Membranotropic Compounds and Alkaloid Accumulation in Plants

Abstract

The biosynthesis of alkaloids is a complex multiphase dynamic process. Its phases are separated spatially, as a rule, at the cellular or subcellular level and, less frequently, at the organ level. In the first two cases, membrane permeability plays a key role in these processes. However, the role of membranotropic compounds (MTC), affecting membrane permeability, in alkaloid biosynthesis is not well studied. Nothing is also known about the effect of the molecular composition of these compounds.The objective of this work was to study the effects of some membranotropic compounds substantially differing in their properties and chemical structure on the accumulation in plants of isoquinolines and indoles, the most widespread types of alkaloids. Five compounds were used to affect the synthesis of isoquinolines; seven compounds, to affect the synthesis of indoles.Experiments were performed with poppy ( Papaver somniferum L., cv. Novinka 198)) and catharanthus ( Catharantus roseus L.) seedlings grown from seeds obtained from the Ukrainian and North Caucasus Experimental Stations of VILR, respectively. Seed germinability in the laboratory was 85 to 95%. Seeds were germinated in petri dishes on ashless filters (red band) moistened with water (control) or aqueous solutions of MTC (treatment) for 4 to 7 days. Catharanthus roseus seeds were germinated in a luminestat at 30 ° C; Papaver somniferum seeds, at room temperature in darkness.After the exposure, a sample of plant material was homogenized with mortar and pestle in ethanol; the homogeneous suspension thus obtained was transferred quantitatively into a 25-ml volumetric flask, the volume was adjusted with ethanol, and the suspension was kept at room temperature for three to five hours under periodic stirring. Then, the suspension was centrifuged at 8000 rpm for 10 min. A 20-ml aliquot of the supernatant was evaporated in a rotor evaporator until dry. The residue was dissolved in 3 to 4 ml of ethyl ether, 10 ml of 2% acetic acid was added, and ether was evaporated. The obtained aqueous acid solution containing the total sum of alkaloids was cooled under water flow or on ice. Sedimented lipophilic ballast compounds were separated by filtration through paper and membrane filters. The pellet on filter was twice washed with 4 ml of 2% acetic acid. The volume of filtrate was adjusted to 25 ml.The filtrate obtained (5-10 ml) was placed in a separatory funnel containing 5 ml of acetate buffer, 2 ml of 0.03% aqueous solution of bromocresol purple was added, and alkaloids were extracted twice with 5 ml of chloroform. Each time, the lower chloroform layer was poured in a 10-ml volumetric flask. The volume of the extract was adjusted to 10 ml. The extract was centrifuged at 2000 rpm for 10 min. An optical density of the supernatant was estimated at 420 nm against the control sample containing only the reagents. The total content of alkaloids was estimated using a calibration curve, which was plotted using tebaine (the major alkaloid of P. somniferum ) or catharantine (the major alk...

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