Cell Suspension Cultures Of Phaseolus Vulgaris Research Articles

Characterization of novel F-box proteins in plants induced by biotic and abiotic stress

Plants protect against pathogen infections by a combination of constitutive and induced strategies. The induction of plant defense involves the recognition of compounds derived from the pathogen or the plant itself, called elicitors. Looking for new genes involved in plant defense responses, we isolated a cDNA clone corresponding to an elicitor-induced mRNA from Phaseolus vulgaris cell suspension cultures. This clone, PvFBS1, encodes a protein with an F-box, therefore a putative component of an SCF ubiquitin ligase complex. PvFBS1 mRNA accumulates in leaves of whole plants in response to wounding or osmotic stress, as well as, following the application of methyl jasmonate (MeJA). salicylic acid (SA) or abscisic acid (ABA). Several sequences related to PvFBS1 were found in the GenBank. In Arabidopsis thaliana there are 4 genomic sequences coding for proteins with similarity to PvFBS1. One of them, AtFBS1, displays a pattern of induction analogous to the one observed for PvFBS1. A yeast two-hybrid assay proved that AtFBS1 was able to interact with ASK1, the component of the SCF complex that binds the F-box. A deletion of the F-box in AtFBS1 abolishes the ability of this protein to interact with ASK1. This demonstrates the functionality of the F-box contained in AtFBS1. Gene fusions to the GUS reporter gene revealed a complex regulation for AtFBS1 expression.

Carbohydrate utilisation by cell suspension cultures of Phaseolus vulgaris

Uptake of sugar by Phaseolus vulgaris cell suspension cultures from a sucrose supplemented medium is predominantly in the hexose form. This is due to a rapid cleavage of the sucrose by an apoplastic acid invertase activity and an apparent very low demand for and uptake of carbon from the medium prior to induction of cell growth and division. Glucose is preferentially taken up, leading to an accumulation of fructose in the medium. However, when the glucose is depleted the cells do take up the fructose at a rate similar to that of glucose. When glucose or fructose is supplied individually to cell cultures, both are utilised very efficiently with growth slightly better on the fructose medium. Hexose uptake is largely an active process with diffusion uptake even at the highest concentrations (> 50 mM) contributing less than 30%. The hexose uptake system of the cells has a greater affinity for glucose (Km= 240 µM) than for fructose (Km= 960 µM) but the maximum uptake (Vmax) is similar. The major difference in the kinetic properties of hexose uptake is that glucose is a strong inhibitor of fructose uptake, while fructose has little effect on glucose uptake. The differences in the kinetic properties of the uptake system for the two hexoses can largely explain the observed pattern of hexose utilisation when both glucose and fructose are present in the medium.

31P NMR Study of Elicitor Treated Phaseolus vulgaris Cell Suspension Cultures

The addition of an elicitor (glucan) to Phaseolus vulgaris cell suspension cultures increased the formation of the phytoalexin phaseollin. Intracellular pH and phosphate concentrations were studied with (31)P nuclear magnetic resonance spectroscopy on elicitor-treated cells which were aerated during the nuclear magnetic resonance measurement. The pH of the vacuole and to a lesser extent the pH of the cytoplasm were affected at 10 minutes after elicitor addition; a decrease in pH from 5.3 to 4.8 was noted in the vacuole and from 7.46 to 7.28 in the cytoplasm. The ratio between the amount of Pi in the vacuole to that in the cytoplasm also changed within 10 minutes after elicitor addition. The signal for ATP (beta-ATP) was low after elicitor addition and was high again 23 hours after elicitation. Forty-eight hours after elicitor addition, vacuolar and cytoplasmic pH had almost returned to their initial values. The rapid change in vacuolar and cytoplasmic pH may cause the change of metabolism that occurs in elicitor-treated P. vulgaris cells.

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.

Effects of growth substances on non-induced and Botrytis cinerea culture filtrate-induced phaseollin production in Phaseolus vulgaris cell suspension cultures

Suspension cultures of Phaseolus vulgaris produced the phytoalexin phaseollin in the absence of added inducers. The concentrations of plant growth substances in the culture medium affected growth of the cultures and the extent of phaseollin production, both in untreated cultures and in response to treatment with sterile culture filtrates from Botrytis cinerea. 2,4-Dichlorophenoxyacetic acid was inhibitory to induced production at concentrations of around 2 × 10 −6 m and above, whereas 1-naphthylacetic acid only inhibited significantly at 2 × 10 −4 m. Gibberellic acid and abscisic acid both stimulated non-induced production, abscisic acid also inhibiting growth of the cultures. High kinetin concentrations were inhibitory to non-induced production, but partially relieved inhibition of induction in culture filtrate-treated cells caused by growth in high concentrations of 2,4-dichlorophenoxyacetic acid. This increased response was associated with higher phenylalanine ammonia-lyase activity during the induction period than observed in cultures grown in low kinetin concentrations. However, as phenylalanine ammonia-lyase activity was often higher in control than in induced cultures, it is unlikely that this enzyme plays a regulatory rôle in phaseollin biosynthesis in this system.

Characterization of components from culture filtrates of Botrytis cinerea which stimulate phaseollin biosynthesis in Phaseolus vulgaris cell suspension cultures

Sterile culture filtrates from shake cultures of Botrytis cinerea stimulated phaseollin biosynthesis in cell suspension cultures of Phaseolus oulgaris. Increased phaseollin levels in induced cultures were observed after about 12 h. The inducing activity in the filtrate was heat stable and soluble in 80% ethanol. Gel-filtration studies showed several peaks of inducing activity, and pronase digestion destroyed the activity of the two highest molecular weight peaks. Treatment of culture filtrates with ribonuclease or deoxyribonuclease did not remove inducing activity and even after inactivation by autoclaving these enzymes were potent inducers of the phytoalexin response.