Cells Of French Bean Research Articles

L-phenylalanine ammonia-lyase from French bean (Phaseolus vulgaris L.). Characterization and differential expression of antigenic multiple Mr forms

L-Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) purified from suspension-cultured cells of French bean (Phaseolus vulgaris) has been further characterized. A number of techniques, including use of an antiserum and affinity probes, have established that all the antigenic polypeptides represent polymorphic Mr forms of the enzyme. These peptides include an apparently higher-Mr (83,000) form which shows different kinetics of induction from the Mr-77000 forms that have been extensively characterized previously. The larger subunit appeared to be PAL by the following criteria: (a) binding to specific affinity and antibody matrices; (b) peptide mapping; (c) active-site labelling; and (d) amino acid composition. The increased Mr of the larger subunit was not completely attributable to glycosylation, although some sugar residues were detected in this Mr-83000 form but not in the other Mr forms. Mr-83000 subunits were also immunoprecipitated from translations in vitro of mRNA from cells that had been stressed for a long period. They were also detected in leaf tissues that were not yet undergoing an extensive wound response. This form of the enzyme may be constitutive and involved in the low-level accumulation of phenolics in most cell types. By contrast, the Mr-77000 forms of PAL were rapidly induced during elicitor action, wounding or cytokinin-induced xylogenesis as a key regulatory enzyme involved in the synthesis of phenolics under stress conditions or during differentiation.

Glutathione S-cinnamoyl transferases in plants

A glutathione S-transferase (GST) which catalyses the conjugation of cinnamic acid with glutathione has been partially characterized from suspension cultured cells of French bean ( Phaseolus vulgaris) and other legume species. The glutathione S-cinnamoyl transferases (GSCTs) appeared to be distinct from other plant GSTs and showed a species-dependent subcellular distribution between the cytosol and microsomes. The enzyme from legumes was inhibited by known inhibitors of plant GSTs, and cross-reacted with an antiserum raised against a cytosolic GST from Zea mays which is involved in herbicide detoxification. Bean GSCT was a cytosolic enzyme with a K m of 330 μM for cinnamic acid. Its activity was unaffected by treating cells with cinnamic acid but was increased two- to three-fold by exposure of the cells to an elicitor preparation from the cell walls of the fungal bean pathogen Colletotrichum lindemuthianum.

Modulation of the elicitation response in cultured french bean cells and its implication for the mechanism of signal transduction

A range of modulators have been used to investigate signal transduction involved in the response of suspension cultured cells of French bean to an elicitor preparation from the fungal pathogen Colletotrichum lindemuthianum. Elicitor action was effectively determined by the induction of phenylalanine ammonia-lyase(PAL) protein and activity. Three types of modulators were used. One group that potentially directly affected calcium mobilization and action had with the exception of caffeine and procaine no consistent effect on elicitor action. A number of ionophores and compounds that affect membrane ion transport weretested and the most effective was found to be monensin. Further studies showed that the inhibition of elicitation by monensin was more likely to be mediated by inhibition of uptake of elicitor than perturbation of ion transport. A third group of modulators were characterized by their ability in animal systems to interact with G proteins. A variety of effects were observed which were consistent with putative G protein involvement as a component of elicitor-induced signal transduction. Modulation of the induction of the synthesis of PAL protein and of PAL activity were both observed. Overall, the results were consistent with elicitor action being potentiated through multicomponent transduction pathways.

Regulation by carbohydrates of the sequential in vitro production of pectic enzymes by Botrytis cinerea

Cultures of Botrytis cinerea in a basal salt medium supplemented with different pectin-related polysaccharides (French bean cell walls; citrus pectin; sodium polygalacturonate) as the only carbon source were examined daily for polygalacturonase activity, type of pectic enzymes present, and mycelial growth. Total polygalacturonase activity and number of enzymes detectable were influenced by type and concentration of the substrate and by the conidial concentration at which the cultures were started. A consistent sequence in the production of pectic enzymes was found. The polygalacturonase PG2 was always the first enzyme present in the culture filtrates and was followed by a number of polygalacturonase and pectinesterase isoenzymes. PG2 was also found in ungerminated conidia. Its production is the expression of a constitutive gene as it was independent of the presence of the substrate and strictly correlated with fungal growth. D-Galacturonic acid at 2 mM induced the production of some of the pectic enzymes. At 10 mM and above, however, it repressed PG2 and the subsequent production of the whole pectic isoenzyme complex, indicating a feedback repression. The results suggest that the pectic isoenzymes produced by B. cinerea constitute a coordinated catabolic pathway for the complete degradation of pectic polysaccharides.

Ultrastructural observations of penetration sites of the cowpea rust fungus in untreated and silicon-depleted French bean cells

The region where the cowpda rust fungus initiates haustorium formation in plant mesophyll cells was compared ultrastructurally in susceptible cowpea plants, nonhost French bean plants given access to silicon (−Si plants), and French bean plants depleted in silicon (−Si plants). In +Si plants, fungal development apparently ceased while the penetration peg was traversing the haustorial mother cell (HMC) wall, often before the peg reached the adjacent silicified wall of the plant. In −Si plants, where silica deposits were absent, HMCs at three out of 10 infection sites had formed a haustorium. At the majority of the remaining sites, fungal growth appeared to have ceased before the initiation of a visible penetration peg and during a stage of development, also seen in susceptible cowpea, where the HMC and the plant wall were bridged by electron-opaque material. It is suggested that this stage represents the incipient degradation of the fungal, and possibly the plant, wall prior to the appearance of a distinguishable penetration peg. The fact that most penetration pegs stopped their development earlier in −Si than in +Si plants supports the previous suggestion that the higher levels of wall-associated phenolic compounds in the former results in a more rapid inhibition of fungal enzymes involved in the formation of the penetration peg. These and other ultrastructural observations suggest that in +Si plants the silicified plant walls may (1) reduce the interchange of materials between plant and fungus so that lesser amounts of phenolic materials are produced, (2) restrict the flow of materials to the HMC that normally prevent its premature senescence, and/or (3) act as a physical barrier if the penetration peg reaches the plant wall.

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.

Stability of the complex formed between French bean ( Phaseolus vulgaris) phenylalanine ammonia-lyase and its transition-state analog

Phenylalanine ammonia-lyase forms trans-cinnamate from l-phenylalanine, and thus stands at a gateway to secondary metabolism in higher plants. l-α-Amino-oxy β-phenylpropanoic acid (L-AOPP), a very effective competitive inhibitor of this enzyme, is most probably a transition-state analog for the elimination reaction. A preparation of phenylalanine ammonia-lyase (PAL), obtained from diluted suspension cultures of French bean cells, was used to investigate the binding of this compound in vitro. After extensive dialysis, the inhibitor remained tightly bound to the enzyme unless both an increased temperature and l-phenylalanine were provided, when the spectrophotometer trace of enzyme activity gradually approached linearity. Under such optimal catalytic conditions (37 °C; 25 m ml-phenylalanine; pH 8.8), dissociation of the enzyme-ligand complex took place with a half-time of approx 10 min. (This is much longer than reported for the enzyme from maize.) The consequences of these findings are discussed for investigations where L-AOPP is applied in vivo. These experiments have shown that the irreversible binding of the transition-state analog under appropriate conditions (0–4 °C, no l-phenylalanine) gave continued protection against attack on the enzyme by an excess of borohydride. By titrating the enzyme with increasing concentrations of analog and measuring the degree of protection afforded, the active-site concentration has been estimated. The turnover number ( k cat = 0.8 s −1) given by this novel approach is of the same order of magnitude as previously reported from extensive purification of enzyme from other species.

Elicitor-mediated induction of chalcone isomerase in Phaseolus vulgaris cell suspension cultures

Approximately fourfold increases in the extractable activity of the enzyme chalcone isomerase (CHI, EC 5.5.1.6) were observed within 24 h of treatment of cell suspension cultures of Phaseolus vulgaris with a crude elicitor preparation heatreleased from the cell walls of the bean pathogen Colletotrichum lindemuthianum. The induction of CHI activity was highly dependent upon elicitor concentration, with maximum induction occurring in two discrete concentration ranges. A basal half-life for CHI>32 h in control cultures was determined by labelling with (2)H from (2)H2O followed by analysis of the equilibrium distribution of enzyme activity in CsCl density gradients. Comparative density labelling indicated that at both the lower and higher effective elicitor concentrations, the induced appearance of CHI activity was the result of an apparent initial activation of pre-existing enzyme followed by an increase in the rate of de-novo synthesis of the enzyme as compared with non-elicited controls. The increased appearance of the enzyme over the first 8 h in elicitor-treated cultures was inhibited by cycloheximide, cordycepin and actinomycin D. The results are discussed in relation to the mechanisms of co-ordinate enzyme induction operating in French-bean cell cultures exposed to fungal elicitors.

Dose responses for Colletotrichum lindemuthianum elicitor-mediated enzyme induction in French bean cell suspension cultures

The induction of L-phenylalanine ammonialyase (PAL, EC 4.3.1.5) and flavanone synthase in French bean cell suspension cultures in response to heat-released elicitor from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum is highly dependent upon elicitor concentration. The elicitor dose-response curve for PAL induction shows two maxima at around 17.5 and 50 μg elicitor carbohydrate per ml culture, whereas the flavanone synthase response shows one maximum at around 100 μg ml(-1). The PAL response is independent of the elicitor concentration present during the lag phase of enzyme induction; if the initial elicitor concentration is increased after 2 h by addition of extra elicitor, or decreased by dilution of the cultures, the dose response curves obtained reflect the concentration of elicitor present at the time of harvest. PAL induction is not prevented by addition of methyl sugar derivatives to the cultures; α-methyl-D-glucoside, itself a weak elicitor of PAL activity, elicits a multiphasic PAL response when increasing concentrations are added in the presence of Colletotrichum elicitor. Eight fractions with different monosaccharide compositions, obtained from the crude elicitor by gel-filtration, each elicit different dose-responses for PAL induction; the response to unfractionated elicitor is not the sum of the response to the isolated fractions. There is no correlation between the ability of the fractions to induce PAL in the cultures and their ability to act as elicitors of isoflavonoid phytoalexin accumulation in bean hypocotyls.

Elicitor modulation of the turnover of l-phenylalanine ammonia-lyase in French bean cell suspension cultures

1. (1) The mechanisms underlying the trasient increase in phenylalanine ammonia-lyase activity during phaseollin accumulation in cell suspension cultures of Dwarf French bean ( Phaseolus volgaris) have been investigated using density labelling with 2H from 2H 2O coupled with residual analysis of the equilibrium distribution of enzyme activity in high-resolution KBr denstiy gradients. 2. (2) The resolution achieved in this system is sufficient to allow quantitative analysis of the relative proportions of light, unlabelled, pre-existing enzynes and heavy, labelled, newly synthesised enzyme. 3. (3) Elicitor released by heat treatment of cell walls of Colletotrichum lindemuthainum, the causal agent of anthracnose disease of French bean, caused a marked but transient increase in phenylalanine ammonia-lyase activity concomitant with the onset of phaseolllin accumulation in the bean cultures. The induction of enzyme activity was highly dependent on elicitor concentration, with maximum induction occurring in two discrete concentration ranges; at an instermediate elicitor concentration, or at supra-optimal elicitor concentrations, no enzyme induction was observed. 4. (4) At low concentrations of elicitor the induction of enzyme was entirely a result of elicitor stimulation of the rate of de novo enzyme production. In contrast, at higher elicitor concentrations the increase in enzyme activity was accompanied by a marked apparent stabilization of the enzyme in vivo, and the rapid but transient increase in enzyme activity was achieved by a programme of reciprocal changes in the rate constant for de novo enzyme production and the rate constant for removal of enzyme activit. Such reciprocal control of the rates of enzyme production and removal may be crucial in determing the magnitude and duration of the phytoalexin defence response. 5. (5) Information on the specific activity of 2H label in the amino acid pools was obtained from analysis of the equilibrium distribution of residual, labelled activity. This showed directly that the turnover of the amino acids pools was fast relative to the turnover of phenylalanine ammonia-lyase and, hence, the rate of enzyme labelling was not limited by the rate of labelling of the amino acid pools. Elicitor treatment had no effect on the specific activity of label in the amino acid pools from which phenylalanine ammonia-lyase is synthesised.

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(-5)M) 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 α-aminooxy-β-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.

Division, expansion and DNA synthesis in meristematic cells of French bean ( Phaseolus vulgaris L.) root-tips invaded by tobacco ringspot virus

The division, expansion and DNA synthesis of meristematic cells of the root-tip of French beans were examined at different times after infection with tobacco ringspot virus. At about the same time that the terminal millimetre of the root was invaded by virus there was a drop in DNA synthesis, followed by a decrease in the mitotic index to about half that of healthy tissue. Cell elongation was unaffected, resulting in a gradual accumulation of expanded cells in the root-tip. Subsequently, the mitotic index of the root-tip returned to a normal level.

Interpretation of ectodesmata in relation to susceptibility to plant viruses

Ectodesmata were demonstrated in the outer walls of epidermal cells of French bean following treatment of leaf tissue with Gilson's solution.There was little change in the number of demonstrable ectodesmata with increasing age of the plant or as a result of increased suction tension of the culture solution. The number of ectodesmata was greater after darkening for 24 h but was not increased further with an additional period of darkening.These results are discussed in relation to studies on susceptibility to plant viruses and the structural evidence for ectodesmata. It is suggested that ectodesmata do not determine susceptibility to plant viruses and are not equivalent to the infectible site.

The infectivity of extracts made from leaves at intervals after inoculation with viruses.

SUMMARY: When leaves are macerated at intervals after being inoculated with plant viruses, the infectivity of the extracts obtained decreases with increasing time until newly produced virus becomes detectable. Infectivity does not start to increase until approximately twice the time apparently needed for virus to multiply in the epidermis and spread from there to the mesophyll. Epidermal cells infected by inoculation seem to produce too few virus particles to be detected by infectivity tests or else the first-formed particles are unstable in vitro. No evidence was obtained, with Rothamsted tobacco necrosis virus (RTNV) in leaves of tobacco and French bean, that the initial decrease in infectivity occurs because of changes in virus particles that succeed in infecting and causing lesions. If such changes occur they are obscured by the inactivation of particles that do not multiply and cause lesions. Washing inoculated leaves removes 95 % of the inoculated virus, but only slightly decreases the numbers of infections, and adding ‘Celite’ to the inoculum greatly increases the numbers of lesions without increasing the amount of virus retained by washed leaves. Neither washing nor adding ‘Celite’ to the inoculum affects the rate at which the infectivity of successive extracts from inoculated leaves decreases. Infectivity continues to decrease after virus appears to have multiplied in and spread from the epidermis. Cells of Nicotiana glutinosa that are infected by tobacco mosaic virus spreading from inoculated epidermal cells die only a few hours after the infectivity of leaf extracts starts to increase: few cells seem to become infected from virus produced in these secondarily infected cells and, at 20°, infectivity reaches a maximum in 2 days. Mesophyll cells of French bean leaves at 22° seem to synthesize new RTNV particles within 5 hr. of becoming infected from the epidermis and to continue synthesizing for another 30 hr., when they probably contain about 106virus particles/cell. Although the cells then die, the virus spreads to further cells and the infectivity of leaf extracts increases for at least five days.