Somatic Hybridization of Nitrate Reductase Deficient Mutants of Nicotiana tabacum by Protoplast Fusion

Protoplasts were isolated from two mutant cell lines of Nicotiana tabacum L. cv. Gatersleben and fused with the aid of polyethylene glycol. Both mutants lacked nitrate reductase and were thus auxotrophic for reduced nitrogen. The fusion resulted in a high frequency of hybrid cells which were detected by their regained ability to grow in media containing nitrate as sole nitrogen source. Thus, the two mutants were found to complement each other in the hybrids. In control experiments, back mutation and cross-feeding were excluded as possible explanations for the occurrence of cell lines utilizing nitrate. A total of 1061 hybrid lines capable of sustained proliferation were isolated. Some of them were further characterized with respect to nitrate reductase activity, chlorate sensitivity, chromosome number, and shoot formation. The results demonstrate that protoplast fusion can be used for the genetic analysis of cell variants of higher plants and that nitrate reductase-deficient mutants provide efficient selective systems for hybrid cells.     

曼陀罗培养细胞的硝酸还原酶突变株

经紫外诱变氯酸钾筛选,得到一个低硝酸还原酶(NADH:硝酸氧化还原酶.EC1.6.6.1.,以下简写为NR)活力的细胞株。其主要特征:NR活力低,约为正常型的1/5;对氯酸钾具有较强的抗性;不适合在单纯以硝酸盐为氮源的培养基上生长,能在以(NH_4)_2SO_4为唯一氮源的培养基上生长。蛋白电泳表明,此细胞株与正常型有不同的蛋白带。这些特征在没有选择压力的培养基上培养二年后,仍保持不变,说明此细胞株是一个遗传型的变异株。     

Isolation of molybdenum cofactor defective cell lines of Nicotiana tabacum

Summary Thirty-nine chlorate resistant cell lines were isolated after plating ethylmethane sulphonate treated allodihaploid cells of Nicotiana tabacum cv. Xanthi on agar medium containing 20 mM chlorate. Thirty-two of these cell lines grew as well on nitrate medium as on amino acid medium and three other cell lines grew well on amino acid medium but poorly on nitrate medium. Four other cell lines, 042, P12, P31 and P47 which could grow on amino acid medium, but not on nitrate medium, were examined further. They lacked in vitro nitrate reductase activity but were able to accumulate nitrate. All lines possessed nitrite reductase activity. Lines 042, P12, and P31 had a cytochrome c reductase species which was the same size as the wild type nitrate reductase associated cytochrome c reductase species, whilst the cytochrome c reductase species in line P47 was slightly smaller. All four lines lacked xanthine dehydrogenase activity and neither nitrate reductase nor xanthine dehydrogenase activity was restored by subculture of the four lines into either nitrate medium or glutamine medium supplemented with 1 mM sodium molybdate. These four lines are different from other molybdenum cofactor defective cell lines so far described in N. tabacum and possess similar properties to certain other cnx mutants described in Aspergillus nidulans.     

A Nitrate Reductase-less Variant Isolated from Suspension Cultures of Datura innoxia (Mill.)

A comparative study has been carried out of the growth of two lines of Datura innoxia (Mill.) cells, designated DI-6 and NR1, their resistance to chlorate, and their ability to assimilate nitrate in sterile culture. The NR1 cell line was isolated from DI-6 cultures by first growing the latter in a nitrate-based medium for 5 days and then transferring the cells to a medium containing 2 grams liter⁻¹ of casein hydrolysate as the sole N source and 49 millimolar KClO₃ for a 6-week incubation period. Cells which survived the chlorate treatment then were transferred to casein hydrolysate medium and have been cultured in the absence of chlorate for more than 18 months (NR1).     

Constitutive nitrate reductase: a dominant conditional marker for plant genetics

We describe the development of a counter-selection system based on the use of an engineered nitrate reductase (NR) gene. The engineered gene consists of an NR cDNA placed under the control of the CaMV 35S promoter (35S-NR). Seedlings and cells derived from transgenic Nicotiana plumbaginifolia plants transformed with 35S-NR are efficiently killed by the selective agent chlorate on medium containing ammonium as the sole nitrogen source. Under these nitrate-free conditions wild-type plants are not affected by chlorate because the endogenous wild-type Niagene is not expressed.     

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 NaClO₃. 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 NaClO₃; 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 NaClO₃. 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.     

Identification and characterization of a chlorate-resistant mutant of Arabidopsis thaliana with mutations in both nitrate reductase structural genes NIA1 and NIA2

Mutant plants defective in the assimilation of nitrate can be selected by their resistance to the herbicide chlorate. In Arabidopsis thaliana, mutations at any one of nine distinct loci confer chlorate resistance. Only one of the CHL genes, CHL3, has been shown genetically to be a nitrate reductase (NR) structural gene (NIA2) even though two NR genes (NIA1 and NIA2) have been cloned from the Arabidopsis genome. Plants in which the NIA2 gene has been deleted retain only 10% of the wildtype shoot NR activity and grow normally with nitrate as the sole nitrogen source. Using mutagenized seeds from the NIA2 deletion mutant and a modified chlorate selection protocol, we have identified the first mutation in the NIA1 NR structural gene. nia1, nia2 double mutants have only 0.5% of wild-type shoot NR activity and display very poor growth on media with nitrate as the only form of nitrogen. The nial-1 mutation is a single nucleotide substitution that converts an alanine to a threonine in a highly conserved region of the molybdenum cofactor-binding domain of the NR protein. These results show that the NIA1 gene encodes a functional NR protein that contributes to the assimilation of nitrate in Arabidopsis.     

Biochemical and genetical characterization of nitrate reductase deficient mutants of Petunia

Four NR^– lines were selected by their resistance to 100 mM chlorate from X-ray irradiated protoplasts of haploid Petunia hybrida var. Mitchell. The four cell lines were characterized by the presence of xanthine dehydrogenase activity and by complementation tests via protoplast fusion. One mutant (line 1) was classified as defective in the NR apoprotein (tentatively, nia-type) and the other three (lines 2, 3, 4) in the molybdenum cofactor (tentatively, cnx-type). Some NR activity (15 %) could be restored by adding unphysiologically high concentrations of molybdate to the culture medium in two of the cnx-lines (lines 3 and 4). The third cnx-line (line 2) had no NR activity. A complementation analysis via protoplast fusion confirmed that the mutants comprised 3 non-allelic groups. From these results it can be concluded that these NR^– mutants are recessive and that two of the cnx-mutants (lines 3, 4) are allelic.Abbreviations MS Murashige and Skoog medium (Murashige and Skoog 1962) - MG Müller and Grafe medium (Müller and Grafe 1978), containing amino acids - V47 protoplast medium (Binding 1974) - MS-413-medium (McCormack and Hanson 1980) - IAA indoleacetic acid - BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - AA amino acids - XDH xanthine dehydrogenase - PEG polyethylene glycol - NR nitrate reductase     

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

Summary 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.     

Toxicity of and mutagenesis by chlorate are independent of nitrate reductase activity in Chlamydomonas reinhardtii

Summary Spontaneous chlorate-resistant (CR) mutants have been isolated from Chlamydomonas reinhardtii wildtype strains. Most of them, 244, were able to grow on nitrate minimal medium, but 23 were not. Genetic and in vivo complementation analyses of this latter group of mutants indicated that they were defective either at the regulatory locus nit-2, or at the nitrate reductase (NR) locus nit-1, or at very closely linked loci. Some of these nit-1 or nit-2 mutants were also defective in pathways not directly related to nitrate assimilation, such as those of amino acids and purines. Chlorate treatment of wild-type cells resulted in both a decrease in cell survival and an increase in mutant cells resistant to a number of different chemicals (chlorate, methylammonium, sulphanilamide, arsenate, and streptomycin). The toxic and mutagenic effects of chlorate in minimal medium were not found when cells were grown either in darkness or in the presence of ammonium, conditions under which nitrate uptake is drastically inhibited. Chlorate was also able to induce reversion of nit ^– mutants of C. reinhardtii, but failed to produce His ⁺ revertants or Ara^r mutants in the BA-13 strain of Salmonella typhimurium. In contrast, chlorate treatment induced mutagenesis in strain E1F1 of the phototrophic bacterium Rhodobacter capsulatus. Genetic analyses of nitrate reductase-deficient CR mutants of C. reinhardtii revealed two types of CR, to low (1.5 mM) and high (15 mM) chlorate concentrations. These two traits were recessive in heterozygous diploids and segregated in genetic crosses independently of each other and of the nit-1 and nit-2 loci. Three her loci and four lcr loci mediating resistance to high (HC) and low (LC) concentrations of chlorate were identified. Mutations at the nit-2 locus, and deletions of a putative locus for nitrate transport were always epistatic to mutations responsible for resistance to either LC or HC. In both nit ⁺ and nit ^– chlorate-sensitive (CS) strains, nitrate and nitrite gave protection from the toxic effect of chlorate. Our data indicate that in C. reinhardtii chlorate toxicity is primarily dependent on the nitrate transport system and independent of the existence of an active NR enzyme. At least seven loci unrelated to the nitrate assimilation pathway and mediating CR are thought to control indirectly the efficiency of the nitrate transporter for chlorate transport. In addition, chlorate appears to be a mutagen capable of inducing a wide range of mutations unrelated to the nitrate assimilation pathway.     

Nitrate Utilization by Nitrate Reductase-deficient Barley Mutants

Two nitrate reductase-deficient barley mutants were studied for growth on nitrate and ammonium sources of nitrogen and for resistance to chlorate. Although nitrate reductase-deficient mutants in some species are chlorate-resistant (unable to reduce chlorate to chlorite), the barley mutants used in these studies when grown on nitrate and treated with chlorate were only slightly more resistant to chlorate than the control. When grown to maturity on vermiculite supplemented with either nitrate or ammonium nutrient solutions, the mutants produced as much dry weight and reduced nitrogen per plant as the control. The in vivo and in vitro nitrate reductase activities in the roots and shoots of the mutants grown on nitrate were consistently less than 10% of the control. To avoid the possibility that the mutants received reduced nitrogen from microbial sources, excised embryos were cultured under sterile conditions. Again the mutants were capable of growth and reduced nitrogen accumulation with nitrate as the sole source of nitrogen. In spite of the low apparent nitrate reductase activity, the nitrate reductase-deficient mutants are capable of substantial nitrate reduction.     

Chlorate toxicity in Aspergillus nidulans. Studies of mutants altered in nitrate assimilation.

Summary It had previously been held that chlorate is not itself toxic, but is rendered toxic as a result of nitrate reductase-catalysed conversion to chlorite. This however cannot be the explanation of chlorate toxicity in Aspergillus nidulans, even though nitrate reductase is known to have chlorate reductase activity. Among other evidence against the classical theory for the mechanism of chlorate toxicity, is the finding that not all mutants lacking nitrate reductase are clorate resistant. Both chlorate-sensitive and resistant mutants lacking nitrate reductase, also lack chlorate reductase. Data is presented which implicates not only nitrate reductase but also the product of the nirA gene, a positive regulator gene for nitrate assimilation, in the mediation of chlorate toxicity. Alternative mechanisms for chlorate toxicity are considered. It is unlikely that chlorate toxicity results from the involvement of nitrate reductase and the nirA gene product in the regulation either of nitrite reductase, or of the pentose phosphate pathway. Although low pH has an effect similar to chlorate, chlorate is not likely to be toxic because it lowers the pH; low pH and chlorate may instead have similar effects. A possible explanation for chlorate toxicity is that it mimics nitrate in mediating, via nitrate reductase and the nirA gene product, a shut-down of nitrogen catabolism. As chlorate cannot act as a nitrogen source, nitrogen starvation ensures.     

Nitrate Nonutilizing Mutants and Vegetative Compatibility Groups in Fusarium poae

Nitrate nonutilizing (nit) mutants were recovered from 24 isolates of Fusarium poae and used to force heterokaryons between these isolates and to determine vegetative compatibility. Between 30 and 90% of the mycelial blocks, cultured on medium containing chlorate, produced nit mutants. The amount of chlorate in the medium altered the frequency and spectrum of nit mutants recovered. Most of the mutants (63%) had lesions at a nitrate reductase structural locus (nit1). Another 30% were mutants at one or more loci that control the production of a molybdenum-containing cofactor necessary for nitrate reductase activity (NitM). A few (6%) of the mutations occurred in a regulatory gene specific for the nitrate reduction pathway (nit3). Pairings between nit1 and NitM mutants were made on minimal medium containing nitrate as the sole nitrogen source. A mutant grows thinly unless it forms a complementary heterokaryon upon contact with another mutant. Heterokaryon formation was indicated by dense growth where the two mutant colonies touched. The 24 isolates could be divided into 13 nonoverlapping vegetative compatibility groups, suggesting that asexual exchange of genetic information within F. poae is subject to significant limitations.     

Nitrate reduction in Aerobacter aerogenes

Summary Mutants of A. aerogenes blocked in aerobic and anaerobic nitrate assimilation and deficient in the reduction of nitrate and chlorate were found to give a positive methylred reaction and no gas formation from glucose. Resting cells, grown anaerobically in minimal medium with glucose, did not show gas production from formate. These results show that these mutants are also deficient in formate hydrogenylase. Revertants could readily be obtained by plating on minimal medium with nitrate as sole nitrogen source, indicating that in these mutants a pleiotropic point mutation is present, which affects both nitrate reductase and formate hydrogeny lase. It is suggested, that these mutants are deficient in the formation of an enzyme complex or particle on which these enzymes are present.     

Identification and characterization of a chlorate-resistant mutant of Arabidopsis thaliana with mutations in both nitrate reductase structural genes NIA1 and NIA2

Mutant plants defective in the assimilation of nitrate can be selected by their resistance to the herbicide chlorate. In Arabidopsis thaliana, mutations at any one of nine distinct loci confer chlorate resistance. Only one of the CHL genes, CHL3, has been shown genetically to be a nitrate reductase (NR) structural gene (NIA2) even though two NR genes (NIA1 and NIA2) have been cloned from the Arabidopsis genome. Plants in which the NIA2 gene has been deleted retain only 10% of the wildtype shoot NR activity and grow normally with nitrate as the sole nitrogen source. Using mutagenized seeds from the NIA2 deletion mutant and a modified chlorate selection protocol, we have identified the first mutation in the NIA1 NR structural gene. nia1, nia2 double mutants have only 0.5% of wild-type shoot NR activity and display very poor growth on media with nitrate as the only form of nitrogen. The nial-1 mutation is a single nucleotide substitution that converts an alanine to a threonine in a highly conserved region of the molybdenum cofactor-binding domain of the NR protein. These results show that the NIA1 gene encodes a functional NR protein that contributes to the assimilation of nitrate in Arabidopsis.     

Constitutive non-inducible expression of the Arabidopsis thaliana Nia 2 gene in two nitrate reductase mutants of Nicotiana Plumbaginifolia

We have isolated a haploid cell line of N. plumbaginifolia, hNP 588, that is constitutive and not inducible for nitrate reductase. Nitrate reductase mutants were isolated from hNP 588 protoplasts upon UV irradiation. Two of these nitrate reductase-deficient cell lines, nia 3 and nia 25, neither of which contained any detectable nitrate reductase activity, were selected for complementation studies. A cloned Arabidopsis thaliana nitrate reductase gene Nia 2 was introduced into each of the two mutants resulting in 56 independent kanamycin-resistant cell lines. Thirty of the 56 kanamycin-resistant cell lines were able to grow on nitrate as the sole nitrogen source. Eight of these were further analyzed for nitrate reductase enzyme activity and nitrate reductase mRNA production. All eight lines had detectable nitrate reductase activity ranging from 7% to 150% of wild-type hNP 588 callus. The enzyme activity levels were not influenced by the nitrogen source in the medium. The eight lines examined expressed a constitutive, non-inducible 3.2 kb mRNA species that was not present in untransformed controls.     

The capability of the algaeChlorella vulgaris andScenedesmus obliquus of utilizing amino acids as the sole nitrogen source

The capability of utilizing 20 amino acids and 2 amides as the sole nitrogen source for growth was studied in two green algae (Chlorophyceae). A comparison was made of the growth rate of algae in a mineral nutrient solution containing nitrate as the nitrogen source, with that in the same solution in which nitrogen in the form of nitrate was substituted by an equivalent nitrogen amount in the form of various amino acids. In addition to this, another series of experiments was carried out in whioh both culture media were supplied with glucose. The results show that both algae utilize a series of amino acids in dependence of their structure (mostly 3-carbon amino acids). The growth rate ofChlorella in the presence of these sources is the same as in nitrate, that ofScenedesmus even much higher. In the cultures containing glucose both algal species exhibit a higher growth rate in the media with the nitrate nitrogen source than in those with amino acids (with the exception of glycine inScenedesmus).     

Nitrogen Metabolism of Lemna minor. II. Enzymes of Nitrate Assimilation and Some Aspects of Their Regulation

In L. minor grown in sterile culture, the primary enzymes of nitrate assimilation, nitrate reductase (NR), nitrite reductase (NiR) and glutamate dehydrogenase (GDH) change in response to nitrogen source. NR and NiR levels are low when grown on amino acids (hydrolyzed casein) or ammonia; both enzymes are rapidly induced on addition of nitrate, while addition of nitrite induces NiR only. Ammonia represses the nitrate induced synthesis of both NR and NiR.NADH dependent GDH activity is low when grown on amino acids and high when grown on nitrate or ammonia, but the activities of NADPH dependent GDH and Alanine dehydro-genase (AIDH) are much less affected by nitrogen source. NADH-GDH and AIDH are induced by ammonia, and it is suggested that these enzymes are involved in primary nitrogen assimilation.     

The isolation and survival characterization of radiation and chemical-mutagen sensitive mutants of Pseudomonas aeruginosa

Chlorate-resistant mutants of Arabidopsis thaliana were isolated in order to find nitrate reductase-less mutants. It appeared that chlorate resistance in higher plants can arise by mutations concerning two different mechanisms: (1) a lower reduction rate of chlorate due to a lower level of nitrate reductase activity; (2) a lower increase in content of chlorate and/or chlorite and of chloride after chlorate treatment. One mutant of the first type and two mutants of the second type are described. The nitrate reductase-less mutant grows poorly on a medium with nitrate as the only nitrogen source but is not blocked in the uptake of nitrate. Both the other mutants exhibit a nitrate reductase activity equal to or higher than that of the wild type, but probably have a much lowered uptake of chlorate. The latter two mutants belong to the same complementation group, whereas the nitrate reductase-less mutant belongs to a different group.     

Reduction of Cytokinin Binding-protein in a Nitrate Reductase Deficient Mutant Cell Line of Datura inoxia

Suspension culture cells initiated from haploid Datura inoxia seedlings were transferred on a paper and were treated with UV. The nitrate reductase (NR) deficient mutants were isolated by selection for chlorate resistance. The NR activity could not be recovered, even though the mutants were transferred into the medium without selective pressure for three years. Isoelectrofocusing gel showed that the gene of NR was not destroyed by the treatment of UV. The mutant cells were defective in the cytokinin binding protein. The cytokinin binding-protein was isolated from wheat seedlings with the aid of 6BA immobilized on the epoxy-sepharose colunm. An addition of binding-protein, together with 6BA, to the medium for synthesis of RNA in vitro brough about an activation of RNA-polymerase. In wild type cells the NR activity was accelerated by the addition of cytokinin to the culture medium. In contrast, cytokinin was of no effect on the synthesis of NR in mutant cells. It is, therefore, suggested that the effect of cytokinin on the RNA synthesis and NR formation was regulated by the content of cytokinin binding-protein in Datura inoxia mutant cells.