Chronic or acute administration of various dialkylnitrosamines enhances the removal of O6-methylguanine from rat liver DNA in vivo

The effect of pretreatment of rats with various symmetrical dialkylnitrosamines on the repair of O⁶-methylguanine produced in liver DNA by a low dose of [¹⁴C]dimethylnitrosamine (DMN) has been examined. DMN, diethylnitrosamine (DEN), dipropylnitrosamine (DPN) or dibutylnitrosamine (DBN) were administered to rats for 14 consecutive weekdays at a daily dose of 5% of the LD₅₀. Animals were given [¹⁴C]DMN 24 h after the last dose and were killed 6 h later. DNA was extracted from the liver and analysed for methylpurine content after mild acid hydrolysis and Sephadex G-10 chromatography. While the amounts of 3-methyladenine and 7-methylguanine were only slightly different from controls, the amounts of O⁶-methylguanine in the DNA of the dialkylnitrosamine pretreated rats were about 30% of those in control rats, indicating a considerable increase in the capacity to repair this base. Liver ribosomal RNA from control and dialkylnitrosamine pretreated rats contained closely similar amounts of O⁶-methylguanine suggesting that the induced enzyme system does not act on this base in ribosomal RNA in vivo. Pretreatment with these dialkylnitrosamines also enhanced the repair of O⁶-methylguanine in liver DNA when they were given as a single dose (50% of the LD₅₀) either 3 or 7 days before the [¹⁴C]DMN. In addition, single low doses of DMN or DEN (5% of the LD₅₀) given either 1 or 6 days before [¹⁴C]DMN increased O⁶-methylguanine repair and the magnitude of the effect after DEN was similar to that produced by the other pretreatment schedules. The possible mechanism(s) of the induction of O⁶-methylguanine repair and its relation to hepatotoxicity, DNA alkylation, carcinogenesis and the adaptive response in Escherichia coli are discussed.     

In vivo replication of carcinogen-modified rat liver DNA: increased susceptibility of O6-methylguanine compared to N-7-methylguanine in replicated DNA to S1-nuclease

In order to characterize rat liver DNA replicated $\text{in}\text{vivo}$ on a carcinogen-damaged template, the replicated DNA was treated with S₁-nuclease and the release of (¹⁴C)-dimethyl-nitrosamine induced 0⁶-methylguanine, a lesion associated with miscoding and N-7-methylguanine, a lesion that does not miscode were monitored. The results indicated that both the methylated guanines became susceptible to S₁-nuclease upon replication. However, a greater percentage of 0⁶-methylguanine (22% of the total 0⁶-methylguanine present in the DNA) compared to N-7-methylguanine (4% of the total N-7-methylguanine present in the DNA) was rendered acid soluble by S₁-nuclease. The preferential release of 0⁶-methylguanine compared to N-7-methylguanine from replicated DNA was interpreted to indicate its occurrence in local denatured regions probably generated as a result of misbase pairing.     

Formation and subsequent removal of O6-methylguanine from deoxyribonucleic acid in rat liver and kidney after small doses of dimethylnitrosamine

1. The amounts of 7-methylguanine and O⁶-methylguanine present in the DNA of liver and kidney of rats 4h and 24h after administration of low doses of dimethylnitrosamine were measured. 2. O⁶-Methylguanine was rapidly removed from liver DNA so that less than 15% of the expected amount (on the basis of 7-methylguanine found) was present within 4h after doses of 0.25mg/kg body wt. or less. Within 24h of administration of dimethylnitrosamine at doses of 1mg/kg or below, more than 85% of the expected amount of O⁶-methylguanine was removed. Removal was most efficient (defined in terms of the percentage of the O⁶-methylguanine formed that was subsequently lost within 24h) after doses of 0.25–0.5mg/kg body wt. At doses greater or less than this the removal was less efficient, even though the absolute amount of O⁶-methylguanine lost during 24h increased with the dose of dimethylnitrosamine over the entire range of doses from 0.001 to 20mg/kg body wt. 3. Alkylation of kidney DNA after intraperitoneal injections of 1–50μg of dimethylnitrosamine/kg body wt. occurred at about one-tenth the extent of alkylation of liver DNA. Removal of O⁶-methylguanine from the DNA also took place in the kidney, but was slower than in the liver. 4. After oral administration of these doses of dimethylnitrosamine, the alkylation of kidney DNA was much less than after intraperitoneal administration and represented only 1–2% of that found in the liver. 5. Alkylation of liver and kidney DNA was readily detectable when measured 24h after the final injection in rats that received daily injections of 1μg of [³H]dimethylnitrosamine/kg for 2 or 3 weeks. After 3 weeks, O⁶-methylguanine contents in the liver DNA were about 1% of the 7-methylguanine contents. The amount of 7-methylguanine in the liver DNA was 10 times that in the kidney DNA, but liver O⁶-methylguanine contents were only twice those in the kidney. 6. Extracts able to catalyse the removal of O⁶-methylguanine from alkylated DNA in vitro were isolated from liver and kidney. These extracts did not lead to the loss of 7-methylguanine from DNA. 7. The possible relevance of the formation and removal of O⁶-methylguanine in DNA to the risk of tumour induction by exposure to low concentrations of dimethylnitrosamine is discussed.     

Cellular reactions of O6-methylguanine, a product of some alkylating carcinogens

Cultures of a purine-requiring mutant of Chinese hamster ovary cells (CHO-104b), randomly bred hamster embryo cells, or Escherichia coli B_s−1 were treated with non-toxic doses of ³H-labelled O⁶-methylguanine. DNA and RNA were isolated and subjected to enzymic digestion to nucleosides at pH8. The products of digestion were analysed by ion-exchange chromatography on columns of Dowex 50 (NH₄⁺ form) at pH8.9. No ³H-labelled O⁶-methylguanosine was detected in nucleic acid digests. ³H-labelled O⁶-methylguanine was O-demethylated yielding [³H]guanine in CHO-104b cells. Radioactivity in nucleic acid digests was associated with thymidine, guanosine, deoxyguanosine and an unidentified early-eluting product. Reports of similar unidentified products from nucleic acids labelled with various agents are discussed.     

Activation of AMP-activated protein kinase by MAPO1 and FLCN induces apoptosis triggered by alkylated base mismatch in DNA

O⁶-Methylguanine produced in DNA by the action of simple alkylating agents, such as N-methyl-N-nitrosourea (MNU), causes base-mispairing during DNA replication, thus leading to mutations and cancer. To prevent such outcomes, the cells carrying O⁶-methylguanine undergo apoptosis in a mismatch repair protein-dependent manner. We previously identified MAPO1 as one of the components required for the induction of apoptosis triggered by O⁶-methylguanine. MAPO1, also known as FNIP2 and FNIPL, forms a complex with AMP-activated protein kinase (AMPK) and folliculin (FLCN), which is encoded by the BHD tumor suppressor gene. We describe here the involvement of the AMPK–MAPO1–FLCN complex in the signaling pathway of apoptosis induced by O⁶-methylguanine. By the introduction of siRNAs specific for these genes, the transition of cells to a population with sub-G₁ DNA content following MNU treatment was significantly suppressed. After MNU exposure, phosphorylation of AMPKα occurred in an MLH1-dependent manner, and this activation of AMPK was not observed in cells in which the expression of either the Mapo1 or the Flcn gene was downregulated. When cells were treated with AICA-ribose (AICAR), a specific activator of AMPK, activation of AMPK was also observed in a MAPO1- and FLCN-dependent manner, thus leading to cell death which was accompanied by the depolarization of the mitochondrial membrane, a hallmark of the apoptosis induction. It is therefore likely that MAPO1, in its association with FLCN, may regulate the activation of AMPK to control the induction of apoptosis triggered by O⁶-methylguanine.     

High performance liquid chromatography studies on the interactions of cis-Pt(NH3)2−(H2O)22+ with guanine and methylated g

Time dependence studies, using high performance liquid chromatography (HPLC), on the reaction between cis-diamminediaquoplatinum and guanine, N1-methylguanine, N7-methylguanine, N9-methylguanine, and N1 ,N7-dimethylguanine are reported. Each reaction gave rise to eight or more compounds; the major components have been prepared and characterization by ¹H and ¹⁹⁵Pt nuclear magnetic resonance has been attempted. Species of the form ((NH₃)₂Pt(NO₃)-(G-H)-(NO₃)-Pt(NH₃)₂)⁺, (NH₃)₂,Pt(G-H)(NO₃) monomer and (NH₃)₂Pt(G-H)(NO₃) dimer, where G-H indicates the guanine monoanion, are postulated.     

Hyperaccumulation of thallium is population-specific and uncorrelated with caesium accumulation in the thallium hyperaccumulator, Biscutella laevigata

Purpose

This study investigated the residual contribution of legume and fertilizer nitrogen (N) to a subsequent crop under the effect of elevated carbon dioxide concentration ([CO₂]).

Methods

Field pea (Pisum sativum L.) was labeled in situ with ¹⁵N (by absorption of a ¹⁵N-labeled urea solution through cut tendrils) under ambient and elevated (700 μmol mol^–1) [CO₂] in controlled environment glasshouse chambers. Barley (Hordeum vulgare L.) and its soil were also labeled under the same conditions by addition of ¹⁵N-enriched urea to the soil. Wheat (Triticum aestivum L.) was subsequently grown to physiological maturity on the soil containing either ¹⁵N-labeled field pea residues (including ¹⁵N-labeled rhizodeposits) or ¹⁵N-labeled barley plus fertilizer ¹⁵N residues.

Results

Elevated [CO₂] increased the total biomass of field pea (21 %) and N-fertilized barley (23 %), but did not significantly affect the biomass of unfertilized barley. Elevated [CO₂] increased the C:N ratio of residues of field pea (18 %) and N-fertilized barley (19 %), but had no significant effect on that of unfertilized barley. Elevated [CO₂] increased total biomass (11 %) and grain yield (40 %) of subsequent wheat crop regardless of rotation type in the first phase. Irrespective of [CO₂], the grain yield and total N uptake by wheat following field pea were 24 % and 11 %, respectively, higher than those of the wheat following N-fertilized barley. The residual N contribution from field pea to wheat was 20 % under ambient [CO₂], but dropped to 11 % under elevated [CO₂], while that from fertilizer did not differ significantly between ambient [CO₂] (4 %) and elevated [CO₂] (5 %).

Conclusions

The relative value of legume derived N to subsequent cereals may be reduced under elevated [CO₂]. However, compared to N fertilizer application, legume incorporation will be more beneficial to grain yield and N supply to subsequent cereals under future (elevated [CO₂]) climates.     

Improved automated solid-phase microsequencing of peptides using DABITC

The methylated purines O⁶-methyl- and 7-methylguanine were isolated from mouse liver DNA hydrolysates by means of a column cleanup employing a Sep Pak C-18 reverse-phase cartridge. The purine bases were eluted from the cartridge with methanol, evaporated to dryness, and then dissolved in mobile phase for liquid chromatographic analysis by normalphase chromatography. The system consisted of a LiChrosorb Si 60 column with a watersaturated mobile phase of 20% methanol in chloroform containing 0.001% H₃PO₄. The two methylated bases eluted before adenine or guanine. For extremely low-level (<300 pmol) quantitation, the peaks corresponding to O⁶-methyl- and 7-methylguanine were collected and then analyzed by reverse-phase chromotography with a LiChrosorb RP-18 column and a mobile phase of 5% methanol in pH 7 phosphate buffer (for 7-methylguanine) or 9.5% methanol/buffer (for O⁶-methylguanine). Comparisons were made with fluorescence detection and with scintillation counting (in animal studies where [¹⁴C]dimethylnitrosamine was used). Minimum detectable levels at 254 nm were about 3 ng (3:1 signal to noise ratio) for each of the title compounds. As low as 10 pmol/mg of each could be detected in DNA hydrolysates. Recoveries of O⁶-methyl- and 7-methylguanine from DNA spiked at 750 pmol/mg were greater than 80%.     

Reversible light-activation of ribulose bisphosphate carboxylase/oxygenase in isolated barley protoplasts and chloroplasts

The enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase displayed near-maximal activity in isolated, intact barley (Hordeum vulgare L. cv. Pennrad) mesophyll protoplasts. The carboxylase deactivated 40 to 50% in situ when protoplasts were dark-incubated 20 minutes in air-equilibrated solutions. Enzyme activity was fully restored after 1 to 2 minutes of light. Addition of 5 millimolar NaHCO₃ to the incubation medium prevented dark-inactivation of the carboxylase. There was no permanent CO₂-dependent activation of the protoplast carboxylase either in light or dark. Activation of the carboxylase from ruptured protoplasts was not increased significantly by in vitro preincubation with CO₂ and Mg²⁺. In contrast to the enzyme in protoplasts, the carboxylase in intact barley chloroplasts was not fully reactivated by light at atmospheric CO₂ levels. The lag phase in carbon assimilation was not lengthened by dark-adapting protoplasts to low CO₂ demonstrating that light-activation of the carboxylase was not involved in photosynthetic induction. Irradiance response curves for reactivation of the the carboxylase and for CO₂ fixation by isolated barley protoplasts were similar. The above results show that there was a fully reversible light-activation of the carboxylase in isolated barley protoplasts at physiologically significant CO₂ levels.     

Carbon footprint of spring barley in relation to preceding oilseeds and N fertilization

Purpose

Carbon footprint of field crops can be lowered through improved cropping practices. The objective of this study was to determine the carbon footprint of spring barley (Hordeum vulgare L.) in relation to various preceding oilseed crops that were fertilized at various rates of inorganic N the previous years. System boundary was from cradle-to-farm gate.

Materials and methods

Canola-quality mustard (Brassica juncea L.), canola (Brassica napus L.), sunflower (Helianthus annuus L.), and flax (Linum usitatissimum L.) were grown under the N fertilizer rates of 10, 30, 70, 90, 110, 150, and 200?kg?N?ha^?1 the previous year, and spring barley was grown on the field of standing oilseed stubble the following year. The study was conducted at six environmental sites; they were at Indian Head in 2005, 2006 and 2007, and at Swift Current in 2004, 2005 and 2006, Saskatchewan, Canada.

Results and discussion

On average, barley grown at humid Indian Head emitted greenhouse gases (GHGs) of 1,003?kg?CO₂eq?ha^?1, or 53% greater than that at the drier Swift Current site. Production and delivery of fertilizer N to farm gate accounted for 26% of the total GHG emissions, followed by direct and indirect emissions of 28% due to the application of N fertilizers to barley crop. Emissions due to N fertilization were 26.6 times the emission from the use of phosphorous, 5.2 times the emission from pesticides, and 4.2 times the emission from various farming operations. Decomposition of crop residues contributed emissions of 173?kg?CO₂eq?ha^?1, or 19% of the total emission. Indian Head-produced barley had significantly greater grain yield, resulting in about 11% lower carbon footprint than Swift Current-produced barley (0.28 vs. 0.32?kg?CO₂eq?kg^?1 of grain). Emissions in the barley production was a linear function of the rate of fertilizer N applied to the previous oilseed crops due to increased emissions from crop residue decomposition coupled with higher residual soil mineral N.

Conclusions

The key to lower the carbon footprint of barley is to increase grain yield, make a wise choice of crop types, reduce N inputs to crops grown in the previous and current growing seasons, and improved N use efficiency.     

The influence of salinity on cell ultrastructures and photosynthetic apparatus of barley genotypes differing in salt stress tolerance

A hydroponic experiment was conducted to elucidate the difference in growth and cell ultrastructure between Tibetan wild and cultivated barley genotypes under moderate (150 mM NaCl) and high (300 mM NaCl) salt stress. The growth of three barley genotypes was reduced significantly under salt stress, but the wild barley XZ16 (tolerant) was less affected relative to cultivated barley Yerong (moderate tolerant) and Gairdner (sensitive). Meanwhile, XZ16 had lower Na⁺ and higher K⁺ concentrations in leaves than other two genotypes. In terms of photosynthetic and chlorophyll fluorescence parameters, salt stress reduced maximal photochemical efficiency (F _v/F _m), net photosynthetic rate (Pn), stomatal conductance (Gs), and intracellular CO₂ concentration (Ci). XZ16 showed relatively smaller reduction in comparison with the two cultivated barley genotypes. The observation of transmission electron microscopy found that fundamental cell ultrastructure changes happened in both leaves and roots of all barley genotypes under salt NaCl stress, with chloroplasts being most changed. Moreover, obvious difference could be detected among the three genotypes in the damage of cell ultrastructure under salt stress, with XZ16 and Gairdner being least and most affected, respectively. It may be concluded that high salt tolerance in XZ16 is attributed to less Na⁺ accumulation and K⁺ reduction in leaves, more slight damage in cell ultrastructure, which in turn caused less influence on chloroplast function and photosynthesis.     

Effect of pretreatment with other dialkylnitrosamines on excision from hepatic DNA of O6-methylguanine produced by dimethylnitrosamine

Pretreatment with diethylnitrosamine or dipropylnitrosamine increased the amount of labelled O⁶-methylguanine found in liver DNA 4 and 24 h after injection of 10 μg/kg [³H] dimethylnitrosamine. Dibutylnitrosamine treatment had a similar, though smaller effect at 4 h but was ineffective when measurements were made 24 h after the dimethylnitrosamine was given. These pretreatments did not affect 7-methylguanine levels in the DNA showing that the metabolic conversion of dimethylnitrosamine into a methylating agent was not altered. Previous studies have shown that O⁶-methylguanine is rapidly removed from hepatic DNA after methylation to a small extent but removal is less efficient after higher amounts of methylation. Therefore, the most probable explanation for the present findings is that these longer dialkylnitrosamines produce a similar product in DNA which interferes with the loss of O⁶-methylguanine. This hypothesis was supported by experiments showing that diethylnitrosamine did give rise to O⁶-ethylguanine which was lost from the DNA at a rate comparable to the observed loss of O⁶-methylguanine in diethylnitrosamine pretreated rats. This method may, therefore, be of value for determination of whether other nitrosamines, not available in a radioactively labelled form, react with DNA at external oxygen atoms. The present results also suggest that different dialkylnitrosamines might have additive effects in prolonging damage to DNA which could be important in carcinogenesis.     

Induction of lactate dehydrogenase isozymes by oxygen deficit in barley root tissue

Lactate dehydrogenase (LDH) activity in attached roots of barley and other cereals increased up to 20-fold during several days of severe hypoxia, reaching a maximum of about 2 micromoles per minute per gram fresh weight. In barley, induction of LDH activity was significant at 2.6% O₂ and greatest at 0.06%, the lowest O₂ concentration tested. Upon return to aerobic conditions, induced LDH activity declined with an apparent half-life of 2 days. The isozyme profile of barley LDH comprised 5 bands, consistent with a tetrameric enzyme with subunits encoded by two different Ldh genes. Changes in staining intensity of the isozymes as a function of O₂ level suggested that one Ldh gene was preferentially expressed in severe hypoxia. When tracer [U-¹⁴C]glucose was supplied to induced roots under hypoxic conditions, lactate acquired label, but much less than either ethanol or alanine. Most of the [¹⁴C] lactate was secreted into the medium, whereas most other labeled anionic products were retained in the root. Neither hypoxic induction of LDH, nor lactate secretion by induced roots, is predicted from the Davies-Roberts hypothesis, which holds that lactate glycolysis ceases soon after the onset of hypoxia due to acidosis brought about by lactate accumulation in the cytoplasm. These results imply a functional significance for LDH beyond that assigned it in this hypothesis.     

The accumulation of O6-methylguanine in the liver and kidney DNA of rats treated with dimethylnitrosamine for a short or a long period

A large dose of dimethylnitrosamine was administered to rats by two different dosing regimens, one being eleven intraperitoneal injections of 5 mg/kg body wt. at 12-h intervals (a dosing regimen strongly carcinogenic for the kidney but not the liver), and the other being a continuous dosing over several weeks by adding 8.5 mg of dimethylnitrosamine to each litre of drinking water giving an approximate daily dose of 0.7 mg/kg body wt. This treatment is known to be strongly carcinogenic for the liver but not the kidney. The accumulation in DNA of liver and kidney of the methylated purines 7-methylguanine and O⁶-methylguanine under each regimen were measured and compared. With the eleven-injection regimen there was a build up of O⁶-methylguanine in the DNA of the susceptible organ, the kidney, whilst in the liver virtually no accumulation was detected. Under the prolonged, low concentration regimen the liver, in spite of its susceptibility to the carcinogen did not accumulate O⁶-methylguanine. The results are discussed in terms of the hypothesis that production of O⁶-methylguanine and its persistence in the DNA of the target organ are responsible for the carcinogenic action of dimethylnitrosamine.     

The nature of single-strand breaks in DNA following treatment of L-cells with methylating agents

DNA from untreated L-cells had a weight average molecular weight (Mw) of 5.7 ± 0.58·10⁸ daltons as measured by sedimentation in an alkaline sucrose gradient. This value was reduced by one half after the cells were treated for 1 h with 8 μg/ml of N-methyl-N-nitrosourea (MNUA), 34 μg/ml of methyl methanesulfonate (MMS) or 0.16 μg/ml of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). That dose of MNUA produced 52 methylations per 5.7·10⁸ daltons DNA. 20% of these were not purine derivatives and were assumed to contain some phosphotriesters. That dose of MMS (above) produced 290 methylations per 5.7·10⁸ daltons DNA and about 14% of these were not purine derivatives. The rates of loss of methylated purines from DNA were 2.3% per hour for 7-methylguanine (7-MeG), 7.4% per hour for 3-methyladenine (3-MeA) and no detectable loss of O⁶-methylguanine (O⁶-MeG) over a 12 h period. Since phosphotriesters are alkali-labile the single-strand breaks probably arose from this structure and did not form within the cell. This conclusion is supported by the following considerations. MNUA was more effective than MMS at reducing the molecular weight of DNA, as measured in alkaline medium. The greater SN₁ character of MNUA would cause a greater formation of phosphotriesters than would MMS.     

The mechanism of alkylation of DNA by 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MIC), a metabolite of DIC (NSC-45388). Non-involvement of diazomethane

Comparison of the nuclear magnetic resonance spectra of chemically synthesized methyl-d₁-methanol with the methanol produced in the solvolytic decompostion of 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MIC) in D₂O under acidic, basic or neutral conditions indicated that no deuterium was exchanged for the hydrogens on the methyl group. Diazomethane can therefore be ruled out as an intermediate in this reaction.The methyl-d₃-guanine isolated after incubation of methyl-d₃-MIC with calfthymus DNA in vitro displayed, on chemical ionization mass spectrometry, a quasimolecular ion (MH⁺) at m/e 169, which was 3 mass units higher than the quasimolecular ion for an undeuterated 7-methylguanine standard. The major fragment ions for 7-methyl-d₃-guanine on electron impact mass spectrometry likewise were situated at positions 3 mass units higher than the fragment ions for 7-methylguanine itself.These data indicate that the methylation of biological macromolecules by MIC must involve the transfer of an intact methyl group.     

Regulated expression of three alcohol dehydrogenase genes in barley aleurone layers

Three genes specify alcohol dehydrogenase (EC 1.1.1.1.; ADH) enzymes in barley (Hordeum vulgare L.) (Adh 1, Adh 2, and Adh 3). Their polypeptide products (ADH 1, ADH 2, ADH 3) dimerize to give a total of six ADH isozymes which can be resolved by native gel electrophoresis and stained for enzyme activity.

Under fully aerobic conditions, aleurone layers of cv Himalaya had a high titer of a single isozyme, the homodimer containing ADH 1 monomers. This isozyme was accumulated by the aleurone tissue during the later part of seed development, and survived seed drying and rehydration. The five other possible ADH isozymes were induced by O₂ deficit. The staining of these five isozymes on electrophoretic gels increased progressively in intensity as O₂ levels were reduced below 5%, and were most intense at 0% O₂.

In vivo³⁵S labeling and specific immunoprecipitation of ADH peptides, followed by isoelectric focusing of the ADH peptides in the presence of 8 molar urea (urea-IEF) demonstrated the following. (a) Aleurone layers incubated in air synthesized ADH 1 and a trace of ADH 2; immature layers from developing seeds behaved similarly. (b) At 5% O₂, synthesis of ADH 2 increased and ADH 3 appeared. (c) At 2% and 0% O₂, the synthesis of all three ADH peptides increased markedly.

Cell-free translation of RNA isolated from aleurone layers, followed by immunoprecipitation and urea-IEF of in vitro synthesized ADH peptides, showed that levels of mRNA for all three ADH peptides rose sharply during 1 day of O₂ deprivation. Northern hybridizations with a maize Adh 2 cDNA clone established that the clone hybridized with barley mRNA comparable in size to maize Adh 2 mRNA, and that the level of this barley mRNA increased 15- to 20-fold after 1 day at 5% or 2% O₂, and about 100-fold after 1 day at 0% O₂.

We conclude that in aleurone layers, expression of the three barley Adh genes is maximal in the absence of O₂, that regulation of mRNA level is likely to be a major controlling factor, and that whereas the ADH system of barley has strong similarities to that of maize, it also has some distinctive features.

     

Gibberellin estimation and biosynthesis in germinating Hordeum distichon

Gibberellic acid (GA₃) and 13-deoxy-gibberellic acid (GA₇) were identified in extracts of germinating barley as their ¹⁴C-methyl esters. The maximal level of GA₃ was estimated by an isotopic dilution procedure to be 1·5 ng per grain. Germinating barley incorporated 2-¹⁴C-mevalonic acid into several terpenes, whose specific radioactivities were measured, but incorporation into GA₃ could not be detected. Cell-free embryo extracts from germinating barley converted 2-¹⁴C-mevalonic acid into isopentenol, dimethylallyl alcohol, farnesol and squalene, while ¹⁴C-isopentenyl pyrophosphate was incorporated into geraniol, farnesol, geranylgeraniol and squalene. There was no detectable incorporation into the gibberellin intermediate ent-kaurene.     

Inhibition of Nitrate Transport by Anti-Nitrate Reductase IgG Fragments and the Identification of Plasma Membrane Associated Nitrate Reductase in Roots of Barley Seedlings

Membrane associated nitrate reductase (NR) was detected in plasma membrane (PM) fractions isolated by aqueous two-phase partitioning from barley (Hordeum vulgare L. var CM 72) roots. The PM associated NR was not removed by washing vesicles with 500 millimolar NaCl and 1 millimolar EDTA and represented up to 4% of the total root NR activity. PM associated NR was stimulated up to 20-fold by Triton X-100 whereas soluble NR was only increased 1.7-fold. The latency was a function of the solubilization of NR from the membrane. NR, solubilized from the PM fraction by Triton X-100 was inactivated by antiserum to Chlorella sorokiniana NR. Anti-NR immunoglobulin G fragments purified from the anti-NR serum inhibited NO₃⁻ uptake by more than 90% but had no effect on NO₂⁻ uptake. The inhibitory effect was only partially reversible; uptake recovered to 50% of the control after thorough rinsing of roots. Preimmune serum immunoglobulin G fragments inhibited NO₃⁻ uptake 36% but the effect was completely reversible by rinsing. Intact NR antiserum had no effect on NO₃⁻ uptake. The results present the possibility that NO₃⁻ uptake and NO₃⁻ reduction in the PM of barley roots may be related.     

Development of a Multi-Species Biotic Ligand Model Predicting the Toxicity of Trivalent Chromium to Barley Root Elongation in Solution Culture

Little knowledge is available about the influence of cation competition and metal speciation on trivalent chromium (Cr(III)) toxicity. In the present study, the effects of pH and selected cations on the toxicity of trivalent chromium (Cr(III)) to barley (Hordeum vulgare) root elongation were investigated to develop an appropriate biotic ligand model (BLM). Results showed that the toxicity of Cr(III) decreased with increasing activity of Ca²⁺ and Mg²⁺ but not with K⁺ and Na⁺. The effect of pH on Cr(III) toxicity to barley root elongation could be explained by H⁺ competition with Cr³⁺ bound to a biotic ligand (BL) as well as by the concomitant toxicity of CrOH²⁺ in solution culture. Stability constants were obtained for the binding of Cr³⁺, CrOH²⁺, Ca²⁺, Mg²⁺ and H⁺ with binding ligand: log K_CrBL 7.34, log K_CrOHBL 5.35, log K_CaBL 2.64, log K_MgBL 2.98, and log K_HBL 4.74. On the basis of those estimated parameters, a BLM was successfully developed to predict Cr(III) toxicity to barley root elongation as a function of solution characteristics.