Expression analysis of genes involved in terpenes biosynthesis in black cumin (Nigella sativa) plants treated with methyl jasmonate

Document Type : Research Paper

Authors

Abstract

Black cumin (Nigella sativa) is an important medicinal plant belongs to the Ranunculaceae which produce large diversity of secondary metabolites including industrial and medicinal monoterpenes/ triterpenes. In the present work, relative gene expression of genes involved in biosynthetic pathway of terpenes including a monoterpene synthase (MTS), geranyldiphosphate synthase (GDS), β- amyrin synthase (βAS) and squaleneepoxidase(SQE) were investigated in black cumin plants treated with methyl jasmonate. The plants were grown in a greenhouse sprayed with 0.1 mM methyl jasmonate. Samples were collected at 0, 4, 8, 24, 48 and 72 hours  after applying the treatments. RT-PCR was performed by gene-specific primers after RNA extraction and cDNA synthesis. Results of semi quantitative RT-PCR showed that the expression pattern of GDS gene differed with the results of other genes. The highest gene expression of GDS was observed at 4 h and its expression remained at the same level till 8 h then dropped at next time periods. The expression patterns of monoterpene synthase, β-amyrin synthase and squaleneepoxidase showed rising trend. Gene expression analysis of the genes at transcript level using semi-quantitative PCR in black cumin leaves treated by methyl jasmonate revealedinduced-geneexpression which might show the roles of terpenes in defense mechanisms and signal transduction. Consequently, methyl jasmonate treatment can be considered to induce the terpene biosynthetic pathway and to study the enzymes/ metabolites associated with terpenoid metabolism.

Keywords


-      Aggarwal, B., Sundaram, C., Malani, N. and Ichikawa, H., 2007. Curcumin: the india solid gold. Advance in Experimental Medicine and Biology, 59: 31-38.
-      Aharoni, A., Jongsma, M.A., Kim, T.Y., Ri, M.B., Giri, A.P., Verstappen, F. W. A., Schwab, W. and Bouwmeester, H.J., 2006. Metabolic engineering of terpeniod biosynthesis in plants. Phytochemistry, 5: 49-58
-      Barmley, P.M., 1997. Isoprenoid metabolism.  In: Dey, P.M., and Hrborne, J.B., " Plant Biochemistry", Academic Press, San Diego, PP. 417-437.
-      Davis, E.M. and Croteau, R., 2000. Cyclization enzymes in the biosynthesis of monoterpenes, sesquiterpenes, and diterpenes. Current Chemistry, 209: p. 54-95.
-      Elyasi, R., Majdi, M., Bahramnejad, B. and Mirzaghaderi, GH., 2015. Expression pattern analysis of genes involved in the biosynthetic pathway of monoterpenes and triterpenes in black cumin (Nigella sativa) plants treated with salicylic acid. Iranian Journal of Rangelands and Forests Plant Breeding and Genetic Research, 23: 164-174
-      Guodong, W. and Dixon, R.A., 2008. Heterodimericgeranyl (geranyl) diphosphate synthase from hop (Humulus lupulus) and the evolution of monoterpene biosynthesis. Proceedings of the National Academy of Sciences, 14: 1-6.
-      Hayashi, H., Huang, P., Takada, S., Obinata, M., Inoue, K. and Shibuya, M., 2004. Biological and Pharmaceutical Bulletin, 27: 1086–1092.
-      Iqbal, M.S., Nadeem, S.H., Mehbood, S.H., Chafoor, A., Rajoka, M.I., Qureshi, A.S. and Niaz, B., 2011. Exploration of genotype specific fingerprinting of Nigella sativa L. using RAPD markers. Turkish Journal Agriculture and Forestry, 35: 1-10.
-      Irmler, S., Schorder, G., St-Pierre, B., Crouch, N.P., Hotze, M., Schmidt, J., Strack, D., Matern, U. and Schroder, J., 2000. Indole alkaloid biosynthesis in Catharan thusroseus: new enzyme activities and identification of cytochrome P-450 CYP72A1 as secologanin synthase. Plant Journal, 24: 797-804.
-      Kozlowski, G., Buchala, A. and M´etraux, J.P., 1999. Methyl jasmonate protects Norway spruce (Picea abies (L.) Karst.) seedlings against Pythium against Pythium ultimumTrow. Physiological and Molecular Plant Pathology, 55: 53-58.
-      Kushiro, T., Shibuya, M. and Ebizuka, Y., 1998. Beta-amyrin synthase-cloning of oxidosqualenecyclase that catalyzes the formation of the most popular triterpene among higher plants. European Journal of Biochemistry, 15: 238-244.
-      Lichtenthaler, H.K., 1999. The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants. Annual Review of Plant Physiology and Plant Molecular Biology, 50:47–65.
-      Lohrasebi, T., Malboobi, M.A., Samaeian, A. and Sanei, V., 2007. Differential expression of Arabidopsis thaliana acid phosphatases in response to abiotic stresses. Iranian Journal of Biotechnology, 5: 130-139.
-      Mangas, S., Bonfill, M., Osuna, L., Moyano, E., Tortoriello, J., Cusido, R.M., Piñol, M.T. and Palazón, J., 2006. The effect of methyl jasmonate on triterpene and sterol metabolisms of Centellaasiatica, Ruscusaculeatus and Galphimia glauca cultured plants. Phytochemistry,  67: 2041-2049.
-      Piotr, C. and Sacchi, N., 2006. The single-step method of RNA isolation by acid guanidiniumthiocyanate-phenol-chloroform extraction: twenty-something years on. Nature Protocols, 1: 581-585.
-      Scholz, M., Lipinski, M., Leupold, M., Luftmann, H., Harving, L., Ofir, R., Fischer, R., Prufer, D. and Muller, K., 2009. Methyl jasmonate induced accumulation of kalapanaxsaponin I in Nigella Sativa. Phytochemistry, 70: 517-522.
-      Shabani, L., Ehsanpour, A.A. and Esmaeili, A., 2010. Assessment of squalene synthase and beta-amyrin synthase gene expression in licorice roots treated with methyl jasmonate and salicylic acid using real-time qPCR. Russian Journal of Plant Physiology, 57: 480-484.
-      ShamsiFard, M.H.,  2012.Transcript analysis of Geranyldiphosphate synthase gene in different tissues of black cumin (Nigella sativa L.). M.Sc. Thesis, Agricultural Biotechnology, Faculty of Agriculture, University of Kurdistan.
-      Suzuki, H., Naoumkina, M.S., Aziz, N., Huhman, G., Sumner, L., Mendes, J.W. and Dixon, R., 2005. Methyl jasmonate and yeast elicitor induce differential transcriptional and metabolic re-programming in cell suspension cultures of the model legume Medicago truncatula. Planta, 220: 696-707.
-      Tholl, D., 2006. Terpene synthases and the regulation, diversity and biological roles of terpene metabolism. Current Opinion in Plant Biology, 9: 1–8.
-      Wasternack, C. and Parthier, B., 1997. Jasmonate-signalled plant gene expression. Trends in Plant Sciences, 2: 302–307.
-      Xu, R., Fazio, G.C. and Matsuda, S.P.T., 2004. On the origins of triterpenoid skeletal diversity. Phytochemistry, 65: 261-291.
-      Zhao, C.L., Cui, X.M., Chen, Y.P. and Liang, Q., 2010. Key enzymes of triterpenoids aponin biosynthesis and the induction of their activities and gene expressions in plants. Natural Products Communication, 5:1147–1158.