ALBERT

Notes: The chlorophyll content increased with time in callus cultures of soybean (Glycine max L., cv. Chippewa) and tobacco (Nicotiana tabacum L., cv. White Gold) grown in the light on a standard medium. Addition of 0.5 mmol/1 glyphosate [N-(phosphonomethyl) glycine] to the medium completely inhibited chlorophyll accumulation in the tobacco callus. Soybean callus required 40 times more glyphosate to initiate inhibitory effects on chlorophyll content. The inhibition was not reversed by simultaneous addition of casein hydrolysate or aromatic amino acids, suggesting that aromatic amino acid levels were not limiting for chlorophyll synthesis. In leaf discs of soybean and tobacco, glyphosate accelerated chlorophyll degradation in the light, but not in the dark; there was little difference in response between the two plant species. Young leaves of tobacco were more sensitive than mature leaves to glyphosate effects with glyphosate-induced net losses of chlorophyll of 24 and 14% respectively after 5 days. Such rates of loss cannot explain the complete inhibition of chlorophyll accumulation in the callus tissue, suggesting that there was an inhibition of chlorophyll synthesis by glyphosate.

Notes: Callus cultures or tobacco (Nicotiana tabacum L. cv. While Gold and N. glauca-langsdorffii, a tumour-forming amphidiploid hybrid) and soybean Glycine max L., cv. Chippewa) were used lo study the effect of glyphosate [N-(phosphonnmethyl) glycine) un growth and interactions with growth hormones. Glyphosate inhibited growth both in the dark and light but showed a greater toxicity in the dark. This was contrary to its effect on chlorophyll degradation which was accelerated by light. The inhibition of growth was not reversed by simultaneous addition of aromatic amino acids to the medium. Thus the results suggest a multiple glyphosate action. The tobacco callus tissue was more sensitive to glyphosate than the soybean callus tissue, confirming a difference in tolerance between plant species. Despite the inhibitory effect of glyphosate. the treated tissue revived after being transferred to a glyphosate-free medium. The glyphosate-induced growth inhibition in soybean and tumour-forming tobacco callus cultures also was reversible by high levels of indole-3-acetic acid (IAA), which itself was inhibitory Theglyphosate-IAA interaction in the tissues which were sensitive to IAA suggests that the inhibition of growth by glyphosate was related to auxin levels in these tissues.

Notes: Peroxidase in tobacco callus tissue differed in extract-ability depending on the subcellular distribution of the enzyme. Based on extractability it consisted of four fractions: freely soluble and less freely soluble in phosphate buffer, KCl-soluble, and insoluble. The latter two fractions were un-extractable by a phosphate buffer alone. The different fractions contained varied proportions of peroxidase isoenzymes. The extractability of indoleacetic acid oxidase was similar. A medium of high ionic strength is essential for quantitative extraction of peroxidase and indoleacetic acid oxidase isoenzymes.For quantitation of isoperoxidase activity on polyacryl-amide gel following electrophoretic separation, benzidine and o-dianisidine were better hydrogen donors than guaiacol and pyrogallol. The optimum pH was 4.5, but a citrate buffer was inhibitory. The optimum conditions included an acetate buffer at pH 4.5, a substrate concentration of 0.03 %, benzidine as the hydrogen donor, and a 3-minute treatment with 7 % acetic acid after staining. The color intensity of the bands remained unchanged for at least three days. With appropriate sample size and reaction time there was a linear relationship between enzyme concentration and activity.

Notes: Studies were conducted with radio-labeled indole-3-acetic acid ([2-14C] IAA) and tobacco callus culture (Nicotiana tabacum L. cv. White Gold) to investigate the mode of action of the herbicide glyphosate (N-phosphonomethylglycine). The tissue was first grown with or without glyphosate for 1 to 14 days and then incubated with [2-14C] IAA for 4 h. Metabolism of [2-14C] IAA in the tissue was studies by solvent fractionation, high performance liquid chromatography and liquid scintillation counting. The tissue grown with 0.2 mM glyphosate had low level of free [2-14C] IAA and high levels of other fractions containing metabolites and conjugates of the labeled IAA. After 1 day of glyphosate treatment the free [2-14C] IAA level in the tissue was reduced by 77% compared to that of the control; after 10 days of treatment the decrease was 96%. The decrease in the free [2-14C] IAA level was not due to inhibition of IAA uptake, but due to enhanced rates of oxidation and conjugate formation of IAA. The increased oxidation of IAA in the treated tissue was not due to a direct effect of glyphosate on IAA-oxidase since glyphosate was inactive on IAA oxidation in a cell-free system in vitro. The glyphosate-induced growth inhibition was partially overcome by addition of 1 μM 2,4-dichlorophenoxyacetic acid to the medium. The results lead to the conclusion that glyphosate inhibits growth by depletion of free IAA through rapid acceleration of both conjugate formation and oxidative degradation of IAA.

Notes: Metabolism of indole-3-acetic acid in soybean [Glycine max (L.) Merr.] was investigated with [1-14C]- and [2-14C]-indole-3-acetic acid (IAA) applied by injection into soybean hypocotyl sections and by incubation with soybean callus. Free IAA and its metabolites were extracted with 80% methanol and separated by high performance liquid chromatography with [3H]-IAA as an internal standard. Metabolism of IAA in soybean callus was much greater than that in tobacco (Nicotiana tabacum L.) callus used for comparison. High performance liquid chromatography of soybean extracts showed at least 10 metabolite peaks including both decarboxylated and undecarboxylated products. A major unstable decarboxylated metabolite was purified. [14C]-indole-3-methanol (IM) was three times more efficient than [2-14C]-IAA as substrate for producing this metabolite. It was hydrolyzable by β-glucosidase (EC 3.2.1.21), yielding an indole and D-glucose. The indole possessed characteristics of authentic IM. Thus, the metabolite is tentatively identified as indole-3-methanol-β-D-glucopyranoside. The results suggest that soybean tissues are capable of oxidizing IAA via the decarboxylative pathway with indole-3-methanol-glucoside as a major product. The high rate of metabolism of IAA may be related to the observed growth of soybean callus with high concentrations of IAA in the culture medium.

Notes: Four-day-old stem segments of Zea mays L. cv. Seneca 60 were treated sequentially with phenolic substances and indole-3-acetic [2-14C] acid ([2-14C]IAA). Formation of bound IAA was rapid, but a pretreatment with p-coumaric acid, ferulic acid or 4-methylumbelliferone decreased the level of bound IAA. The decrease is not likely related to the effect of the phenolics on enzymic oxidation of IAA, since the level of free IAA was not limiting and the activity of ferulic acid in enzymic oxidation of IAA is different from that of p-coumaric acid and 4-methylum-belliferone. Apparently these compounds inhibited the formation of bound IAA and consequently caused an accumulation of free IAA. In contrast, caffeic acid, protocatechuic acid and 2,3-dihydro-2, 2-dimethyl-7-benzofuranol had little effect.After the uptake of IAA there was a slow but steady incorporation of the radioactivity into the 80% ethanol-insoluble, 1 M NaOH-soluble fraction. Phenolic substances also affected this process. The compounds which are cofactors of IAA-oxidase increased the incorporation while those which are inhibitors of IAA-oxidase decreased the incorporation. Results suggest that the phenolics also affected the enzymic oxidation of IAA in vivo in the same way as in vitro.

Notes: Hydroxyhenzoic acids were tested for their effects on oxidation of the reduced nicotinamide adenine dinucleotide (NADH) in the absence of added H2O2 and Mn2* by an enzyme preparation from tobacco leaves (Nicotiana tabacum, var. White Gold). For comparison, a commercial horseradish peroxidase was also used. The rate of NADH oxidation was followed spectruphotometrically at 340 nm.Mono- and dihydroxybenzoic acids exerted significant effect on the rate of NADU oxidation, yet their effectiveness was determined by the number and position of the hydroxyl group on the ring. 4-Hydroxybenzoic acid was very effective in stimulating the reaction. Shifting the hydroxyl from the 4- to the 3-position and from the 3- to the 2-position decreased activity. 2,4- And 2,5-dihydroxybenzoic aeids were more active than the other dihydroxy-iscuners in stinulating oxidation of NADH. the dihydroxybenzoic acids with the hydroxyls in adjacent positions were less effective, and their activity was affected by other phenolic activators. In the presence of 4-hydroxybenzoic acid which enhanced oxidation of NADH, 2,4- and 2,5-dihydroxybenzoic acids further stimulated the reaction, but 3,4-, 2,3- and 2,6-dibydoxybenzoic acids were inhibitory. The inhibition by 3,4- and 2,3-dihydroxybenzoic aciils was non-competitive.The enzymes extracted by a L-cysteine-containing buffer showed lower NADH-oxidase activity.The enzyme preparation possessed peroxidase activity. The activity of NADH-oxidase inereased when H2O2 and Mi2* were present in addition to 4-hydroxy-benzoic acid. The effect of the position and number of hydroxyl substitution on the rate of NADH oxidation by borseradish peroxidase was also significant. This suggests the involvement of peroxidase in the NADH-oxidase system of tobacco leaves. However, a combination of the inactivated enzyme solution and active horseradish peroxidase with peroxidase activity equivalent to that of the enzyme preparation from tobacco leaves did not reconstitute the NADH-oxidase activity of tobacco leaves. This and other evidence suggests that the soluble NADH-oxidizing zyme system of tobacco leaves is more complicated than peroxidase.

Notes: Abstract A comparison study was conducted on the effect of glyphosate (N-[phosphonomethyl]glycine) on indole-3-[2-14C]acetic acid (IAA) metabolism, ethylene production, and growth of 7-day-old seedlings of different plants. The plants tested were American germander (Teucrium canadense L.), soybean (Glycine max L. Merr.), pea (Pisum sativum L. cv. Alaska and Little marvel), mungbean (Vigna radiata L.), and buckwheat (Fagopyrum esculentum Moench). A spray with 2 mM glyphosate affected IAA metabolism to a varied degree. The induced increase of IAA metabolism was greater in buckwheat, Alaska pea, and mungbean than soybean, Little marvel pea, and American germander. The increased IAA metabolism was correlated with the inhibition of growth and with the decrease of ethylene production. The natural rate of IAA metabolism was markedly different among the plant species and cultivars tested and appeared to be related to the sensitivity of the plants to glyphosate. American germander and Little marvel pea with high rates of IAA metabolism were more tolerant to glyphosate than buckwheat and Alaska pea, which had low rates of IAA metabolism. Plants with a high natural rate of IAA metabolism were probably less dependent on IAA and thus less susceptible to glyphosate.