A new method for the rapid and quantitative fluorometric determination of callose is described. In suspension-cultured cells of Glycine max, synthesis of callose starts within 20 minutes of treatment with chitosan and parallels over hours the accumulation of 1,3-linked glucose in the wall. Poly-L-lysine also elicits callose synthesis. The effect of chitosan is enhanced by Polymyxin B at low concentrations; this antibiotic alone at higher concentrations can also induce callose synthesis. Callose synthesis is immediately stopped when external Ca2l is bound by ethylene glycolbis-(2-aminoethyl ether)-N,N'-tetraacetate or cation exchange beads, and partly recovers upon restoration of 15 micromolar Ca21.Callose synthesis is observed only when membrane perturbation causing electrolyte leakage from the cells is induced by one of the above treatments. It does not appear to be due to de novo synthesis or proteolytic activation of 1,3-#-D-glucan synthase. It is concluded that this Ca24-dependent enzyme is directly activated by the influx of Ca24 occurring concomitantly with the leakage of cell constituents. This suggestion is also discussed in conjunction with the chitosan-induced synthesis of phytoalexin in the same cells.

Treatment of suspension-cultured Glycine max cv Harosoy 63 cells with soluble chitosan (20-500 micrograms per milliliter) increased membrane permeability as shown by leakage of electrolytes, protein, and UV absorbing material. Severe damage to the cell membrane by chitosan (100 and 500 ug/ml) was also indicated by reduced staining with fluorescein diacetate and the leakage of fluorescein from preloaded cells. Other basic polymers (poly-L-lysine, histone, DEAE-dextran, protamine sulfate, and glycol chitosan) also increased permeability, whereas the basic monomers L-lysine and D-glucosamine, and acidic or neutral polymers were not active. Chitosan-induced leakage was inhibited by divalent cations, the order of effectiveness being Ba2 > Ca2+ > Sr2 > Mg2+. Na polygalacturonate and Na poly-L-aspartate also reduced polycation-induced leakage, probably by formation of polycation-polyanion complexes. A chitosan-polygalacturonate complex precipitated on mixing solutions of the two polymers containing approximately equal numbers of galacturonate and glucosamine residues, but not with either polymer in excess. A similar concentrationdependent precipitation of chitosan by Na poly-L-aspartate was found. Leakage from Phaseolus vulgaris cv Grandessa cells was also induced by chitosan, and was inhibited by Ca2+ and Na polygalacturonate.Chitosan (,8-1,4- MATERIALS AND METHODSCell Cultures. The cell suspension culture of Glycine max cv Harosoy 63 was a gift from J. Ebel, Freiburg University, Germany and that of Phaseolus vulgaris cv Grandessa (seeds from Bruno Nebelung, Munster, Germany) was initiated by inoculation of callus derived from sterile hypocotyl explants of 6-d-old seedlings. Suspension cultures were grown at 26°C in the dark on a 1.5-cm radius rotary shaker at 120 rpm in Erlenmeyer flasks containing B5 medium (9) which was modified by using 50 jLM FeSO4-EDTA as the source of iron (8), and subcultured at 6-and 12-d intervals, respectively.Chemicals. Chitosan from crab shells (Sigma) was dissolved in 90 ml 0.1 N acetic acid/g chitosan by stirring overnight, centrifuged at 27,000g for 20 min to remove insoluble material, then precipitated by neutralization to pH 8.0 with 5 N NaOH. The precipitate was washed extensively with distilled H20 by centrifugation and freeze-dried. The glucosamine content of this purified preparation was estimated to be 100%o by the method of Ride and Drysdale (23). Aqueous solutions of purified chitosan and glycol chitosan (Sigma) were prepared for use by dissolving 100 mg in 18 ml 0.1 N acetic acid and dialyzing four times against 2 liters of distilled H20. Poly-L-lysine hydrobromide (mol wt, >70,000), DEAE-dextran (approximate mol wt, 500,000), histone (calf thymus, type II), Na polygalacturonate (grade II), Na poly-L-aspartate (mol wt, 20,000-50,000), glucosamine hydrochloride, L-lysine monohydrochloride, L-aspartic acid, FDA, and BSA (fraction V) were from Sigma. Protamine sulfate and galacturonic acid were from Merck (Darmstadt, Germany). Solutions of aspartic acid and galacturonic...

In homogenates from suspension‐cultured soybean cells, 1,3‐β‐d‐glucan synthase activity is increased through preincubation with trypsin or due to action of an endogenous enzyme which presumably is a protease as it is inhibited by soybean trypsin inhibitor. The 1,3‐β‐d‐glucan synthase in untreated membrane preparations is also reversibly stimulated by Ca2+. This Ca2+‐dependence is lost on proteolytic activation. Regardless as to whether the enzyme was rendered active by preincubation with trypsin or by the presence of Ca2+, it is inhibited by calmidazolium, trifluoperazine and polymyxin B, suggesting that the activation by Ca2+ is not mediated by calmodulin.

SummaryAs nitric oxide (NO) is a key messenger in many organisms, reliable techniques for the detection of NO are essential. Here, it is shown that a combination of membrane inlet mass spectrometry (MIMS) and restriction capillary inlet mass spectrometry (RIMS) allows for the fast, speci®c, and non-invasive online detection of NO that has been emitted from tissue cultures of diverse organisms, or from whole plants. As an advantage over other NO assays, MIMS/RIMS discriminates nitrogen isotopes and simultaneously measures NO and O 2 (and other gases) from the same sample. MIMS/RIMS technology may thus help to identify the source of gaseous NO in cells, and elucidate the relationship between primary gas metabolism and NO formation. Using RIMS, it is demonstrated that the novel fungicide F 500 j triggers NO production in plants.

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