Occurrence of phytoalexins and other putative defense-related substances in uninfected parsley plants

Considerable amounts of the following substances were found in uninfected parsley (Petroselinum crispum) cotyledons: furanocoumarins, the putative phytoalexins of this and some related plant species, two enzymes of the furanocoumarin pathway (S-adenosyl-L-methionine: xanthotoxol and S-adenosyl-L-methionine: bergaptol O-methyltransferases), two hydrolytic enzymes (1,3--glucanase, EC 3.2.1.39, and chitinase, EC 3.2.1.14), and pathogenesis-related proteins. The furanocoumarins and the methyltransferase activities reached their highest levels at the onset of cotyledon senescence as the hydrolytic enzymes increased from low to relatively high activity values. The relative amounts of pathogenesis-related proteins 1 and 2, as well as the corresponding mRNAs, also increased markedly. Two enzymes of general phenylpropanoid metabolism, L-phenylalanine ammonia-lyase and 4-coumarate: CoA ligase, decreased in activity in a biphasic fashion during cotyledon development. At all developmental stages, the levels of these putative defense-related agents in total cotyledon extracts were too high to enable detection of, possibly, additional changes upon infection with zoospores of Phytophthora megasperma f. sp. glycinea, a fungal pathogen to which parsley shows a non-host, hypersensitive resistance response.Abbreviations BMT S-adenosyl-L-methionine: bergaptol O-methyltransferase (EC 2.1.1.-) - 4CL 4-coumarate: CoA ligase (EC 6.1.1.12) - CMT S-adenosyl-L-methionine: caffeate O-methyltransferase (EC 2.1.1.-) - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5) - PR pathogenesis-related - XMT S-adenosyl-L-methionine: xanthotoxin O-methyltransferase (EC 2.1.1.-)     

Differential regulation and tissue-specific distribution of enzymes of phenylpropanoid pathways in developing parsley seedlings

Characteristic enzymes of general phenylpropanoid metabolism (phenylalanine ammonialyase) and of the flavonoid-glycoside and furanocoumarin branch pathways (chalcone synthase and S-adenosyl-l-methionine: bergaptol O-methyltransferase, respectively) were localized immuno-histochemically in cross-sections of various aerial parts of parsley (Petroselinum crispum) at different stages of seedling development. Phenylalanine ammonia-lyase occurred predominantly in epidermal and oil-duct epithelial cells, but was also detectable in other tissue parts. The two pathway-specific enzymes were localized in the epidermis (chalcone synthase) and in oil ducts (bergaptol O-methyl-transferase). High chalcone-synthase concentrations occurred very early in leaf development and then declined. High levels of the methyltransferase were present at all times investigated. The temporal and spatial at all times investigated. The temporal and spatial distribution of all three enzymes is in agreement with the time courses and sites of accumulation of the biosynthetic end products.Abbreviations BMT S-adenosyl-l-methionine: bergaptol O-methyltransferase - CHS chalcone synthase - PAL phenylalanine ammonia-lyase     

Biphasic Temporal and Spatial Induction Patterns of Defense-Related mRNAs and Proteins in Fungus-Infected Parsley Leaves

Previous experiments using in situ RNA hybridization have shown that the mRNAs encoding phenylalanine ammonia-lyase, 4-coumarate:coenzyme A ligase, and pathogenesis-related protein 1 accumulated transiently around fungal infection sites in parsley (Petroselinum crispum) leaf buds. These studies have now been extended by (a) analyzing different stages of the infection process and (b) monitoring the timing of appearance and the spatial distribution of the proteins as well as the corresponding mRNAs. An early and short period of mRNA induction throughout a large portion of the infected leaf was followed by a longer period, during which the mRNA levels remained high in a more localized area around the site of fungal penetration with sharp borders toward the surrounding tissue. This biphasic pattern of mRNA accumulation was followed after some delay by the same pattern of protein accumulation.     

In Situ Localization of Phenylpropanoid Biosynthetic mRNAs and Proteins in Parsley (Petroselinum crispum)

Using in situ RNA/RNA hybridization, enzyme immunolocalization, and histochemical techniques, several phenylpropanoid biosynthetic activities and products were localized in tissue sections from various aerial parts of parsley (Petroselinum crispum) plants at different developmental stages. The enzymes and corresponding mRNAs analyzed included two representatives of general phenylpropanoid metabolism: phenylalanine ammonia-lyase (PAL) and 4-coumarate: CoA ligase (4CL), and one representative each from two distinct branch pathways: chalcone synthase (CHS; flavonoids) and S-adenosyl-L-methionine: bergaptol O-methyltransferase (BMT; furanocoumarins). In almost all cases, the relative timing of accumulation differed greatly for mRNA and protein and indicated short expression periods and short half-lives for all mRNAs as compared to the proteins. PAL and 4CL occurred almost ubiquitously in cell type-specific patterns, and their mRNAs and proteins were always coordinately expressed, whereas the cell type-specific localization of flavonoid and furanocoumarin biosynthetic activities was to a large extent mutually exclusive. However, the distribution patterns of CHS and BMT, when superimposed, closely matched those of PAL and 4CL in nearly all tissues analysed, suggesting that the flavonoid and furanocoumarin pathways together consituted a large majority of the total phenylpropanoid biosynthetic activity. Differential sites of synthesis and accumulation indicating intercellular translocation were observed both for flavonoids and for furanocoumarins in oil ducts and the surrounding tissue. The widespread occurrence of both classes of compounds, as well as selected, pathway-specific mRNAs and enzymes, in many cell types of all parsley organs including various flower parts suggests additional functions beyond the previously established roles of flavonoids in UV protection and furanocoumarins in pathogen defence.     

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.     

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 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.     

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     

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)     

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     

Chromosomal localization of parsley 4-coumarate: CoA ligase genes by in situ hybridization with a complementary DNA

A near full-length cDNA (1.9 kb) was used as probe forin situ hybridization to assign one of the two highly homologous 4-coumarate: CoA ligase genes in parsley (Petroselinum crispum) to the short arm of a submetacentric chromosome. The results suggest, but do not definitely prove, that the second gene is located on a metacentric chromosome and is thus unlinked from the other.Abbreviations CHS chalcone synthase - 4CL 4-coumarate:CoA ligase - Kb kilobases (kilobasepairs)     

Molecular cloning, induction and taxonomic distribution of caffeoyl-CoA 3-O-methyltransferase, an enzyme involved in disease resistance

Trans-Caffeoyl-CoA 3-O-methyltransferase is involved in the reinforcement of the plant cell wall under conditions that trigger the disease resistance response (Pakusch, A.-E., Kneusel, R.E., and Matern, U. (1989) Arch. Biochem. Biophys. 271, 488-494). Partial amino acid sequences of the enzyme from cultured parsley cells that had been treated with a crude elicitor were identified (Pakusch, A.-E., Matern, U., and Schiltz, E. (1991) Plant Physiol. 95, 137-143), and corresponding degenerated oligonucleotides of 29- and 30-nucleotide length were synthesized. Northern hybridizations with these probes revealed one specific RNA band, and the amount of this RNA appeared to be transiently induced upon elicitation of the cells. De novo enzyme synthesis was confirmed by Western blotting experiments using a specific antiserum. The time course of induction closely followed the pattern observed for phenylalanine ammonia-lyase and suggested the operational coordination of the methyltransferase with the general phenylpropanoid pathway in vivo. Full size cDNA of 1.258 kilobases was isolated in lambda gt11, sequenced and found to contain a remarkably long 5'-untranslated leader sequence followed by an open reading frame that codes for a 241-residue polypeptide representing the 27-kDa subunit of the native, dimeric parsley enzyme. Almost no homology was found to protein sequences filed in data banks. Southern hybridization with genomic DNA suggested that only one or two copies of the respective gene(s) are present in the parsley genome. Caffeoyl-CoA-specific methyltransferase activity was demonstrated in taxonomically widely diverse plants such as Dianthus caryophyllus (Caryophyllaceae), Carthamus tinctorius (Asteraceae) or Daucus carota, and Ammi majus (Apiaceae) where it is commonly induced by elicitor treatment. In Northern blots with RNA from Ammi or Daucus, parsley cDNA hybridized specifically to one band comparable in size to the parsley RNA, whereas Dianthus and Carthamus appear to code for slightly larger RNAs (roughly 1.45 and 1.3 kilobases, respectively). Slot-blot hybridizations revealed in all instances the rapid and transient increase of mRNA levels in response to elicitation. This emphasizes the integral role of the enzyme in disease resistance expression in plants far beyond parsley and also illustrates a new physiological context for the induction of 4-coumarate:CoA ligase.     

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)     

Cellular localization of nonhost resistance reactions of parsley (Petroselinum crispum) to fungal infection

The response of parsley seedlings (Petroselinum crispum) inoculated with zoospores of the soybean-pathogenic fungus, Phytophthora megasperma f. sp. glycinea, ranged from immunity to physiological susceptibility depending on the post-inoculation environmental conditions. Typical nonhost resistance reactions, hypersensitive cell death and the formation of small local lesions, occurred under high relative humidity and 16 h illumination per day. Localized biochemical reactions were monitored using fluorescence microscopy combined with histochemical and immunohistochemical methods. The rapid accumulation of furanocoumarin phytoalexins, wall-bound phenolics and callose was detected around infection sites. Indirect antibody staining of frozen tissue sections demonstrated the local accumulation of phenylalanine ammonia-lyase, a key enzyme of general phenylpropanoid metabolism, and S-adenosyl-L-methionine: bergaptol O-methyltransferase, a specific enzyme of the furanocoumarin pathway. The results indicate that phenylpropanoid derivatives are synthesized de novo at infection sites.Abbreviations BMT S-adenosyl-L-methionine:bergaptol O-methyltransferase - PAL phenylalanine ammonia-lyase - PBS phosphate-buffered saline