Abstract
Cloning of OSCs required for triterpene synthesis from legume species that are amenable to molecular genetics will provide tools to address the importance of triterpenes and their derivatives during normal plant growth and development and also in interactions with symbionts and pathogens. Here we report the cloning and characterization of a total of three triterpene synthases from the legume species Medicago truncatula and Lotus japonicus. These include a β-amyrin synthase from M. truncatula (MtAMYI) and a mixed function triterpene synthase from Lotus japonicus (LjAMY2). A partial cDNA predicted to encode a β-amyrin synthase (LjAMY1) was also isolated from L. japonicus. The expression patterns of MtAMY1, LjAMY1 and LjAMY2 and of additional triterpene synthases previously characterised from M. truncatula and pea differ in different plant tissues and during nodulation, suggesting that these enzymes may have distinct roles in plant physiology and development.
Similar content being viewed by others
References
Abe, I., Ebizuka, Y. and Sankawa, U. 1988. Purification of 2,3-oxidosqualene;cycloartenol cyclase from pea seedlings. Chem. Pharm. Bull. 36: 5031–5034.
Abe. I., Sankawa, U. and Ebizuka, Y. 1989. Purification of 2,3-Oxidosqualene – beta amyrin cyclase from pea seedlings. Chem. Pharm. Bull. 37 (2): 536–538.
Abe, I., Rohmer, M. and Prestwich, G.D. 1993. Enzymatic cyclization of squalene and oxidosqualene to sterols and triterpenes. Chem. Rev. 93: 2189–2206.
Abe, I. and Prestwich, G.D. 1995. Identification of the active site of vertebrate oxidosqualene cyclase. Lipids 30: 231–234.
Ali, M.S., Ahmad, F., Ahmad, V.U., Azhar, I. and Usmanghani, K. 2001. Unusual chemical constituents of Lotus garcinii (Fabaceae). Turkish J.Chem. 25: 107–112 2001.
Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. 1997. Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acid Res. 25: 3389–3402.
Baisted, D.J. 1971. Sterol and triterpene synthesis in the developing and germinating pea seed. Biochem. J. 124: 375–383.
Cook, D.R. 1999. Medicago truncatula – a model in the making. Curr. Opin. Plant Biology 2: 301–304.
Corey, E.J., Matsuda, S.P.T. and Bartel, B. 1993. Isolation of an Arabidopsis thaliana gene encoding cycloartenol synthase by functional expression in a yeast mutant lacking lanosterol synthase by the use of a chromatographic screen. Proc. Natl. Acad. Sci. USA 90: 11628–11632.
Dellaporta, S., Wood, L.G. and Hincks, J.B. 1983. A plant DNA minipreparation. Version II. Plant. Mol. Biol. Report 1: 19–21.
deVries, S.C., Springer, J. and Wessels, J.H.G. 1982. Diversity of abundant messenger RNA sequences and patterns of protein synthesis in etiolated and greened pea seedlings. Planta 156: 129–135.
Felsenstein, J. 1996. Inferring phylogenies from protein sequences by parsimony, distance, and likelihood methods. Methods Enzymol. 266, 418–427.
Gamas, P., Niebel, F.D.C., Lescure, N. and Cullimore, J.V. 1996. Use of a subtractive hybridization approach to identify new Medicago truncatula genes induced during root nodule development. Mol. Plant-Microbe Interact. 9: 233–242.
Gogorcena, Y., Gordon, A.J., Escuredo, P.R., Minchin, F.R., Witty, J.F., Moran, J.F. and Becana, M. 1997. Nitrogen fixation, carbon metabolism, and oxidative damage in nodules of dark-stressed common bean plants. Plant Physiol. 113: 1193–1201.
Grandmougin-Ferjani, A., Dalpe, Y., Hartmann, M.A., Laruelle, F. and Sancholle, M. 1999. Sterol distribution in arbuscular mycorrhizal fungi. Phytochemistry 50: 1027–1031.
Haralampidis, K., Trojanowska, M. and Osbourn, A.E. 2001a. Biosynthesis of triterpenoid saponins in plants. Adv. Biochem. Eng./Biotechnology 75: 31–49.
Haralampidis, K., Bryan, G., Qi, X., Papadopoulou, K., Bakht, S., Melton, R. and Osbourn, A. 2001b. A new class of oxidosqualene cyclases directs synthesis of antimicrobial phytoprotectants in monocots. Proc. Natl. Acad. Sci. USA 98: 13431–13436.
Hayashi, H., Huang, P.Y., Kirakosyan, A., Inoue, K., Hiraoka, N., Ikeshiro, Y., Kushiro, T., Shibuya, M. and Ebizuka, Y. 2001a. Cloning and characterization of a cDNA encoding beta-amyrin synthase involved in glycyrrhizin and soyasaponin biosynthesis in liquorice. Biol. and Pharm. Bull. 24: 912–916.
Hayashi, H., Huang, P.Y., Kirakosyan, A., Inoue, K., Hiraoka, N., Ikeshiro, Y., Yazaki, K., Tanaka, S., Kushiro, T., Shibuya, M. and Ebizuka, Y. 2001b. Molecular cloning and characterization of isomultiflorenol synthase, a new triterpene synthase from Luffa cylindrica, involved in biosynthesis of bryonolic acid. Eur. J. Biochem. 268: 6311–6317.
Hernandez, L.E. and Cooke, D.T. 1996. Lipid composition of symbiosomes from pea root nodules. Phytochemistry 42: 341–346.
Herrera, J.B.R., Bartel, B., Wilson, W.K. and Matsuda, S.P.T. 1998. Cloning and characterization of the Arabidopsis thaliana lupeol synthase gene. Phytochemistry 49: 1905–1911.
Hoffmann, B., Trinh, T.H., Leung, J., Kondorosi, A. and Kondorosi, E. 1997. A new Medicago truncatula line with superior in vitro regeneration, transformation, and symbiotic properties isolated through cell culture selection. Mol. Plant-Microbe Interact. 10: 307–315.
Hostettmann, K.A. and Marston, A. 1991. Saponins (Cambridge Univ. Press, Cambridge, UK)
Huhman, D.V. and Sumner, L.W. 2002. Metabolic profiling of saponins in Medicago sativa and Medicago truncatula using HPLC coupled to an electrospray ion-trap mass spectrometer. Phytochemistry 59: 347–360.
Husselstein-Muller, T., Schaller, H. and Benveniste, P. 2001. Molecular cloning and expression in yeast of 2,3-oxidosqualenetriterpenoid cyclases from A rabidopsis thaliana. Plant Mol. Biol. 45: 75–92.
Jurzysta, M., Burda, S., Oleszek, W., Ploszynski, M., Small, E. and Nozzolillo, C. 1992. Chemical-composition of seed saponins as a guide to the clasification of Medicago species. Can. J. Bot. 70: 1384–1387.
Kushiro, T., Shibuya, M. and Ebizuka, Y. 1998a. Beta-amyrin synthase – Cloning of oxidosqualene cyclase that catalyzes the formation of the most popular triterpene among higher plants. Eur. J. Biochem. 256: 238–244.
Kushiro, T., Shibuya, M. and Ebizuka, Y. 1998b. Molecular cloning of oxidosqualene cyclase cDNA from Panax ginseng. The isogene that encodes β-amyrin synthase. Towards Natural Medicine Research in the 21st Century, Excerpta Medica International Congress Series 1157 (Ageta, H., Aimi, N., Ebizuka, Y., Fujita, T. & Honda, G., eds.), pp. 421–427. Elsevier ScienceBV, Amsterdam, the Netherlands.
Kushiro, T., Shibuya, M. and Ebizuka, Y. 1999a. Chimeric triterpene synthasse. A possible model for multifunctional triterpene synthase. J. Am Chem. Soc. 121: 1208–1216.
Kushiro, T., Shibuya, M. and Ebizuka, Y. 1999b. Cryptic regiospeci-ficity in deprotonation step of triterpene biosynthesis catalyzed by new members of lupeol synthase. Tetrahedron Letts. 40: 5553–5556.
Kushiro, T., Shibuya, M., Masuda, K. and Ebizuka, Y. 2000a. A novel multifunctional triterpene synthase from Arabidopsis thaliana. Tetrahedron Letts. 41: 7705–7710.
Kushiro, T., Shibuya, M., Masuda, K. and Ebizuka, Y. 2000b. Mutational studies on triterpene synthases: Engineering lupeol synthase into beta-amyrin synthase. J. Am. Chem. Soc. 122: 6816–6824.
Matsuda, S.P.T. 1998. On the diversity of oxidosqualene cyclases. Biochemical Principles and Mechanisms of Biosynthesis and Biodegradation of Polymers. (A. Steinbüchel, ed), pp. 300–307. Wiley-VCH, Weinheim.
Morita, M., Shibuya, M., Kushiro, T., Masuda, K. and Ebizuka, Y. 2000. Molecular cloning and functional expression of triterpene synthases from pea (Pisum sativum) – New alpha-amyrinproducing enzyme is a multifunctional triterpene synthase. Eur. J. Biochem. 267 (12): 3453–3460.
Morrissey, J.P. and Osbourn, A.E. 1999. Fungal resistance to plant antibiotics as a mechanism of pathogenesis. Microbiol. Mol. Biol. Revs. 63: 708–724.
Nes, W. D. and Heftmann, E. 1981. A comparison of triterpenoids with steroids as membrane components. J. Nat. Prod. 44, 377–400.
Nes, W.R. and McKean, M.L. 1977. Biochemistry of steroids and other isoprenoids, University Park Press, Baltimore.
Ohana, P., Delmer, D.P., Carlson, R.W., Glushka, J., Azadi, P., Bacic, T. and Benziman, M. 1998. Identification of a novel triterpenoid saponin from Pisum sativum as a specific inhibitor of the diguanylate cyclase of Acetobacter xylinum. Plant and Cell Physiol. 39: 144–152.
Oleszek, W., Jurzysta, M., Ploszynski, M., Colquhoun, I.J., Price, K.R., and Fenwick, G.R. 1992. Zahnic acid tridesmoside and other dominant saponins from alfalafa (Medicago sativa L.) aerial parts. J. Ag. Food Chemistry 40: 191–196.
Palmer, M.A. and Bowden, B.N. 1977. Variation in sterol and triterpene content of developing Sorghum bicolor grain. Phytochemistry 16: 459–463.
Papadopoulou, K., Melton, R.E., Leggett, M., Daniels, M.J. and Osbourn, A.E. 1999. Compromised disease resistance in saponindeficient plants. Proc. Natl. Acad. Sci. USA 96: 12923–12928.
Poralla, K., Hewelt, A., Prestwich, G.D., Abe, I., Reipen, I. and Sprenger, G. 1994. A specific amino acid repeat in squalene and oxidosqualene cyclases. Trends Biochem. Sci. 19: 157–8.
Price, K.R., Johnson, I.T. and Fenwick, G.R. 1987. The Chemistry and biological significance of saponins in food and feedingstuffs. Crit. Rev. Food Sci. Nutr. 26: 27–135
Rahman, A., Ahamed, A., Amakawa, T., Goto, N. and Tsurumi, S. 2001. Chromosaponin I specifically interacts with AUX1 protein in regulating the gravitropic response of Arabidopsis roots. Plant Physiol. 125: 990–1000.
Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, Ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Segura, M.J.R., Meyer, M.M. and Matsuda, S.P.T. 2000. Arabidopsis thaliana LUP1 converts oxidosqualene to multiple triterpene alcohols and a triterpene diol. Org. Letts. 2: 2257–2259.
Shibuya, M., Zhang, H., Endo, A., Shishikura, K., Kushiro, T. and Ebizuka, Y. 1999. Two branches of the lupeol synthase gene in the molecular evolution of plant oxidosqualene cyclases. Eur. J. Biochem. 266: 302–307.
Stougard, J. 2001. Genetics and genomics of root symbiosis. Curr. Opin. Plant Biology 4: 328–335.
Threlfall, D. and Whitehead, I.M. 1990. Redirection of terpenoid biosynthesis in elicitor-treated plant cell suspension cultures. In: Plant Lipid Biochemistry (P.J. Quinn and J.L. Harwood, eds.,), Portland Press, London, pp. 344–346.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Iturbe-Ormaetxe, I., Haralampidis, K., Papadopoulou, K. et al. Molecular cloning and characterization of triterpene synthases from Medicago truncatula and Lotus japonicus . Plant Mol Biol 51, 731–743 (2003). https://doi.org/10.1023/A:1022519709298
Issue Date:
DOI: https://doi.org/10.1023/A:1022519709298