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

Abstract

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 lignification 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.Phenylalanine ammonia-lyase (PAL; L-phenylalanine ammonia-lyase, EC 4.3.1.5) catalyzes the first step in the synthesis of a diverse array of plant natural products based on the phenylpropane skeleton (1). PAL activity is highly regulated during development associated with the synthesis of flavonoid pigments and during xylogenesis with the cell wall polymer lignin and by environmental cues for the synthesis of these and other protective compounds (2). Distinct isoforms of the PAL enzyme subunit are encoded by a small family of differentially regulated genes (3-6). Transgenic plants containing the reporter gene p-glucuronidase under the control of the bean PAL2 promoter show tissue-and cell-type-specific expression associated with xylem differentiation during vascular development, in pigmented regions of petals, in root hairs, and in root and shoot meristems (7,8). Moreover, in stems the PAL2 promoter is activated in epidermal and subepidermal layers by illumination of dark-adapted plants and in the epidermis and perivascular parenchyma in response to wounding (7). Nuclear run-off transcription experiments have shown that the activation of PAL transcription by environmental stimuli is characteristically rapid but transient (9).These data indicate that PAL transcription is a major control site regulating the induction of PAL activity and initiation of phenylpropanoid biosynthesis in response to developmental and environmental cues. In the present paper, we have used a gene transfer approach to perturb this key step of phenylpropanoid biosynthesis. MATERIALS AND METHODS PAL2 ExpressionVectors. An expression cassette pYE1, consisting of a cauliflower mosaic virus 35S enhancer element and nopaline synthase 3' flanking sequences was constructed from pBI221 (10) cut with EcoRV and Sac I and ligated to the Sal I (filled-in with Klenow)/Sac I fragment of the pUC19 polylinker. The 5.3-kilobase (kb) PAL2 gene (4) including 1170 base pairs (bp) of 5' flanking sequence was used to generate two exo...

Coordinated Activation of Programmed Cell Death and Defense Mechanisms in Transgenic Tobacco Plants Expressing a Bacterial Proton Pump.

“…Our results and those of others (Takahashi et al, 1989;Bachmairet al, 1990;Elkind et al, 1990;Dietrich et al, 1994) suggest that perturbation of cellular homeostasis in higher plants may result in the activation of acell death pathway. Such perturbation may be the outcome of an environmental insult, a pathogen infection, or expression of a particular transgene.…”

Transgene-Mediated and Elicitor-Induced Perturbation of Metabolic Channeling at the Entry Point into the Phenylpropanoid Pathway

“…PAL -overexpressing tobacco lines, resulting from constitutive expression of a bean PAL2 gene, overproduce phenylpropanoid compounds (Howles et al, 1996), whereas underexpressing lines, resulting from epigenetic gene silencing (Elkind et al, 1990;Bate et al, 1994), have reduced levels of phenylpropanoid compounds. We initiated callus and then cell suspension cultures from PAL -overexpressing transgenic tobacco; constitutive PAL activities in the cell suspension cultures were at least threefold higher than in comparable cultures derived from wild-type plants.…”

“…These results indicate that externally added unlabeled transcinnamic acid does not dilute isotope incorporation from 3 H-Phe into phenylpropanoid compounds in tobacco cell cultures, whereas externally added unlabeled 4-coumaric acid does, suggesting a specific metabolic compartmentalization of trans -cinnamic acid. This does not directly prove channeling or necessarily indicate the presence of more than one endogenous pool of trans -cinnamic acid in the absence of an externally fed compound.PAL -overexpressing tobacco lines, resulting from constitutive expression of a bean PAL2 gene, overproduce phenylpropanoid compounds (Howles et al, 1996), whereas underexpressing lines, resulting from epigenetic gene silencing (Elkind et al, 1990;Bate et al, 1994), have reduced levels of phenylpropanoid compounds. We initiated callus and then cell suspension cultures from PAL -overexpressing transgenic tobacco; constitutive PAL activities in the cell suspension cultures were at least threefold higher than in comparable cultures derived from wild-type plants.…”

“…Tobacco represents such a system. We have generated a series of transgenic tobacco plants containing a heterologous bean PAL2 gene, in which the levels of extractable PAL activity are increased or decreased when compared with wild-type plants (Elkind et al, 1990;Howles et al, 1996;Sewalt et al, 1997). These plants allow assessment of the effects of quantitative and qualitative changes in a component of a potential metabolic channel on the operation of that channel.…”

Differential Extraction and Protein Sequencing Reveals Major Differences in Patterns of Primary Cell Wall Proteins from Plants

“…Tomato is important commercially and was the subject of the first successful attempts to modify the expression of wall proteins by transformation (10). Tobacco is an important model plant for attempts to modify cellulose extractability by modifying lignification (11).…”