Chlorate Resistance Research Articles

Thiosulfate reductase as a chlorate reductase inSalmonella typhimurium

The contribution of thiosulfate reductase to chlorate sensitivity in Salmonella typhimurium was examined. Electrophoresed extracts of nitrate-grown cells of both the wild type and a chlC mutant were shown to contain chlorate reductase activity of the same relative mobility as a thiosulfate reductase activity which was present in the chlC mutant, but not in the wild-type grown under these conditions. A mutation is phs, which is essential for thiosulfate reductase by S. typhimurium, was shown to confer some chlorate resistance in the wild-type background and to increase the chlorate resistance obtained with a chlC mutation. Finally, thiosulfate in the anaerobic growth medium was shown to protect a chlC mutant growing in the presence of chlorate, but it did not protect the wild type. The results are consistent with a picture in which thiosulfate reductase can function as a chlorate reductase in both the wild-type and chlC backgrounds, although its capacity to reduce thiosulfate is diminished by the presence of an active nitrate reductase encoded by chlC.

Involvement of a low-molecular-weight substance in in vitro activation of the molybdoenzyme respiratory nitrate reductase from a chlB mutant of Escherichia coli.

The soluble subcellular fraction of a chlB mutant contains an inactive precursor form of the molybdoenzyme nitrate reductase, which can be activated by the addition to the soluble fraction of protein FA, which is thought to be the active product of the chlB locus. Dialysis or desalting of the chlB soluble fraction leads to the loss of nitrate reductase activation, indicating that some low-molecular-weight material is required for the activation. The protein FA-dependent activation of nitrate reductase can be restored to the desalted chlB soluble fraction by the addition of a clarified extract obtained after heating the chlB soluble fraction at 100 degrees C for 8 min. The heat-stable substance present in this preparation has a molecular weight of approximately 1,000. This substance is distinct from the active molybdenum cofactor since its activity is unimpaired in heat-treated extracts prepared from the organism grown in the presence of tungstate, which leads to loss of cofactor activity. Mutations at the chlA or chlE locus, which are required for molybdenum cofactor biosynthesis, similarly do not affect the activity of the heat-treated extract in the in vitro activation process. Moreover, the active material can be separated from the molybdenum cofactor activity by gel filtration. None of the other known pleiotropic chlorate resistance loci (chlD, chlG) are required for the expression of its activity. Magnesium ATP appears to have a role in the formation of the active substance. We conclude that a low-molecular-weight substance, distinct from the active molybdenum cofactor, is required to bestow activity on the molybdoenzyme nitrate reductase during its biosynthesis.

Variation in nitrate utilization and chlorate resistance amongst parents and progeny of Phytophthora drechsleri

A preliminary genetical study of nitrogen source utilization and chlorate resistance in Phytophthora drechsleri is described. Whilst the ability to utilize nitrate as sole nitrogen source may be under the control of a single gene, the response to chlorate in the presence of a complex nitrogen source appeared to be under polygenic control.

Host Preference Correlated with Chlorate Resistance in Macrophomina phaseolina

Host Preference Correlated with Chlorate Resistance in Macrophomina phaseolina

Improved in vitro selection of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia

The use of increasing knowledge on regulation of nitrate reductase activity in Nicotiana cell cultures is the basis for the described optimization of in vitro selection for nitrate reductase-deficient mutants by screening for chlorate resistance. Selection was carried out on haploid mesophyll protoplast-derived cell cultures of Nicotiana plumbaginifolia. It is demonstrated that revised selection results in high variant detectability and increased variant confirmability in comparison with the hitherto used selection scheme.

Variable Chlorate Resistance inMacrophomina phaseolinafrom Corn, Soybean, and Soil

Variable Chlorate Resistance in<i>Macrophomina phaseolina</i>from Corn, Soybean, and Soil

Nitrate reductase deficient cell lines from diploid cell cultures and lethal mutant M2 plants of Arabidopsis thaliana

Cell suspensions of diploid Arabidopsis thaliana were screened for resistance to chlorate on a medium with ammonium nitrate as the nitrogen source, and after plating on filters to increase the plating efficiency. Thirty-nine lines were selected, four of which were still resistant after two years of subculturing on non-selective medium. Of the latter lines three were nitrate reductase deficient but exhibited some residual nitrate reductase activity; the fourth line showed a high level of enzyme activity. Screening M2-seeds for callus production on selective medium with amino acids as the nitrogen source and chlorate revealed resistant calli in 17 out of 483 M2-groups. Nine well-growing lines, all but one (G3) exhibiting no detectable in vivo nitrate reductase activity, were classified as defective in the cofactor. Two lines (G1 and G3) could be analysed genetically at the plant level. Chlorate resistance was monogenic and recessive. Sucrose gradient fractionation of callus extracts of G1 revealed that a complete enzyme molecule can be assembled. Nitrate reductase activity in G1 could partly be restored by excess molybdenum. It is suggested that G1 is disturbed in the catalytic properties of the cofactor. It appeared that G1 is neither allelic with another molybdenum repairable mutant (B73) nor with another cofactor mutant (B25). Wilting of intact G1 plants could be ascribed to non-closing stomata.

The Combination of a Nitrate Reductase Deficient Nuclear Genome with a Streptomycin Resistant Chloroplast Genome, in Nicotiana tabacum, by protoplast Fusion

It has been possible to isolate streptomycin resistant and chlorate resistant/nitrate reductase deficient plants of Nicotiana tabacum by fusing γ-irradiated protoplasts of streptomycin resistant N. tabacum (SRI) with protoplasts of nitrate reductase deficient N. tabacum (nia-130). Five green colonies were recovered from 1.6 × 10(5) colonies, derived from a protoplast fusion experiment, in a selection medium containing streptomycin and chlorate. All five colonies regenerated plants carrying the mutant traits. Two of the plants had the normal chromosome number and were fertile. Reciprocal crossing to the wild type showed that chlorate resistance was due to recessive nuclear mutation and streptomycin resistance was inherited maternally. One of these plants was morphologically identical to the nia-130 parent. Another plant possessed nuclear encoded traits of the irradiated SRI parent in addition to the chloroplast from this mutant.

Isolation of biochemical mutants using haploid mesophyll protosplasts of Hyoscyamus muticus

Four nitrate non-utilizing clones of Hyoscyamus muticus, obtained by a total isolation method, possess all the known characteristics of cnx-type nitrate-reductase-deficient variants: 1) strict dependence on a reduced nitrogen source such as a mixture of amino acids; 2) chlorate resistance; 3) normal nitrate uptake; 4) lack of nitrate-reductase and xanthine-dehydrogenase activities, but presence of cytochrome-c-reductase and nitrite-reductase activities; 5) in vitro nitrate-reductase complementation with a molybdenum cofactor source. Two of the clones (MA-2 and I2D12) are molybdate repairable in vivo, whilst the other two clones (VIC2 and XIVE9) are not.

Regeneration of fully nitrate reductase — deficient mutants from protoplast culture of Nicotiana plumbaginifolia (Viviani)

Protoplast-derived colonies of haploid N. plumbaginifolia were selected for by chlorate resistance in media supplemented with casamino acids. Eighty resistant lines were confirmed by a second passage on a higher concentration of chlorate. Frequency of spontaneous mutation ranged from 10(-5) to 10(-6). Fifty of the resistant lines could be regenerated into plants, and 30 were characterized biochemically. Ninety percent were fully deficient for nitrate reductase activity. The lines were further tested for xanthine dehydrogenase activity and subsequently classified as defective in the apoenzyme (nia type, 26 lines) or the cofactor (cnx type, 4 lines). Two groups had been identified up until now within the cnx type by growth tests on high concentrations of molybdate supplied to the medium. Nitrate reductase deficiency was stably and continously expressed in both variant cell cultures and regenerants. Genetic analysis demonstrated that nitrate reductase deficiency was inherited as a single recessive nuclear gene.

Chlorate-resistant variants of Nicotiana tabacum L.

In somatic hybrids of each of four chlorate-resistant cell lines of Nicotiana tabacum L., two of which were also nitrate-nonutilizing, and a common chlorate-sensitive parent, nitrate utilization and/or chlorate resistance were found to be recessive traits. Complementation for nitrate utilization was observed in somatic hybrids of the two nitrate-nonutilizing cell lines. No chlorate-resistant or nitrate-nonutilizing seedlings were detected among a large number of progeny resulting from the self-fertilization of a somatic hybrid plant one of whose parents was one of the nitrate-nonutilizing cell lines.

Analysis of distributions of mutants in clones of plant-cell aggregates

The organization of plant cells (or any other genetic elements, such as organelles) into aggregates modifies the expected distribution of mutants in clones. The reason for the modification, and its effect on the use of the Luria-Delbrück fluctuation test, are discussed. The Luria-Delbriick test was used to show that the trait for chlorate resistance in cultured rose-cell aggregates appeared spontaneously and in the absence of chlorate ion.

Involvement of a gene of the chl E locus in the regulation of the nitrate reductase operon.

A strain of E. coli carrying a Mudl insertion leading to chlorate resistance was found to lack nitrate reductase and formate dehydrogenase activities, but to synthesize b-type cytochrome constitutively. Introduction of this insertion mutation into a strain bearing a fusion between the nitrate reductase operon (chl C, chl I) and the lac structural genes resulted in the constitutive expression of the lac genes of this last fusion. Identical results were found when the Mudl was eliminated promoting a deletion in the original insertion site. This mutation was located midway between gal and aro A, at the Chl E locus. Study of a chl E strain already described revealed similar behaviour. Absence of nitrate reductase activity in these strains which constitutively express the structural genes of the nitrate reductase operon was tentatively attributed to the simultaneous lack of a cofactor of the nitrate reductase terminal enzyme, possibly cofactor Mo-X, and of a repressor of the operon.

Isoleucine-requiring Nicotiana plant deficient in threonine deaminase

Well characterized auxotrophic mutants would greatly assist the genetic manipulation of flowering plants, but such mutants are rare1–3. Moreover, auxotrophic mutants isolated at the plant level4–8 and some of those isolated in cell cultures9 are leaky. The isolation of the first non-leaky auxotrophic mutant, requiring a source of nitrogen in reduced form, was possible by direct selection for chlorate resistance of this rare mutant type10. A different approach, the individual testing of colonies grown from mutagenized haploid cells of Datura11 and Hyoscyam us12, has resulted in cell lines with absolute nutritional requirement. We report here the isolation of an isoleucine-requiring line from haploid cell cultures of Nicotiana plumbaginifolia and the regeneration of diploid plants. These plants have no detectable activity of L-threonine deaminase (EC 4.2.1.16), the first enzyme in isoleucine biosynthesis.

ISOLATION AND PARTIAL CHARACTERIZATION OF NITRATE ASSIMILATION MUTANTS OF DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

ABSTRACTFifteen nitrate assimilation‐deficient mutants of the euryhaline green alga, Dunaliella tertiolecta Butcher were selected by their chlorate resistance. Ten mutants, unable to grow on NO3− but able to grow on NO2−, had no detectable nitrate reductase activity. Five mutants, unable to grow on either NO3− or NO2−, had depressed levels of both nitrate and nitrite reductase. A method for assaying methyl viologen‐nitrate reductase in the presence of nitrite reductase is described.

Induction and Characterization of Chlorate-resistant Strains of Rosa damascena Cultured Cells

The sensitivity of Rosa damascena cultured cells to chlorate was measured by plating samples of suspensions in agar containing NaClO(3). This sensitivity depended on the age of the cultures that were plated. Chlorate-resistant colonies isolated from 5- to 7-day cultures retained their resistance through many generations of growth in medium lacking NaClO(3); they also retained resistance when mixed with sensitive cells. Treating cell aggregates with ultraviolet (UV) light (254 nanometers), or UV light (360 nanometers) in the presence of 4'-methoxymethyltrioxsalen, increased the proportion that was resistant to NaClO(3). However, the amount of increase was low (three times) and required very specific doses of UV light. The UV treatments did not select for chlorate-resistant cells over chlorate-sensitive cells. The data suggested that UV had induced mutations leading to chlorate resistance. Approximately 15% of the resistant strains did not grow on medium containing nitrate as the sole nitrogen source. These strains lacked ability to reduce chlorate to chlorite. This observation supports the current idea that chlorate toxicity depends on the activity of nitrate reductase. Approximately 85% of the resistant strains grew on medium containing nitrate as the sole nitrogen source. These strains lost catalase activity following chlorate treatment, indicating that they took up and reduced chlorate. These strains have a mechanism for tolerating chlorate and its reduction products, rather than avoiding them.

Chlorate Resistance and Nitrate Assimilation in Industrial Strains of Penicillium chrysogenum

Several chlorate-resistant mutants of Penicillium chrysogenum were isolated and analysed; all were affected in nitrate assimilation. Nine loci were recognized by complementation analysis and these appear to be equivalent to the niaD (nitrate reductase structural gene), nirA (control locus) and seven cnx loci (responsible for the biosynthesis of a cofactor for nitrate reductase) of Aspergillus nidulans. The organization of the nitrate assimilation genes appears to be similar in both organisms, even to the extent of having contiguous genes coding for the nitrate and nitrite reductase enzymes.

Phylogenetic Differentiation of Cultivated Rice, XXII. Numerical Evaluation of the Indica-Japonica Differentiation

Principal component analysis of the data for 11 characters of 89 cultivars sampled at random from a large collection of Asian origin has reconfirmed the previous assertion by the junior author that the Indica-Japonica diferentiation of rice cultivars is recogni-zable on the basis of character-association patterns, although there is no key character discriminating between the two types. The probability of misclassification by a single character ranged from about 3 percent by the potassium chlorate resistance of seedling to about 40 percent by the length/width ratio of spikelets. Discriminant functions combining 3 or 4 character values gave lower probabilities of misclassification, although there still remained a few intermediate forms having recombined characteristics. The F1 sterility relationships were almost useless for the purpose of classification. About a half of the total character variance among the cultivars was attributable to Indica-Japonica differenti-ation. The normalized genetic identity between the two types was estimated to be 0.62 from data for 28 alleles at 12 Ioci. A great number of genic differences seem to be involved in the Indica-Japonica differentiation.

Free-living and symbiotic characteristics of chlorate resistant mutants of Rhizobium.

This work investigated the usefulness of chlorate resistance as a method for the selection of nitrate reductase negative (NR-) strains from Rhizobium japonicum (61A76) and evaluated the symbiotic, characteristics of these strains. Chlorate resistent strains were selected from populations seeded on CS 7 agar containing 10 or 20 mM KC10, and incubated in 2% air- 98% N2-CO2 (95:5). Over 200 resistant strains were isolated, 58% of which lacked the dissimilatory nitrate reductase. In 12 selected isolates, some strains had also lost the assimilatory nitrate reductase, but all retained hydrogenase activity. Chlorate resistant strains inoculated to soybean seedlings were equal to or better than the parent strain in terms of nodule mass and acetylene reduction. Those strains lacking both assimilatory and dissimilatory nitrate reductase showed the best symbiotic characteristics, suggesting that chlorate resistance in R. japonicum could be a useful method for the selection of strains with superiod nitrogen-fixing characteristics.

Биохимические мутан ты высших растений ка к объекты для химических и физиологических исс ледований. — Обзор

A report is given on biochemical mutants in higher plants with emphasis on metabolic mutants. Since the discovery of thiamine-defective mutants inArabidopsis thaliana andLycopersicon esculentum some other mutants in different species became available. The first higher plant with an absolute requirement for an amino acid is a proline-deficient mutant inZea mays. Inchloronerva ofLycopersicon esculentum synthesis of nicotianamine is blocked. This is the first instance where a hitherto unknown function is attributed to an already known compound by work with a mutant of higher plants. Nicotianamine is distributed throughout all vascular plants and appears to be linked with short-distance iron transport. The wilty mutantflacca inL. esculentum lacks abscisic acid with some consequences to different parts of metabolism. In the same species the mutant T 3238btl/btl, fer/fer carries two separate, independent mutations which affect the transport of boron and iron, respectively. Selection for chlorate resistance led to the discovery of mutants with impaired nitrate reductase activity and nitrate uptake inArabidopsis thaliana and in consequence added new information to our knowledge of the regulation of nitrate assimilation. Other mutants of similar kind were detected inNicotiana tabacum andHordeum vulgare. Mutants with defects in the photosynthetic system were discovered inZea mays andHordeum vulgare. Plastome mutants inOenothera revealed a lack of ribulose-1,5-biphosphate carboxylase activity and showed interference between the genetic system of the plastids and that of the nucleus.