CO-ORDINATED INHIBITION OF SQUALENE SYNTHETASE AND INDUCTION OF ENZYMES OF SESQUITERPENOID PHYTOALEXIN BIOSYNTHESIS IN CULTURES OF NICOTIANA TABACUM

Article abstract of DOI:10.1016/0031-9422(88)87028-6

Treatment of log-phase cell suspension cultures of Nicotiana tabacum with the biotic elicitor cellulase completly inhibited cellular growth within a few hours and causes a rapid accumulation of the sesquiterpenoid phytoalexins capsidiol and debneyol.Acetosyringone (an unrelated phenolic compound) was also produced by the elicited cultures.Capsidiol levels fell to low concentrations after the accumulation period had ceased whereas the level of debneyol was maintained and that of acetosyringone was bi-phasic.Growth of the elicited cultures resumed after 24 hr and paralleled that of control cultures.When capsidiol or debneyol were supplied to unelicited cultures they were rapidly catabolized to low levels.Elicitor treatment inhibited the catabolism of capsidiol within three hr but did not affect the metabolism of debneyol.A cell-free preparation obtained from cells 12 hr after elicitation efficiently catalysed the formation of two uncharacterised sesquiterpenoids (one of which may be an eremophilane hydrocarbon) when incubated with either <1-14C>IPP or <1-3H2>FPP as substrate.In the presence of NADPH and O2, the cell-free system catalysed the formation of capsidiol and debneyol with the amount of unknown I being proportionately reduced.None of the above biosynthetic capabilities were present in cell-free extracts from unelicited cells.The total amount of radioactivity incorporated into capsidiol, debneyol and the two unknows provided an indirect estimate of the activity of FPP-carbocyclase, the enzyme catalysing the first commited step in the biosynthesis of these compounds.The time course of the rate of synthesis of capsidiol and debneyol in cell-free preparations showed that the synthesis of the phytoalexins was detectable within two after elicitation of the cultures with cellulase.Thereafter, the rate of synthesis of both phytoalexins rose rapidly with the maximum rate for each phytoalexin preceding the maximum level of its accumulation.The synthesis of unknowns I and II was detectable at all time points after elicitation.Squalene synthetase (EC 2.5.1.21) activity underwent a rapid decline over the first six hr period of elicitation and remained low until about 48 hr.The apparent K_m values (FPP as substrate) for squalene synthetise from unelicited cultures and for capsidiol, debneyol, unknown I and unknown II biosynthesis in elicited cultures were 10, 2, 12, 10 and 10 μm respectively.The rapid inhibition of squalene synthetase in elicited cultures may operate to channel FPP away from sterol biosynthesis (which is required for cellular growth) and towards the FPP-carbocyclase involved in the biosynthesis of the two sesquiterpenoid phytoalexins or may be part of a more general response of plant cells to treatment with elicitors.The levels of debneyol which accumulated in the cultures in response to cellulase were lower than those of capsidiol.These differences are thought to be due to the inhibitory effect of cellulase on the catabolism of capsidiol but not that of debneyol.The results indicate that a coordina