Identification of the metabolites of Indole-3-acetic acid in growing hypocotyls of Lupinus albus

The products of indole-3-acetic acid (IAA) metabolism by incubating hypocotyl sections and decapitated seedlings of Lupinus albus were investigated. Single treatments using [1-¹⁴C]-IAA, [2-¹⁴C]-IAA or [5-³H]-IAA and double treatments using [1-¹⁴C]-IAA+[5-³H]-IAA were carried out. Extracts from treated plant material were analyzed by paper chromatography (PC), Thin layer chromatography (TLC), and high performance liquid chromatography (HPLC). When hypocotyl sections were incubated in [2-¹⁴C]-IAA, several IAA decarboxylation products including indole-3-aldehyde (IA1), indole-3-methanol (IM), 3-hydroxymethyloxindole (HMOx), methyleneoxindole (MOx) and 3,3-bisindolylmethane (BIM) were detected in the 95% ethanol extract; a latter extraction with 1M NaOH rendered IAA, IM and BIM, suggesting that conjugated auxins were formed in addition to conjugated IM. In sections incubated with [1-¹⁴C]-IAA, the 1M NaOH extraction also produced IAA so confirming the formation of conjugated auxins. The same decarboxylation products and two conjugated auxins, indole-3-acetylaspartic acid (IAAsp) and 1-O-(indole-3-acetyl)--D-glucose (IAGlu), were detected in the acetonitrile extracts from decapitated seedlings treated with [5-³H]-IAA. After a double isotope treatment ([1-¹⁴C]-IAA+[5-³H]-IAA) of decapitated seedlings, the ratio ¹⁴C/³H measured in the HPLC fractions of the acetonitrile extracts confirmed the presence of decarboxylation products as well as conjugated auxins.     

Effects of auxins,cytokinins, carbohydrates and amino acids on somatic embryogenesis induction from shoot apices of pea

Studies were conducted to test the effects of various auxins, cytokinins, carbohydrates and amino acids on somatic embryogenesis from shoot apices of pea (Pisum sativum L.) cultured on a sole medium. Picloram (4.5 M) and 4-chlorophenoxyacetic acid (45 M) were the most effective auxins. Addition of cytokinins (benzyladenine, zeatin, kinetin) to auxin-containing medium reduced embryo production. Amino acids (glutamine, alanine, proline) did not improve somatic embryogenesis. Carbohydrate seemed to be a critical factor. Embryogenic efficiency and embryo development were promoted by high carbohydrate concentration. The best results were obtained with fructose (252–504 mM); the number of somatic embryos per cultured explant was 3- to 4-fold higher compared to the control (84 mM sucrose). From these results, an optimized induction medium is proposed.Abbreviations BA benzyladenine - 4-CPA 4-chlorophenoxyacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - MS Murashige & Skoog - EE embryogenic explants - G globular somatic embryos     

Auxin binding to corn coleoptile membranes: Kinetics and specificity

Summary Detailed examination of binding over the range 10^-7–10^-6 M suggests that membrane preparations from coleoptiles of Zea mays L., cv Kelvedon 33 contain at least two sets of high affinity binding sites for 1-naphthylacetic acid (NAA), with dissociation constants of 1.8×10^-7 M (site 1) and 14.5×10^-7 M (site 2). Similar studies with 3-indolylacetic acid (IAA) also indicate two sets of binding sites, whose concentrations are closely comparable to those deduced for NAA. A substantial proportion of the total binding activity is retained in a detergent-solubilized preparation. Using [¹⁴C]NAA the interactions of a range of analogues with each of the binding sites have been examined with the aid of double reciprocal plots. The specificity of site 2 is compatible with that expected for an auxin receptor, in that only active auxins, antiauxin transport inhibitors are able to compete with [¹⁴C]NAA for the binding sites. Site 1 on the other hand is less specific, since it appears to bind all compounds tested, including physiologically inactive analogues.Abbreviations NAA 1-naphthylacetic acid - IAA 3-indolylacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,6-D 2,6-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - 2-CPIB -(2-chlorophenoxy)-isobutyric acid - 2,4-B 2,4-dichlorobenzoic acid - 2,6-B 2,6-dichlorobenzoic acid - TIBA 2,3,5-triiodobenzoic acid - NPA 1-N-naphthylphthalamic acid     

l-Ascorbic-acid biosynthesis in the euryhaline diatom Cyclotella cryptica

l-Ascorbic acid (AA) production in cells of Cyclotella cryptica Reimann, Lewin, Guillard (Bacillariophyceae) is enhanced when darkadapted cells are exposed to light.Heterotrophically grown cells incubated with d-[6-³H,6-¹⁴C]glucose and d-[1-³H,6-¹⁴C]glucose (2 h in dark followed by 15 h light) produced labeled AA with significantly different ratios of ³H and ¹⁴C. Comparisons of labeling patterns in AA and chitin-derived d-glucosamine support a path of conversion in Cyclotella from d-glucose to AA that inverts the carbon chain of the sugar. This process resembles similar conversions found in AA-synthesizing animals and species from two other algal classes.Abbreviations AA l-Ascorbic acid - glc d-glucose - glcN d-glucosamine     

Acifluorfen Enhancement of Cryptochrome-Modulated Sporulation following an Inductive Light Pulse

Acifluorfen enhancement of blue-light mediated phototropism suggested that this diphenyl-ether herbicide augments the light reaction (TY Leong, WR Briggs 1983 Plant Physiol 70: 875-881). The separation of the possible direct interaction of acifluorfen with light reactions from interactions with dark pathways has been elucidated in this paper with Trichoderma harzianum. Acifluorfen at 30 micromolar, given for 5 hours in the growth medium, stimulated the conidiation of Trichoderma in response to blue light without apparently affecting growth. Enhanced conidiation could be elicited by dipping cultures into medium with acifluorfen both before as well as 0.5 hour after inductive blue light. This postphotoinduction stimulation indicates that acifluorfen does not directly augment the effect of light by interacting with cryptochrome(s) in Trichoderma. Instead, acifluorfen most probably interacted with the dark reactions following photoinduction.     

The specificity of auxin transport in intact pea seedlings (Pisum sativum L.)

Summary When eight ¹⁴C-labelled auxin and non-auxin compounds were applied to the apical buds of intact dwarf pea seedlings (Pisum sativum L.), only [1-¹⁴C]indoleacetic acid ([¹⁴C]IAA) and -[1-¹⁴C] naphthaleneacetic acid ([¹⁴C]NAA) underwent appreciable basipetal transport during the first 24 h; over a longer period (72 h) considerable basipetal transport of the auxin [1-¹⁴C]2,4-dichlorophenoxyacetic acid ([¹⁴C]2,4-D) also occurred, but at a very much lower velocity (ca. 1.4–2.2 mm·h^-1). The movement of 2,4-D possessed many of the characteristics of a typical auxin transport. During uptake and transport IAA and NAA were extensively metabolised to the corresponding aspartates, and to ethanol-insoluble/NaOH-soluble compounds; little metabolism of 2,4-D was observed. None of the non-auxin compounds applied (sorbose, sucrose, leucine, adenine and kinetin) underwent appreciable basipetal transport from the apical bud. All but sorbose were extensively metabolised by the apical tissues. Little metabolism of sorbose itself was detected.The results suggest that the long-distance basipetal auxin transport system from the apical bud of intact plants is specific for auxins; the specificity may result from the affinity of auxins for specific transport sites.     

Plant regeneration from shoot apex explants of foxtail millet

Green and etiolated shoot apices of foxtail millet (Setaria italica L.) cv. Nese 2A were cultured on Murashige and Skoog medium with four concentrations of 2,4-dichlorophenoxyacetic acid or 2,4,5-trichlorophenoxyacetic acid. In all treatments, embryogenic calli capable of plant regeneration were induced after ten weeks in culture. Calli induced on 2 mg l^-1 of 2,4-d from green apices gave a higher rate of plant regeneration in comparison with etiolated apices on the other treatments. Plant regeneration was obtained from one year-old cultures. Regenerated plants were successfully established in soil, reached maturity and produced seeds.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - EC embryogenic calli - NE nonembryogenic calli - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

Applicability of the chemiosmotic polar diffusion theory to the transport of indol-3yl-acetic acid in the intact pea (Pisum sativum L.)

The transport of exogenous indol-3yl-acetic acid (IAA) from the apical tissues of intact, light-grown pea (Pisum sativum L. cv. Alderman) shoots exhibited properties identical to those associated with polar transport in isolated shoot segments. Transport in the stem of apically applied [1-¹⁴C]-or [5-³H]IAA occurred at velocities (approx. 8–15 mm·h^-1) characteristic of polar transport. Following pulse-labelling, IAA drained from distal tissues after passage of a pulse and the rate characteristics of a pulse were not affected by chases of unlabelled IAA. However, transport of [1-¹⁴C]IAA was inhibited through a localised region of the stem pretreated with a high concentration of unlabelled IAA or with the synthetic auxins 1-napthaleneacetic acid and 2,4-dichlorophenoxyacetic acid, and label accumulated in more distal tissues. Transport of [1-¹⁴C]IAA was also completely prevented through regions of the intact stem treated with N-1-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid.Export of IAA from the apical bud into the stem increased with total concentration of IAA applied (labelled+unlabelled) but approached saturation at high concentrations (834 mmol·m^-3). Transport velocity increased with concentration up to 83 mmol·m^-3 IAA but fell again with further increase in concentration.Stem segments (2 mm) cut from intact plants transporting apically applied [1-¹⁴C]IAA effluxed 93% of their initial radioactivity into buffer (pH 7.0) in 90 min. The half-time for efflux increased from 32.5 to 103.9 min when 3 mmol·m^-3 NPA was included in the efflux medium. Long (30 mm) stem sections cut from immediately below an apical bud 3.0 h after the apical application of [1-¹⁴C]IAA effluxed IAA when their basal ends, but not their apical ends, were immersed in buffer (pH 7.0). Addition of 3 mmol·m^-3 NPA to the external medium completely prevented this basal efflux.These results support the view that the slow long-distance transport of IAA from the intact shoot apex occurs by polar cell-to-cell transport and that it is mediated by the components of IAA transmembrane transport predicted by the chemiosmotic polar diffusion theory.Abbreviations IAA indol-3yl-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - NPA N-1-naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid     

Studies on the evolution of auxin carriers and phytotropin receptors: Transmembrane auxin transport in unicellular and multicellular Chlorophyta

The characteristics of transmembrane transport of ¹⁴C-labelled indol-3yl-acetic acid ([1-¹⁴C]IAA) were compared in Chlorella vulgaris Beij., a simple unicellular green alga, and in Chara vulgaris L., a branched, multicellular green alga exhibiting axial polarity and a high degree of cell and organ specialization. In Chara thallus cells, three distinguishable trans-plasmamembrane fluxes contributed to the net uptake of [1-¹⁴C]-IAA from an external solution, viz.: a non-mediated, pH-sensitive influx of undissociated IAA (IAAH); a saturable influx of IAA; and a saturable efflux of IAA. Both saturable fluxes were competitively inhibited by unlabelled IAA. Association of [³H]IAA with microsomal preparations from Chara thallus tissue was competitively inhibited by unlabelled IAA. Results indicated that up-take carriers occurred in the membranes at a much higher density than efflux carriers. The efflux component of IAA net uptake by Chara was not affected by several phytotropins (N-1-naphthylphthalmic acid, NPA; 2-(1-pyrenoyl)benzoic acid; and 5-(2-carboxyphenyl)-3-phenylpyrazole), which are potent non-competitive inhibitors of specific auxin-efflux carriers in more advanced plant groups, and no evidence was found for a specific association of [³H]NPA with Chara microsomal preparations. It was concluded that Chara lacked phytotropin receptors. Net uptake of [1-¹⁴C]IAA also was unaffected by 2,3,5-triiodobenzoic acid except at concentrations ( 10^–1 mol · m^–3) high enough to depress cytoplasmic pH (determined by uptake of 5,5-dimethyloxazolidine-2,4-dione). Chlorella cells accumulated [1-¹⁴C]IAA from an external solution by pH-sensitive diffusion of IAA across the plasma membrane and anion (IAA^–) trapping, but no evidence was found in Chlorella for the occurrence of IAA carriers. These results indicate that carrier systems capable of mediating the transmembrane transport of auxins appeared at a very early stage in the evolution of green plants, possibly in association with the origin of a differentiated, multicellular plant body. Phytotropin receptors evolved independently of the carriers.Abbreviations CPP 5-(2-carboxyphenyl)-3-phenylpyrazole - DMO 5,5-dimethyloxazolidine-2,4-dione - IAA indol-3yl-acetic acid - NPA N-1-naphthylphthalamic acid - PBA 2-(1-pyrenoyl)benzoic acid - TIBA 2,3,5-triiodobenzoic acid We thank the Nuffield Foundation for the award of an Undergraduate Research Bursary to J.E.D.-F., Dr. G.F. Katekar, C.S.I.R.O., Canberra, Australia for generous gifts of phytotropins, and Mrs. R.P. Bell for technical support.     

Regulation of auxin transport in pea (Pisum sativum L.) by phenylacetic acid: inhibition of polar auxin transport in intact plants and stem segments

The transport of [¹⁴C]phenylacetic acid (PAA) in intact plants and stem segments of light-grown pea (Pisum sativum L. cv. Alderman) plants was investigated and compared with the transport of [¹⁴C]indiol-3yl-acetic acid (IAA). Although PAA was readily taken up by apical tissues, unlike IAA it did not undergo long-distance transport in the stem. The absence of PAA export from the apex was shown not to be the consequence of its failure to be taken up or of its metabolism. Only a weak diffusive movement of PAA was observed in isolated stem segments which readily transported IAA. When [1-¹⁴C]PAA was applied to a mature foliage leaf in light, only 5.4% of the ¹⁴C recovered in ethanol extracts (89.6% of applied ¹⁴C) had been exported from the leaf after 6.0 h. When applied to the corresponding leaf, [¹⁴C]sucrose was readily exported (46.4% of the total recovered ethanol-soluble ¹⁴C after 6.0 h). [1-¹⁴C]phenylacetic acid applied to the root system was readily taken up but, after 5.0 h, 99.3% of the recovered ¹⁴C was still in the root system.When applied to the stem of intact plants (either in lanolin at 10 mg·g^-1, or as a 10^-4 M solution), unlabelled PAA blocked the transport through the stem of [1-¹⁴C]IAA applied to the apical bud, and caused IAA to accumulate in the PAA-treated region of the stem. Applications of PAA to the stem also inhibited the basipetal polar transport of [1-¹⁴C]IAA in isolated stem segments. These results are consistent with recent observations (C.F. Johnson and D.A. Morris, 1987, Planta 172, 400–407) that no carriers for PAA occur in the plasma membrane of the light-grown pea stem, but that PAA can inhibit the carrier-mediated efflux of IAA from cells. The possible functions of endogenous PAA are discussed and its is suggested that an important role of the compound may be to modulate the polar transport and-or accumulation by cells of IAA.Abbreviations IAA indol-3yl-acetic acid - NPA N-1-naphthylphthalamic acid - PAA phenylacetic acid - IIBA 2,3,5-triiodobenzoic acid     

Citrus Tissue Culture : AUXINS IN RELATION TO ABSCISSION IN EXCISED PISTILS

An in vitro bioassay for chemicals that affect Citrus abscission was used to identify three inhibitors of stylar abscission in lemon pistil explants incubated on defined nutrient media. The three inhibitors (picloram, 4-chlorophenoxyacetic acid, and 3,5,6-trichloropyridine-2-oxyacetic acid) are all auxins, and the most potent of them (i.e. picloram) was found to be at least 10 times more active in the bioassay than 2,4-dichlorophenoxyacetic acid. Picloram (2 micromolar) also was shown to be effective in inhibiting stylar abscission in pistil explants from other Citrus cultivars such as mandarin, Valencia, and Washington navel oranges and grapefruit. To study the physiology of auxins active as abscission inhibitors versus inactive auxins in lemon pistils, the transport and metabolism of [1-¹⁴C]-2,4-dichlorophenoxyacetic acid was compared with that of [2-¹⁴C]indole-3-acetic acid, which is without effect in the bioassay over the range from 0.1-100 micromolar. Insignificant quantities of labeled indole-3-acetic acid and/or labeled derivatives were found to reach the presumptive zone of stylar abscission under the test conditions. Labeled 2,4-dichlorophenoxyacetic acid and/or labeled derivatives also were transported slowly through pistils, but some radioactivity could be detected in the stylar abscission zone as early as 24 hours after the start of incubation. Extensive conversion of [2-¹⁴C]indole-3-acetic acid to labeled compounds tentatively considered to be glycoside and cellulosic glucan derivatives was found with the use of solvent extraction methodology. A significantly smaller percentage of the radioactivity in pistils incubated on [1-¹⁴C]-2,4-dichlorophenoxyacetic acid was found in fractions corresponding to these derivatives. Both transport and metabolism appear to be important factors affecting the activity of auxins as abscission inhibitors in the bioassay.     

Effects of Acifluorfen on Endogenous Antioxidants and Protective Enzymes in Cucumber (Cucumis sativus L.) Cotyledons

The herbicide acifluorfen (2-chloro-4-(trifluoromethyl)phenoxy-2-nitrobenzoate) causes strong photooxidative destruction of pigments and lipids in sensitive plant species. Antioxidants and oxygen radical scavengers slow the bleaching action of the herbicide. The effect of acifluorfen on glutathione and ascorbate levels in cucumber (Cucumis sativus L.) cotyledon discs was investigated to assess the relationship between herbicide activity and endogenous antioxidants. Acifluorfen decreased the levels of glutathione and ascorbate over 50% in discs exposed to less than 1.5 hours of white light (450 microeinsteins per square meter per second). Coincident increases in dehydroascorbate and glutathione disulfide were not observed. Acifluorfen also caused the rapid depletion of ascorbate in far-red light grown plants which were photosynthetically incompetent.

Glutathione reductase, dehydroascorbate reductase, superoxide dismutase, ascorbate oxidase, ascorbate free radical reductase, peroxidase, and catalase activities rapidly decreased in acifluorfen-treated tissue exposed to white light. None of the enzymes were inhibited in vitro by the herbicide. Acifluorfen causes irreversible photooxidative destruction of plant tissue, in part, by depleting endogenous antioxidants and inhibiting the activities of protective enzymes.

     

Biosynthesis and metabolism of indole-3-ethanol and indole-3-acetic acid by Pinus sylvestris L. needles

Combined gas chromatography-mass spectrometry has been used to identify indole-3-ethanol (IEt) in a purified extract from needles of Pinus sylvestris L. Quantitative estimates obtained by high-performance liquid chromatography with fluorescence detection, corrected for samples losses occurring during purification, indicate that Pinus needles contain 46±4 ng g^-1 IEt. This compares with 24.5±6.5 ng g^-1 indole-3-acetic acid (IAA) and 2.3±0.4 ng g^-1 indole-3-carboxylic acid (ICA) (Sandberg et al. 1984, Phytochemistry, 23, 99–102). Metabolism studies with needles incubated in a culture medium in darkness revealed that both [3-¹⁴C]-tryptophan and [2-¹⁴C]tryptamine mine are converted to [¹⁴C]IEt. It was also shown that [3-¹⁴C]IEt acted as a precursor of [¹⁴C]IAA. The observed metabolism appears to be enzymic in nature. The [2-¹⁴C]IAA was not catabolised to [¹⁴C]ICA in detectable quantities implying that, at best, only a minor portion of the endogenous ICA pool in the Pinus needles originates from IAA.Abbreviations DEAE diethylaminoethyl - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - ICA indole-3-carboxylic acid - IEt indole-3-ethanol - PVP polyvinylpyrrolidone     

Assimilation of exogenous 2′-14C-indole-3-acetic acid and 3′-14C-tryptophan exposed to the roots of three wheat varieties

This study was conducted to determine if plants can assimilate indole-3-acetic acid (IAA) from rooting media and if exogenous L-tryptophan (L-TRP) can be assimilated and converted by plants into auxins. The addition of 2-¹⁴C-IAA (3.7 kBq plant^-1) to wheat (Triticum aestivum L.) seedlings of three varieties grown in nutrient solution resulted in the uptake (avg.=7.6%) of labelled IAA. Most of the label IAA was recovered in the shoot (avg.=7.2%) with little accumulation in the root (avg.=0.43%). A portion of the assimilated IAA-label in the plant was identified by co-chromatography and UV spectral confirmation as IAA-glycine and IAA-aspartic acid conjugates. Little of the assimilated IAA label was found as free IAA in the wheat plants. These same assimilation patterns were observed when 2-¹⁴C-IAA was added to wheat plants grown in sterile and nonsterile soil. In contrast, the wheat varieties assimilated considerably less (avg.=1.3%) of the added microbial IAA precursor, 3-¹⁴C-L-TRP (3.7 kBq plant^-1) and thus much lower amounts of IAA conjugates were detected. Glasshouse soil experiments revealed that 2 out of 3 wheat varieties had increased growth rates and increased yields when L-TRP (10^-5 and 10^-7 M) was added to the root zone. It is surmised that this positive response is a result of microbial auxin production within the rhizosphere upon the addition of the precursor, L-TRP. The amino acid composition of the root exudates plays a critical role in microbial production of auxins in the rhizosphere. This study showed that wheat roots can assimilate IAA from their rooting media, which will supplement the endogenous IAA levels in the shoot tissue and may positively influence plant growth and subsequent yield.     

Fatty Acid Transport Through the Blood-Brain Barrier

Across the cerebral capillaries, the anatomical locus of the blood-brain barrier, the unidirectional influxes of the saturated fatty acids, octanoic and myristic acids, and the unsaturated essential fatty acid, linoleic acid, were measured. Employing an in situ rat brain perfusion technique that allows control of perfusate composition and accurate measurement of perfusate-to-brain fatty acid transport, we found that both [¹⁴C]octanoic and [¹⁴C]myristic acids were transported through the blood-brain barrier in vivo, in large part, by a specific, probenecid-sensitive transport system. However, the transport of [¹⁴C]linoleic acid was not probenecid sensitive. With 0.5 μM fatty acid but no plasma proteins in the perfusate, the permeability-surface area constant was higher for myristic acid (4.8 × 10^--²× s^-1) than for octanoic and linoleic acids (1.5 and 1.2 × 10^--²× s^-1, respectively). Approximately 70, 30, and 25% of the [¹⁴C]myristic, [¹⁴C]octanoic, or [¹⁴C]linoleic acids, respectively, were extracted from the perfusate.     

Identification of oxindole-3-acetic acid,and metabolic conversion of indole-3-acetic acid to oxindole-3-acetic acid in Pinus sylvestris seeds

Oxindole-3-acetic acid (OxIAA) has been identified in germinating seeds of Scots pine (Pinus sylvestris) using gas chromatography-mass spectrometry. Seeds germinated for 5 d contained 2.7 ng OxIAA·g^-1 (dry weight) whereas ungerminated seeds contained 0.2 ng·g^-1. Isotopically labelled OxIAA was formed in seeds incubated with [1-¹⁴C]-, [2-¹⁴C]- or [²H₅]indole-3-acetic acid.Abbreviations DDC sodium diethyldithiocarbamate - GC gas chromatography - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - MS mass spectrometry - OxIAA oxindole-3-acetic acid - PVP polyvinylpyrrolidone - TMS trimethylsilyl     

Biodegradation of 2,4,5-trichlorophenoxyacetic acid in liquid culture and in soil by the white rot fungus Phanerochaete chrysosporium

Summary Extensive biodegradation of [¹⁴C]-2,4,5-trichlorophenoxyacetic acid ([¹⁴C]-2,4,5-T) by the white rot fungus Phanerochaete chrysosporium was demonstrated in nutrient nitrogen-limited aqueous cultures and in [¹⁴C]-2,4,5-T-contaminated soil inoculated with this fungus and supplemented with ground corn cobs. After incubation of [¹⁴C]-2,4,5-T with aqueous cultures of the fungus for 30 days, 62.0%±2.0% of the [¹⁴C]-2,4,5-T initially present was degraded to ¹⁴CO₂. Mass balance analysis demonstrated that water soluble metabolites were formed during degradation, and HPLC and thin layer chromatography (TLC) of methylene chloride-extractable material revealed the presence of polar and non-polar [¹⁴C]-2,4,5-T metabolites. It was also shown that only 5% of the [¹⁴C]-2,4,5-T initially present in cultures remained as undegraded [¹⁴C]-2,4,5-T. In incubations composed of [¹⁴C]-2,4,5-T-contaminated soil, ground corn cobs, and 40% (w/w) water, 32.5%±3.6% of the [¹⁴C]-2,4,5-T initially present was converted to ¹⁴CO₂ after 30 days of incubation. These results suggest that it may be possible to develop practical systems based on the use of this fungus to detoxify 2,4,5-T-contaminated water and soil.     

1,25-Dihydroxyvitamin D3 or dexamethasone modulate arachidonic acid uptake and distribution into glycerophospholipids by normal adult human osteoblast-like cells

The effects of treatment with the osteotropic steroids 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), 17β-estradiol, or dexamethasone on [1-¹⁴C]arachidonic acid (AA) uptake and distribution into glycerophospholipid classes by normal adult human osteoblast-like (hOB) cells were investigated. Total uptake of [1-¹⁴C]AA was decreased in cells treated with dexamethasone when assayed after a 24-, 48-, or 96-h exposure to the hormone. Specific radiolabel incorporation into phosphatidylcholine was reduced by a 48-h treatment with dexamethasone with a concurrent increase in the radiolabeling of phosphatidylethanolamine. However, these changes were transient, and by 96 h of dexamethasone treatment the distribution of the radiolabeled fatty acid had reequilibrated to resemble the pattern found for vehicle treated samples. Total uptake of [1-¹⁴C]AA was diminished by 96-h treatment with 1,25(OH)₂D₃ (79 ± 3% of control, P < 0.01); at that time point, a significant decrease in the proportional radiolabeling of the phosphatidylinositol pool was identified (92 ± 2% of control, P < 0.05). The 1,25(OH)₂D₃-dependent decrease in total uptake and in phosphatidylinositol incorporation of [1-¹⁴C]AA were found to be hormone dose dependent. Treatment with 24,25(OH)₂D₃ was without effect on either total [1-¹⁴C]AA uptake or the specific [1-¹⁴C]AA radiolabeling of the phosphatidylinositol pool. 1,25(OH)₂D₃ treatment decreased hOB cell uptake of [1-¹⁴C]oleic acid and decreased its proportional incorporation into the phosphatidylinositol pool. Gas chromatographic analyses revealed no 1,25(OH)₂D₃-dependent effects on total phosphatidylinositol lipid mass or on the mole percent of arachidonic acid within the phosphatidylinositol pool, leaving the mechanism of the effects of the secosteroid on hOB cell AA metabolism unexplained. 17β-Estradiol had no effects on the parameters of AA metabolism measured. As a consequence of their modulation of arachidonic acid uptake and its distribution into hOB cellular phospholipids, steroids might alter the biological effects of other hormones whose actions include the stimulated production of bioactive AA metabolites, such as prostaglandins or the various lipoxygenase products.     

Chloroplast-Diphenyl Ether Interactions II

Acifluorfen, a p-nitrodiphenyl ether herbicide, is inhibitory to those photosynthetic functions that require a functioning chloroplast envelope. Functions involving the stroma are also affected. Acifluorfen does not lyse intact spinach chloroplasts, yet does increase the sensitivity of CO₂-dependent O₂ evolution to exogenous inorganic phosphate without directly affecting the function of the phosphate translocator. Acifluorfen penetrates into the chloroplast stroma in a light-independent fashion. Once inside, it causes the inactivation of light and dithiothreitol-activated fructose 1,6-bisphosphatase. Light-activated glyceraldehyde-3-phosphate dehydrogenase (NADP) is also inactivated by acifluorfen.

These data suggest that acifluorfen stimulates a pathway for inactivation of fructose 1,6-bisphosphatase and glyceraldehyde 3-phosphate dehydrogenase (NADP) which uses oxygen as a terminal oxidant and which involves thioredoxin and ferredoxin-thioredoxin reductase.

     

Properties of auxin binding sites in different subcellular fractions from maize coleoptiles

In-vitro binding of labeled auxins to sedimentable particles was tested in subcellular fractions from homogenates of maize (Zea mays L.) coleoptiles. The material was fractionated by differential centrifugation or on sucrose density gradients. It was confirmed that the major saturable binding activity (site I) for 1-naphthyl[1-¹⁴C]acetic acid is associated with vesicles derived from the endoplasmatic reticulum. A second type of specific auxin binding (site II) could be distinguished by several criteria, e.g. by the low affinity towards phenylacetic acid. The particles carrying site II could be clearly separated from markers of the endoplasmatic reticulum, the plasmalemma, the mitochondria and the nuclei, while their density as well as sedimentation velocity correlated with particle-bound acid phosphatase, indicating a localization at the tonoplast. In contrast to site I, binding at site II was hardly affected by a supernatant factor and by sulfhydryl groups. However, the specificity pattern of site II towards auxins and auxin analogs was very similar to that of site I tested in the presence of supernatant factor. The existence of a third auxin receptor localized in plasma membrane-rich gradient fractions was indicated by a preferential in-vitro binding of 2,4-dichlorophenoxyacetic acid.Abbreviations 1-NAA 1-naphthyl acetic acid - 2-NAA 2-naphthyl acetic acid - IAA 3-indolyl acetic acid - PAA phenyl acetic acid - 2,4-D 2,4-D-dichlorophenoxy acetic acid - D-2,4-DP dichlorophenoxy isopropionic acid - NPA 1-N-naphthyl phthalamic acid - ER endoplasmatic reticulum - SF supernatant factor