Polysaccharide Elicitor Research Articles

Elicitor-induced metabolic changes in cell cultures of chickpea (Cicer arietinum L.) cultivars resistant and susceptible to Ascochyta rabiei

Cell-suspension cultures of Ascochyta rabiei-resistant (ILC 3279) and -susceptible (ILC 1929) chickpea (Cicer arietinum L.) cultivars were compared with regard to their elicitor-induced accumulation of pterocarpan phytoalexins and increases in the activities of biosynthetic enzymes. The growth performances and protein patterns of the two cell-culture lines were essentially identical. Treatment of cell cultures with a polysaccharide elicitor from A. rabiei induced fivefold-higher amounts of the phytoalexins medicarpin and maackiain in the cells of the resistant than in the susceptible cultivar. Glucose 6-phosphate dehydrogenase and eight enzymes representing the general phenylpropanoid pathway, the flavonoid-forming steps and the pterocarpanspecific branch of phytoalexin biosynthesis were found to be elicitor-induced. Phenylalanine ammonia-lyase and chalcone synthase reached sharp, transient optima some 8 h after elicitor application in the cells of both cultivars. The activities of isoflavone 2'- and 3'-hydroxylases were only induced in cells of the resistant cultivar with a maximum after 8 h. Cinnamic acid 4-hydroxylase, chalcone isomerase, 2'-hydroxyisoflavone reductase and pterocarpan synthase showed a later or no sharp optimum. The isoflavone-specific 7-O-glucosyltransferase was not induced in either cell-culture line. Cells of the susceptible cultivar failed to induce significant activities of isoflavone 2'-hydroxylase and these cells produced only very low amounts of phytoalexins. Isoflavone 2'-hydroxylase is postulated to be the main limiting enzyme for pterocarpan biosynthesis in cells of the susceptible cultivar. The pterocarpan biosynthetic pathway in chickpea cells represents a suitable model for investigations of differential gene activation in connection with the expression of antimicrobial defence reactions.

Rapid changes in gene expression in response to microbial elicitation

Treatment of cell suspension cultures of French bean ( Phaseolus vulgaris ) with polysaccharide elicitor molecules from cell walls of the anthracnose fungus, Colletotrichum lindemuthianum , results in the rapid accumulation of isoflavonoid phytoalexins, deposition of wall-bound phenolic compounds and synthesis of hydroxyproline-rich glycoproteins. These changes are dependent upon a highly selective induction of gene products, including the enzymes L-phenylalanine ammonia-lyase, cytochrome P450-dependent cinnamic acid 4-hydroxylase, chalcone synthase, chalcone isomerase, prolyl hydroxylase and protein: arabinosyl transferase. Use of in vivo labelling, in vitro translation and RNA blot hybridization techniques has shown that these elicitormediated changes arise from rapid but transient induction of enzyme synthesis, resulting from the accumulation of specific mRNAs. Similar phenomena are observed in bean hypocotyls at the onset of phytoalexin synthesis in response to infection by incompatible and compatible races of C. lindemuthianum . In bean, both L-phenylalanine ammonia-lyase and chalcone synthase are encoded by multigene families and, at the protein level, both exhibit subunit and intact enzyme polymorphism. A number of less than full-length phenylalanine ammonialyase copy DNAs containing identical open reading frames have been produced from mRNA from elicitor-induced bean cells. Analysis of phenylalanine ammonia-lyase genomic clones predicts the presence of enzyme forms of differing amino acid sequence. In cultured bean cells, elicitor differentially induces the two apparent phenylalanine ammonia-lyase iso-forms with the lowest K m values. In addition to transcriptional control of the appearance of specific gene products, post-translational processes may result in increased subunit polymorphism for phenylalanine ammonia-lyase, and in the activation of chalcone isomerase. Changes in endogenous phenylpropanoid intermediate pools may signal the rapid removal of phenylalanine ammonia-lyase activity, in addition to exerting less specific inhibitory effects on the formation and/or activity of the mRNAs encoding phenylalanine ammonia-lyase and other phytoalexin biosynthetic enzymes.

L-Phenylalanine ammonia-lyase from Phaseolus vulgaris: partial degradation of enzyme subunits in vitro and in vivo

L-Phenylalanine ammonia-lyase (EC 4.3.1.5) has been purified from suspension cultured cells of French bean ( Phaseolus vulgaris L.) which had been exposed to polysaccharide elicitor preparations from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum. After preliminary purification by ammonium sulphate fractionation and gel filtration, the enzyme was further purified by (a) ion-exchange chromatography followed by chromatofocussing, (b) chromatography on rabbit anti-(phenylalanine ammonia-lyase) IgG, or (c) affinity chromatography on L-aminooxy( p-hydroxyphenyl)propionic acid (or L-tyrosine) linked to epoxy-activated Sepharose 6B via the phenolic hydroxyl group. The purified enzyme preparations exhibited subunit M r values of 77 000, 70 000 and 53 000, the relative proportions of these depending upon the enzyme source, length of time taken for purification, and inclusion of freeze-thaw steps. Four forms of the enzyme, differing in p I value, were resolved by chromatofocussing, although all forms from the same preparation consisted of similar proportions of the different subunit M r forms. Peptide mapping and freeze-thaw studies indicate that the M r 77 000 native phenylalanine ammonia-lyase subunit is inherently unstable in vitro and breaks down to yield the lower M r partial degradation products. Such products could also be observed following in vitro translation of phenylalanine ammonia-lyase mRNA. Pulse-chase experiments indicated that the 77 000 → 70 000 → 53 000 subunit interconversion also occurs in vivo.