Cell Suspension as a Tool to Study the Biosynthesis of Pilocarpine in Jaborandi

Abstract

Jaborandi (Pilocarpus microphyllus) is a species that naturally occurs in the North and Northeast of Brazil, whose leaves produce pilocarpine (an imidazole alkaloid that has been used to treat glaucoma and xerostomy), the biosynthesis of which is still uncertain. The aim of this work was to establish cell lineages and select them according to an alkaloid profile similar to the one from Jaborandi leaves. The induction of callus was done in different culture media and growth regulators. Calluses from primary cultures or those subcultured several times were used as explants for the obtainment of six cell lineages. Alkaloids content analyses and growth curves showed that lines obtained from primary cultures produced more alkaloids and a better development. Cell lines from 12 subcultures presented a decrease in pilocarpine and pilosine production. After 24 subcultures, the production of alkaloids remained constant. ESI-MS analysis showed that cell culture extracts have the same alkaloid composition as extracts made from leaves. The results indicate that cell suspensions can be used as a model to study the biosynthesis of the imidazole alkaloid in P. microphyllus.

P. microphyllus - pilosine - pilocarpine - alkaloids.

References

• 1 Abreu I. N., Sawaya A. C. H. F., Eberlin M. N., Mazzafera P.. Production of pilocarpine in callus of jaborandi (Pilocarpus microphyllus Stapf).  In Vitro Cellular Developmental Biology Plant. (2005);  41 806-811
• 2 Andrade Neto M., Mendes P. H., Silveira E. R.. An imidazole alkaloid and other constituents from Pilocarpus trachyllophus.  Phytochemistry. (1996);  42 885-887
• 3 Avancini G., Abreu I. N., Saldana M. D. A., Mazzafera P.. Induction of pilocarpine formation in Jaborandi leaves by salicylic acid and methyljasmonate.  Phytochemistry. (2003);  63 171-175
• 4 Bourgaud F., Gravot A., Milesi S.. Production of plant secondary metabolites: a historical perspective.  Plant Science. (2001);  16 839-851
• 5 Collin H. A.. Secondary product formation in plant tissue cultures.  Plant Growth Regulation. (2001);  34 119-134
• 6 Deus-Neumann B., Zenk M. H.. Instability of indole alkaloid production in Catharanthus roseus cell suspension cultures.  Planta Medica. (1984);  50 427-431
• 7 Dias A. C. P., Tomas-Barberan F. A., Fernandes-Ferreira M., Ferreres F.. Unusual flavonoids produced by Callus of Hypericum perforatum.  Phytochemistry. (1998);  48 1165-1168
• 8 Facchini P. J.. Alkaloid biosynthesis in plants: biochemistry, cell biology, molecular regulation, and metabolic engineering applications.  Annual Review of Plant Physiology and Plant Molecular Biology. (2001);  52 29-66
• 9 Hobbs M. C., Yeoman M. M.. Effect of light on alkaloid accumulation in cell - cultures of Nicotiana species.  Journal of Experimental Botany. (1991);  42 1371-1378
• 10 Lindsey K., Yeoman M. M.. The relationship between growth rate, differentiation and alkaloid accumulation in cell cultures.  Journal of Experimental Botany. (1983);  34 1055-1065
• 11 Link H., Bernauer K.. Über die Synthese der Pilocarpus-Alkaloide Isopilosin und Pilocarpin, sowie die absolute Konfiguration des (+)Isopilosins.  Helvetica Chimica Acta. (1972);  55 1053-1062
• 12 Morris P., Rudge K., Cresswell R., Fowler M. W.. Regulation of product synthesis in cell cultures of Catharanthus roseus. V. Long-term maintenance of cells on a production medium.  Plant Cell Tissue and Organ Culture. (1989);  17 79-90
• 13 Murashige T., Skoog F.. A revised medium for rapid growth and bioassays with Tobacco tissue cultures.  Physiologia Plantarum. (1962);  15 473-497
• 14 Pinheiro C. U. B.. Jaborandi (Pilocarpus sp., Rutaceae): a wild species and its rapid transformation into a crop.  Economic Botany. (1997);  51 49-58
• 15 Roberts S. C., Naill M., Gibson D., Shuler M. L.. A simple method for enhancing paclitaxel release from Taxus canadensis cell suspension cultures utilizing cell wall digesting enzymes.  Plant Cell Reports. (2003);  21 1217-1220
• 16 Schenk R. U., Hildebrandt A. C.. Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant-cell cultures.  Canadian Journal of Botany. (1972);  50 199-204
• 17 Sierra M. I., Van der Heijden R., Ven der Leer T., Verpoorte R.. Stability of alkaloid production in cell suspension cultures of Tabernaemontana divaricata during long term subculture.  Plant Cell Tissue and Organ Culture. (1992);  28 59-68
• 18 Simola L. K., Nieminen S., Huhtikangas A., Ylinen M., Naaranlahti T., Lounasmaa M.. Tropane alkaloids from Atropa-Elladonna. 2. Interaction of origin, age, and environment in alkaloid production of callus-cultures.  Journal of Natural Products. (1988);  51 234-242
• 19 Tabata M.. Transport and secretion of natural products in plant cell cultures.  Planta Medica. (1991);  57 S21-S27
• 20 Tabata M., Mizukami H., Hirakoa H., Konoshim M.. Pigment formation in callus cultures of Lithospermun erythrorhizon.  Phytochemistry. (1974);  13 927-932
• 21 Tabata M., Yamamoto H., Hirakoa H., Marumoto Y., Konoshim M.. Regulation of nicotina production in Tabacco tissue cultura by plant growth regulators.  Phytochemistry. (1971);  10 723-729
• 22 Val J.. Modelling the physiology of plant cells in suspension culture. Thesis, University of Leiden. (1993)
• 23 Vanhengel A. J., Harkes M. P., Wichers H. J., Hesselink P. G. M., Buitelaar R. M.. Characterization of callus formation and camptothecin production by cell lines of Camptotheca acuminate.  Plant Cell Tissue and Organ Culture. (1992);  28 11-18
• 24 Vazquez-Flota F., De Luca V., Carrillo-Pech M., Canto-Flick A., Miranda-Ham M. D.. Vindoline biosynthesis is transcriptionally blocked in Catharanthus roseus cell suspension cultures.  Molecular Biotechnology. (2002);  22 1-8
• 25 Viehweger K., Dordschbal B., Roos W.. Elicitor-activated phospholipase A₂ generates lysophosphatidylcholines that mobilize the vacuolar H⁺ pool for pH signaling via the activation of Na⁺-dependent proton fluxes.  Plant Cell. (2002);  14 1509-1525
• 26 Voigtlander H. W., Balsam G., Engelhardt M.. Epiisopiloturin, ein neues Pilocarpus-Alkaloid.  Archiv der Pharmazie. (1978);  311 927-935
• 27 Yu F., Zhang D. Y., Bai F. W., An L. J.. The accumulation of isocamptothecin A and B in suspension cell culture of Camptotheca acuminata. .  Plant Cell Tissue and Organ Culture. (2005);  81 159-163
• 28 Zenk M. H., El-Shagi H., Schulte U.. Anthraquinone production by cell suspension cultures of Morinda citrifolia.  Planta Medica. (1975);  79-101

Editor: E. Pichersky