Tyrosine and Phenylalanine Ammonia Lyase Activities during Shoot Initiation in Tobacco Callus Cultures

Both phenylalanine ammonia lyase and tyrosine ammonia lyase were detected in tobacco (Nicotiana tabacum L. Wisconsin 38) callus. The enzymes were separated from each other by Sephadex G-200 column chromatography. Increased activity of tyrosine ammonia lyase was observed during culture of tobacco callus under shoot-forming conditions, while activity of phenylalanine ammonia lyase increased during culture under non-organ-forming conditions. Confirmation of these findings was obtained by examining the incorporation of [¹⁴C]tyrosine and [¹⁴C]phenylalanine into p-coumarate and trans-cinnamate, respectively.     

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.     

Differential counteraction of ethylene effects by gibberellin a(3) and n(6)-benzyladenine in senescing citrus peel

Treatment of mature citrus fruit (Citrus sinensis) with ethylene induced rapid chlorophyll destruction, a rise in respiration, a release of free amino acids, an accumulation of reducing sugars, and an appearance of phenylalanine ammonia lyase activity. Gibberellin A₃ (GA₃) and N₆-benzyladenine (BA) opposed the effects of ethylene on chlorophyll, amino acids, and to a lesser extent, reducing sugar levels. The ethylene-induced respiratory rise was only slightly modified by GA₃ and BA. Phenylalanine ammonia lyase activity was not affected by GA₃.     

Specificity of deoxyribonucleic Acid intercalating compounds in the control of phenylalanine ammonia lyase and pisatin levels

Compounds with planar triple ring systems such as acridine orange, 9-amino acridine, 9-amino-1,2,3,4-tetrahydroacridine (tacrine), 6,9-diamino-2-ethoxyacridine lactate monohydrate (DE-acridine), 6-chloro-9-(3′-diethylamino-2′-hydroxypropylamino) -2-methoxyacridine·2 HCl (CDM-acridine), quinacrine, 6-chloro-9-(4′-diethylamino-1′-methylbutylamino) -2-methoxy-1,10-diazaanthracene (CDM 1,10-diazaanthracene), thionine, azure A, methylene blue, and pyronine Y when applied to excised pea pods were potent inducers of phenylalanine ammonia lyase or of pisatin, or of both. Compounds with an array of structural variation around the planar three-ring system were tested for their ability to induce these responses in pea tissue. In general, dimethylamino, diethylamino, or amino substitutions at position 2 and 6 or an amino (with or without an aliphatic side chain) substitution at position 9 of the three-ring system augmented induction potential. Methyl green, methylene blue, 2,7-diaminofluorene, nile blue, neutral red, pyrogallol red, ethidium bromide, nogalamycin, quinine, chloroquine, spermine, 8-azaguanine, gliotoxin, chromomycin A₃, actinomycin D, and mitomycin C were also potent inducers. The inhibition of phenylalanine ammonia lyase induction by the application of actinomycin D (300 micrograms per milliliter) or 6-methylpurine (1 milligram per milliliter) within 1 hour after inducer application indicated that newly synthesized RNA is necessary for induction. Phenylalanine ammonia lyase induction was also inhibited by cycloheximide (150 micrograms per milliliter).     

Effect of Elicitors on the Plasmamembrane of Petunia hybrida Cell Suspensions : Role of DeltapH in Signal Transduction

Primary processes during elicitation of the phenylpropanoid pathway (PPP) were studied in Petunia hybrida cell suspensions. We tested the hypothesis that decrease of the proton gradient across the plasma membrane activates the PPP. Induction of the PPP was determined by measuring phenylalanine ammonia lyase activity. A variety of ATPase inhibitors and ionophores were tested for the ability to elicit the PPP. The ATPase inhibitors orthovanadate and N,N′-dicyclohexylcarbodiimide and the ionophores carbonyl cyanide-4-trifluoromethoxyphenylhydrazone and nigericin were all effective elicitors. Carbonyl cyanide-4-trifluoromethoxyphenylhydrazone and nigericin elicit also when used in combination with N,N′-dicyclohexylcarbodiimide. Valinomycin had little effect on phenylalanine ammonia lyase activity. Treatment with orthovanadate or nigericin led to the formation of lignin. Alkalinization of the external medium by N,N′-dicyclohexylcarbodiimide, carbonyl cyanide-4-trifluoromethoxyphenylhydrazone, and nigericin was observed directly with the use of a sensitive pH electrode and internal acidification was deduced from the changes in emission intensity of the fluorescent probe bis[3-propyl-5-oxoisoxazol-4-yl] pentamethineoxonol. These data indicate that changes in the activity of the plasmamembrane H⁺-ATPase, and subsequent decrease of the proton gradient (particularly of the pH gradient) by itself are sufficient to influence phenylalanine ammonia lyase activity of P. hybrida cells and are therefore important intermediates in signal transduction.     

Phenol metabolism, phytoalexins, and respiration in potato tuber tissue treated with Fatty Acid

Potato (solanum tuberosum L. cv Katahdin) tuber discs treated with arachidonic acid become necrotic and accumulate sesquiterpenoid phytoalexins. The arachidonic acid also causes increases in both phenylalanine ammonia lyase and lignin, but no change in total alcohol-soluble phenols. Linoleic acid does not alter any of these parameters. A high concentration of nonanoic acid promotes both necrosis and accumulation of low levels of phytoalexins, but decreased levels of phenols, phenylalanine ammonia lyase, and lignin. The respiration of the control discs and those treated with linoleic acid declines by 24 hours after treatment, but the respiration of arachidonic acid-treated discs remains constant for at least 48 hours.     

Two phenylalanine ammonia lyase isoforms are involved in the elicitor-induced response of rice to the fungal pathogen Magnaporthe oryzae

Suspension cultured cells of a blast-resistant rice genotype (Oryza sativa L. cv. Gigante Vercelli) were treated with cell wall hydrolysates prepared from the fungal pathogen Magnaporthe oryzae. As a consequence, a complex pattern of phenylalanine ammonia lyase time course specific activity levels was evident. Ion-exchange chromatographic fractionation of crude extracts suggested that the early (6 h) and the late (48-72 h after elicitation) increase of activity relied upon the sequential induction of two different isoenzymes. The relative expression levels of 11 genes putatively coding for a phenylalanine ammonia lyase were measured by semi-quantitative capillary gel electrophoresis of RT-PCR products. Two genes were indeed found to be induced by treatments with the hydrolysate, and data were validated by real-time PCR. Conversely, only the early-responsive enzyme form was observed following elicitation in a blast-sensitive rice genotype (cv. Vialone nano). Therefore, the late-responsive isoform may represent a candidate gene to select for decreased sensitivity to blast.     

Phaseollin production in cell suspensions and whole plants of Phaseolus vulgaris

Production of phaseollin was measured in cell suspension cultures and whole plants of Phaseolus vulgaris. In suspension cultures phaseollin appeared when there was no further increase in cell mass. Cells transferred to a medium without auxins yielded three times higher phaseollin concentrations than cells grown in their presence. Addition of autoclaved fungal mycelia or polysaccharides as elicitors resulted in an increased phaseollin concentration in the cell suspension.In whole plants phaseollin could be detected only after the plants were challenged by a fungus which caused lesions (browning) of the upper root neck region, Rhizoctonia solani. Treatment of non-infected plants with autoclaved fungal mycelia or other elicitors did not induce phaseollin production. However, when they were added before or together with the pathogenic fungus, the elicitors further increased phaseollin concentration in the root neck regions of the plants. This indicated that the pathogenic fungus was important for the penetration of the elicitors to inner plant tissues where phaseollin (and probably other phytoalexins) is produced.     

Fungal elicitor preparations and methyl jasmonate enhance rosmarinic acid accumulation in suspension cultures of Coleus blumei

bstract Suspension cultures of Coleus blumei (Lamiaceae) treated with either an elicitor preparation from the culture medium of the phytopathogenic oomycete Pythium aphanidermatum or with methyl jasmonate enhanced accumulation of rosmarinic acid approximately threefold. The specific activities of phenylalanine ammonia lyase and rosmarinic acid synthase were also enhanced after addition of the fungal elicitor. The addition of methyl jasmonate transiently increased activities of phenylalanine ammonia lyase and hydroxyphenylpyruvate reductase, whereas the activity of rosmarinic acid synthase was not stimulated and the activity of tyrosine aminotransferase was slightly and constantly enhanced. Methyl jasmonate stimulated rosmarinic acid accumulation not only when added directly to the culture medium, but also when it could reach the cells only via the gas phase. Received: 2 April 1997 / Revision received:16 June 1997 / Accepted: 15 September 1997     

Metabolism of lunularic acid in liverworts

The presence, in Conocephalum conicum, of the enzymes phenylalanine ammonia lyase, trans-cinnamic acid-4-hydroxylase and lunularic acid decarbo     

Influence du piclorame sur l'activité phénylalanine-ammoniac lyase de Nicotiana tabacum induite par la réaction d'hypersensibilité au virus de la mosaïque du tabac

In Nicotiana tabacum var. Xanthi n.c. phenylalanine ammonia lyase (PAL) activity, which increases significantly during the hypersensitivity reaction to tobacco mosaic virus (TMV), is only partially affected when plants are treated with piclorame, which is known to be an inhibitor of PAL in vivo. Using piclorame together with TMV, it has been possible to distinguish between that increase in PAL activity due to the virus and that dependent on the photoperiod.     

Induced defense-related proteins in soybean (Glycine max L. Merrill) plants by Carnobacterium sp. SJ-5 upon challenge inoculation of Fusarium oxysporum

The aim of the present study was to analyze induced expression of defense-related proteins in the soybean plants by rhizobacterial stain Carnobacterium sp. SJ-5 upon challenge inoculation with Fusarium oxysporum. Determination of the enzymatic activity of the different defense-related enzymes, phenylalanine ammonia lyase (PAL), lipoxygenase (LOX), peroxidase (POD) and polyphenol oxidase (PPO) was performed in the major parts of Glycine max L. Merrill using spectrophotometric method. Native-polyacrylamide gel electrophoresis analysis of the POD and PPO was employed followed by activity staining to find out the isoforms of respective enzymes. Activities of the PAL, LOX, POD and PPO were found to be highest in the bacterized root tissue of the soybean plants challenged with F. oxysporum. Isoform analysis revealed that PPO1, PPO4 and POD2 isoforms were expressed at higher levels in bacterized soybean root tissues challenge inoculated with the pathogen. Conclusively it was found that bacterial strain Carnobacterium sp. SJ-5 protect soybean plants from wilt disease caused by F. oxysporum by elicitation of the defense-related enzymes.     

Ethylene: indicator but not inducer of phytoalexin synthesis in soybean

Cell wall preparations (elicitors) from Phytophthora megasperma var. sojae increase C₂H₄ formation, phenylalanine ammonia lyase activity, and glyceollin accumulation in soybean cotyledons within about 1.5, 3, and 6 hours after treatment, respectively. The immediate precursor of C₂H₄, 1-aminocyclopropane-1-carboxylic acid, stimulates C₂H₄ formation like the elicitor within 1.5 hours after administration, whereas phenylalanine ammonia lyase activity and glyceollin concentration remain unchanged. Aminoethoxyvinylglycine, a specific inhibitor of C₂H₄ formation in higher plants, inhibits elicitor-induced C₂H₄ formation by about 95% but has no effects on phenylalanine ammonia lyase or glyceollin accumulation. It was concluded that C₂H₄ is a signal accompanying the specific recognition process which finally leads to the induction of phytoalexin formation, but it is not functioning as a link or messenger in the induction sequence of glyceollin accumulation.     

Concomitant Induction of Systemic Resistance to Pseudomonas syringae pv. lachrymans in Cucumber by Trichoderma asperellum (T-203) and Accumulation of Phytoalexins

Most studies on the reduction of disease incidence in soil treated with Trichoderma asperellum have focused on microbial interactions rather than on plant responses. This study presents conclusive evidence for the induction of a systemic response against angular leaf spot of cucumber (Pseudomonas syringae pv. lachrymans) following application of T. asperellum to the root system. To ascertain that T. asperellum was the only microorganism present in the root milieu, plants were grown in an aseptic hydroponic growth system. Disease symptoms were reduced by as much as 80%, corresponding to a reduction of 2 orders of magnitude in bacterial cell densities in leaves of plants pretreated with T. asperellum. As revealed by electron microscopy, bacterial cell proliferation in these plants was halted. The protection afforded by the biocontrol agent was associated with the accumulation of mRNA of two defense genes: the phenylpropanoid pathway gene encoding phenylalanine ammonia lyase (PAL) and the lipoxygenase pathway gene encoding hydroxyperoxide lyase (HPL). This was further supported by the accumulation of secondary metabolites of a phenolic nature that showed an increase of up to sixfold in inhibition capacity of bacterial growth in vitro. The bulk of the antimicrobial activity was found in the acid-hydrolyzed extract containing the phenolics in their aglycone form. High-performance liquid chromatography analysis of phenolic compounds showed a marked change in their profile in the challenged, preelicited plants relative to that in challenged controls. The results suggest that similar to beneficial rhizobacteria, T. asperellum may activate separate metabolic pathways in cucumber that are involved in plant signaling and biosynthesis, eventually leading to the systemic accumulation of phytoalexins.     

Isolation and Characterization of Cell Lines Resistant to Crude Culture Filtrate of Fusarium in Chickpea

Cell lines resistant to Fusarium oxysporum f. sp. Ciceri crude culture filtrate have been selected In Cicer arietinum cv C235. Phenol content and the activities of tyrosine ammonia lyase (TAL), phenylalanine ammonia lyase (PAL) and ß-1, 3-glucanase were Increased by the elicitor In callus cultures of Fusarium susceptible cultlvar C235, resistant cultlvar WR315 and a stable Fusarium resistant cell clone FC-3. The Increase In phenols and glucanase activity, was more In selected cell line than In ev C235 and ev WR315, while the increase In activities of TAL and PAL showed reverse correlation.     

Application of benzothiadiazole and Trichoderma harzianum to control faba bean chocolate spot disease and their effect on some physiological and biochemical traits

Chocolate spot disease is the most prevalent and important disease in the major faba bean growing regions in the world. Different concentrations of the abiotic inducer (0.3 and 0.5 mM benzothiadiazole) and the biotic inducer (1 × 10⁷ and 2 × 10⁷ spore/ml Trichoderma harzianum) were used alone or in combination to study their efficiency against faba bean chocolate spot disease caused by Botrytis fabae and Botrytis cinerea and their effect on some chemical analyses (phenylalanine ammonia lyase activity, total flavonoids and peroxidase isozymes, pectin and lignin content and total chlorophyll content). Application of the tested inducers as foliar treatment significantly reduced the severity of chocolate spot disease as compared with untreated infected plants. The reduction in disease severity was associated with a gradual increase in phenylalanine ammonia lyase activity. Maximum increase was recorded at 72 h after inoculation with B. fabae and B. cinerea. In addition, the levels of flavonoids in induced infected leaves recorded a sharp increase at 24 h after inoculation with B. fabae or B. cinerea. Also, pectin and lignin contents in the cell wall of induced infected plants were significantly increased as compared with untreated infected plants. Beside the induction of resistance, the tested inducers markedly increased total chlorophyll content in treated infected plants as compared with untreated infected plants. Isozymes analysis revealed that new peroxidase bands were induced only in treated faba bean leaves in response to infection with B. fabae or B. cinerea.     

Expression of phenolics and defence-related enzymes in relation to red root rot disease of tea plants

Red root rot caused by Poria hypolateritia is a dreadful disease in tea plant due to sudden death of bushes. In response to fungal pathogen, variation in the defence-related enzymes was investigated. The infected tea root was undertaken to study about various defence-related and pathogen-related enzymes. The infected root, as a prime response to disease attack, was subjected to the analysis of phenolics, phenylalanine ammonia lyase, tyrosine ammonia lyase, peroxidase, polyphenol oxidase, catalase, chitinase, β-1,3-glucanase and protease were assayed. The results on assay of defence-related enzymes revealed that the activity was significantly higher in infected roots when compared with healthy roots. Phenolics were accumulated more in infected roots. The sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis further confirmed the presence of induced pathogenesis-related proteins in the infected root tissues. The activity of all enzymes was increased up to threefold amount when compared with normal ones. The accumulation of defence enzymes in plants revealed the virulence of root pathogen in stimulating induced systemic resistance of tea plants and phytopathogenicity causing pathogenesis. This study exemplify to recognise underlying processes in causing infection and to identify the existence of host–pathogen relationship.     

Activity of enzymes involved in lignin biosynthesis in swede root disks

The activity of nine enzymes involved in the biosynthesis of lignin precursors has been studied during the ageing of swede root disks in the presence and absence of ethylene. Peroxidase, aromatic alcohol dehydrogenase and phenylalanine transaminase show very little change in activity during ageing under either ageing condition. O-methyl transferase, shikimate dehydrogenase and ferulyl CoA reductase show only a 2–3 fold increase on ageing and are relatively insensitive to ethylene treatment. A third group (comprising phenylalanine ammonia lyase, cinnamic acid-4-hydroxylase and hydroxycinnamate CoA ligase) show 20–30 fold increase on ageing and are most sensitive to ethylene treatment. Phenylalanine ammonia lyase and cinnamic acid-4-hydroxylase behave very similarly in respect of their time course of ageing and in their responses to metabolic inhibitors such as cycloheximide, puromycin and actinomycin D. In addition the properties of the O-methyl transferase of swede root tissue are described.     

Induction of defense responses in cultured parsley cells by plant cell wall fragments

Cell suspension cultures of parsley (Petroselinum crispum) accumulated coumarin phytoalexins and exhibited increased β-1,3-glucanase activity when treated with either a purified α-1,4-d-endopolygalacturonic acid lyase from Erwinia carotovora or oligogalacturonides solubilized from parsley cell walls by endopolygalacturonic acid lyase. Coumarin accumulation induced by the plant cell wall elicitor was preceded by increases in the activities of phenylalanine ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL) and S-adenosyl-l-methionine:xanthotoxol O-methyltransferase (XMT). The time courses for the changes in these three enzyme activities were similar to those observed in cell cultures treated with a fungal glucan elicitor. The plant cell wall elicitor was found to act synergistically with the fungal glucan elicitor in the induction of coumarin phytoalexins. As much as a 10-fold stimulation in coumarin accumulation above the calculated additive response was observed in cell cultures treated with combinations of plant and fungal elicitors. The synergistic effect was also observed for the induction of PAL, 4CL, and XMT activities. These results demonstrate that plant cell wall elicitors induce at least two distinct biochemical responses in parsley cells and further support the role of oligogalacturonides as important regulators of plant defense.