Induction of biosynthetic enzymes for avenanthramides in elicitor-treated oat leaves

The accumulation of oat (Avena sativa L.) phytoalexins, avenanthramides, occurred in leaf segments treated with oligo-N-acetylchitooligosaccharides. The amount of avenanthramide A, the major oat phytoalexin, reached a maximum 36–48 h after elicitor treatment. This accumulation was preceded by a marked increase in enzyme activities of phenylpropanoid pathway members, including phenylalanine ammonia-lyase (EC 4.3.1.5), cinnamate 4-hydroxylase (EC 1.14.13.11) and 4-coumarate:CoA ligase (EC 6.2.1.12). These enzyme activities reached a maximum 6–12 h after elicitor treatment, when the avenanthramides were produced most rapidly. Both phenylalanine ammonia-lyase and 4-coumarate:CoA ligase activities decreased thereafter to undetectable levels 72 h after treatment, while cinnamate 4-hydroxylase activity showed a second increase 48 h after treatment. Among the chitooligosaccharides tested, tetra- and pentasaccharides most effectively induced these enzyme activities in a dose-dependent manner. The elicitor-induced 4-coumarate: CoA ligase accepted all hydroxycinnamic acids occurring in the avenanthramides as substrates, with the exception of avenalumic acid. These findings indicate that accumulation of the avenanthramides results from de-novo synthesis through the general phenylpropanoid pathway and that early biosynthetic enzymes function as regulatory points of carbon flow to the avenanthramides. Received: 3 December 1998 / Accepted: 27 January 1999     

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.     

Benzoic acid biosynthesis in cell cultures of<Emphasis Type="Italic"> Hypericum androsaemum</Emphasis>

Biosynthesis of benzoic acid from cinnamic acid has been studied in cell cultures of Hypericum androsaemum L. The mechanism underlying side-chain shortening is CoA-dependent and non-beta-oxidative. The enzymes involved are cinnamate:CoA ligase, cinnamoyl-CoA hydratase/lyase and benzaldehyde dehydrogenase. Cinnamate:CoA ligase was separated from benzoate:CoA ligase and 4-coumarate:CoA ligase, which belong to xanthone biosynthesis and general phenylpropanoid metabolism, respectively. Cinnamoyl-CoA hydratase/lyase catalyzes hydration and cleavage of cinnamoyl-CoA to benzaldehyde and acetyl-CoA. Benzaldehyde dehydrogenase finally supplies benzoic acid. In cell cultures of H. androsaemum, benzoic acid is a precursor of xanthones, which accumulate during cell culture growth and after methyl jasmonate treatment. Both the constitutive and the induced accumulations of xanthones were preceded by increases in the activities of all benzoic acid biosynthetic enzymes. Similar changes in activity were observed for phenylalanine ammonia-lyase and the xanthone biosynthetic enzymes benzoate:CoA ligase and benzophenone synthase.     

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.     

Salicylic acid has a dual role in the activation of defence-related genes in parsley

Systemic acquired resistance is an inducible plant defence state, the activation of which depends mostly on the accumulation of salicylic acid (SA). During the past several years, it has been demonstrated that pretreatment of cultured parsley cells with SA potentiates the elicitation of several defence responses that are local in whole plants, including the accumulation of phenylpropanoid products. Here it is reported that while anionic peroxidase and mannitol dehydrogenase encoding genes are directly responsive to SA, pretreating parsley cells with SA not only enhances elicitation of the phenylpropanoid genes phenylalanine ammonia-lyase and 4-coumarate:CoA ligase but also of genes for PR-10 and a hydroxyproline-rich glycoprotein. Enhanced induction of these genes was seen at low levels of endogenous free SA. Enhancement of phenylalanine ammonia-lyase gene activation was proportional to the length of SA pretreatment. Furthermore, the ability of SA analogues to both potentiate elicited and directly induce defence gene activation correlated with their biological activity to promote plant disease resistance. In summary, these results emphasize that SA has at least a dual role in plant defence gene activation.     

Differential responses of five cherry tomato varieties to water stress: changes on phenolic metabolites and related enzymes

Different tomato cultivars (Solanum lycopersicum L.) with differences in tolerance to drought were subjected to moderate water stress to test the effects on flavonoids and caffeoyl derivatives and related enzymes. Our results indicate that water stress resulted in decreased shikimate pathway (DAHP synthase, shikimate dehydrogenase, phenylalanine ammonium lyase, cinnamate 4-hydroxylase, 4-coumarate CoA ligase) and phenolic compounds (caffeoylquinic acid derivatives, quercetin and kaempferol) in the cultivars more sensitive to water stress. However, cv. Zarina is more tolerant, and registered a rise in querc-3-rut-pent, kaempferol-3-api-rut, and kaempferol-3-rut under the treatment of water stress. Moreover, this cultivar show increased activities of flavonoid and phenylpropanoid synthesis and decreased in degradation-related enzymes. These results show that moderate water stress can induce shikimate pathway in tolerant cultivar.     

Mapping candidate genes in Eucalyptus with emphasis on lignification genes

We used the single-strand conformation polymorphism (SSCP) technique to map eight genes on Eucalyptus urophylla and Eucalyptus grandis linkage maps. These included four genes involved in the common phenylpropanoid pathway (caffeic acid 3-0-methyltransferase, caffeoyl CoA 3-O-methyltransferase, 4-coumarate CoA ligase and phenylalanine ammonia-lyase), two genes involved in the `lignin specific' pathway (cinnamoyl CoA reductase and cinnamyl alcohol dehydrogenase), and two symbiosis regulated genes (EgHypar and EgTubA1). A novel source of variation which affects the SSCP pattern, i.e. the presence or absence of electrophoresis buffer upon loading the samples into the polyacrylamide gel, was found. The placement of these genes on the Eucalyptus maps was carried out using an interspecific hybrid mapping population. This will further facilitate the identification or exclusion of `positional' candidate genes for characterizing quantitative trait loci (QTL) for wood quality and vegetative propagation related traits.     

Coordinated changes in enzyme activities of phenylpropanoid metabolism during the growth of soybean cell suspension cultures

Variations in teh activities of several enzymes of phenylpropanoid metabolism were studied in fermenter-grown cell suspension cultures of soyben (Glycine max).Concomitant large increases and subsequent decreases in the activities of phenylalanine ammonina-lyase (EC 4.3.1.5), cinnamic acid 4-hydroxylase, and two isoenzymes of p-coumarate:CoA ligase occurred prior to the stationary phase of the cell cultures. These findings represent a further example of an interdependent regulation of these enzymes of the general phenylpropanoid metabolism.The increases in all of these enzyme activities could be further enhanced by illunination of the cells.No comparable light effects and no significant changes were observed for the specific activity of an S-adenosylmethionine:o-dihydric phenol m-O-mehyltransferase and for the overall rate of the two-step reduction of feruloyl-CoA to coniferyl alcohol. These enzymatic reactions therefore appear to be regulated independently of the enzymes of the general phenylpropanoid metabolism.     

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.     

Induction of enzymes of phytoalexin synthesis in cultured soybean cells by an elicitor from Phytophthora megasperma f. sp. glycinea

The glucan elicitor from cell walls of the fungal pathogen, Phytophthora megasperma f. sp. glycinea, induced rapid but transient increases in enzyme activities of general phenylpropanoid metabolism (phenylalanine ammonia-lyase and 4-coumarate: CoA ligase) and of the flavonoid pathway (chalcone synthase) in cell suspension cultures of soybean (Glycine max). After transferring cells into fresh medium, two peaks of inducibility for the enzymes by elicitor were observed, one shortly after transfer (stage I), and one at the end of the linear growth phase (stage II). Only one of the two isoenzymes of 4-coumarate: CoA ligase (isoenzyme 2), for which a specific involvement in flavonoid biosynthesis has been postulated, was affected by the elicitor. For two of the induced enzymes, phenylalanine ammonia-lyase and chalcone synthase, the changes in activity at stage I were shown to be preceded by large changes in their rates of synthesis, as determined by in vivo labelling with [³⁵S] methionine and immunoprecipitation.Abbreviations Pmg Phytophthora megasperma f. sp. glycinea - glyceollin is a term used to designate the 3 isomers which accumulate in challenged soybean tissue (Moesta and Grisebach 1981b)     

Responses of cultured parsley cells to elicitors from phytopathogenic fungi : timing and dose dependency of elicitor-induced reactions

Cultured parsley cells (Petroselinum crispum) responded to treatment with heat-released soluble cell-wall fragments (elicitors) from several different phytopathogenic fungi by forming coumarin derivatives (phytoalexins). This response was preceded in all cases by large but transient increases in the activities of two enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL). The activities of two hydrolytic enzymes, chitinase and 1,3-β-glucanase, also increased strongly in elicitor-treated cells, whereas the activities of three enzymes participating in primary metabolism were affected differently by the elicitor treatment. Glucose-6-phosphate dehydrogenase increased, phosphofructokinase remained almost constant, and pyrophosphate:fructose-6-phosphate phosphotransferase declined sharply in activity. Different amounts of cell-wall preparations from various phytopathogenic fungi were required for maximum elicitor activity. While three oomycetes (Phytophthora spp.) yielded the most active elicitors studied (maximum coumarin accumulation at concentrations of about 10 microgram per milliliter), cell-wall preparations from an ascomycete and three deuteromycetes gave comparable results only at 10 to 100 times higher concentrations. Optimal induction of PAL, 4CL, and chitinase with Phytophthora elicitor required only about 1 microgram per milliliter, whereas 1,3-β-glucanase induction showed a dose dependence similar to that observed for coumarins. The elicitor concentration had pronounced effects not only on the extent, but also on the timing of all induced reactions.     

Enzyme Chnages During Lignogenesis in Pea Shoots Induced by Illumination

When the epicotyls of etiolated 7 d pea seedlings were illuminated,there was a rapid rise in the lignin content of the apical shoot(consisting of the third internode with its terminal bud), togive a 10-fold increase after 24 h; there was no change in theunilluminated controls. About 80% of the lignin in the apicalshoot was found in the stem internode, both before and afterillumination. Over this period, the activities of phenylalanineammonia-lyase, cinnamate 4-hydroxylase, and 4-coumarate: CoAligase increased co-ordinately up to five times those of thedark controls over 12 h, when lignification was most rapid,and then declined to about half their maximum activity. Allthree enzymes showed the same 1.5 h lag period before increasing.By comparison, no increases were observed in the later enzymesof lignin biosynthesis, namely S-adenosylmethionine: caffeateO-methyltransferase, cinnamoyl-CoA reductase, NADP+-cinnamylalcohol dehydrogenase, and cell-wall peroxidase, but the proportionsof these enzymes in buds and stem internodes were close to thedistribution of lignin between them, both before and after illumination.Two forms of 4-coumarate: CoA ligase were found; despite theirdifferent activities and substrate specificities, both formsshowed substantial changes. The results suggest that lignogenesisis initiated by an increase in the activities of the three enzymesof general phenylpropanoid metabolism in cells already containingenzymes catalysing the later stages of lignin synthesis; theyare discussed in relation to biochemical and anatomical differentiationwithin plant organs generally. Key words: Lignification, pea shoots, phenylalanine ammonia-lyase, phenylpropanoid enzymes, Pisum sativum     

Tissue-distribution of secondary phenolic biosynthesis in developing primary leaves of Avena sativa L.

Primary leaves of oats (Avena sativa L.) have been used to study the integration of secondary phenolic metabolism into organ differentiation and development. In particular, the tissue-specific distribution of products and enzymes involved in their biosynthesis has been investigated. C-Glucosylflavones along with minor amounts of hydroxycinnamic-acid esters constitute the soluble phenolic compounds in these leaves. In addition, considerable amounts of insoluble products such as lignin and wall-bound ferulic-acid esters are formed. The tissue-specific activities of seven enzymes were determined in different stages of leaf growth. The rate-limiting enzyme of flavonoid biosynthesis in this system, chalcone synthase, together with chalcone isomerase (EC 5.5.1.6) and the terminal enzymes of the vitexin and isovitexin branches of the pathway (a flavonoid O-methyltransferase and an isovitexin arabinosyltransferase) are located in the leaf mesophyll. Since the flavonoids accumulate predominantly (up to 70%) in both epidermal layers, an intercellular transport of products is postulated. In contrast to the flavonoid enzymes, L-phenylalanine ammonia-lyase (EC 4.3.1.5), 4-coumarate: CoA ligase (EC 6.2.1.12), and S-adenosyl-L-methionine: caffeate 3-O-methyltransferase (EC 2.1.1.-), all involved in general phenylpropanoid metabolism, showed highest activities in the basal leaf region as well as in the epidermis and the vascular bundles. We suggest that these latter enzymes participate mainly in the biosynthesis of non-flavonoid phenolic products, such as lignin in the xylem tissue and wall-bound hydroxycinnamic acid-esters in epidermal, phloem, and sclerenchyma tissues.Abbreviations CHI chalcone isomerase - CHS chalcone synthase - 4CL 4-coumarate: CoA ligase - CMT S-adenosyl-L-methionine:caffeate 3-O-methyltransferase - FMT S-adenosyl-L-methionine:vitexin 2-O-rhamnoside 7-O-methyltransferase - HPLC high-performance liquid chromatography - IAT uridine 5-diphosphate L-arabinose:isovitexin 2-O-arabinosyltransferase - PAL L-phenylalanine ammonia-lyase     

Activities of some enzymes of lignin formation in reaction wood of Thuja orientalis,Metasequoia glyptostroboides and Robinia pseudoacacia

The activities of the following five enzymes which are involved in the formation of lignin have been compared in reaction wood and in opposite wood: phenylalanine ammonia lyase (EC 4.3.1.5), caffeate 3-O-methyltransferase (EC 2.1.1.-), p-hydroxycinnamate: CoA ligase (EC 6.2.1.12), cinnamyl alcohol dehydrogenase (EC 1.1.1.-) and peroxidase (EC 1.11.1.7). The activities of the four first-named enzymes in the compression wood of Thuja orientalis L. and Metasequoia glyptostroboides Hu et Cheng were 2.8±1.4-fold and 2.6±1.5-fold higher than those in opposite wood, respectively, whereas peroxidase had the same level of activity in either type of wood. On the other hand, no differences were observed in the activities of the five enzymes between tension and opposite woods of Robinia pseudoacacia L. These findings are well in accord with the chemical structure of lignin in the compression and tension woods of the three species studied: high content of lignin rich in condensed units in compression wood, and little difference in lignin between tension and opposite woods.Abbreviations CAD cinnamyl alcohol dehydrogenase (EC 1.1.1.-) - OMT caffeate O-methyltransferase (EC 2.1.1-) - PAL phenylalanine ammonia lyase (EC 4.3.1.5) - PCL p-hydroxycinnamate: CoA ligase (EC 6.2.1.12) - PO peroxidase (EC 1.11.1.7)     

Dependence of the level of phytoalexin and enzyme induction by fungal elicitor on the growth stage of Petroselinum crispum cell cultures

The extent of induction of some metabolic activities in cultured parsley cells (Petroselinum crispum) by an elicitor preparation from Phytophthora megasperma f. sp. glycinea varied with the growth stage of the cell culture. On the basis of cell fresh weight, the induction of phytoalexin accumulation was high until cell mass reached a maximum, and then declined to a low level which was indistinguishable from a level caused by an endogenous mechanism operating at this late growth stage. The induction of phenylalanine ammonia-lyase and 4-coumarate:CoA ligase activities by the elicitor showed a high degree of coordination and a sharp maximum preceding the stage of maximal cell mass. 1,3--Glucanase activity was induced to about the same level throughout all growth stages, with a large contribution by an endogenous mechanism at late stages.Abbreviations PAL Phenylalanine ammonia-lyase (EC 4.3.1.5) - 4CL 4-Coumarate:CoA ligase (EC 6.2.1.12)     

Plant ERD2-like proteins function as endoplasmic reticulum luminal protein receptors and participate in programmed cell death during innate immunity

Plant secondary metabolites, such as those derived from the phenylpropanoid pathway, have a beneficial effect on human health. Manipulation of metabolic flux in the phenylpropanoid pathway is important for achieving enhanced production of compounds such as anthocyanins, flavonoids and isoflavonoids. Here, we describe the development of a high-throughput molecular evolution approach that can be used for catalytic improvement of at least four key phenylpropanoid pathway enzymes, within the context of the metabolic pathway. This method uses yeast cells that express plant phenylpropanoid pathway enzymes, leading to formation of a colored intermediate that can be used as a readout in high-throughput screening. Here we report the identification of improved tomato peel 4-coumarate:CoA ligase variants using this approach. We found that the wild-type enzyme is strongly allosterically inhibited by naringenin, a downstream product of the pathway. Surprisingly, at least two of the improved variants are completely insensitive to feedback inhibition by naringenin. We suggest that this inhibition is exerted through a unique and previously unrecognized allosteric domain.     

Hypersensitive lignification response as the mechanism of non-host resistance of wheat against oat crown rust

Seven-day-old seedlings of the near-isogenic wheat ( Triticum aestivum L.) lines Prelude and Prelude-Sr5, susceptible and resistant to wheat stem rust, respectively, were inoculated with uredospores of the oat crown rust fungus Puccinia coronata Cda. f. sp. avenae Fraser & Led. Fluorescence microscopy revealed that the majority of colonies developed intercellular infection structures including haustorial mother cells and haustoria after penetration of wheat mesophyll cells. All penetrated cells became necrotic, and exhibited bright yellow autofluorescence. This autofluorescence was not extractable with alkali, and fluorescent cells stained positively with phloroglucinol/HCI, suggesting that hypersensitive cell death was correlated with cellular lignification. Accordingly, the lignin biosynthetic enzymes phenylalanine ammonia-lyase (EC4.3.1.5). 4-coumarate:CoA ligase (EC6.2.1.12), cinnamyl-alcohol dehydrogenase (EC1.1.1.149), and peroxidases (EC1.11.1.7) increased in activity during the expression of resistance. The induced pattern of peroxidase iso/ymes closely resembled that observed for highly incompatible wheat/wheat stem rust interactions. Furthermore, an elieitor was extracted from oat crown rust germlings. which induces lignification when injected into the intercellular space of wheat leaves. This elieitor appears to be functionally similar to that isolated from wheat stem rust germlings. The results suggest that the non-host resistance of wheat to the xenopara-site oat crown rust closely resembles the race/cullivar-speeific resistant mechanism of highly resistant wheat varieties to wheat stem rust.