Abstract
Cell suspension cultures of Ruta graveolens L. produce a variety of acridone alkaloids, and the accumulation can be stimulated by the addition of fungal elicitors. Acridone synthase, the enzyme catalyzing the synthesis of 1,3-dihydroxy-N-methylacridone from N-methylanthraniloyl-CoA and malonyl-CoA, had been isolated from these cells, and the partial enzyme polypeptide sequence, elucidated from six tryptic fragments, revealed homology to heterologous chalcone synthases. Poly(A)+ RNA was isolated from Ruta cells that had been treated for 6 h with a crude cell wall elicitor from Phytophthora megasperma f. sp. glycinea, and a cDNA library was constructed in λ2AP. Clones harboring acridone synthase cDNA were isolated from the library by screening with a synthetic oligonucleotide probe complementary to a short stretch of sequence of the enzyme peptide with negligible homology to chalcone synthases. The identity of the clones was substantiated by DNA sequencing and by recognition of five additional peptides, determined previously from tryptic acridone synthase digests, in the translated sequence. An insert of roughly 1.4 kb encoded the complete acridone synthase, and alignments at both DNA and protein levels corroborated the high degree of homology to chalcone synthases. Expression of the enzyme in vector pET-11c in the Escherichia coli pLysS host strain proved the identity of the cloned cDNA. The heterologous enzyme in the crude E. coli extract exhibited high acridone but no chalcone synthase activity. The results were fully supported by northern blot hybridizations which revealed that the specific transcript abundance did not increase but rather decreased upon white light irradiation of cultured Ruta graveolens L. cells, a condition that commonly induces the abundance of chalcone synthase transcripts.
Similar content being viewed by others
Abbreviations
- SDS:
-
sodium dodecyl sulfate
- ACS:
-
acridone synthase (EC 2.3.1.)
- CHS:
-
chalcone synthase (EC 2.3.1.74)
- STS:
-
stilbene synthase (EC 2.3.1.)
- LB:
-
Luria Bertoni
- IPTG:
-
isopropyl 1-thio-β-D-galactopyranoside
References
Ahsan M, Gray AI, Leach G, Waterman PG: Quinolone and acridone alkaloids from Boronia lanceolata. Phytochemistry 33: 1507–1510 (1993).
Baumert A, Hieke M, Gröger D: N-Methylation of anthranilic acid to N-methylanthranilic acid by cell-free extracts of Ruta graveolens tissue cultures. Planta Med 48: 258–262 (1983).
Baumert A, Gröger D: Synthesis of 1,3-dihydroxy-N-methylacridone by cell-free extracts of Ruta graveolens cell suspension cultures. FEBS Lett 187: 311–313 (1985).
Baumert A, Gröger D, Schmidt J, Kuzovkina IN, Mügge C: Alkaloids and other constituents from tissue cultures of Ruta graveolens Fitoterapia 49: 83–88 (1988).
Baumert A, Maier W, Schumann B, Gröger D: Increased accumulation of acridone alkaloids by cell suspension cultures of Ruta graveolens in response to elicitors. J Plant Physiol 139: 224–228 (1991).
Baumert A, Maier W, Gröger D, Deutzmann R: Purification and properties of acridone synthase from cell suspension cultures of Ruta graveolens L. Z Naturforsch 49c: 26–32 (1994).
Baumert A, Maier W, Matern U, Schmidt J, Schumann B, Gröger D: Acridone alkaloids from cell suspension cultures of Thamnosma montana. Planta Med 60: 143–145 (1994).
Chou TC, Tzeng CC, Wu TS, Watanabe KA, Su TL: Inhibition of cell growth and macromolecule biosynthesis of human promyelocytic leukemic cells by acridone alkaloids. Phytother Res 3: 237–242 (1989).
Eilert U, Ehmke A, Wolters B: Elicitor-induced accumulation of acridone alkaloid epoxides in Ruta graveolens suspension cultures. Planta Med 50: 508–512 (1984).
Eilert U, Wolters B, Nahrstedt A, Wray V: Hydroxyrutacridone epoxide, a new acridone alkaloid from Ruta graveolens. Z Naturforsch 37c: 132–133 (1982).
Eilert U, Wolters B, Constabel F: Ultrastructure of acridone alkaloid idioblasts in roots and cell cultures of Ruta graveolens. Can J Bot 64: 1089–1096 (1986).
Fliegmann J, Schröder G, Schanz S, Britsch L, Schröder J: Molecular analysis of chalcone and dihydropinosylvin synthases from Scots pine (Pinus sylvestris), and differential regulation of these and related enzyme activities in stressed plants. Plant Mol Biol 18: 489–503 (1992).
Gröger D: Vorkommen und Biochemie der Acridon-Alkaloide: ein Fortschrittsbericht. Pharmazie 43: 815–826 (1988).
Kozack M: The scanning model for translation: an update. J Cell Biol 108: 229–241 (1989).
Kuzovkina IN, Chernysheva TP, Al'Terman IE: Characteristics of a rutacridone-producing strain of rue callus tissue. Fiziol Rast 26: 492–500 (1979).
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).
Lanz T, Tropf S, Marner FJ, Schröder J, Schröder G: The role of cysteines in polyketide synthases. Site-directed mutagenesis of resveratrol and chalcone synthases, two key enzymes in different plant-specific pathways. J Biol Chem 266: 9971–9976 (1991).
Lehrach H, Diamond D, Wozney JM, Boedtker H: RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16: 4743–4751 (1977).
Lütcke HA, Chow KC, Mickel FS, Moss KA, Kern HF, Scheele GA: Selection of AUG initiation codons differs in plant and animals. EMBO J 6: 43–48 (1987).
Maier W, Baumert A, Schumann B, Furukawa H, Gröger D: Synthesis of 1,3-di-hydroxy-N-methylacridone and its conversion to rutacridone by cell-free extracts of Ruta graveolens cell cultures. Phytochemistry 32: 691–698 (1993).
Martin CR: Structure, function, and regulation of the chalcone synthase. Int Rev Cytol 147: 233–284 (1993).
Melchior F, Kindl H: Grapevine stilbene synthase cDNA only slightly differing from chalcone synthase cDNA is expressed in Escherichia coli into a catalytically active enzyme. FEBS Lett 268: 17–20 (1990).
Michael JP: Quinoline, quinazoline, and acridone alkaloids. Nat Prod Rep 11: 163–172 (1994).
Miyada CG, Wallace RB: Oligonucleotide hybridization techniques. Meth Enzymol 154: 94–107 (1987).
Nevins JR: The pathway of eucaryotic mRNA formation. Annu Rev Biochem 52: 441–466 (1983).
Palmer BD, Rewcastle GW, Atwell GJ, Baguley BC, Denny WA. Potential antitumor agents. 54. Chromophore requirements for in vivo antitumor activity among the general class of linear tricyclic carboxamides. J Med Chem 31: 707–712 (1988).
Palmiter RD: Magnesium precipitation of ribonucleoprotein complexes. Expedient techniques for the isolation of undegraded polysomes and messenger ribonucleic acid. Biochemistry 13: 3606–3615 (1974).
Paulini H, Schimmer O: Mutagenicity testing of rutacridone epoxide and rutacridone alkaloids in Ruta graveolens L., using the Salmonella and microsome assay. Mutagenesis 4: 45–50 (1989).
Queener SF, Fujioka H, Nishiyama Y, Furukawa H, Bartlett MS, Smith JW: In vitro activities of acridone alkaloids against Pnemocystis carinii. Antimicrobial Agents Chemother 35: 377–379 (1991).
Sambrook J, Fritsch EF, Maniatis R: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).
Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).
Scharlemann W: Acridin-Alkaloide aus Kalluskulturen von Ruta graveolens L. Z Naturforsch 27b: 806–808 (1972).
Schlemper B, Siegers DJ, Paxton JW, Robertson IG: Rat hepatocyte-mediated metabolism of the experimental anti-tumor agent N-[2′-(dimethylamino)ethyl]-acridine-4-carboxamide. Xenobiotica 23: 361–371 (1993).
Schröder G, Brown JWS, Schröder J: Molecular analysis of resveratrol synthase. cDNA, genomic clones and relationship with chalcone synthase. Eur J Biochem 172: 161–169 (1988).
Schröder G, Schröder J: A single change of histidine to glutamine alters the substrate preference of a stilbene synthase. J Biol Chem 267: 20558–20560 (1992).
Schröder J, Heller W, Hahlbrock K: Flavone synthase: simple and rapid assay for the key enzyme of flavonoid biosynthesis. Plant Sci Lett 14: 281–286 (1979).
Schröder J, Schröder G: Stilbene and chalcone synthases: related enzymes with key functions in plant-specific pathways. Z Naturforsch 45c: 1–8 (1990).
Tabor S, Richardson CC: DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci USA 84: 4767–4771 (1987).
Tropf S, Lanz T, Rensing SA, Schröder J, Schröder G: Evidence that stilbene synthases have developed from chalcone synthases several times in the course of evolution. J Mol Evol 38: 610–618 (1994).
Vieira PC, Kubo I, Kujime H, Yamagiwa Y, Kamikawa T: Molluscicidal acridone alkaloids from Angostura paniculata: isolation, structures, and synthesis. J Nat Prod 55: 1112–1117 (1992).
Wallace RB, Miyada CG: Oligonucleotide probes for the screening of recombinant DNA libraries. Meth Enzymol 152: 432–442 (1987).
Wijeratne EMK, Bandara BMR, Gunatilaka AAL, Tezuka Y: Kikuchi T: Chemical constituents of three Rutaceae species from Sri Lanka. J Nat Prod 55: 1261–1269 (1992).
Wolters B, Eilert U: Accumulation of acridone epoxides in callus cultures of Ruta graveolens increased by coculture with non-host-specific fungi. Z Naturforsch 37c: 575–583 (1982).
Yamamoto N, Furukawa H, Ito Y, Yoshida S, Maeno K, Nishiyama Y: Anti herpesvirus activity of citrusinine-I, a new acridone alkaloid, and related compounds. Antiviral Res 12: 21–36 (1989).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Junghanns, K.T., Kneusel, R.E., Baumert, A. et al. Molecular cloning and heterologous expression of acridone synthase from elicited Ruta graveolens L. cell suspension cultures. Plant Mol Biol 27, 681–692 (1995). https://doi.org/10.1007/BF00020222
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00020222