Specific inhibition of lignification breaks hypersensitive resistance of wheat to stem rust

When highly resistant wheat (Triticum aestivum L.) varieties are infected by an avirulent race of the stem rust fungus (Puccinia graminis Pers. f. sp. tritici Erics. and E. Henn.), penetrated host cells undergo rapid necrotization. This hypersensitive cell death is correlated with cellular lignification which efficiently restricts further fungal growth. Three competitive inhibitors of phenylalanine ammonia-lyase, the first enzyme of the general phenylpropanoid pathway and, thus, of lignin biosynthesis, namely α-aminooxyacetate, α-aminooxy-β-phenylpropionic acid, and (1-amino-2-phenylethyl)phosphonic acid, and two highly specific irreversible suicide inhibitors of the lignification-specific enzyme cinnamyl-alcohol dehydrogenase, namely N(O-aminophenyl)sulfinamoyl-tertiobutyl acetate and N(O-hydroxyphenyl)sulfinamoyl-tertiobutyl acetate, were applied to genetically resistant wheat plants prior to inoculation with stem rust. Treatment with any of these inhibitors decreased the frequency of lignified necrotic host cells and concomitantly led to increased fungal growth. The cinnamyl-alcohol dehydrogenase inhibitors were generally more effective than the phenylalanine ammonia-lyase inhibitors, occasionally allowing some sporulation to occur on the resistant wheat leaves. These results clearly point to a causal relationship between the formation of lignin precursors and the resistance of wheat to stem rust.     

Phenolics and the activities of phenylalanine ammonia-lyase, phenol-β-glucosyltransferase and β-glucosidase in cucumber roots as affected by phenolic allelochemicals

Seven-day-old seedlings of cucumber (Cucumis sativus L.) cv. Wisconsin were treated with 0.1 mM solutions of cinnamic acid (ferulic and p-coumaric acids) and benzoic acid (p-hydroxybenzoic and vanillic acids) derivatives as stressors. The content of free and glucosylated soluble phenols and the activity of phenylalanine ammonia-lyase (E.C.4.3.1.5), phenol-β-glucosyltransferase (E.C.2.4.1.35.), and β-glucosidase (E.C.3.2.1.21.) in seedling roots as well as their length and fresh weight were examined. Changes in glucosylated phenolic content and phenol-β-glucosyltranspherase activity were observed under the influence of all phenolics applied. Treatment with ferulic and p-coumaric acids stimulated the increase of phenylalanine ammonia-lyase and β-glucosidase activity and slightly inhibited cucumber root growth.     

The estimation of phenylalanine ammonia-lyase (PAL) activity in intact cells of higher plant tissue

A previously described procedure for the estimation of relative activities of phenylalanine ammonia-lyase (EC 4.3.1.5) in intact plant cells (Amrhein et al. (1976) Planta 131, 33–40) was reexamined for its specificity and its applicability to various tissues. In buckwheat hypocotyl segments ³H is stereospecifically released from the pro-3S-position of L-[2,3-³H]phenylalanine and is thus due to phenylalanine ammonia-lyase activity. In buck wheat and sunflower leaf disks, however, ³H release occurs from both the 2- and 3-positions of the labeled substrate and can only partially be attributed to phenylalanine ammonia-lyase activity.Abbreviations AOA -aminooxyacetic acid - L-AOD L-aminoacid oxidase (EC 1.4.3.2) - D-AOD D-amino-acid oxidase (EC 1.4.3.3) - L-AOPP L--aminooxy--phenylpropionic acid - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - TAL tyrosine ammonia-lyase     

Differential expression of phenylalanine ammonia-lyase genes in barley induced by fungal infection or elicitors

Gene-specific probes were used to assess the expression patterns of four different phenylalanine ammonia-lyase ( pal ) genes in infected or elicitor-treated leaves and suspension-cultured cells of barley. Genes corresponding to hpal2 , hpal3 , hpal4 , and hpal6 were all induced by mercuric chloride and fungal infection by Bipolaris sorokiniana Sacc. (Shoem.) in barley ( Hordeum vulgare L. cv. Pokko) leaves, but with considerable variation in their expression level and timing. The expression patterns of hpal2 and hpal6 were similar, both showing a rapid, strong induction after treatment with mercuric chloride and a slower induction after fungal inoculation, whereas the more divergent hpal3 was induced at a later time and at a lower level after both treatments. Hpal4 was expressed with timing like that of hpal2 and hpal6 in infected or mercuric chloride-treated leaves but its expression was much weaker. Hpal2 and hpal4 were induced in elicitor-treated, suspension-cultured barley cells, whereas the expression of hpal3 was nearly undetectable, and hpal6 was strongly and constitutively present. All pal genes except hpal4 were developmentally regulated, but differentially expressed in various barley tissues. The results suggest that the four pal genes studied here might be responsible for the activation of different branches in the phenylpropanoid biosynthesis of barley.     

Polyamine Sparing May Be Involved in the Prolongation of Cell Division Due to Inhibition of Phenylpropanoid Synthesis in Cytokinin-Starved Soybean Cells

Effects on growth, mostly of an inhibitory nature, have been attributed to phenolic compounds in vivo and in vitro. This suggests that l-α-aminooxy-β-phenylpropionic acid (l-AOPP), a competitive inhibitor of phenylalanine ammonia-lyase (PAL), the enzyme controlling the first step in phenylpropanoid synthesis, might stimulate growth in soybean suspension cultures (Glycine max, cv. Acme). The promotive effect of l-AOPP, measured as an increase in cell number, was more clearly detected in the growth-limiting condition of cytokinin starvation. At least one more cell division cycle was completed in the presence of l-AOPP before growth by division ceased and growth continued by expansion only. Phenolic acids are known to conjugate with polyamines, modulating the free levels of these plant growth substances. Thus, the effect of l-AOPP on the titers of free and conjugated polyamines (putrescine, spermidine, and spermine) was investigated by high performance liquid chromatography in the course of cytokinin starvation. An increased level of free putrescine was detected in the presence of l-AOPP relative to controls, especially in the initial period before growth became restricted to cell expansion. The decrease in free putrescine associated with the cessation of cell division was temporarily delayed, suggesting that an interaction between phenolic acids and polyamines is involved in the mechanism of growth promotion by l-AOPP. Received July 30, 1996; accepted January 28, 1997     

Induction of de novo synthesis of phenylalanine ammonia-lyase by L-α-aminooxy-β-phenylpropionic acid in suspension cultures ofDaucus carota L.

Application of L--aminooxy--phenylpropionic acid (L-AOPP), a potent and specific competitive inhibitor of L-phenylalanine ammonia-lyase (PAL), to an anthocyanin-producing cell suspension culture ofDaucus carota results in a dramatic increase in extractable PAL activity and an accumulation of phenylalanine (Noé et al., 1980, Planta149, 283–287). Using an immunoprecipitation technique, evidence was obtained that the increase in PAL activity the result of de-novo synthesis. The activity of the other enzymes of the general phenylpropanoid metabolism, e.g., trans-cinnamate 4-hydroxylase and hydroxycinnamate: CoA ligase, were not affected by L-AOPP. This result strongly supports the view that PAL is regulated independently.Abbreviations CAH trans-cinnamate 4-hydroxylase - L-AOPP L--aminooxy--phenylpropionic acid - PAL L-phenylalanine ammonia-lyase     

Metabolism of p-fluorophenylalanine in p-fluorophenylalanine sensitive and resistant tobacco cell cultures

The metabolism of D- and L-p-fluorophenylalanine (PFP) in DL-PFP resistant and sensitive tobacco cell cultures (Nicotiana tabacum), cell lines TX4 and TX1, respectively, has been compared. The amino acid analogue was taken up at a lower rate by the resistant cell line TX4. Incorporation of PFP into protein was also considerably reduced in TX4 cells, compared to TX1 cells. This, however, resulted mainly from a diminished availability of PFP due to a more rapid conversion of PFP by TX4 cells. TX1 cells and TX4 cells converted PFP qualitatively in the same way. The only detectable metabolite of D-PFP was N-malonyl-D-PFP, while all metabolites of L-PFP were identified as sequent products of the initial deamination of L-PFP by the enzyme phenylalanine ammonia-lyase (PAL). As TX4 cells were endowed with higher PAL-activity than TX1 cells, the resistant cells were able to metabolize L-PFP more rapidly to give, e.g., p-fluorocinnamoyl glucose ester and p-fluorocinnamoyl putrescine. In the presence of the specific PAL-inhibitor -aminooxy--phenylpropionic acid TX4 cells were slightly more sensitive to PFP. This suggests that the better detoxification contributes to the acquired resistance. The use of PFP as specific indicator for cell lines with increased PAL-activity, and hence increased levels of phenolic compounds, is discussed.Abbreviations AOPP -aminooxy--phenylpropionic acid - MCW methanol:chloroform:water - PAL phenylalanine ammonia-lyase - PFP p-fluorophenylalanine - Phe phenylalanine     

Rapid Response of Suspension-cultured Parsley Cells to the Elicitor from Phytophthora megasperma var. sojae: INDUCTION OF THE ENZYMES OF GENERAL PHENYLPROPANOID METABOLISM

Large and rapid increases in the activities of two enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase and 4-coumarate:CoA ligase, occurred in suspension-cultured parsley cells (Petroselinum hortense) treated with an elicitor preparation from Phytophthora megasperma var. sojae. Highest enzyme activities were obtained with an elicitor concentration similar to that required for maximal phenylalanine ammonialyase induction in cell suspension cultures of soybean, a natural host of the fungal pathogen.     

The influence of lignification on the development of vascular tissue inVigna radiata L.

Summary During the early development of mungbean seedlings, treatment with L--aminooxy--phenylpropionic acid (AOPP), a potent specific inhibitor of phenylalanine ammonia-lyase, results in an inhibition of anthocyanin and lignin synthesis. The xylem vessels of the hypocotyl and root of AOPP treated seedlings collapse, and the cellulose microfibrils of the unlignified secondary wall are separated from one another and lie disorganized in the lumen of the mature xylem cell. The differentiation of the secondary cell wall appears unaffected by AOPP treatment, as does the ultrastructure of the wall of the mature phloem fibers of the root which is also lignified in untreated tissue. The results are discussed in the light of current thinking on the role and development of lignification in the xylem vessel.Abbreviation AOPP L-aminooxy--phenylpropionic acid     

Modulation of L-phenylalanine ammonia-lyase by pathway intermediates in cell suspension cultures of dwarf French bean (Phaseolus vulgaris L.)

The increase in extractable phenylalanine ammonia-lyase (PAL;EC 4.3.1.5.) activity induced in French bean cell suspension cultures in response to treatment with autoclaved ribonuclease A was inhibited by addition of the phenylpropanoid pathway intermediates cinnamic acid, 4-coumaric acid or ferulic acid. The effectiveness of inhibition was in the order cinnamic acid>4-coumaric acid>ferulic acid. Cinnamic acid also inhibited the PAL activity increase induced by dilution of the suspensions into an excess of fresh culture medium. Addition of low concentrations (<10^-5M) of the pathway intermediates to cultures at the time of application of ribonuclease gave variable responses ranging from inhibition to 30–40% stimulation of the PAL activity measured at 8 h. Following addition of pathway intermediates to cultures 4–5 h after ribonuclease treatment, rapid increases followed by equally rapid declines in PAL activity were observed. The cinnamic acid-stimulated increase in enzyme activity was unaffected by treatment with cycloheximide at a concentration which gave complete inhibition of the ribonuclease-induced response. However, cycloheximide completely abolished the subsequent decline in enzyme activity. Treatment of induced cultures with -aminooxy--phenylpropionic acid (AOPPA) resulted in increased but delayed rates of enzyme appearance when compared to controls not treated with the phenylalanine analogue. The results are discussed in relation to current views on the regulation of enzyme levels in higher plants.Abbreviations AOPPA -aminooxy--phenylpropionic acid - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5) - AOA -aminooxyacetic acid     

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

Tomato (Lycopersicon esculentum Mill.) cell suspension cultures accumulated wall-bound phenolic materials in response to inoculation with Verticillium 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 (K_m = 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.     

Interactions between parallel pathways during biosynthesis of rosmarinic acid in cell suspension cultures of Coleus blumei

Phenylalanine ammonia-lyase from an over-producer strain of Coleus blumei Benth. cell cultures accumulating high levels of rosmarinic acid (RA) has been shown to possess no special feed-back sensitivity to RA or its precursors. No tyrosine-3-hydroxylase activity could be detected in culture extracts and no specific inhibitors of tyrosine incorporation into RA were found. L--aminooxy--phenyl propionic acid, however, was effective in specifically blocking phenylalanine incorporation. This block also led to an accumulation of label from tyrosine in 4-hydroxyphenyllactic acid rather than in 3,4-dihydroxyphenylalanine (DOPA) or 3,4-dihydroxyphenyllactic acid. These observations require a re-evaluation of the possible role of DOPA as a major biogenic precursor to RA.Abbreviations AOPP -aminooxy--phenylpropionic acid - DOPA 3,4-dihydroxyphenylalanine - RA rosmarinic acid (-O-caffeoyl-3,4-dihydroxyphenyllactic acid) - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5)     

Stress Responses in Alfalfa (Medicago sativa L.) : V. Constitutive and Elicitor-Induced Accumulation of Isoflavonoid Conjugates in Cell Suspension Cultures

The isoflavonoid conjugates medicarpin-3-O-glucoside-6″-O-malonate (MGM), afrormosin-7-O-glucoside (AG), and afrormosin-7-O-glucoside-6″-O-malonate (AGM) were isolated and characterized from cell suspension cultures of alfalfa (Medicago sativa L.), where they were the major constitutive secondary metabolites. They were also found in alfalfa roots but not in other parts of the plant. The phytoalexin medicarpin accumulated rapidly in suspension cultured cells treated with elicitor from Colletotrichum lindemuthianum, and this was subsequently accompanied by an increase in the levels of MGM. In contrast, net accumulation of afrormosin conjugates was not affected by elicitor treatment. Labeling studies with [¹⁴C]phenylalanine indicated that afrormosin conjugates were the major de novo synthesized isoflavonoid products in unelicited cells. During elicitation, [¹⁴C]phenylalanine was incorporated predominantly into medicarpin, although a significant proportion of the newly synthesized medicarpin was also conjugated. Treatment of ¹⁴C-labeled, elicited cells with l-α-aminooxy-β-phenylpropionic acid, a potent inhibitor of PAL activity in vivo, resulted in the initial appearance of labeled medicarpin of very low specific activity, suggesting that the phytoalexin could be released from a preformed conjugate under these conditions. Our data draw attention to the involvement of isoflavone hydroxylases during the constitutive and elicitor-induced accumulation of isoflavonoids and their conjugates in alfalfa cell cultures.     

Phenylalanine Ammonia-lyase Activity in Barley After Infection with Bipolaris sorokiniana or Treatment with its Purified Xylanase

Phenylalanine ammonia-lyase (PAL) activity was determined from leaves and roots of two barley (Hordeum vulgare L.) cultivars after infection with a necrotrophic pathogen, Bipolaris sorokiniana (Sacc.) Shoem., and treatment with its purified xylanase. PAL activity increased in leaves of both cultivars 16 h after fungal inoculation but two phases, with activity peaks at 24–32 h and 40 h, were recorded only for the more resistant cultivar, Agneta. Attempts to use a PAL inhibitor, χ-amin, ooxyacetic acid, to increase susceptibility to B. sorokiniana in barley leaves were unsuccessful. Treatments of leaves with purified xylanase resulted in more rapid (4–12 h after injection), although reduced, induction of PAL compared with fungal injection. The higher the concentration of xylanase applied the earlier the activity peaks were detected. Fungal inoculation only slightly increased PAL activity in barley roots while xylanase treatment had no effect. The basal level of PAL was however much higher in roots than in leaves. In wheat, Triticum aestivum L. resistant to B. sorokiniana, the time-course of PAL induction after fungal infection and xylanase treatment resembled that for cv. Agneta, while in oats, Avena sativa L. (non-host) PAL activity did not change after the treatments. The results suggest that the second phase of PAL induction, associated only with responses of barley cv. Agneta and wheat, is linked with their resistance to B. sorokiniana infection. The possible role of xylanase as an elicitor of PAL is discussed.     

Phytoalexin synthesis in soybean cells: elicitor induction of phenylalanine ammonia-lyase and chalcone synthase mRNAs and correlation with phytoalexin accumulation

A glucan elicitor from cell walls of the fungus Phytophthora megasperma f. sp. glycinea, a pathogen of soybean (Glycine max), induced large and rapid increases in the activities of enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase, and of the flavonoid pathway, acetyl-CoA carboxylase and chalcone synthase, in suspension-cultured soybean cells. The changes in phenylalanine ammonia-lyase and chalcone synthase activities were correlated with corresponding changes in the mRNA activities encoding these enzymes, as determined by enzyme synthesis in vitro in a mRNA-dependent reticulocyte lysate. The time courses of the elicitor-induced changes in mRNA activities for both enzymes were very similar with respect to each other. Following the onset of induction, the two mRNA activities increased significantly at 3 h, reached highest levels at 5 to 7 h, and subsequently returned to low values at 10 h. Similar degrees of induction of mRNA activities and of the catalytic activities of phenylalanine ammonia-lyase and chalcone synthase were observed in response to three diverse microbial compounds, the glucan elicitor from P. megasperma, xanthan, an extracellular polysaccharide from Xanthomonas campestris, and endopolygalacturonase from Aspergillus niger. However, whereas the glucan elicitor induced the accumulation of large amounts of the phytoalexin, glyceollin, in soybean cells, endopolygalacturonase induced only low, albeit significant, amounts; xanthan did not enhance glyceollin accumulation under the conditions of this study. This result might imply that enzymes other than phenylalanine ammonia-lyase or chalcone synthase exert an important regulatory function in phytoalexin synthesis in soybean cells.     

Activities of enzymes of polyphenol metabolism inPhaseolus aureus seedlings germinated in the presence of 2-Chloroethylphosphonic acid

The plant growth regulator 2-ohloroethylphosphonic acid inhibited the elongation of growth inPhaseolus aureus seedlings. In comparison to the control, the polyphenol oxidase and peroxidase activity of treated seedlings was low up to 24 and 48 h of germination, respectively and that of phenylalanine ammonia-lyase and tyrosine ammonia-lyase was slightly less at 120 h and that of α- and β-glucosidases were less at 48 and 72 h, respectively. At other stages of germination, it greatly stimulated the activities of these enzymes. Part of Ph. D. dissertation submitted by Y. K. Arora to Punjab Agricultural University, Ludhiana, India     

Comparison of secondary product accumulation in photoautotrophic,photomixotrophic and heterotrophic Nicotiana tabacum cell suspension cultures

Summary Photoautotrophic, photomixotrophic and heterotrophic Nicotiana tabacum cell suspension cultures were compared for the constitutive accumulation of secondary metabolites and the elicitor-induced formation of the phytoalexin capsidiol. Nicotine and chlorogenic acid were found in high amounts in the heterotrophic cultures and in moderate concentrations in photomixotrophic but not in photoautotrophic cells. Nicotinic acid-N-glucoside occured in all culture types; in photoautotrophic and photomixotrophic cells the formation of N-methylnicotinic acid (trigonelline) was also observed. Treatment with a fungal elicitor led to substantial accumulation of capsidiol in heterotrophic and photomixotrophic cells and in only low levels in photoautotrophic cultures. Elicitor-treated photomixotrophic cells showed a pronounced increase in cell wall-bound phenolics. The levels of nicotine, nicotinic acid-N-glucoside and trigonelline were not affected by elicitation.Abbreviations hcc heterotrophic cell culture - mcc photomixotrophic cell culture - pcc photoautotrophic cell culture - fr.wt. freshweight - nic-N-glc nicotinic acid-N-glucoside - PMG Phytophthora megasperma f. sp. glycínea - HPLC high performance liquid chromatography - GC gas chromatography - TLC thin layer chromatography - 2,4D 2,4-dichlorophenoxyacetic acid - Kin kinetin - BAP 6-benzylaminopurine - NAA -naphthylacetic acid     

Effects of competitive inhibitors of phenylalanine ammonia-lyase on the levels of phenylpropanoid enzymes in Solatium tuberosum

Abstract The accumulation of chlorogenic acid in illuminated discs of Solanum tuberosum tuber tissue is accompanied by rapid but transient increases in the activity levels of the biosynthetic enzymes phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase and hydroxycinnamoyl-CoA : quinate hydroxycinna-moyl transferase. Exogenous D-phenylalanine and L-α-aminooxy-β-phenylpropionic acid, competitive inhibitors of phenylalanine ammonia-lyase, inhibit the accumulation of chlorogenic acid and presumably reduce the endogenous pools of pathway intermediates such as cinnamic acid. These treatments prolong the phase of increase in phenylalanine ammonia-lyase and cinnamic acid 4-hydroxylase activities and indicate that product feedback modulation is important in maintaining the interrelationship between the levels of these two enzymes during the later stages of induction. In contrast,L-α-aminooxy-β-phenylpropionic acid inhibits the development of hydroxycinnamoyl transferase in illuminated discs supporting the idea that the light-stimulated increase in phenylalanine ammonia-lyase activity causes an increase in cinnamic acid production which mediates the light-stimulated increase in hydroxycinnamoyl transferase activity.     

Elicitor-stimulated biosynthesis of hydroxycinnamoyltyramines in cell suspension cultures of Solanum tuberosum

Treatment of suspension-cultured potato cells (Solanum tuberosum L. cv. Desirée) with an elicitor from Phytophthora infestans induced increased incorporation of 4-hydroxybenzaldehyde, 4-hydroxybenzoate, and N-4-coumaroyl- and N-feruloyltyramine into the cell␣wall and secretion of N-4-coumaroyl- and N-feruloyltyramine into the culture medium. Induced metabolite accumulation was preceded by rapid and transient increases in activities of phenylalanine ammonia-lyase (EC 4.3.1.5) and tyrosine decarboxylase (TyrDC; EC 4.1.1.25), exhibiting maximal activities 5–10 h after initiation of elicitor treatment. Activities of hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase (EC 2.3.1.110), catalyzing the formation of N-4-coumaroyl- and N-feruloyltyramine, increased later and remained at high levels. The phenolic defense compounds appear to be involved in cell wall reinforcement and may further directly affect fungal growth in the apoplastic space. Received: 26 July 1997 / Accepted: 9 September 1997