Yvette Lienart

Purification of cell-wall β-d-xylanases from Acacia cultured cells

Abstract An improved technique for the purification of β- d -xylanases from Acacia in vitro cultured cells is described, involving 4 M LiCl extraction anion-exchange chromatography, gel filtration chromatography and flat-bed electro-focusing steps. The procedure had been efficient since it resulted in preparations each containing a cell-wall xylanase with slight α-amylase and 1.4-β- d -glucanase contaminant activities. It also yielded a highly active homogeneous xylanase with a molecular weight of 55-kilodalton and an isoelectric point, pI of 5.70.

Wall-bound 1,3-β-d-glucan: Orthophosphate glucosyltransferase activity from acacia cultured cells

Abstract A wall-bound enzyme has been identified from Acacia verek suspended cells. It exhibited a 1,3-β- d -glucan: orthophosphate glucosyltransferase activity (EC 2.4.1.97), the characteristics of which are reported. In 50 mM citrate-phosphate buffer (pH 6.5), the phosphorolytic cleavage of laminarin and laminaribiose can be observed. In 0.2 M imidazole buffer (pH 6.5), the phosphorylase equilibrium lies in the direction of the synthesis reaction, α- d -glucose-1-phosphate as glucosyl donor being incorporated into a dimer laminaribiose. Glucose released from exo laminarinase activity (EC 3.2.1.58), similarly bound to cell walls, can compete phosphorylase action, suggesting that this has an in vivo regulatory role.

Silica-bound N-acetylglucosaminyl residues elicit laminarinase activity in Rubus protoplasts

Abstract Treatment of Rubus protoplasts with glycosylated silica-beads triggers rapid and transient activation of laminarinase activity. The response of freshly prepared protoplasts and intact suspended-cell to silica-bound N -acetylglucosaminyl residues was followed as a function of elecitation time and elicitor dose. The data strongly suggest that the plant cell wall is unlikely to be involved in the perception of such immobilized carbohydrate signals, and are compatible with the hypothesis that specific receptors exist on plasmalemma.

Modulation of laminarinase activity by Ca2+, dcAMP, phorbol-ester and diacylglycerol in plant cells

Abstract Reagents known to modulate the intracellular level of Ca 2+ , CAMP or Ca 2+ phospholipid-dependent protein kinase activity efficiently regulated laminarinase activity in suspended Rubus cells . Thus second messengers or related factors could alter the cell response to a sugar elicitor. Furthermore, two proteins of 15 and 35 000 in enzyme extracts of cells incubated with liposomes containing diacylglycerol and phosphatidylserine could be phosphorylated while no change occurred in extracts from control cells. The results suggest that protein kinases may play a major role in the control of laminarinase activity.

Immobilized sugars as abiotic inducers of β-d-glycanohydrolases in plant cells

Abstract Silica beads coated with various sugars were used to induce β- d -glycanohydrolases in Rubus-cultured cells. Laminarase, xylanase and chitosanase activities were strongly increased after the incubation of cells for 1 h with the immobilized sugars. The chemical structure and the dose of immobilized sugars affect the nature and the extent of the induced activities. Optimal induction of enzymes required only about 100 nmol of sugar per g cells and occurred within 1 h cell-treatment. The activation of β- d -glycanohydrolases could be modulated in presence of Ca2+ or K+ regulators. The findings suggest that the immobilized sugars mimic some effects of biotic elicitoes derived from pathogen or plant cell-walls.

High Performance Liquid Chromatography and Photodiode Array Detection of Phenylpropanoids and Benzoates in Rubus Protoplasts Elicited by Kinetin

Reverse-phase high performance liquid chromatography with photodiode array detection (PAD) has beendeveloped for simultaneous separation, identification and quantification of picomoles of phenylalanine ammonia-lyase (PAL) reaction products. Separation has been achieved in 30 min on a reverse-phase silica column usingmethanol:water gradient elution, and PAD provided sensitive and specific determination of phenolics. Themethod has been applied to biological samples to study kinetin-induced changes in phenol pathways withinprotoplasts of Rubus fruticosus(raspberry).

Chitosan-elicited laminarinase activity in Rubus cells or protoplasts

Abstract The oligomers of fully deacetylated chitosan with a degree of polymerization (DP) within 2 and 29 and the polymers up to DPv 3850 were assayed

Hormone-mediated response to immobilized sugars in Rubus cells

Abstract This work investigated the changes in response of Rubus fruticosus L. suspension cells when exposed to nanomoles of immobilized sugars in the presence of 2,4-D and kinetin. Silica beads coated with various sugars (α- l -fucosyl (1 → 2)-β- d -galactosyl (1 → 3)-β- d -N- acetylglucosamine trisaccharide H(t 1 ) and derivatives) can induce, within 60 min, laminarinase activity in treated cells. The structural features of the sugars required for the inhibition or amplification of 2,4-D (or kinetin) effects are reported. The extent of laminarinase activation is dependent on the dose levels of both sugar and hormone. These findings suggest that the trisaccharide H(t1)-recognition system is highly discriminating.