Studying Morphine Biosynthesis Using Transgenic Opium Poppy (Papaver somniferum L.)

Opium poppy (Papaver somniferum L.) produces a large variety of isoquinoline alkaloids. The aim of this investigation is to understand the regulation of biosynthesis and the ecological function of the alkaloids in the plant. Agrobacterium-mediated transformations of opium poppy were used to introduce the berberine bridge enzyme cDNA bbe 1 in the antisense orientation into seedling explants. After induction of callus on an appropriate medium, embryos were developed via somatic embryogenesis. After the embryos were developed into plantlets with leaves and roots they were transferred to soil. In this way, forty-nine phenotypically normal T0 plants were produced. Forty-six plants produced viable seeds and were used to produce T1 plants. These plants were then analyzed for the presence of the bbe 1 transgene and for the content of alkaloid in latex and root. Selected plants showed a differential alkaloid pattern in latex compared to the wild type. In this paper, the results of a plant with an altered alkaloid profile, heritable at least to the T2 generation, is presented. This represents the first example of metabolic engineering of the alkaloid pathways in opium poppy. INTRODUCTION Opium poppy is one of the most important industrial medicinal plants. It serves as the sole renewable source of the analgesic and narcotic drugs morphine and codeine, as well as the muscle relaxant papaverine, and the antitumoric agent noscapine. Although morphine biosynthesis is well understood at the enzymatic level (reviewed in Kutchan, 1998), the regulation and ecological function of the morphinan alkaloids in plants is still unknown. Opium poppy was regenerated via organogenesis (Nessler and Mahlberg, 1979; Kamo et al., 1982; Staba et al., 1982) as well as via somatic embryogenesis (Nessler, 1982; Schuchmann and Wellmann, 1983; Yoshikawa and Furuya, 1983; Larkin et al., 1999). In 1997, Belny and co-workers succeeded in the transformation of opium poppy from a cell suspension culture. They developed first a callus from the hypocotyl segments, which was maintained as a suspension culture. This cell culture was later cocultivated with an Agrobacterium tumefaciens strain. The bacterium contained a binary vector, in which sam 1 has been cloned, a gene coding for S-adenosyl-L-methionine synthase. The control vector contained uidA as a reporter gene. In one out of five transformed cell lines Belny et al. (1997) found a 1.8 fold increase in S-adenosyl-Lmethionine synthase activity. This study proved for the first time that opium poppy is transformable with an Agrobacterium strain as well as a gene from primary metabolism. Because of the heterogeneity of their results the authors believe, that there are unknown factors in their system, which influence the transformation efficiency considerably. In only one suspension culture they could find a small increase in sam synthase activity. Similar results were shown for the frequency of three independent transient expressions of the uidA reporter gene (Belny et al., 1997). In all regeneration methods of opium poppy described so far, a callus was always necessary. Proc. WOCMAP III, Vol.6: Traditional Medicine & Nutraceuticals Eds. U.R. Palaniswamy, L.E. Craker and Z.E. Gardner Acta Hort. 680, ISHS 2005 38 Recently a protocol was published where intact opium poppy plants had been transformed via shoot organogenesis. The cotyledons were co-cultivated with an Agrobacterium strain, which contained the uidA reporter gene. After the development of shoots, they first must be rooted and subsequently planted into soil. In all the plants regenerated with this procedure, GUS enzyme activity could be detected. The cytohistochemical evidence of GUS activity in different plant organs confirmed the transformation of the regenerated poppy plants (Park and Facchini, 2000). An established protocol (Larkin et al., 1999) for the stable genetic transformation of opium poppy using a disarmed strain of A. tumefaciens and the efficient regeneration of poppy plants via somatic embryogenesis was used to transform opium poppy explants with a binary vector containing antisense bbe. One transgenic plant where the alkaloid profile in the latex is different in comparison to the wild type are reported in this study. MATERIALS AND METHODS Production of Transgenic Plants and Harvest of Latex Transgenic plants were regenerated according to Larkin et al. (1999). The poppy flower was self-pollinated on the day the petals opened. Two days after the petals were dropped by the plant, latex was collected by incising the capsule longitudinally with a scalpel. The exuded latex was collected with a pipette and stored in 200 μL collection buffer (100 mM potassium phosphate pH 7.2, 500 mM D-mannitol, and 20 mM ascorbic acid) and frozen in liquid nitrogen. The samples were stored at -80°C until analysis. HPLC Analysis of Benzylisoquinoline Alkaloids Latex samples of wild type or transgenic plants were thawed on ice and treated for 6 min with ultrasound at 4°C. The sample was centrifuged for 30 min at 14,000 rpm at 4°C. The supernatant was used to determine the protein concentration and the pellet extracted with 70% ethanol. After a second centrifugation, an aliquot of the supernatant was mixed with an internal standard (dihydrocodeine). Samples were analyzed by HPLC on a liquid chromatography system (Agilent Technologies, Waldbronn, Germany) using a reverse phase column (LiChrospher 60 RP-select B, 4.0 x 250 mm, 5 μm). The alkaloids were separated at a flow rate of 1 mL min and using the gradient H2O : acetonitrile (0-25 min 0-46% acetonitrile, 25-26 min 46-100% acetonitrile, 26-33 min 100% acetonitrile) containing 0.01% (v/v) phosphoric acid. Peaks were routinely identified from their UV spectra and by comparison of their retention times to those of authentic standards. Subsequently the identity of the peaks was confirmed by liquid chromatography-mass spectroscopy. Liquid Chromatography-Mass Spectroscopy The positive electrospray ionisation (ESI) mass spectra were obtained with a Mariner-TOF instrument (Applied Biosystems, Weiterstadt, Germany) using a turbulon spray [spray tip potential 5200 V, SCIEX heater 275°C, nozzle potential 140 V, nebulizer and auxillary gas (nitrogen) coupled to a modified Agilent 1100-LC equipped with a Lichrospher 60 RP-select B column (2 x 125 mm i.d.; 5 μm)]. The analysis was performed with a gradient system consisting of H2O CH3CN (each containing 0.2% HCOOH) as described above. Retention times and (M+H) ions were as follows: morphine (7.4 min; m/z 286.1); dihydrocodeine (11.2 min; m/z 302.2); codeine (11.5 min; m/z 300.2); oripavine (13.1 min; m/z 298.1); reticuline (15.5 min; m/z 328.2); thebaine (18.1 min; m/z 312.2). RESULTS AND DISCUSSION The full-length opium poppy BBE (Fig. 1) coding region (Facchini et al., 1996; Huang and Kutchan, 2000) was isolated and cloned into pGEM-T vector (Promega). The blunt-ended SpeI/SacII restriction fragment was ligated into the SnaBI digested vector pPLEX X002 and named pPOP19. This construct (Fig. 2) was used to transform opium