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-α-aminooxy-phenylpropionic acid (AOPP), a potent inhibitor of PAL activity (and therefore cinnamate production) in vivo, 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.

The extractable activity ofl-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in cell suspension cultures of bean (Phaseolus vulgaris) is greatly induced following exposure to an elicitor preparation from the cell walls of the phytopathogenic fungusColletotrichum lindemuthianum. Following exogenous application oftrans-cinnamic acid (the product of the PAL reaction) to elicitor-induced cells, the activity of the enzyme rapidly declines. Loss of enzyme activity is accompanied by inhibition of the rate of synthesis of PAL subunits, as determined by [35S]methionine pulse-labelling followed by specific immunoprecipitation; this is insufficient to account for the rapid loss of PAL enzyme activity. Pulse-chase and immune blotting experiments indicate that cinnamic acid does not affect the rate of degradation of enzyme subunits, but rather mediates inactivation of the enzyme. A non-dialysable factor from cinnamicacid-treated bean cells stimulates removal of PAL activity from enzyme extracts in vitro; this effect is dependent on the presence of cinnamic acid. Such loss of enzyme activity in vitro is accompanied by an apparent loss or reduction of the dehydroalanine residue of the enzyme's active site, as detected by active-site-specific tritiation, although levels of immunoprecipitable enzyme subunits do not decrease. Furthermore, cinnamic-acid-mediated loss of enzyme activity in vivo is accompanied, in pulse-chase experiments, by a greater relative loss of35S-labelled enzyme subunits precipitated by an immobilised active-site affinity ligand than of subunits precipitated with anti-immunoglobulin G. It is therefore suggested that a possible mechanism for cinnamic-acid-mediated removal of PAL activity may involve modification of the dehydroalanine residue of the enzyme's active site.

Treatment of cell suspension cultures of French bean (Phaseolus vulgaris) with polysaccharide elicitor molecules from cell walls of the anthracnose fungus, Colletotrichum lindemuthianum, results in the rapid accumulation of isoflavonoid phytoalexins, deposition of wall-bound phenolic compounds and synthesis of hydroxyproline-rich glycoproteins. These changes are dependent upon a highly selective induction of gene products, including the enzymes L-phenylalanine ammonia-lyase, cytochrome P450-dependent cinnamic acid 4-hydroxylase, chalcone synthase, chalcone isomerase, prolyl hydroxylase and protein: arabinosyl transferase. Use of in vivo labelling, in vitro translation and RNA blot hybridization techniques has shown that these elicitormediated changes arise from rapid but transient induction of enzyme synthesis, resulting from the accumulation of specific mRNAs. Similar phenomena are observed in bean hypocotyls at the onset of phytoalexin synthesis in response to infection by incompatible and compatible races of C. lindemuthianum. In bean, both L-phenylalanine ammonia-lyase and chalcone synthase are encoded by multigene families and, at the protein level, both exhibit subunit and intact enzyme polymorphism. A number of less than full-length phenylalanine ammonialyase copy DNAs containing identical open reading frames have been produced from mRNA from elicitor-induced bean cells. Analysis of phenylalanine ammonia-lyase genomic clones predicts the presence of enzyme forms of differing amino acid sequence. In cultured bean cells, elicitor differentially induces the two apparent phenylalanine ammonia-lyase iso-forms with the lowest Km values. In addition to transcriptional control of the appearance of specific gene products, post-translational processes may result in increased subunit polymorphism for phenylalanine ammonia-lyase, and in the activation of chalcone isomerase. Changes in endogenous phenylpropanoid intermediate pools may signal the rapid removal of phenylalanine ammonia-lyase activity, in addition to exerting less specific inhibitory effects on the formation and/or activity of the mRNAs encoding phenylalanine ammonia-lyase and other phytoalexin biosynthetic enzymes.

Changes in the rates of synthesis of three enzymes of phenyl‐propanoid biosynthesis in Phaseolus vulgaris L. (dwarf French bean) have been investigated by immunoprecipitation of [35S]methionine‐labeled enzyme subunits with mono‐specific antisera. Elicitor causes marked, rapid but transient co‐ordinated increases in the rate of synthesis of phenyl‐alanine ammonia‐lyase, chalcone synthase and chalcone isomerase concomitant with the phase of rapid increase in enzyme activity at the onset of accumulation of phenyl‐propanoid‐derived phytoalexin antibiotics in suspension cultures of P. vulgaris. Co‐ordinate induction of enzyme synthesis is also observed in hypocotyl tissue during race:cultivar‐specific interactions with Colletotrichum lindemuthianum, causal agent of anthracnose. In an incompatible interaction (host resistant) there are early increases apparently localized to the initial site of infection prior to the onset of phytoalexin accumulation and expression of hypersensitive resistance. In contrast, in a compatible interaction (host susceptible) there is no induction of synthesis in the early stages of infection, but a delayed widespread response at the onset of lesion formation associated with attempted lesion limitation. It is concluded that expression of the phytoalexin defense response in biologically stressed cells of P. vulgaris characteristically involves co‐ordinate induction of synthesis of phytoalexin biosynthetic enzymes.

DNAs complementary to a size-selected fraction of poly(A)(+) RNA present in elicitor-treated cells of bean (Phaseolus vulgaris L.) were inserted into pAT153 and used to transform Escherichia coli strain C600. Five clones were identified by hybrid-selected translation and cross-hybridization that contained sequences complementary to mRNA encoding phenylalanine ammonia-lyase (EC 4.3.1.5), which catalyzes the first reaction of phenylpropanoid biosynthesis. The longest insert contained a single open reading frame of 1520 base pairs together with 223 base pairs of 3' untranslated sequence. RNA blot hybridization showed that elicitor caused a rapid, marked but transient increase in phenylalanine ammonia-lyase mRNA that was closely correlated with changes in translatable mRNA activity in vitro and enzyme synthesis in vivo. Blot hybridization of newly synthesized mRNA purified by organomercurial affinity chromatography following in vivo pulse-labeling with 4-thiouridine indicates that elicitor caused a rapid stimulation of phenylalanine ammonia-lyase mRNA synthesis as an early in the defense response leading to accumulation of phenylpropanoid-derived phytoalexins.

l‐Phenylalanine ammonia‐lyase (EC 4.3.1.5) has been purified over 200‐fold from cell cultures of bean (Phaseolus vulgaris L.) exposed to elicitor heat‐released from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum. Four forms of the enzyme, with identical Mr but differing apparent pI values of 5.4, 5.2, 5.05 and 4.85, were observed following the final chromatofocussing stage of the purification. A preparation (purified 43‐fold by ammonium sulphate precipitation, gel‐filtration and ion‐exchange chromatography) containing all four forms exhibited apparent negative rate cooperativity with respect to substrates. However, the individual forms displayed normal Michaelis‐Menten kinetics, with Km values of 0.077 mM, 0.122 mM, 0.256 mM and 0.302 mM in order of decreasing apparent pI value. A preparation purified 200‐fold and containing all four forms was used to immunise rabbits for the production of anti‐(phenylalanine ammonia‐lyase) serum. The antiserum was characterised by: (a) immunotitration experiments; (b) solid phase enzyme‐linked immunosorbent assays; (c) comparison of immunoprecipitates of 35S‐labelled phenylalanine ammonia‐lyase subunits (synthesized both in vivo and in vitro) on both one‐dimensional and two‐dimensional polyacrylamide gels after immunoprecipitation with the bean antiserum or antisera raised against pea and parsley phenylalanine ammonia‐lyase preparations and (d) immune blotting. Experiments involving (a) immunoprecipitation followed by analysis on SDS/polyacrylamide gels and (b) SDS/polyacrylamide gel electrophoresis followed by immune blotting, indicated that the Mr of newly synthesized (in vivo and in vitro) bean phenylalanine ammonia‐lyase subunits is 77000; a 70000‐Mr form is readily generated as a partial degradation product during purification. Immunoprecipitates of bean phenylalanine ammonia‐lyase synthesized both in vivo and in vitro showed the presence of multiple subunit types of identical Mr but differing in pI. Furthermore, treatment of bean cultures with Colletotrichum elicitor resulted in a 10‐fold increase in phenylalanine ammonia‐lyase extractable activity within 8 h, and chromatofocussing analysis indicated that this was associated with differential increased appearance of the high‐pI, low‐Km forms as compared to the two higher Km forms. This differential induction was further confirmed by immune blotting of crude extracts subjected to isoelectric focussing.