network of elements associated with the phloem throughout much of the intact plant. Latex is the combined cytoplasm of fused laticifer vessels, and contains numerous large alkaloid vesicles in which latex-associated poppy alkaloids are sequestered. The formation of alkaloid vesicles, the subcellular compartmentation of alkaloid biosynthesis, and the tissue-specific localization and control of these processes are important unresolved problems in plant cell biology. Alkaloid biosynthesis in opium poppy is an excellent model system to investigate the developmental regulation and cell biology of complex metabolic pathways, and the relationship between metabolic regulation and cell-type specific differentiation. In this review, we summarize the literature on the roles of cellular differentiation and plant development in alkaloid biosynthesis in opium poppy plants and tissue cultures.     

Genetic parameters and correlations of collar rot resistance with important biochemical and yield traits in opium poppy (Papaver somniferum L.)

Collar rot, caused by Rhizoctonia solani Kühn, is one of the most severe fungal diseases of opium poppy. In this study, heritability, genetic advance and correlation for 10 agronomic, 1 physiological, 3 biochemical and 1 chemical traits with disease severity index (DSI) for collar rot were assessed in 35 accessions of opium poppy. Most of the economically important characters, like seed and capsule straw yield per plant, oil and protein content of seeds, peroxidase activity in leaves, morphine content of capsule straw and DSI for collar rot showed high heritability as well as genetic advance. Highly significant negative correlation between DSI and seed yield clearly shows that as the disease progresses in plants, seed yield declines, chiefly due to premature death of infected plants as well as low seed and capsule setting in the survived population of susceptible plants. Similarly, a highly significant negative correlation between peroxidase activity and DSI indicated that marker-assisted selection of disease-resistant plants based on high peroxidase activity would be effective and survived susceptible plants could be removed from the population to stop further spread.     

Molecular genetic diversity and association mapping of morphine content and agronomic traits in Turkish opium poppy (<Emphasis Type="Italic">Papaver somniferum</Emphasis>) germplasm

As the sole plant source of many potent alkaloids, opium poppy (Papaver somniferum L.) is an important medicinal crop. Nevertheless, few studies have characterized opium poppy germplasm with crop-specific molecular markers. Because Turkey is a diversity center for opium poppy, Turkish germplasm is a valuable genetic resource for association mapping studies aimed at identifying QTLs controlling morphine content and agronomic traits. In this study, the morphological diversity and molecular diversity of 103 Turkish opium poppy landraces and 15 cultivars were analyzed. Potentially useful morphological variation was observed for morphine content, plant height, and capsule index. However, the landraces exhibited limited breeding potential for stigma number, and seed and straw yields. Both morphological and molecular analyses showed distinct clustering of cultivars and landraces. In addition, a total of 164 SSR and 367 AFLP polymorphic loci were applied to an opium poppy association mapping panel composed of 95 opium poppy landraces which were grown for two seasons. One SSR and three AFLP loci were found to be significantly associated with morphine content (P < 0.01 and LD value (r ²) = 0.10–0.32), and six SSR and 14 AFLP loci were significantly associated with five agronomic traits (plant height, stigma number, capsule index, and seed and straw yields) (P < 0.01 and LD value (r ²) = 0.08–0.35). This is the first report of association mapping in this crop. The identified markers provide initial information for marker-assisted selection of important traits in opium poppy breeding.     

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.     

Molecular cloning and functional expression of codeinone reductase: the penultimate enzyme in morphine biosynthesis in the opium poppy Papaver somniferum.

The narcotic analgesic morphine is the major alkaloid of the opium poppy Papaver somniferum. Its biosynthetic precursor codeine is currently the most widely used and effective antitussive agent. Along the morphine biosynthetic pathway in opium poppy, codeinone reductase catalyzes the NADPH-dependent reduction of codeinone to codeine. In this study, we have isolated and characterized four cDNAs encoding codeinone reductase isoforms and have functionally expressed them in Escherichia coli. Heterologously expressed codeinone reductase-calmodulin-binding peptide fusion protein was purified from E. coli using calmodulin affinity column chromatography in a yield of 10 mg enzyme l-1. These four isoforms demonstrated very similar physical properties and substrate specificity. As least six alleles appear to be present in the poppy genome. A comparison of the translations of the nucleotide sequences indicate that the codeinone reductase isoforms are 53% identical to 6'-deoxychalcone synthase from soybean suggesting an evolutionary although not a functional link between enzymes of phenylpropanoid and alkaloid biosynthesis. By sequence comparison, both codeinone reductase and 6'-deoxy- chalcone synthase belong to the aldo/keto reductase family, a group of structurally and functionally related NADPH-dependent oxidoreductases, and thereby possibly arise from primary metabolism.     

Effect of triacontanol on photosynthesis,alkaloid content and growth in opium poppy (Papaver Somniferum L.)

The influence of different foliar applications of Triacontanol (Tria.) on growth, CO₂ exchange, capsule development and alkaloid accumulation in opium poppy was studied in glasshouse conditions. Plant height, capsule number and weight, morphine content, CO₂ exchange rate, total chlorophyll and fresh and dry weight of the shoot were significantly maximum at 0.01 mg/l Tria. At the highest concentration (4 mg/l) total chlorophyll, CO₂ exchange rate and plant height were significantly inhibited. Thebaine and codeine contents remained unaffected at all the concentrations. The concentration of Fe, Mn, Cu in shoots were maximum at .01 and Zn at 0.1 mg/l Tria. Increase in shoot weight, leaf area ratio and chlorophyll content were significantly correlated with morphine content.CIMAP Communication No. 839.     

Metabolic engineering with a morphine biosynthetic P450 in opium poppy surpasses breeding

Morphine biosynthesis was genetically engineered in an industrial elite line of the opium poppy (Papaver somniferum L.), to modify the production of alkaloids in plants. The cytochrome P-450-dependent monooxygenase (S)-N-methylcoclaurine 3'-hydroxylase (CYP80B3) lies on the pathway to the benzylisoquinoline alkaloid branch point intermediate (S)-reticuline. Overexpression of cyp80b3 cDNA resulted in an up to 450% increase in the amount of total alkaloid in latex. This increase occurred either without changing the ratio of the individual alkaloids, or together with an overall increase in the ratio of morphine. Correspondingly, antisense-cyp80b3 cDNA expressed in opium poppy caused a reduction of total alkaloid in latex up to 84%, suggesting that the observed phenotypes were dependent on the presence of the transgene. This study found compelling evidence, that cyp80b3 is a key regulation step in morphine biosynthesis and provides practical means to genetically engineer valuable secondary metabolites in this important medicinal plant.     

Sanguinarine biosynthesis is associated with the endoplasmic reticulum in cultured opium poppy cells after elicitor treatment

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.     

Timing of reproductive and vegetative development in Saxifraga oppositifolia in an alpine and a subnival climate

Morphogenesis of floral structures, dynamics of reproductive development from floral initiation until fruit maturation, and leaf turnover in vegetative short-stem shoots of Saxifraga oppositifolia were studied in three consecutive years at an alpine site (2300 m) and at an early- and late-thawing subnival site (2650 m) in the Austrian Alps. Marked differences in the timing and progression of reproductive and vegetative development occurred: individuals of the alpine population required a four-month growing season to complete reproductive development and initiate new flower buds, whereas later thawing individuals from the subnival sites attained the same structural and functional state within only two and a half months. Reproductive and vegetative development were not strictly correlated because timing of flowering, seed development, and shoot growth depended mainly on the date of snowmelt, whereas the initiation of flower primordia was evidently controlled by photoperiod. Floral induction occurred during June and July, from which a critical day length for primary floral induction of about 15 h could be inferred. Preformed flower buds overwinter in a pre-meiotic state and meiosis starts immediately after snowmelt in spring. Vegetative short-stem shoots performed a full leaf turnover within a growing season: 16 (+/-0.8 SE) new leaves per shoot developed in alpine and early-thawing subnival individuals and 12 (+/-1.2 SE) leaves in late-thawing subnival individuals. New leaf primordia emerged continuously from snowmelt until late autumn, even when plants were temporarily covered with snow. Differences in the developmental dynamics between the alpine and subnival population were independent of site temperatures, and are probably the result of ecotypic adaptation to differences in growing season length.     

Timing of exposure to ozone affects reproductive sensitivity and compensatory ability in Brassica campestris

It is well established that exposure to ozone (O₃) may impair vegetative growth and reproductive development in plants, although the consequences for yield depend on the effectiveness of the compensatory processes induced. This study examined the effects of exposing the terminal inflorescence of Brassica campestris L. to 100 ppb O₃ for 6 h d⁻¹ on four consecutive days during early flowering while the vegetative organs received charcoal-filtered air. The ordered predictability of development in B. campestris is ideal for studies of the impact of abiotic stress factors such as O₃ on reproductive development and seed production. Effects on reproductive development and seed yield characteristics were determined for floral sites exposed at different developmental stages. Flower and pod numbers on the terminal raceme were unaffected by exposure, but effects on pod length varied depending on the developmental stage of floral sites during exposure. Increased ovule abortion and precocious seed germination in the pods of O₃-treated plants reduced mature seed number pod⁻¹. Although the individual weight of mature seeds was slightly greater in O₃-treated plants, seed yield pod⁻¹ and seed yield plant⁻¹ were reduced due to the lower seed number pod⁻¹. Seed from O₃-treated plants germinated more rapidly than control seed irrespective of the stage of floral development during exposure. The results show that exposure to realistic O₃ episodes during the early stages of flowering significantly reduced seed yield without impairing the vigour of the seeds produced. The physiological origins of these effects are discussed.     

滇鼠刺花的形态发生（鼠刺科）

在扫描电镜下 ,观察了滇鼠刺 (IteayunnanensisFranch .)花的形态发生。花 3朵一束 ,排成总状花序。花器官为轮状结构 ,向心发生 ;花萼以 2 /5螺旋式相继发生 ,5个花瓣原基几乎同步地在花萼内侧与其互生的位置发生。雄蕊单轮对萼。当雄蕊发生后 ,花顶中心的分生组织开始凹陷 ,成为浅锅状 ;在其周围出现一个环状的分生组织 ,随之 ,2心皮原基产生 ,进而发育为马蹄形。初期的心皮相互分离 ,随着进一步发育 ,心皮内卷 ,彼此靠近、紧贴 ,逐渐于腹面合生 ,形成 2室的中轴胎座 ;花柱的腹维管束通过薄壁组织连通 ;花期柱头融合 ,因此该种为合生心皮。对鼠刺属 (Itea)及相关类群花发育性状和花结构进行了比较 ,支持把鼠刺属提升为鼠刺科 (Iteaceae)的观点。     

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.