Phytochemistry, Vol. 34, No. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 1,pp. 139-145, 1993 0031-9422/93 $6.00+0.00 Printed in Great Britain. 0 1993 Pergamon Press Ltd zyxwvutsrq INHIBITION OF PHYTOSTEROL BIOSYNTHESIS IN ELICITOR-TREATED CULTURES OF AMMI zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPO M AJUS DANIEL C. FULTON, PAUL A. KROON, ULRICH MATERN,* DAVID R. THRELFALLt and IAN M. WHITEHEAD~: Department of Applied Biology, University of Hull, Hull HU6 7RX, U.K.;* Department of Plant Biochemistry, Biological Institute II, University of Freiburg, Schanzelstrasse 1, D-7800 Frieburg, F.R.G. (Received 8 February 1993) IN HONOUR OF PROFESSOR JEFFREY HARBORNE’S SIXTY-FIFTH BIRTHDAY zyxwvutsrqponmlkjihgfed Key Word Index-Ammi mujus; Apiaceae; Bishop’s weed; cell suspension cultures; phytosterol biosynthesis; inhibition. Abstract-The elicitor-mediated induction of coumarin phytoalexin production by cell-suspension cultures of Ammi majus is accompanied by the inhibition of both biomass production and phytosterol biosynthesis [from mevalonic acid (MVA)]. However, cell-free preparations of cells from elicitor-treated cultures are able to support the synthesis of squalene from MVA, isopentenyl diphosphate (IPP) or (2E, 6E)-farnesyl diphosphate (FPP), even though radioactive squalene is not found following administration of [2-14C]MVA to elicitor-treated cultures. The preparations, as expected, also exhibit umbelliferone:O-dimethylallyl transferase activity. These results indicate that in uiuo there is a specific inhibition of one of the enzymes on the MVA to dimethylallyl diphosphate (DMAPP) span of the cytosolic microsomal pathway of terpenoid biosynthesis in elicitor-treated cultures, and that in cell-free preparations this deficiency is made good by the corresponding plastid enzyme. The finding that the coumarin phytoalexins are not labelled on feeding either [2-14C]acetate or [2-‘4C]MVA to elicitor-treated cultures precluded us from establishing whether the inhibition of phytosterol biosynthesis is a means of increasing the input of cytosolic IPP into the plastid for coumarin phytoalexin biosynthesis (in which case cytosolic IPP isomerase would be the enzyme inhibited in elitiitor- treated cultures) or whether the cytosolic and plastid pathways of terpenoid biosynthesis are not linked and the site of inhibition (between MVA and DMAPP) is unrelated to the provision of plastid IPP for phytoalexin biosynthesis. The feeding experiments with [2-14C]acetate suggested that, somewhat unexpectedly, polar lipid biosynthesis is not repressed in elicitor-treated cultures. INTRODlJCTlON The cells of dark-grown cell suspension cultures of Ammi mujus L. excrete large quantities of coumarin phytoalex- ins in response to treatment of the cultures with either fungal cell wall fractions [l] or Difco yeast extract (DYE) (U. Matern, unpublished work). The major coumarins excreted are umbelliferone, isopiminellin, (S)-marmesin, (R)-ammirin and two butenyl ethers of umbelliferone. The umbelliferone:6-C- and 7-0-dimethylallyl transferase [dimethylallyl diphosphate (DMAPP):umbelliferone di- methylallyl transferase (EC 2.5.1.3)] activities needed for the biosynthesis of some of the linear dihydrofurano- coumarins and furanocoumarins, and of the butenyl ethers of umbelliferone, respectively, are both elicitor- induced enzymes [2]. The finding that the umbelliferone:dimethylallyl trans- ferases are apparently peripheral proteins of the endo- t Author to whom correspondence should be addressed. 1 Present address: Biotechnology Department, Firmenich SA, La Jon&on, 1 Route des Jeunes, Geneva, Switzerland. plasmic reticulum [2] led us to examine the possibility that in elicitor-treated cultures of A. majus, as in elicitor- treated cultures of potato [3], tobacco [4,5] and Taber- naemontana dioaricata [6], there is a redirection of the cytosolic-microsomal pathway of terpenoid biosynthesis away from phytosterol biosynthesis and towards the biosynthesis of phytoalexins. In A. majus, the redirection of the pathway would be brought about by the inhibition of (2E,6E)-farnesyl diphosphate (FPP) synthetase (EC 2.5.1.1), i.e. the constitutive enzyme which would be in direct competition with the induced umbelliferone:C- and 0-dimethylallyl transferases for cytosolic DMAPP. RESULTS Biomass production in control and dicitor-treated cultures The biomass of the control (unelicited) cultures, after a short lag phase, showed a three-fold increase over the 11 day period of the experiment (Fig. 1A). Biomass produc- tion (as measured by dry wt) was completely inhibited 139