Phenylalanine Ammonia-Lyase from Tomato Cell Cultures Inoculated with Verticillium albo-atrum

Abstract

Tomato (Lycopersicon esculentum Mill.) cell suspension cultures accumulated wall-bound phenolic materials in response to inoculation with VerticiliIum albo-atrum Reinke et Berth. in a fashion analogous to that observed in whole plants. Both monomeric and polymeric materials were recovered. Deposition of phenolics into the cell walls of inoculated tomato cell cultures was inhibited by the phenylalanine ammonia-lyase (PAL) inhibitor, 2-amino-2-indanephosphate. Tomato PAL activity was induced over 12-fold by fungal inoculation, with a concomitant increase in the corresponding mRNA. The enzyme was purified >3400-fold, to apparent homogeneity, by anion-exchange chromatography, chromatofocusing, and gel filtration. The holoenzyme had a molecular mass of 280 to 320 kilodaltons, comprising 74-kilodalton subunits, and displayed an isoelectric point of 5.6 to 5.7. Induced PAL displayed apparent Michaelis-Menten kinetics (Km = 116 micromolar) and was not appreciably inhibited by its product cinnamic acid. Chromatographic analysis did not reveal multiple forms of the enzyme in either inoculated or uninoculated cultures. In an effort to further understand the role of phenylpropanoid metabolism in the interaction between tomato and V. albo-atrum, we have developed an in vitro cocultivation system (3) in which tomato cell suspension cultures are inoculated with spores of V. albo-atrum. Our initial reports focused on the induction of phenylpropanoid biosynthetic activity and the accumulation of specific soluble phenolic metabolites (3,4). We report here the accumulation of phenylpropanoidderived metabolites in the cell walls of Verticillium-inoculated tomato suspension cultures as correlated with the induction of PAL both at the enzyme and mRNA levels, and the purification of tomato PAL to apparent homogeneity.

Changes in biomass allocation and phenolic compounds accumulation due to the effect of light and nitrate supply in Cecropia peltata plants

“…In this respect, it is known that PAL is the key enzyme in phenolic-compound biosynthesis, and it has been reported that it is activated and its expression induced in response to different stress conditions, among these, an increase in light (Hemm et al 2004;Jaakola et al 2004) and nitrate deficiency (Fritz et al 2006). Different studies mention that PAL-activity increases during the first days of stress, and once the required compounds have been synthesized, the enzyme's activity decreases (Bernards and Ellis 1991;Kováčik and Bačkor 2007). When C. peltata plants were submitted to different treatments, PAL activity could have increased to a maximum in days prior to the analysis, with which phenolics were synthesized and later diminished to levels observed at the end of the treatments (d28).…”

“…Under conditions of high photosynthesis rate or reduction of available nitrogen (N), production of C-based defense compounds will be favored, whereas under the opposite conditions, N-based defense compound synthesis will be carried out (Coviella et al 2002;Fritz et al 2006;Grechi et al 2007). According to these predictions, it has been demonstrated that the production of phenolics can be stimulated by low nitrate levels (Armstrong et al 1970;Fritz et al 2006;Palumbo et al 2007;Le Bot et al 2009), high light (Hemm et al 2004;Jaakola et al 2004;Mosaleeyanon et al 2005) and other biotic and abiotic stresses, e.g., pathogen attack, mechanic wounding, ultraviolet (UV) radiation, low temperatures and nutrient deficit (Bernards and Ellis 1991;Kováčik and Bačkor 2007;Clé et al 2008;Jansen et al 2008). Phenylalanine ammonia-lyase enzyme (PAL) catalyzes the conversion of phenylalanine into trans-cinnamic acid; therefore, this enzyme causes the flux of primary into secondary metabolites in the phenolics pathway.…”

Tomato Phenylalanine Ammonia-Lyase Gene Family, Highly Redundant but Strongly Underutilized

“…Other studies on the phenylalanine ammonia lyase enzyme in tomato, although limited, are consistent with the current analyses. Fungal inoculations of tomato cell cultures have been reported not to result in multiple forms of the enzyme (32).…”

Biotechnological production and applications of microbial phenylalanine ammonia lyase: a recent review

“…It plays a major role in the catabolism of L-phenylalanine. In higher plants, PAL has been extensively studied because of its role in plant development and its response to a wide variety of environmental stimuli (Bernards & Ellis, 1991;Havir & Hanson, 1973;Jones, 1984;Rosler et al, 1997;Santiago et al, 2009). In microorganisms, PAL was demonstrated for the first time by Ogata et al (1966).…”