Phymcktiwy. Vol. 26. No. 5, pp. 1273-1277. 1987. 0031~9422/87 S3.OO+O.W Printed in Great Britain. Q 1987 Pcrgamoo JoumPls Ltd. EFFECTS OF LIPOXYGENASE INHIBITORS ON THE FORMATION OF VOLATILE COMPOUNDS IN WHEAT T. R. HAMILTON-KEMP,R. A. ANDERSEN’, D. F. HILDERRANDt, J. H. LOUGHRIN and P. D. FLEMING* Department of Horticulture, University of Kentucky. Lexington, KY 40496, USA; l United States Department of Agriculture and Department of Agronomy, University of Kentucky, Lexington, KY 40546, U.S.A.; department of Agronomy, University of Kentucky, Lexington, KY 40546, U.S.A. (Reuised received 28 September 1986) Key Word Index-rriticum aestivwn; Gramineae; whear lipoxygenase inhibitors; acetonylacetone bis- phenylhydrazonc; volatiles. Abstract-An inhibitor of lipoxygenase, acetonylacetone his-phenylhydrazone (AABPH), markedly reduced the formation of the major volatile Ce products isolated by reduced pressure steam distillation-hexane extraction of wheat plant homogenates. The compound was active on plant extracts containing lipoxygenase activity and completely inhibited activity at a concentration of 2.5 PM. Two other lipoxygenase inhibitors, phenidone and nordihydroguaiaretic acid, were not as active as AABPH. Linoleate added prior to homogenization increased the quantities of certain lipoxygenase derived volatiles, whereas heat treatment caused a marked reduction in the production of all volatile compounds. INTRODUCTION Extensive studies have been conducted on the isolation and chemical identification of volatile compounds pro- duced by plants [l-3]. Although large numbers of compounds have been identified, many questions remain about the synthesis of these compounds and their pre- cursors in plant tissue. Information on the generation of volatiles may provide a basis for altering quantities of biologically active volatiles such as flavour and off-&our components, and compounds involved in ‘plant host-parasite interactions. Enzyme-specific inhibitors may be used to determine the origin of volatiles. Recently, a ketone hydrazone was discovered which blocks the action of arachidonic acid lipoxygenase in blood platelets; this enzyme may be involved in the inflammatory response in animal systems [43. Subsequently, several additional ketone hydrazones were found to inhibit soybean lipoxygenase type 1, a seed- derived enzyme also involved in lipid peroxidation reac- tions [S]. Gallaird and coworkers isolated lipoxygenase from cucumber fruit and showed that it was the initiator enzyme in a multi-component enzyme system capable of producing volatile compounds from long chain un- saturated fatty acids [6, fl. The fatty acid hydroperoxides formed by lipoxygenase were cleaved by hydroperoxide lyase to produce volatile aldehydes. The cucumber-fruit- derived lipoxygen-lyase system produced mainly vol- atile C9 compounds whereas that from tea leaves pro- duced volatile C6 compounds [g]. Approximately 35 volatile compounds from wheat plants were isolated and identified [%ll] as part of a study of the interaction of wheat with fungal pathogens which cause rust disease. Many were thought to be formed through the combined action of lipoxygenase and hydro- peroxide lyase. An investigation was made on the effects of some known inhibitors of lipoxygenase and cyclooxyg- enase on the generation of wheat plant volatiles [12]. Volatile compounds have been shown to promote germi- nation of spores [ I33 and to influence differentiation and growth [I43 of the fungal pathogens which cause rusts. In the present study a comparison was made of the effects of three relatively potent inhibitors of lipoxygenase on wheat volatiles thought to be formed via lipoxygenase action. Subsequently, experiments were undertaken on tissue extracts to determine if an inhibitor, acetonyl- acetone bis-phenylhydrazone, would block the enzyme activity in oitro. This information was used to helpconfirm that the action of the inhibitor was on lipoxygenase per se. The effects of substrate addition to the homogenate and heat denaturation of enzymes on the volatiles formed by wheat plants were also evaluated. RESULTS AND DISCUSSION Acetonylacetone his-phenylhydrazone (AABPH) was synthesized and purified by a method previously de- scribed [S]. The inhibitor was dissolved in methanol and then added to water prior to homogenization of fresh wheat plants. Volatiles were isolated by reduced pressure steam distillation-extraction, separated on a fused silica capillary GC column and quantitated relative to haxa- methylbenzene. The effect of AABPH on the generation of several volatile compounds from wheat plants is shown in Table 1. Compounds listed comprised ca 90 % of the total yield of volatiles (60 ppm) obtained from fresh young plant homogenates by steam distillation-hexane extrac- tion. The most marked change was the reduction in amount of 2-hexenal, which was the principal wheat volatile. The quantities of other C6 compounds were also reduced following treatment with the inhibitor. The maximum inhibition of C6 volatile production was ob- served at AABPH concentrations of co 40 PM and above. 1273