Phenylpropanoid pathway intermediates regulate transient expression of a chalcone synthase gene promoter.

A chimeric gene construct containing a bean chalcone synthase (CHS) promoter fused to the chloramphenicol acetyltransferase (CAT) reporter gene was strongly expressed when electroporated into alfalfa protoplasts that were then exposed to a fungal elicitor. Low concentrations (5 x 10(-6) to 10(-4) M) of exogenously applied trans-cinnamic acid (CA), the first intermediate of the phenylpropanoid pathway, slightly stimulated elicitor-induced CAT expression, whereas high concentrations (greater than 10(-4) M) severely reduced expression to below the levels observed in the absence of elicitor. In contrast, trans-p-coumaric acid (4-CA, the second intermediate in the pathway) stimulated expression from the CHS promoter up to 4.5-fold at 5 x 10(-4) M. Expression of CAT driven by the promoters of other elicitor-inducible defense response genes was not markedly affected by CA or 4-CA. Stimulation of CHS promoter expression by low concentrations of CA and 4-CA was completely abolished by 5' deletion to position -130, but not -174. When the -180 to -130 region of the CHS15 promoter was coelectroporated into elicited protoplasts on a separate plasmid along with the intact -326 CHS-CAT construct, the decreased CAT expression as a function of CA or 4-CA concentration was consistent with the coelectroporated sequence competing in trans with the intact promoter for the binding of a factor(s) involved in the up regulation of CHS transcription by 4-CA and low concentrations of CA. Our data support the hypothesis that phenylpropanoid compounds may act as natural and specific regulators of plant gene expression and define the location of a cis-acting element in the CHS15 promoter involved in the induction by phenylpropanoid pathway intermediates.

Abnormal plant development and down-regulation of phenylpropanoid biosynthesis in transgenic tobacco containing a heterologous phenylalanine ammonia-lyase gene.

Biosynthesis of phenylpropanoid natural products in tobacco was perturbed by introduction of a heterologous (bean) phenylalanine ammonia-lyase (PAL; L-phenylalanine ammonia-lyase, EC 4.3.1.5) gene, modified by inclusion of cauliflower mosaic virus 35S enhancer sequences in its promoter. These transgenic plants can exhibit a series of unusual phenotypes including localized fluorescent lesions, altered leaf shape and texture, reduced signification in xylem, stunted growth, reduced pollen viability, and altered flower morphology and pigmentation. Genetic analysis of a transformant with severe symptoms showed that symptom development was inherited as a single, partially dominant trait and cosegregated with reduced levels of PAL activity and soluble phenylpropanoid products. Accumulation of transcripts encoded by the endogenous tobacco PAL genes was suppressed. We conclude that the transgene disrupts PAL regulation and that some of the phenotypes reflect interference with putative signals dependent on phenylpropanoid biosynthesis.

Effects of trans-Cinnamic Acid on Expression of the Bean Phenylalanine Ammonia-Lyase Gene Family.

Using DNA probes specific for the three members of the bean (Phaseolus vulgaris L.) phenylalanine ammonia-lyase (PAL) gene family, we have studied the effects of the product of the PAL reaction, trans-cinnamic acid (CA), on the appearance of individual PAL transcripts in suspension cultured bean cells. Concentrations of CA in excess of 10(-4) molar inhibited appearance of elicitor-induced transcripts encoding PAL1, PAL2, and PAL3 when added to the cells at the same time as fungal elicitor. Addition of CA 4 hours postelicitation caused a major reduction in the levels of all three PAL transcripts, but with different kinetics and subsequent rates of recovery. The inhibition of accumulation of PAL1, PAL2, or PAL3 transcripts, measured 3 hours after exposure to elicitor, as a function of the time of addition of CA postelicitation reflected the different rates of appearance of the three PAL transcripts in the presence of elicitor alone. The inhibitory effects of CA as seen on PAL transcripts were not observed for the constitutively expressed transcript H1, or the elicitor-inducible 1,3-beta-D-glucanase. Analysis of in vitro translated polypeptides showed that some elicitor-induced mRNA activities were not down-regulated by CA, and that a number of other mRNA activities were induced by CA, thus providing further evidence for specificity in the action of CA on bean cells. Treatment of elicited cells with L-alpha-aminooxy-beta-phenylpropionic acid, a potent and specific inhibitor of PAL activity, resulted in maintenance of elevated PAL transcript levels beyond 12 hours postelicitation, this effect being greatest for PAL transcripts 2 and 3. Our results are consistent with a model in which CA, or a derivative, could act as a component in a regulatory feedback system operating at the level of phenylpropanoid gene transcription.

Defense gene expression in elicitor-treated cell suspension cultures of french bean cv. Imuna.

Cell suspension cultures of bean (Phaseolus vulgaris) cv. Imuna accumulated isoflavonoid phytoalexins on exposure to elicitor from the phytopathogenic fungus Colletotrichum lindemuthianum (CL). This was preceeded by rapid increases in the activities of phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS). However, the patterns of expression of PAL and CHS genes differed from those observed in cultures of a previously studied bean cultivar. The relative levels of transcripts from individual members of the CHS multigene family differed significantly at 1.5 h compared to 22.5 h after elicitation. More strikingly, three PAL genes were expressed in cultivar Imuna in response to fungal elicitor, whereas two are expressed in elicitor-treated cell cultures of cultivar Canadian Wonder.