Phytochemistry, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Vol. 27, No. 7, pp. 2109-2117, 1988. 003 l-9422/88 $3.00 + 0.00 Printed in Great Britain. 0 1988Pergamon Press pk. zyxwvutsrq INHIBITION OF mRNA LEVELS AND ACTIVITIES BY TRANS-CINNAMIC ACID IN ELICITOR-INDUCED BEAN CELLS G. PAUL BOLWELL,*~$ MEHRDAD MAVANDAD,t DAVID J. MILLAR,~ KEITH J. EDWARDS& WOLFGANG SCHUCH§ and RICHARD A. DIXON*~(( TDepartment of Biochemistry, Royal Holloway and Bedford New College, (University of London), Egham Hill, Egham, Surrey TW20 OEX, U.K.; §Imperial Chemical Industries plc, Corporate Biosciences Laboratory, The Heath, Runcorn, Cheshire WA7 4QE, U.K. $Present address: Department of Biology, City of London Polytechnic, Calcutta House, Old Castle Street, London El 7NT, U.K. 11 Present address: Plant Biology Division, The Samuel Roberts Noble Foundation Inc., 2510 Highway 199 East, P.O. Box 2180, Ardmore, OK 73402, U.S.A. (Revised receioed 16 November 1987) Key Word Index-Phaseohs uulgaris; Leguminosae; elicitor; gene expression; phenylalanine ammonia-lyase; phenylpropanoid synthesis; transcription. Abstract-Addition of trans-cinnamic acid to bean cell suspension cultures after their treatment with fungal elicitor resulted in the loss of induced phenylalanine ammonia-lyase (PAL) enzyme activity. In contrast elicitor-induced chalcone synthase (CHS) activity was arrested but did not decline, whereas chalcone isomerase (CHI) activity was relatively unaffected. However, translational activities of extracted polysomal mRNAs encoding these three enzymes were depressed by cinnamate treatments as were levels of PAL and CHS polysomal mRNAs and rates of transcription of these genes measured in isolated nuclei. Treatment of elicitor-induced cultures with L-a-aminooxy-phenylpropionic acid (AOPP), a potent inhibitor of PAL activity (and therefore cinnamate production) in ho, resulted in increased production of PAL and CHS mRNAs. Addition of cinnamate to elicitor-treated cultures inhibited the appearance of a number of polypeptides translated in vitro from polysomal mRNA, although at least nine polypeptides were specifically induced by cinnamate treatment. We conclude that cinnamic acid potentially could act as an in vivo modulator of the synthesis of phenylpropanoid pathway enzymes although it is not yet fully possible to rule out less specific inhibitory effects. INTRODUCTION L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) cata- lyses the first reaction in the biosynthesis of plant phe- nolic compounds from L-phenylalanine [ 11. Treatment of suspension cultured bean cells wtih elicitor macro- molecules heat-released from cell walls of the fungal phytopathogen Coktotrichum lindemuthianium leads to rapid induction of enzyme activities involved in the ac- cumulation of isoflavonoid phytoalexins derived from L-phenylalanine [2-61. This response is characterized by rapid increases in PAL and chalcone synthase (CHS) mRNA levels and translational activities leading to in- creased rates of enzyme synthesis [2, 4, 7, 81, and the differential induction of multiple forms of active PAL differing in pI and K, values [9]. The elicitor-induced increase in PAL activity is rapidly reversed by addition of trans-cinnamic acid, the product of the PAL reaction W I. Density labelling studies with ‘H from ‘H,O have indicated that the loss of PAL activity observed on application of exogenous cinnamate to pea epicotyl sec- tions is mediated by a dual mechanism involving a decrease in the rate of PAL synthesis and an increase in the rate of PAL removal [ll]. Confirmation of the cinnamate-mediated inhibition of PAL synthesis has *Authors to whom correspondence should be addressed. been obtained from studies on the incorporation of 35S- methionine into immunoprecipitable PAL subunits in oivo in elicitor-treated bean cultures [12]. Furthermore, a mechanism has recently been proposed for the cinna- mate-mediated removal of PAL activity involving an irreversible inactivation of the enzyme in the absence of an increased rate of subunit degradation [12]. A number of studies have suggested that endogenous cinnamate pools may act in uivo to modulate PAL turn- over. Central to this argument is the observed super- induction of PAL enzyme activity by treatments, such as application of the potent PAL inhibitor AOPP, which prevent the accumulation of endogenous cinnamate in uivo [lo, 13-161. That cinnamate potentially may act to regulate the flux through the phenylpropanoid pathway at sites other than PAL is suggested by the induction of the activities of chalcone isomerase (CHI) [16] and hyd- roxycinnamoyl CoA: quinate hydroxycinnamoyl trans- ferase [17], in cinnamate-treated tissues of bean and potato respectively. In the present paper we extend previous observations on the inhibition of PAL synthesis by exogenously ap- plied cinnamate to consider transcriptional ‘events. We also describe the effects of cinnamate on mRNAs en- coding other phenylpropanoid pathway enzymes. The results are discussed in relation to the selectivity and possible involvement in uivo of cinnamate as a modulator of plant gene expression. 2109