Metabolism of diclofop-methyl in cell cultures of Avena sativa and Avena fatua

The metabolism of the herbicide, diclofop-methyl (methyl-2-[4-(2', 4'-dichlorophenoxy) phenoxy]propanoate), in cell suspension cultures of Avena sativa L. (cv. Garry) and in callus of Avena fatua L. (transferred to liquid) was determined as a function of time (8 h to about 3 weeks) and was compared to previous metabolism data from intact plants. A. fatua metabolized ¹⁴C-labeled diclofop-methyl more rapidly than A. sativa, but the metabolites formed were similar if not identical. Within 2 days, approximately 50% of the total ¹⁴C recovered was in A. fatua cells whereas less than 15% was in A. sativa cells. In older cultures of A. fatua, the amounts of ¹⁴C in the cells and in the medium were about 45% each; 10 to 12% was in the non-extractable cell residue. The ¹⁴C recovered from A. sativa cells increased to a maximum of about 35% at 7 days and then slowly decreased to about 18% by 21 days, whereas the ¹⁴C in the medium of A. sativa decreased to about 60% at 7 days and then increased to over 75% by 21 days. The nonextractable ¹⁴C residue was 5% or less even after 21 days. Major metabolites in methanolic extracts of cells of both A. sativa and A. fatua were diclofop (2-[4-(2', 4'-dichlorophenoxy)phenoxy] propanoate), diclofop hydroxylated at an undetermined position on the 2,4-dichlorophenyl ring (ring OH-diclofop), and conjugates of diclofop and ring-OH diclofop.     

Mode of action of glyphosate in relation to metabolism of indole-3-acetic acid

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 m M 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 μ M 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.     

A plasma membrane-bound enzyme oxidizes l-tryptophan to indole-3-acetaldoxime

The in vitro conversion of [¹⁴C]-tryptophan to [¹⁴C]-indole-3-acetaldoxime (IAOX) by microsomal membranes of Chinese cabbage (Brassica campestris ssp. pekinensis cv. Granat) has been studied. The reaction product was identified by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC). Furthermore. IAOX was identified as an endogenous compound of Chinese cabbage by mass spectroscopy. The tryptophan-oxidizing enzyme (TrpOxE) was characterized. MnCl₂ was required as cofactor, H₂O₂, and 2,4-dichlorophenol (DCP) stimulated the reaction. The enzyme showed a pH optimum at pH 8–9 and a K_m for l -tryptophan of 20 μ M . The membranes containing TrpOxE activity were identified as plasma membranes by means of aqueous polymer two-phase partitioning. The TrpOxE from Chinese cabbage was purified 3-fold from plasma membranes by solubilization followed by (NH₄)₂SO₄-fractionation, affinity-chromatography with concanavalin A, and native gel electrophoresis. Enzyme activity was reduced by a tunicamycin pretreatment. Several other plant species, e.g. maize (Zea mays L. Inrakorn), sunflower (Helianthus annuus L. cv. Hohes Sonnengold), tobacco (Nicotiana tabacum L. cv. White Burley), and pea (Pisum sativum L. cv. Krombeck) showed a similar conversion of [¹⁴C]-tryptophan to [¹⁴C]-IAOX by phase-partitioned plasma membranes.     

Maternal effects in progeny of field pansy (Viola arvensis) subjected to different herbicide and nitrogen rates

Observations were made on the effects of reduced rates of herbicide and nitrogen on naturally occurring populations of Viola aruensis. Progeny arising from these plants were grown in a uniform environment and monitored through to maturity. The size and number of reproductive structures produced by the maternal plants were positively correlated with the maternal plant weight. Herbicide dose affected the plant height of offspring and the effects were more pronounced in those from plants which had received 160 kg N ha^-1than 40 kg N ha^-1. Although the maternal effects on offspring size diminished with time, the number of reproductive structures in the offspring was significantly correlated with plant height during early development. Increased nitrogen availability to the maternal plant in the absence of herbicide may therefore increase the overall productivity of the subsequent generation.     

Metabolism of Cysteine in Astroglial Cells: Synthesis of Hypotaurine and Taurine

Abstract: The synthesis of hypotaurine and taurine was investigated in astroglia-rich primary cultures obtained from brains of neonatal Wistar rats using ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. Cell extracts of astroglial cultures analyzed by ¹H NMR spectroscopy show prominent signals of hypotaurine. To identify cysteine as precursor for hypotaurine and taurine synthesis in astroglial cells, primary cultures were incubated with [3-¹³C]cysteine for 24 or 72 h. Cell extracts and incubation media were then analyzed with ¹³C NMR spectroscopy. Labeled hypotaurine, taurine, glutathione, and lactate were identified in the cell extracts. Within 72 h, 35.0% of the total intracellular hypotaurine and 22.5% of taurine were newly synthesized from [3-¹³C]cysteine. The presence of [1-¹³C]hypotaurine and [1-¹³C]taurine in the incubation medium proves the release of those products of cysteine metabolism into the medium. Minor amounts of the [3-¹³C]cysteine were used for the synthesis of glutathione in astroglial cells or metabolized to [3-¹³C]lactate, which was found in cell extracts and media. These results indicate that the formation of hypotaurine and taurine is a major pathway of cysteine metabolism in astroglial cells.     

Comparative studies of sucrose and mannitol utilization in celery (Apium graveolens)

Utilization of sucrose and mannitol, the major forms of translocatable assimilate in celery ( Apium graveolens L. cv. Giant Pascal), was investigated in intact plants, excised leaves and leaf discs by estimating the soluble carbohydrate pools, starch levels and oxidation of [¹⁴C]-sucrose or mannitol in the light and after extended dark treatments. In detached mature fully-expanded leaves, mannitol pools remained constant, while sucrose decreased during a 48 h dark treatment. In attached leaves on plants trimmed to a single compound leaf, however, mannitol levels decreased after a dark treatment. In leaf discs floated on bathing solutions containing [¹⁴C]-sucrose or [¹⁴C]-mannitol, oxidation of mannitol was restricted to young leaf tissues, whereas sucrose was metabolized to CO₂ regardless of leaf age. Uptake of labelled mannitol, however, was greater than that of sucrose in the light in leaves of every age. Although both mannitol and sucrose are translocated out of leaf tissues, leaf age differences indicate that, unlike sucrose, mannitol utilization is restricted to active sink tissues. The results suggest different roles for mannitol and sucrose with mannitol representing a more rigorously sequestered transport carbohydrate.     

Metabolic fate of nicotinamide in higher plants

Metabolism of [carbonyl-¹⁴C]nicotinamide was surveyed in various plant materials including the model plants, Arabidopsis thaliana , Oryza sativa and Lotus japonicus . In all plants studied, nicotinamide was used for the pyridine (nicotinamide adenine) nucleotide synthesis, probably after conversion to nicotinic acid. Radioactivity from [carbonyl-¹⁴C]nicotinamide was incorporated into trigonelline (1- N -methylnicotinic acid) and/or into nicotinic acid 1 N -glucoside (Na-Glc). Trigonelline is formed mainly in leaves and cell cultures of O. sativa and L. japonicus and in seedlings of Trifolium incarnatum , Medicago sativa and Raphanus sativus . Trigonelline synthesis from nicotinamide is generally greater in leaves than in roots. Na-Glc was formed as the major nicotinic acid conjugate in A. thaliana and in tobacco Bright Yellow-2 cells. In seedlings of Chrysanthemum coronarium and Theobroma cacao , both trigonelline and Na-Glc were synthesized from [carbonyl-¹⁴C]nicotinamide. Trigonelline is accumulated in some seeds, mainly Leguminosae species. The pattern of formation of the nicotinic acid conjugates differs between species and organs.     

Effects of desiccation on the 77°K fluorescence properties of the liverwort Porella navicularis and the isolated lichen green alga Trebouxia pyriformis

The uptake of the auxin type herbicide 2,4-D into rice seedlings ( Oryza sativa L. cv. Dunghan Shali) and its effects on the K⁺, NH⁺₄ and NO₃ ion uptake and the K⁺ content were investigated at different pH values. A short incubation of the roots in 0.01 m M 2,4-D caused a marked ion uptake inhibition only at low pH. The non-auxin type herbicide benthiocarb did not produce such an inhibitory effect. Lowering of the pH in the external medium led to an increased 2,4-D uptake by the roots. These results can be explained by the increased H⁺ permeability of the membranes, allowing a more rapid entrance of 2,4-D into the root cells, thereby inhibiting the active ion uptake. Rice roots not subjected to 2,4-D treatment responded to H⁺ stress with an increased anomalous K⁺ uptake and a decreased K⁺ content. With reference to the effects of pH changes on the ion and 2,4-D uptake, possible transport mechanism of NH⁺₄ and 2,4-D are briefly discussed.     

Evaluation of the in vitro genetic toxicity of 4-(2, 4-dichlorophenoxy)butyric acid

The herbicide 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB) is principally used in the USA on peanuts, soybeans and alfalfa. In Europe, it is used on undersown spring barley and grassland (with clover). The genetic toxicity in vitro of the dimethylamine salt of 2,4-DB was examined by employing a range of end points including gene mutation in bacteria (Ames test) and mammalian cell cultures (CHO/HGPRT assay), cytogenetic abnormalities in mammalian cells (CHO/chromosomal aberration assay), and induction of DNA damage and repair in rat hepatocytes. There were no indications of genotoxic potential for 2,4-DB in the first three of these assays. One of the two criteria for a positive response in the UDS assay was exceeded but the increases did not exceed the second criteria for a positive response. The test material was therefore evaluated as weakly active in this assay. The weight of the evidence clearly indicates that 2, 4-DB is not genotoxic to mammals and are consistent with the reported lack of carcinogenic potential for 2,4-DB in both mice and rats.     

Exogenous Glutamate Concentration Regulates the Metabolic Fate of Glutamate in Astrocytes

Abstract: The metabolic fate of glutamate in astrocytes has been controversial since several studies reported >80% of glutamate was metabolized to glutamine; however, other studies have shown that half of the glutamate was metabolized via the tricarboxylic acid (TCA) cycle and half converted to glutamine. Studies were initiated to determine the metabolic fate of increasing concentrations of [U-¹³C]glutamate in primary cultures of cerebral cortical astrocytes from rat brain. When astrocytes from rat brain were incubated with 0.1 m M [U-¹³C]glutamate 85% of the ¹³C metabolized was converted to glutamine. The formation of [1,2,3-¹³C₃]glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. When astrocytes were incubated with 0.2–0.5 m M glutamate, ¹³C from glutamate was also incorporated into intracellular aspartate and into lactate that was released into the media. The amount of [¹³C]lactate was essentially unchanged within the range of 0.2–0.5 m M glutamate, whereas the amount of [¹³C]aspartate continued to increase in parallel with the increase in glutamate concentration. The amount of glutamate metabolized via the TCA cycle progressively increased from 15.3 to 42.7% as the extracellular glutamate concentration increased from 0.1 to 0.5 m M , suggesting that the concentration of glutamate is a major factor determining the metabolic fate of glutamate in astrocytes. Previous studies using glutamate concentrations from 0.01 to 0.5 m M and astrocytes from both rat and mouse brain are consistent with these findings.     

Effect of carbon dioxide enrichment on chlorophyll content, starch content and starch grain structure in Trifolium subterraneum leaves

Trifolium subterraneum (cv. Dinninup) responds to enriched atmospheric CO₂ in a manner similar to that described by Madsen (1968 and 1976) for tomato. In immature leaves, the total chlorophyll content per unit dry weight and the chlorophyll a:b ratio are significantly lower in plants grown at 0.10 vol% CO₂. Although fully expanded mature leaves partially overcome the deficit in chlorophyll content, the chlorophyll a:b ratio remains substantially lower in these high CO₂ grown plants. The large amount of starch accumulated as irregularly shaped grains appears to disrupt normal chloroplast structure in clover plants grown in enriched atmospheric CO₂. These results indicate the chlorotic appearance of leaves from high CO₂ grown clover plants is due to a decrease in chlorophyll content per dry weight possibly resulting from large starch grains and starch accumulation altering normal chloroplast structure and function.     

Photoinhibition of white clover seed germination at low water potential

Photosensitivity of germination of white clover ( Trifolium repens L. cv. Podkowa) seeds was studied under water deficit (low water potential) conditions at 25°C. The seeds showed negative photoblastism, which was most pronounced at -0.03 MPa polyethylene glycol solution. Inhibition was observed at two different wavelength bands with maxima at 660 nm (R) and around 730 nm (FR). Red light acted identically to white light (maximum inhibition ca 50%). The effect of far-red illumination was less inhibitory (20–30%). The photoresponse required long illuminations (3 h exposures); saturation level was at 0.1 W m⁻², independently of the light quality. White clover seed germination showed no reversibility of the effects of R and FR light. Prolonged illumination with R and FR increased the inhibition, and intermittent illumination had a higher effect than a continuous one. It was concluded that the photoinhibition of germination of seeds of Trifolium repens involves a reaction dependent on the rate of phytochrome interconversion, a property that is characteristic for the high irradiance reaction.     

Incorporation of label from root-applied N6[8-14C]furfiuryIadenine into the guanine nucleotide fraction of pea bud ribonucleic acid

The pattern of incorporation of label into the nucleotides of axillary bud ribonucleic acid was investigated in Pisum sativum L. cv. Meteor following the application of N ⁶[8-^I4C]furfuryladenine or of [8-¹⁴C]adenine to the root system of decapitated plants and to cultured excised buds. When N ⁶[8-¹⁴C]furifaryladenine was applied to the root system label was confined to the guanine nucleotide moiety of the axillary bud ribonucleic acid; label from [8-¹⁴C]adenine was incorporated preferentially into adenine nucleotide in the molar ratio adenine nucleotide/guanine nucleotide = 3.23. When isolated buds were incubated in media containing [8-¹⁴C]adenine or N ⁶[8-¹⁴C]furfuryladenine, label was incorporated into both purine moieties of the ribonucleic acid. However, the relative incorporation into the guanine nucleotide fraction was considerably greater for N ⁶[8-^I4C]furfuryladenine (adenine nucleotide/guanine nucleotide = 2.23) than for [8-¹⁴C]adenine (ratio = 4.67).
It was concluded that the pattern of metabolism of adenine to guanine and its incorporation into the guanine nucleotide moiety of pea axillary bud ribonucleic acid, is influenced by the presence of a substitution in the N ⁶ position of the adenine base.     

Degradation of 2,4-DB in Argentinean agricultural soils with high humic matter content

The dissipation of 4-(2,4-dichlorophenoxy) butyric acid (2,4-DB) in high-humic-matter-containing soils from agricultural fields of the Argentinean Humid Pampa region was studied, employing soil microcosms under different experimental conditions. The added herbicide was dissipated almost completely by soils with and without history of herbicide use by day 28. At 500 ppm, both soils showed the same degradation rates; but at 5-ppm concentration, the chronically exposed soil demonstrated a faster degradation of the herbicide. 2,4-DB addition produced increases in herbicide-degrading bacteria of three and 1.5 orders of magnitude in soils with and without history of herbicide use, respectively, in microcosms with 5 ppm. At 500-ppm concentration, the increase in 2,4-DB degraders was five orders of magnitude after 14 days, independent of the history of herbicide use. No differences were observed in either 2,4-DB degradation rates or in degrader bacteria numbers in the presence and absence of alfalfa plants, in spite of some differential characteristics in patterns of 2,4-DB metabolite accumulation. The main factor affecting 2,4-DB degradation rate would be the history of herbicide use, as a consequence of the adaptation of the indigenous microflora to the presence of herbicides in the field.     

Inhibiton of fatty acid biosynthesis in isolated chloroplasts by cycloxydim and other cyclohexane-1,3-diones

Kobek, K., Focke, M., Lichtenthaler, H.K., Retzlaff, G. and Würzer, B. 1988. Inhibiton of fatty acid biosynthesis in isolated chloroplasts by cycloxydim and other cyclohexane-1,3-diones. - Physiol. Plant. 72: 492–498.
The effect of the three cyclohexane-1,3-dione herbicides cycloxydim, sethoxydim and clethodim (proposed common name) on the de novo fatty acid biosynthesis of isolated chloroplasts as test system was investigated with intact chloroplasts isolated from sensitive grasses (Poaceae) and tolerant dicotyledonous plants. All three herbicides blocked the de novo fatty acid biosynthesis ([¹⁴C]-acetatc incorporation into total fatty acid fraction) in Avena sativa L. cv. Flämingnova chloroplasts in a dose-dependent manner. The I₅₀-values are lower for cycloxydim and clethodim than for sethoxydim. The rate of de novo fatty acid biosynthesis in isolated, intact and photosynthetically active Avena chloroplasts was higher in the light than in the dark, which appeared to be due to the light-dependent regeneration of the cofactors ATP and NADPH. The de novo fatty acid biosynthesis by isolated chloroplasts from the tolerant dicotyledonous species pea ( Pisum savivum L. cv. Kleine Rheinländerin), spinach ( Spinacea oleracea L. cv. Matador) and tobacco ( Nicotiana tabacum L. cv. su/su) was insensitive to the three herbicides. It is assumed that one of the enzymes of the fatty acid biosynthesis is modified in the dicotyledonous plants and not accessible to the cyclohexane-1,3-dione herbicides. In the case of Poa annua L., which as a whole plant is tolerant towards sethoxydim, the tolerance seems not to lie in the chloroplasts but in properties of the cytoplasm, since the isolated chloroplasts are sensitive to the herbicide.     

Effects of Hormones on Morphogenesis and Cold Resistance in Berseem Clover (Trifolium alexandrinum L.)

Plants of berseem clover (Trifolium alexandrinum L.) cv. Taborwere raised under conditions inhibiting the acquisition of coldhardiness (non-hardened) or inducing cold hardiness (hardened).All non-hardened plants developed an elongated shoot and exhibitedconsiderable frost sensitivity, as measured by the extent ofthe reduction in yield of variable chlorophyll fluorescenceafter exposure to sub-zero temperature. Hardened plants developeda shorter shoot, with fewer leaves and a greater percentageof dry matter in the root system. These parameters were associatedwith a marked increase in frost resistance. Exogenous applicationof ABA to plants effected similar morphological modificationsin both hardening and non-hardening temperature regimes; plantsdeveloped a shorter primary shoot axis and leaves exhibiteda marked increase in frost hardiness. In berseem clover ABAcan thus substitute, at least partially, for the low temperaturetreatment required to induce cold hardiness. Spraying plantsraised under hardening conditions with gibberellic acid reversedthe effects of the hardening treatment, since they developedan elongated shoot and exhibited frost sensitivity comparableto non-treated plants grown under non-hardening conditions.It is concluded that these endogenous hormones are directlyinvolved in triggering changes in morphogenesis which accompanyphysiological and metabolic events associated with the inductionof plant cold hardiness. Key words: Frost resistance, morphogenesis, abscisic acid, giberellic acid, Trifolium alexandrinum     

Distribution and metabolism of root-applied cytokinins in Pisum sativum

When N ⁶ [8–¹⁴C] furfuryladenine was applied to the intact root system of Pisum sativum L. cv. Meteor seedlings it was almost completely metabolised to other compounds within 24 h. Of the total activity recovered from the plants 94.5% was retained in the root system itself. ¹⁴C was recovered in a number of ethanol-soluble compounds and in ribonucleic acid, deoxyribonucleic acid and protein fractions of roots, stems, leaves and axillary buds. In rapidly growing axillary buds released from apical dominance by removal of the shoot apex the combined nucleic acid fractions accounted for 63.3% of the total ¹⁴C recovered from these organs. Xylem exudate collected from decapitated plants 0 to 12 h after supplying N ⁵[8–¹⁴C]furfuryladenine to the roots consistently contained a single major ¹⁴C-labelled compound which, in three different solvent systems, had the same Rf values as a major endogenous cytokinin isolated from the xylem of unlabelled plants. The content of N ⁶ [8–¹⁴C] furfuryladenine itself in the xylem exudate was always low and in some experiments it could not be detected.
It is suggested that part of the label from N ⁶ [8- ¹⁴CJfurfuryladenine taken up by the intact root system may have become incorporated in an endogenous cylokinin before export to the shoot.     

Availability of neurotransmitter glutamate is diminished when β-hydroxybutyrate replaces glucose in cultured neurons

Ketone bodies serve as alternative energy substrates for the brain in cases of low glucose availability such as during starvation or in patients treated with a ketogenic diet. The ketone bodies are metabolized via a distinct pathway confined to the mitochondria. We have compared metabolism of [2,4-¹³C]β-hydroxybutyrate to that of [1,6-¹³C]glucose in cultured glutamatergic neurons and investigated the effect of neuronal activity focusing on the aspartate–glutamate homeostasis, an essential component of the excitatory activity in the brain. The amount of ¹³C incorporation and cellular content was lower for glutamate and higher for aspartate in the presence of [2,4-¹³C]β-hydroxybutyrate as opposed to [1,6-¹³C]glucose. Our results suggest that the change in aspartate–glutamate homeostasis is due to a decreased availability of NADH for cytosolic malate dehydrogenase and thus reduced malate–aspartate shuttle activity in neurons using β-hydroxybutyrate. In the presence of glucose, the glutamate content decreased significantly upon activation of neurotransmitter release, whereas in the presence of only β-hydroxybutyrate, no decrease in the glutamate content was observed. Thus, the fraction of the glutamate pool available for transmitter release was diminished when metabolizing β-hydroxybutyrate, which is in line with the hypothesis of formation of transmitter glutamate via an obligatory involvement of the malate–aspartate shuttle.     

Isoprenoid biosynthesis in bacteria: Two different pathways?

Abstract The biosynthesis of isopentenylpyrophosphate, a central intermediate of isoprenoid formation, was investigated in six different bacterial organisms. Cell-free extracts of Myxococcus fulvus, Staphylococcus carnosus, Lactobacillus plantarum and Halobacterium cutirubrum converted [¹⁴C]acetyl-CoA or [¹⁴C]hydroxymethylglutaryl-CoA to [¹⁴C]mevalonic acid. Furthermore, [¹⁴C]mevalonic acid, [¹⁴C]mevalonate-5-phosphate and [¹⁴C]mevalonate-5-pyrophosphate were metabolized to [¹⁴C]isopentenylpyrophosphate in bacteria. In contrast, no intermediates of this reaction sequence could be detected using cell-free extracts of Zymomonas mobilis and Escherichia coli . These results indicate that at least two different pathways for the biosynthesis of isopentenylpyrophosphate are present in bacteria.