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Tobacco plants over-expressing L-phenylalanine ammonia-lyase (PAL(+)) produce high levels of chlorogenic acid (CGA) and exhibit markedly reduced susceptibility to infection with the fungal pathogen Cercospora nicotianae, although their resistance to tobacco mosaic virus (TMV) is unchanged. Levels of the signal molecule salicylic acid (SA) were similar in uninfected PAL(+) and control plants and also following TMV infection. In crosses of PAL(+) tobacco with tobacco harboring the bacterial NahG salicylate hydroxylase gene, progeny harboring both transgenes lost resistance to TMV, indicating that SA is critical for resistance to TMV and that increased production of phenylpropanoid compounds such as CGA cannot substitute for the reduction in SA levels. In contrast, PAL(+)/NahG plants showed strongly reduced susceptibility to Cercospora nicotianae compared to the NahG parent line. These results are consistent with a recent report questioning the role of PAL in SA biosynthesis in Arabidopsis, and highlight the importance of phenylpropanoid compounds such as CGA in plant disease resistance.

Gene silencing of phenylalanine ammonia-lyase (PAL) in transgenic tobacco (Nicotiana tabacum L.) is manifested phenotypically by reduced growth, altered leaf morphology, and reduced levels of phenylpropanoid compounds. Here we report the rare event of somatic reversion from silencing to PAL over-expression in tobacco harboring the bean PAL2 gene. This phenomenon allows the comparison of the effects of PAL under- and over-expression in the same plant. A comparison of gene silenced and revertant tissues reveals striking differences in lignin content and monomer composition, and both qualitative and quantitative differences in soluble and cell wall bound phenylpropanoid compounds in the recovered sector.

Pre-inoculation of plants with a pathogen that induces necrosis leads to the development of systemic acquired resistance (SAR) to subsequent pathogen attack [1]. The phenylpropanoid-derived compound salicylic acid (SA) is necessary for the full expression of both local resistance and SAR [2] [3]. A separate signaling pathway involving jasmonic acid (JA) is involved in systemic responses to wounding and insect herbivory [4] [5]. There is evidence both supporting and opposing the idea of cross-protection against microbial pathogens and insect herbivores [6] [7]. This is a controversial area because pharmacological experiments point to negative cross-talk between responses to systemic pathogens and responses to wounding [8] [9] [10], although this has not been demonstrated functionally in vivo. Here, we report that reducing phenylpropanoid biosynthesis by silencing the expression of phenylalanine ammonialyase (PAL) reduces SAR to tobacco mosaic virus (TMV), whereas overexpression of PAL enhances SAR. Tobacco plants with reduced SAR exhibited more effective grazing-induced systemic resistance to larvae of Heliothis virescens, but larval resistance was reduced in plants with elevated phenylpropanoid levels. Furthermore, genetic modification of components involved in phenylpropanoid synthesis revealed an inverse relationship between SA and JA levels. These results demonstrate phenylpropanoid-mediated cross-talk in vivo between microbially induced and herbivore-induced pathways of systemic resistance.

Transgenic tobacco (Nicotiana tabacum L.) plants overexpressing the enzyme L-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) were grown from seeds of a primary transformant containing the bean PAL2 gene, which had shown homology-dependent silencing of the endogenous tobacco PAL genes. Analysis of endogenous and transgene-encoded PAL transcripts and protein in the primary transformant (T0) and first-generation (T1) overexpressor plants indicated that the transgene-encoded PAL is the cause of the greater than wild-type levels of PAL activity (up to 5- and 2-fold greater in leaf and stem tissue, respectively) in the T1 plants. Leaves of PAL-overexpressing plants contained increased levels of the hydroxycinnamic acid ester chlorogenic acid but not of the flavonoid rutin, indicating that PAL is the key control point for flux into chlorogenic acid. In addition, levels of the glucoside of 4-coumaric acid increased in the overexpressing plants, suggesting that the 4-coumarate:coenzyme A ligase or coumarate hydroxylase reactions might have become limiting. These results help to define the regulatory architecture of the phenylpropanoid pathway and indicate the possibility of engineering-selective changes in this complex metabolic pathway by overexpression of a single early pathway gene.

We analyzed lignin content and composition in transgenic tobacco (Nicotiana tabacum) lines altered in the expression of the early phenylpropanoid biosynthetic enzymes L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase (C4H). The reduction of C4H activity by antisense expression or sense suppression resulted in reduced levels of Klason lignin, accompanied by a decreased syringyl/guaiacyl monomer ratio as determined by pyrolysis gas chromatography/mass spectrometry Similar reduction of lignin levels by down -regulation of L-phenylalanine ammonia-lyase, the enzyme preceding C4H in the central phenylpropanoid pathway, did not result in a decreased syringyl/guaiacyl ratio. Rather, analysis of lignin methoxyl content and pyrolysis suggested an increased syringyl/guaiacyl ratio. One possible explanation of these results is that monolignol biosynthesis from L-phenylalanine might occur by more than one route, even at the early stages of the core phenylpropanoid pathway, prior to the formation of specific monolignol precursors.

Pharmacological evidence implicates trans-cinnamic acid as a feedback modulator of the expression and enzymatic activity of the first enzyme in the phenylpropanoid pathway, l-phenylalanine ammonia-lyase (PAL). To test this hypothesis independently of methods that utilize potentially non-specific inhibitors, we generated transgenic tobacco lines with altered activity levels of the second enzyme of the pathway, cinnamic acid 4-hydroxylase (C4H), by sense or antisense expression of an alfalfa C4H cDNA. PAL activity and levels of phenylpropanoid compounds were reduced in leaves and stems of plants in which C4H activity had been genetically down-regulated. However, C4H activity was not reduced in plants in which PAL activity had been down-regulated by gene silencing. In crosses between a tobacco line over-expressing PAL from a bean PAL transgene and a C4H antisense line, progeny populations harboring both the bean PAL sense and C4H antisense transgenes had significantly lower extractable PAL activity than progeny populations harboring the PAL transgene alone. Our data provide genetic evidence for a feedback loop at the entry point into the phenylpropanoid pathway that had previously been inferred from potentially artifactual pharmacological experiments.

Introduction of a bean phenylalanine ammonia-lyase (PAL) transgene into tobacco plants results in epigenetic post-transcriptional gene silencing which is unstable, such that after self-pollination first generation progeny may become PAL over-expressors. The change from gene silencing to PAL over-expression is accompanied by a loss of cytosine methylation of the PAL transgene and reduced methylation of the endogenous tobacco PAL2 gene, but not the PAL1 gene. These changes are associated with the appearance of high levels of bean PAL and tobacco PAL2 transcripts in the total RNA fraction from PAL over-expressing plants. However, tobacco PAL2 transcripts are inefficiently recruited into polysomes, and tobacco PAL2 protein is not detected in leaves of PAL over-expressing or wild-type lines. Thus, in spite of the post-transcriptionally controlled increase in tobacco PAL2 transcripts in PAL over-expressors, the increased PAL activity is primarily the result of the increase in bean PAL transcripts and corresponding enzymatic activity. These results reveal a complex cross-talk between expression of the PAL transgene and the corresponding endogenous PAL genes at the levels of transcription, transcript stability and polysomal recruitment during sense transgene-mediated silencing and subsequent over-expresson of PAL in tobacco.

The photocontrol of l -phenylalanine ammonia-lyase (EC 4.3.1.5) activity in discs of potato (Solanum tuberosum) tuber parenchyme has been investigated by density labelling with 2H from 2H2O. Labelling of enzyme was measured by analysis of the equilibrium distribution of enzyme activity in CsCl density gradients as a function of buoyant density corrected with respect to the distribution of the external marker enzyme β-galctosidase (β- d -galactoside galactohydrolase, EC 3.2.1.23). It is demonstrated that the white-light mediated stimulation of phenylalanine ammonia-lyase activity involves stimulation of the rate of de novo production of active enzyme.

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.

1. (1) The synthesis and removal of phenylalanine ammonia-lyase (EC 4.3.1.5) in illuminated discs of potato (Solanum tuberosum cv King Edward) tuber tissue has been investigated by density labelling with deuterium (2H) from deuterium oxide (2H2O) followed by centrifugation to equilibrium in a CsCl density gradient. 2. (2) Temporal changes in enzyme level have been described in terms of the equation (dE/dt) = ks − kdE where (dE/dt) is the rate of change of enzyme level per unit of tissue (E) with respect to time (t), ks is the rate constant for synthesis of the enzyme and kd is the rate constant for the removal of active enzyme. 3. (3) The optimal concentration of 2H2O was determined by analysis of the relationship between 2H2O concentration, development of enzyme activity and the magnitude of the increase in buoyant density of phenylalanine ammonialyase. A concentration of 2H2O of about 40% (v/v) was found to be optimal, allowing achievement of maximal or near maximal increases in the buoyant density of the enzyme without inhibition of the development of enzyme activity, thereby circumventing the major drawback of 2H2O as a source of density label. 4. (4) The overlapping distribution profiles of enzyme activity after density gradient centrifugation were resolved by an iterative method of best fit which allows estimation of the proportions of pre-existing, unlabelled enzyme and newly synthesised, labelled enzyme at the end of the labelling period. This technique has been developed to obtain the rate constants for enzyme synthesis and for removal of active enzyme throughout the period of rapid change in enzyme level. 5. (5) It is demonstrated that the initial rapid increase in phenylalanine ammonia-lyase activity in illuminated discs reflects an increase in the rate constant for enzyme synthesis in the absence of activation of pre-existing enzyme and in the absence of removal of active enzyme. The abrupt transition to a phase of decline in enzyme activity is caused by (a) a reduction in the rate constant for enzyme synthesis and (b) a dramatic increase in the rate constant for removal of active enzyme. The subsequent stabilisation of the enzyme level is caused by decay of both rate constants to relatively low levels. 6. (6) The results are consistent with the hypothesis that rapid modulation of enzyme levels during tissue differentiation is achieved by simultaneous changes in the rate constants for both enzyme synthesis and for removal of active enzyme.

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and cinnamic acid 4-hydroxylase (CA4H, EC 1.14.13.11) undergo concomitant increases in activity after a 2 h lag following disc preparation and illumination. The increases in PAL and CA4H activity can be inhibited by actinomycin-D, cordycepin and cycloheximide. It is demonstrated that the concomitant increases in enzyme activity stem from the simultaneous stimulation of the respective actinomycin-D-sensitive steps followed by parallel transmission of these stimuli through the cordycepin- and cycloheximide-sensitive steps. The events between the actinomycin-D- and cordycepin-sensitive steps are rate limiting in the expression of the stimulations in terms of increased enzyme activities. Delayed transfer of discs to actinomycin-D or cordycepin 'superinduces' PAL but not CA4H activity. This superinduction is related to the inhibition by cycloheximide of the subsequent decline in PAL activity and is interpreted in terms of the transcription and translation of the mRNA coding for an inactivator-protein of PAL.

l-Phenylalanine ammonia-lyase (EC 4.3.1.9, which catalyses the first reaction in the biosynthesis of plant phenylpropanoid products, undergoes a transient increase in activity in excised sections of pea (Pisum sativum) epicotyl tissue. Exogenous supplies of pathway intermediates inhibit the initial development of phenylalanine ammonia-lyase activity and, if added at the time of high enzyme levels, cause a rapid decay in enzyme activity. The inhibitory effect of exogenous cinnamic acid, the immediate product of the enzyme, is expressed very rapidly (lag < 1 h) with the initial rate of decay of enzyme activity and the final steady-state level of enzyme activity being dependent on the concentration of cinnamic acid. The response has been studied by density labelling in vivo with 2H from 2H2O with analysis of the equilibrium distribution of enzyme activity in high-resolution KBr density gradients. Cinnamic acid, at a concentration of 1 mM completely inhibits further incorporation of label and also markedly stimulates the removal of pre-existing, unlabelled enzyme. Inhibition of de novo production and stimulation of removal occur at concentrations of cinnarnic acid as low as 10 μM. The initial increase in enzyme activity reflects an increase in the rate of de novo enzyme production against a low background rate of enz:yme removal. The subsequent decay in enzyme activity is a result of a decline in production of the enzyme concomitant with a marked increase in the rate of removal of enzyme activity. In the presence of α- aminooxy-β-phenylpropionic acid, which specifically inhibits phenylalanine ammonia-lyase and hence endogenous generation of cinnamic cid, high rates of enzyme production and low rates of enzyme removal are maintained leading to high levels of zyme activity in the later stages of enzyme induction. These data indicate the operation of dual feedback modulation of phenylalanine ammonia-lyase in vivo following endogenous production of cinnamic acid. Such dual control by a product over both production and removal of an enzyme may be of general importance in slowly growing plant cells for the rapid removal of biosynthetic enzymes no longer required by the cell.

l -Phenylalanine ammonia-lyase (EC 4.31.5) undergoes a transient increase in activity in illuminated discs of Solanum tuberosum tuber parenchyme. Cycloheximide and cordycepin inhibit the initial increase in enzyme activity, but if addition of these anti-metabolites is delayed until the time of maximum enzyme levels, the subsequent decline in enzyme activity is inhibited (Lamb, C.J. (1977) Planta, 135, 169–175). The effect of delayed treatment with cycloheximide or cordycepin on the turnover of phenylalanine ammonia-lyase has been analysed by density labelling with 2H from 2H2O. Delayed introduction of cycloheximide or cordycepin reduces the rate of labelling of phenylalanine ammonia-lyase whilst preventing the decay in enzyme activity observed in controls not treated with inhibitor, and this labelling pattern cannot be accounted for by effects of cycloheximide or cordecypin on the labelling of amino acid pools. It is concluded that delayed treatment with cycloheximide or cordycepin leads to the maintenance of high levels of phenylalanine ammonia-lyase by inhibition of the removal of active enzyme rather than by maintenance of high rates of enzyme synthesis.

1. (1) The regulation of the accumulation of the isoflavonoid-derived phytoalexin phaseollin in cell suspension cultures of Dwarf French Bean (Phaseolus vulgaris) has been investigated. 2. (2) An elicitor preparation from cell walls of Colletotrichum lindemuthianum, the causal agent of anthracnose disease of French bean, caused a marked accumulation of phaseollin in the cultures. The elicitor induced phaseollin accumulation to a level of 60% that obtained with the artificial elicitor autoclaved ribonoclease A and was maximally active at a concentration (weight basis) of at least 50 times lower than required for maximall response to ribonuclease. 3. (3) Elicitor preparations from cell walls of Phytophthora megasperma var. sojae, a fungal pathogen of soybean, and Botrytis cinerea, the common grey mould, were much less effective than the C. lindemuthianum wall-released elicitor. 4. (4) There was a marked but transient increase in the extractable activity of phenylalanine ammonia-lyase, the enzyme catalysing the first reaction in the biosynthesis of phaseollin from l -phenylalanine, in response to the elicitor from C. lindemuthianum. 5. (5) Comparative density labelling with 2H from 2H2O indicated that the elicitor stimulates de novo synthesis of phenylalanine ammonia-lyase, and that this is an early event in the phytoalexin defence response. 6. (6) These findings provide the basis of a scheme for elicitor induction of phytoalexin accumulation.