Induction of Flavonoid Synthesizing Enzymes by Light in Etiolated Pea (Pisum sativum cv. Midfreezer) Seedlings

Etiolated pea (Pisum sativum cv. Midfreezer) seedlings respond to illumination with white light by changes in the activity of phenylpropanoid and flavonoid synthesizing enzymes. Unlike in cell cultures, changes in enzyme activity in pea seedlings are not concerted. Phenylalanine ammonia-lyase (EC 4.3.1.5) activity peaked approximately 18 hours after onset of illumination. The phenylacetate path did not interfere with the measurement of phenylalanine ammonia-lyase activity. Activity of cinnamic acid 4-hydroxylase (EC 1.14.13.11) showed an early peak after 8 hours illumination, declined thereafter sharply, then gradually increased during the remainder of the experiment. Activities of chalcone synthase and UDP glucose:flavonol 3-O-glucosyltransferase (EC 2.4.1.91) increased steadily and reached a plateau after approximately 70 hours illumination time. Activity of 4-hydroxycinnamate:coenzyme A ligase (EC 6.2.1.12) remained relatively unchanged, whereas that of chalcone isomerase (EC 5.5.1.6) declined steadily during the course of the experiment. The relative in vitro enzyme activities suggest that the rate-limiting step for the phenylpropanoid path is the cinnamic acid 4-hydroxylase, that of the flavonoid pathway is the chalcone synthase. Integration of enzyme activity curves, however, show that only the curve deriving from phenylanine ammonia-lyase activity matches closely the production of the flavonol glycosides.     

Phenylpropanoid Metabolism in Suspension Cultures of Vanilla planifolia Andr. : II. Effects of Precursor Feeding and Metabolic Inhibitors

Feeding of cinnamic acid and ferulic acid to non-treated and chitosan-treated cell suspension cultures of Vanilla planifolia resulted in the formation of trace amounts of p-hydroxy benzoic acid (5.2 micrograms per gram fresh weight of cells) and vanillic acid (6.4 micrograms per gram fresh weight of cells), respectively. Addition of a 4-hydroxycinnamate: CoA-ligase inhibitor, 3,4-(methylenedioxy)-cinnamic acid (MDCA), resulted in a reduced biosynthesis of ligneous material with a simultaneous significant increased vanillic acid formation (around 75 micrograms per gram fresh weight of cells). A K₁ of 100 micromolar for 4-hydroxycinnamate: CoA-ligase in a crude preparation was estimated for this inhibitor. It is suggested that the conversion of cinnamic acids into benzoic acids does not involve cinnamoyl CoA esters as intermediates. Feeding of ¹⁴C-cinnamic acid and ¹⁴C-ferulic acid to cells treated with MDCA indicate that cinnamic acid, but not ferulic acid, is a precursor of vanillic acid in these cultivated cells of V. planifolia.     

Stress Responses in Alfalfa (Medicago sativa L.): I. Induction of Phenylpropanoid Biosynthesis and Hydrolytic Enzymes in Elicitor-Treated Cell Suspension Cultures

Alfalfa (Medicago sativa L.) cell suspension cultures accumulated high concentrations of the pterocarpan phytoalexin medicarpin, reaching a maximum within 24 hours after exposure to an elicitor preparation from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum. This was preceded by increases in the extractable activities of the isoflavonoid biosynthetic enzymes l-phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, 4-coumarate coenzyme A-ligase, chalcone synthase, chalcone isomerase, and isoflavone O-methyltransferase. Pectic polysaccharides were weak elicitors of phenylalanine ammonia-lyase activity but did not induce medicarpin accumulation, whereas reduced glutathione was totally inactive as an elicitor in this system. The fungal cell wall extract was a weak elicitor of the lignin biosynthetic enzymes, caffeic acid O-methyltransferase and coniferyl alcohol dehydrogenase, but did not induce appreciable increases in the activities of the hydrolytic enzymes chitinase and 1,3-β-d-glucanase. The results are discussed in relation to the activation of isoflavonoid biosynthesis in other legumes and the development of the alfalfa cell culture system as a model for studying the enzymology and molecular biology of plant defense expression.     

Elicitor induction of furanocoumarin biosynthetic pathway in cell cultures ofRuta graveolens

Treatment with an autoclaved culture homogenate of the yeastRhodotorula rubra induces rapid accumulation of acridone epoxides, furoquinolines and furanocoumarins in cell cultures ofRuta graveolens (L). The increased accumulation is preceeded by an induction of enzymes of the biosynthetic pathways. In the case of furanocoumarins induction was shown for phenylalanine ammonia-lyase (PAL), 4-coumarate: CoA ligase (4-CL) and S-adenosyl-l-methionine: xanthotoxol O-methyltransferase (XOMT). For PAL and 4-CL time courses of induced activity showed an early maximum, 8–12 h after treatment, whereas XOMT was found to reach its maximum later, about 36–42 h after treatment. The elicitor dose-response curve showed saturation at an elicitor concentration of 1%. At any time during the whole culturing period cells responded to elicitiation but the maximum enzyme activities induced were lower at the late stages. Experiments with different suspension culture strains, a shoot teratoma culture and hydroponically grown sterile photomixotrophic plants were performed to assess the influence of differentiation on constitutive activities of these enzymes and their inducibility by elicitation. Constitutive furanocoumarin accumulation was positively correlated with the level of differentiation. Although induction of PAL, 4-CL and XOMT activity always accompanied induced furanocoumarin accumulation no absolute correlation existed between induced enzyme activities and the induced product level or relative product increase.Abbreviations 4-CL 4-coumarate:CoA ligase - COMT S-adenosyl-l-methionine:caffeic acid 3-O-methyltransferase - PAL phenylalanine:ammonia-lyase - XOMT S-adenosyl-l-methionine:xanthotoxol O-methyltransferase     

Distribution and roles of p-hydroxycinnamate: CoA ligase in lignin biosynthesis

p-Hydroxycinnamate:CoA ligases were extracted from the xylems of angiosperms and gymnosperms, and the substrate specificities toward ferulate and sinapate were examined. Most of angiosperm and gymnosperm CoA ligases examined were active with ferulate but not with sinapate; however, the enzymes of Erythrina crista-galli, Robinia pseudoacacia and bamboo showed considerable activity with sinapate. The other enzymes, although inactive with sinapate, showed no inhibitory effect on the Erythrina CoA ligase reaction with sinapate. The K_ms for sinapate and ferulate of the Erythrina enzyme were 1.0 and 2.1 μM, respectively, and p-hydroxycinnamate was the best substrate among cinnamates examined. The MW of the CoA ligase was 40 000 and the pH optimum was between 7.2 and 7.6. The possible roles of p-hydroxycinnamate:CoA ligase in lignin biosynthesis are discussed.     

Tissue compartmentation of phenylpropanoid metabolism in tomatoes during growth and maturation

Marked changes in the metabolism of hydroxycinnamic acid derivatives were observed in pulp and pericarp of tomato fruit (Lycopersicon esculentum var. cerasiforme) during its development. During fruit growth, biosynthesis and accumulation of chlorogenic acid were especially active in the pulp, whereas the formation of glucose derivatives occurred during maturation in the pericarp. There was a clear difference between the two compartments of the fruit concerning hydroxycinnamate: CoA ligase, O-methyltransferase and glucosyltransferase activities. The first two enzymes were high in the pulp during growth and the latter one was high in the pericarp during maturation. Of all the enzymes studied, only the glucosyltransferase showed increasing activity during maturation; it may be considered, along with the glucosylated derivatives, as a biochemical marker of maturation in tomato.     

3-Hydroxybenzoate:coenzyme A ligase and 4-coumarate: coenzyme A ligase from cultured cells of Centaurium erythraea

3-Hydroxybenzoate:coenzyme A ligase, an enzyme involved in xanthone biosynthesis, was detected in cell-free extracts from cultured cells of Centaurium erythraea Rafn. The enzyme was separated from 4-coumarate:coenzyme A ligase by fractionated ammonium sulphate precipitation and hydrophobic interaction chromatography. The CoA ligases exhibited different substrate specificities. 3-Hydroxybenzoate:coenzyme A ligase activated 3-hydroxybenzoic acid most efficiently and lacked affinity for cinnamic acids. In contrast, 4-coumarate:CoA ligase mainly catalyzed the activation of 4-coumaric acid but did not act on benzoic acids. The two enzymes were similar with respect to their relative molecular weight, their pH and temperature optima, their specific activity and the changes in their activity during cell culture growth. Received: 23 September 1996 / Accepted: 28 November 1996     

Photocontrol of chlorogenic acid biosynthesis in potato tuber discs

The appearance of phenylalanine ammonia-lyase activity and the accumulation of chlorogenic acid in potato tuber discs are stimulated by illumination with white light, whereas the appearance of cinnamic acid 4-hydroxylase activity is unaffected by illumination. The photosensitive step in chlorogenic acid biosynthesis may be by-passed by treatment of discs with exogenous supplies of cinnamic acid, whereas treatment of discs with phenylalanine does not isolate the photosensitive step. Therefore, the site of photocontrol of chlorogenic acid biosynthesis in potato tuber discs is the reaction catalysed by phenylalanine ammonia-lyase. Cinnamic acid 4-hydroxylase activity in vitro is unaffected by p-coumaric acid, caffeic acid or chlorogenic acid. Phenylalanine ammonia-lyase activity in vitro is sensitive to inhibition by cinnamic acid. The in vitro properties of the two enzymes are also consistent with the hypothesis that phenylalanine ammonia-lyase rather than cinnamic acid 4-hydroxylase is important in the regulation of chlorogenic acid biosynthesis in potato tuber discs.     

Xanthone biosynthesis in Hypericum perforatum cells provides antioxidant and antimicrobial protection upon biotic stress

Xanthone production in Hypericum perforatum (HP) suspension cultures in response to elicitation by Agrobacterium tumefaciens co-cultivation has been studied. RNA blot analyses of HP cells co-cultivated with A. tumefaciens have shown a rapid up-regulation of genes encoding important enzymes of the general phenylpropanoid pathway (PAL, phenylalanine ammonia lyase and 4CL, 4-coumarate:CoA ligase) and xanthone biosynthesis (BPS, benzophenone synthase). Analyses of HPLC chromatograms of methanolic extracts of control and elicited cells (HP cells that were co-cultivated for 24 h with A. tumefaciens) have revealed a 12-fold increase in total xanthone concentration and also the emergence of many xanthones after elicitation. Methanolic extract of elicited cells exhibited significantly higher antioxidant and antimicrobial competence than the equivalent extract of control HP cells indicating that these properties have been significantly increased in HP cells after elicitation. Four major de novo synthesized xanthones have been identified as 1,3,6,7-tetrahydroxy-8-prenyl xanthone, 1,3,6,7-tetrahydroxy-2-prenyl xanthone, 1,3,7-trihydroxy-6-methoxy-8-prenyl xanthone and paxanthone. Antioxidant and antimicrobial characterization of these de novo xanthones have revealed that xanthones play dual function in plant cells during biotic stress: (1) as antioxidants to protect the cells from oxidative damage and (2) as phytoalexins to impair the pathogen growth.     

Fungal elicitor-mediated responses in pine cell cultures

A tissue culture system has been developed to examine phenylpropanoid metabolism induced in pine tissues by an ectomycorrhizal symbiont. An elicitor preparation from the ectomycorrhizal fungus Thelephora terrestris Fr. induced enhanced phenolic metabolism in suspension cultured cells of Pinus banksiana Lamb., as indicated by tissue lignification and accumulation of specific methanol-extractable compounds in the cells. Induction of lignification was observed as early as 12 h after elicitation. The activity of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), the entry-point enzyme into phenylpropanoid metabolism, also increased within the same time-frame in elicited cells. Significant increases in PAL activity were evident by 6 h after elicitation, and, by 12 h after elicitation, PAL activity in elicited cells was ten times greater than that in the corresponding controls. Lignification of the elicited tissue was also accompanied by an increase in the activity of other enzymes associated with lignin synthesis, including caffeic acid O-methyl transferase (EC 2.1.1.46), hydroxycinnamate:CoA ligase (EC 6.2.1.12), cinnamyl alcohol dehydrogenase (EC 1.1.1.-), coniferin glucosidase (EC 3.2.1.21) and peroxidase (EC 1.11.1.7). The increase in total peroxidase activity was associated with a change in the pattern of soluble peroxidase isoforms. The pine cell culture-ectomycorrhizal elicitor system provides a good model for molecular analysis of the process of lignification in an economically important softwood species.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 4CL hydroxycinnamate:Coenzyme A ligase (EC 6.2.1.12) - CAD cinnamyl alcohol dehydrogenase (EC 1.1.1.-) - COMT S-adenosyl-l-methionine:caffeate O-methyl transferase (EC 2.1.1.46) - HPLC high-pressure liquid chromatography - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - TGA thioglycolic acid To whom correspondence should be addressedFinancial assistance for this work was provided by the Natural Sciences and Engineering Research Council of Canada.     

Elicitor-inducible 3-hydroxy-3-methylglutaryl coenzyme a reductase activity is required for sesquiterpene accumulation in tobacco cell suspension cultures

Addition of cell wall fragments from Phytophthora species or cellulase from Trichoderma viride, but not pectolyase from Aspergillus japonicus, to tobacco (Nicotiana tabacum) cell suspension cultures induced the accumulation of the extracellular sesquiterpenoid capsidiol. Pulse-labeling experiments with [¹⁴C]acetate and [³H]mevalonate suggested that enzymatic steps preceding mevalonate were limiting capsidiol biosynthesis in the pectolyase-treated cell cultures. Treatment of the cell cultures with either Phytophthora cell wall fragments or cellulase induced 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and sesquiterpene cyclase activities, enzymes of the sesquiterpene biosynthetic pathway, and phenylalanine ammonia lyase activity, an enzyme of the general phenylpropanoid pathway. Pectolyase treatment induced sesquiterpene cyclase and phenylalanine ammonia lyase activities, but not HMGR activity. These results corroborate the importance of inducible HMGR enzyme activity for sesquiterpene accumulation.     

Phenylalanine ammonia-lyase-mediated biosynthesis of 2-hydroxy-4-methoxybenzaldehyde in roots of Hemidesmus indicus

The fragrant rootstocks of Hemidesmus indicus are known to accumulate 2-hydroxy-4-methoxybenzaldehyde (MBALD), yet, the enzymatic route to this hydroxybenzoate is not known. Therefore, root organs of H. indicus hold promises to unravel the biosynthesis related to this phenolic fragrance. Chitosan treatment at 200mg/L concentration to the excised roots effectively increased phenolic accumulation in both the cortex and cork tissues reaching a peak after 24h treatment and decreasing thereafter. The activity of phenylalanine ammonia-lyase (PAL) enzyme in excised roots also increased upon chitosan elicitation, and the maximum specific activity was recorded after 12h of elicitation. Suppression of PAL in vivo by using a specific irreversible inhibitor aminooxyacetic acid (AOAA) resulted in the decrease in MBALD content, indicating its formation via phenylpropanoid pathway.     

Differential response of cultured parsley cells to elicitors from two non-pathogenic strains of fungi. 2. Effects on enzyme activities

Parsley cell cultures produce linear furanocoumarins and the linear benzodipyrandione, graveolone, in response to treatment with an elicitor from either Phytophthora megasperma or Alternaria carthami. Activities of enzymes involved in general phenylpropanoid metabolism, phenylalanine ammonia-lyase and 4-coumarate: CoA ligase, as well as of an enzyme involved specifically in furanocoumarin biosynthesis, dimethylallyl diphosphate: umbelliferone dimethylallyltransferase, were monitored over several days after treatment with A. carthami elicitor. In addition, the activities of chalcone synthase, an enzyme involved in flavonoid formation, and of glucose-6-phosphate: NADP 1-oxidoreductase were also monitored. The lyase and the ligase activities increased steadily for 48 h and the dimethylallyltransferase activity for 54 h, while the synthase activity was not altered and the oxidoreductase activity decreased gradually. In some experiments, phenylalanine ammonia-lyase activity reached a maximum value of 250 mukat/kg, twice the maximal activity observed previously in parsley cells after treatment with either ultraviolet light or an elicitor preparation from P. megasperma. In crude extracts, phenylalanine ammonia-lyase activity was shown to be inhibited by unidentified small-molecular-weight compounds which were formed in proportion to the elicitor treatment. While phenylalanine ammonia-lyase and dimethylallyl diphosphate: umbelliferone dimethylallyltransferase are known to be required for furanocoumarin biosynthesis, the involvement of 4-coumarate: CoA ligase is as yet unclear. The concomitant increase and decrease of the ligase activity with the activities of the lyase and the dimethylallyltransferase, as well as its similar response to elicitor concentrations, suggest that CoA esters of cinnamic acids play a role in the biosynthesis of furanocoumarins.     

Metabolic Regulation during Glyceollin Biosynthesis in Green Soybean Hypocotyls

The accumulation of the isoflavonoid phytoalexin, glyceollin, occurs in hypocotyls of green soybean seedlings (Glycine max L. Merr. cv Harosoy 63) in response to the injection of a glucan elicitor isolated from the mycelial walls of the fungus, Phytophthora megasperma f. sp. glycinea. This accumulation, which levels off after 24 hours, is preceded by a dramatic, transient rise in extractable activities of two early enzymes in the biosynthetic pathway, phenylalanine ammonia-lyase (PAL) and p-coumaryl CoA ligase (pCL). The maximum amount of extractable activity occurs 12 to 16 hours after elicitor treatment and is coincident with the most rapid period of glyceollin accumulation. These results suggest a regulatory role for these early enzymes in the biosynthesis of this secondary metabolite. High performance liquid chromatography analysis of the early intermediates in the pathway further corroborates this hypothesis. The relative pool size and rate of turnover of p-coumaric acid, an early intermediate in glyceollin production, increase during the period of rapid increases in enzyme activities. Removal of cotyledons from elicitor-treated seedlings reduces glyceollin accumulation approximately 70%. This limitation of phytoalexin accumulation by cotyledon removal is correlated with a similar cotyledon effect on reduction of extractable activities of both PAL and pCL as well as a decrease in the flux of carbon through the p-coumaric acid pool. This research further supports the hypothesis that early enzymic steps in a biosynthetic pathway diverting carbon from primary to secondary metabolites function as regulatory control points.     

A novel process for obtaining pinosylvin using combinatorial bioengineering in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Pinosylvin as a bioactive stilbene is of great interest for food supplements and pharmaceuticals development. In comparison to conventional extraction of pinosylvin from plant sources, biosynthesis engineering of microbial cell factories is a sustainable and flexible alternative method. Current synthetic strategies often require expensive phenylpropanoic precursor and inducer, which are not available for large-scale fermentation process. In this study, three bioengineering strategies were described to the development of a simple and economical process for pinosylvin biosynthesis in Escherichia coli. Firstly, we evaluated different construct environments to give a highly efficient constitutive system for enzymes of pinosylvin pathway expression: 4-coumarate: coenzyme A ligase (4CL) and stilbene synthase (STS). Secondly, malonyl coenzyme A (malonyl-CoA) is a key precursor of pinosylvin bioproduction and at low level in E. coli cell. Thus clustered regularly interspaced short palindromic repeats interference (CRISPRi) was explored to inactivate malonyl-CoA consumption pathway to increase its availability. The resulting pinosylvin content in engineered E. coli was obtained a 1.9-fold increase depending on the repression of fabD (encoding malonyl-CoA-ACP transacylase) gene. Eventually, a phenylalanine over-producing E. coli consisting phenylalanine ammonia lyase was introduced to produce the precursor of pinosylvin, trans-cinnamic acid, the crude extraction of cultural medium was used as supplementation for pinosylvin bioproduction. Using these combinatorial processes, 47.49 mg/L pinosylvin was produced from glycerol.     

A Simple Method for Enzymatic Synthesis of Unlabeled and Radiolabeled Hydroxycinnamate-CoA

Hydroxycinnamate coenzyme A (CoA) thioesters are substrates for biosynthesis of lignin and hydroxycinnamate esters of polysaccharides and other polymers. Hence, a supply of these substrates is essential for investigation of cell wall biosynthesis. In this study, three recombinant enzymes, caffeic acid 3-O-methyltransferase, 4-coumarate-CoA ligase 1, and 4-coumarate-CoA ligase 5, were cloned from wheat, tobacco, and Arabidopsis, respectively, and were used to synthesize ¹⁴C-feruloyl-CoA, caffeoyl-CoA, p-coumaroyl-CoA, feruloyl-CoA, and sinapoyl-CoA. The corresponding hydroxycinnamoyl-CoA thioesters were high-performance liquid chromatography purified, the only extraction/purification step necessary, with total yields between 88–95%. Radiolabeled ¹⁴C-feruloyl-CoA was generated from caffeic acid and S-adenosyl-¹⁴C-methionine under the combined action of caffeic acid 3-O-methyltransferase and 4-coumarate-CoA ligase 1. About 70% of ¹⁴C-methyl groups from S-adenosyl methionine were incorporated into the final product. The methods presented are simple, fast, and efficient for the preparation of the hydroxycinnamate thioesters.     

Rapid Activation of Phenylpropanoid Metabolism in Elicitor-Treated Hybrid Poplar (Populus trichocarpa Torr. & Gray x Populus deltoides Marsh) Suspension-Cultured Cells

Elicitor induction of phenylpropanoid metabolism was investigated in suspension-cultured cells of the fast-growing poplar hybrid (Populus trichocarpa Torr. & Gray × Populus deltoides Marsh) H11-11. Treatment of cells with polygalacturonic acid lyase or two fungal elicitors resulted in rapid and transient increases in extractable l-phenylalanine ammonia lyase and 4-coumarate:coenzyme A ligase enzyme activities. The substrate specificity of the inducible 4-coumarate:coenzyme A ligase enzyme activity appeared to differ from substrate specificity of 4-coumarate:coenzyme A ligase enzyme activity in untreated control cells. Large and transient increases in the accumulation of l-phenylalanine ammonia-lyase and 4-coumarate:coenzyme A ligase mRNAs preceded the increases in enzyme activities and were detectable by 30 minutes after the start of elicitor treatment. Chalcone synthase, cinnamyl alcohol dehydrogenase, and coniferin β-glucosidase enzyme activities were unaffected by the elicitors, but a large and transient increase in β-glucosidase activity capable of hydrolyzing 4-nitrophenyl-β-glucoside was observed. Subsequent to increases in l-phenylalanine ammonialyase and 4-coumarate:coenzyme A ligase enzyme activities, cell wall-bound thioglycolic acid-extractable compounds accumulated in elicitor-treated cultures, and these cells exhibited strong staining with phloroglucinol, suggesting the accumulation of wall-bound phenolic compounds.     

Substrates and enzyme activities related to biotransformation of resveratrol from phenylalanine by Alternaria sp. MG1

To identify the substrates and enzymes related to resveratrol biosynthesis in Alternaria sp. MG1, different substrates were used to produce resveratrol, and their influence on resveratrol production was analyzed using high performance liquid chromatography (HPLC). Formation of resveratrol and related intermediates was identified using mass spectrum. During the biotransformation, activities of related enzymes, including phenylalanine ammonia-lyase (PAL), trans-cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL), were analyzed and tracked. The reaction system contained 100 mL 0.2 mol/L phosphate buffer (pH 6.5), 120 g/L Alternaria sp. MG1 cells, 0.1 g/L MgSO₄, and 0.2 g/L CaSO₄ and different substrates according to the experimental design. The biotransformation was carried out for 21 h at 28 °C and 120 rpm. Resveratrol formation was identified when phenylalanine, tyrosine, cinnamic acid, and p-coumaric acid were separately used as the only substrate. Accumulation of cinnamic acid, p-coumaric acid, and resveratrol and the activities of PAL, C4H, and 4CL were identified and changed in different trends during transformation with phenylalanine as the only substrate. The addition of carbohydrates and the increase of phenylalanine concentration promoted resveratrol production and yielded the highest value (4.57 μg/L) when 2 g/L glucose, 1 g/L cyclodextrin, and phenylalanine (4.7 mmol/L) were used simultaneously.     

Linoleic acid-induced expression of defense genes and enzymes in tobacco

Linoleic acid (LA) is a naturally occurring fatty acid (FA) found to elicit induced systemic resistance (ISR) of tobacco against the bacterial soft rot pathogen, Pectobacterium carotovorum subsp. carotovorum (PCC). In this study, we examined effects of six doses of exogenous LA on the induction of defense genes and enzymes. The optimum ISR activity was observed in plants treated with 0.1 mM LA where the effect of LA on membrane permeability was minimal. The application of LA as a root drench enhanced the activity of defense enzymes such as phenylalanine ammonia-lyase (PAL), peroxidase (POD), and polyphenol oxidase (PPO) and induced the expression of β-glucuronidase (GUS). PAL and POD activities were increased in a concentration dependent manner while the maximum PPO activity was observed after treatment with 0.01 mM LA. An RT-PCR analysis of the defense-related genes, Coi1, NPR1, PR-1a and PR-1b, of tobacco plants treated with 0.1 mM LA revealed an association of LA with elicitation of ISR in tobacco.