Nitrate Reductase-deficient Mutants Research Articles

Improved sensitivity of genetic complementation of nitrate reductase deficient mutants via protoplast fusion

An improved rapid assay for complementation testing of mutants of Nicotiana tabacum deficient in nitrate reductase is described. The test is based on measurement of in vivo nitrate reductase activity in 7 to 10 day old cultures derived from fusion-treated protoplast mixtures of the respective mutants as a criterion for complementation. It allows to detect complementing hybrids induced by the conventional droplet fusion technique in small numbers of protoplasts per assay (8×10(4)).

Intergeneric asymmetric hybrids between Nicotiana plumbaginifolia and Atropa belladonna obtained by ?gamma-fusion?

Asymmetric nuclear hybrids have been obtained by fusion of cells from a nitrate-reductase deficient mutant of Nicotiana plumbaginifolia (cnx20) and gamma irradiated protoplasts of Atropa belladonna (irradiation doses tested were 10, 30, 50 and 100 krad). The hybrid formation frequency following selection for genotypic complementation in the NR function was in the range of 0.7%-3.7%. Cytogenetic studies demonstrated that all hybrid plants tested possessed multiple (generally tetra- or hexaploid) sets of N. plumbaginifolia (n = 10) chromosomes along with 6-29 Atropa chromosomes (n = 36), some of which were greatly deleted. Besides the cnxA gene (the selection marker), additional material of the irradiated partner was expressed in some of the lines, as shown by analyses of multiple molecular forms of enzymes. Surprisingly, rDNA genes of both parental species were present and amplified in the majority of the hybrids. Whenever studied, the chloroplast DNA in the hybrids was derived from the Nicotiana parent. Regenerants from some lines flowered and were partially fertile. It is concluded that irradiation of cells of the donor parent before fusion can be used to produce highly asymmetric nuclear hybrid plants, although within the dose range tested, the treatment determined the direction of the elimination but not the degree of elimination of the irradiated genome.

Genetic analysis of nitrate reductase deficient mutants of Nicotiana plumbaginifolia: Evidence for six complementation groups among 70 classified molybdenum cofactor deficient mutants

A total of 70 cnx mutants have been characterized from a collection of 211 nitrate reductase deficient (NR-) mutants isolated from mutagenized Nicotiana plumbaginifolia protoplast cultures after chlorate selection and regeneration into plants. They are presumed to be affected in the biosynthesis of the molybdenum cofactor since they are also deficient for xanthine dehydrogenase activity but contain NR apoenzyme. The remaining clones were classified as nia mutants. Sexual crosses performed between cnx mutants allowed them to be classified into six independent complementation groups. Mutants representative of these complementation groups were used for somatic hybridization experiments with the already characterized N. plumbaginifolia mutants NX1, NX24, NX23 and CNX103 belonging to the complementation groups cnxA, B, C and D respectively. On the basis of genetic analysis and somatic hybridization experiments, two new complementation groups, cnxE and F, not previously described in higher plants, were characterized. Unphysiologically high levels of molybdate can restore the NR activity of cnxA mutant seedlings in vivo, but cannot restore NR activity to any mutant from the other cnx complementation groups.

Selection and characterization of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia

Nitrate reductase mutants are a tool to study nitrate reductase (NR), a key enzyme of nitrogen assimilation in higher plants (Review by Kleinhofs, 1985).

Chromosomes in somatic hybrids between Nicotiana plumbaginifolia and a monoploid potato

Leaf mesophyll protoplasts of the monohaploid potato (Solanum tuberosum L.) clone H7322 were fused with callus protoplasts of nitrate reductase deficient (NR−) mutants Cnx 20 and NA 36 of Nicotiana plumbaginifolia. Somatic hybrid lines were selected for nitrate reductase proficiency. All callus lines tested appeared to be stable for the retention of the potato chromosome carrying the compensating NR gene when grown for over 1.5 years in the absence of nitrate. Shoots were regenerated from six different fusion lines of Cnx 20 + H7322 24 months after fusion. Chromosomal analysis in callus cultures revealed that in both fusion combinations 40–120 N. plumbaginifolia chromosomes were present, as were 9–20 potato chromosomes. Cells with 17 potato chromosomes in combination with a relatively small number (31) of N. plumbaginifolia chromosomes were found in one line. Preferential loss of species-specific chromosomes was not observed. Analysis of regenerating tissue from three lines of Cnx 20 + H7322 revealed that after 24 months of culture intra- and intergeneric translocations, fragments and deletions were present. Elimination of the potato and N. plumbaginifolia chromosomes had taken place before and after genome doubling.

Isolation and characterization of Nicotiana plumbaginifolia nitrate reductase-deficient mutants: genetic and biochemical analysis of the NIA complementation group

Two hundred and eleven nitrate reductase-deficient mutants (NR-) were isolated from mutagenized Nicotiana plumbaginifolia protoplast cultures by chlorate selection and regenerated into plant. More than 40% of these clones were classified as cnx and presumed to be affected in the biosynthesis of the molybdenum cofactor, the remaining clones being classified as nia mutants. A genetic analysis of the regenerated plants confirmed this proportion of nia and cnx clones. All mutants regenerated were found to carry monogenic recessive mutations that impaired growth on nitrate as sole nitrogen source. Mutants propagated by grafting on N. tabacum systematically displayed a chlorotic leaf phenotype. This chlorosis was therefore related to the NR deficiency. The observation of leaves with NR- chlorotic sectors surrounded by NR+ wild-type tissues suggests that an NR deficiency is not corrected by diffusible factors. Periclinal chimeras between wild-type tobacco and the NR- graft were also observed. In this type of chimeric tissue chlorosis was no longer detectable when NR+ cells were in the secondmost (L2) layer, but was still detectable when NR- cells were in the secondmost layer. The genetic analysis of nia mutants revealed that they belong to a single complementation group. However three nia mutants were found to complement some of the other nia mutants. The apoenzyme of nitrate reductase was immunologically detected in several nia mutants but not in other members of this complementation group. Some of the nia mutants, although they were NR-, still displayed methylviologen-nitrate reductase activity at a high level. These data show that the nia complementation group corresponds to the structural gene of nitrate reductase. Some of the mutations affecting this structural gene result in the overproduction of an inactive nitrate reductase, suggesting a feedback regulation of the level of the apoenzyme in the wild type.

Somatic Hybridization between Nicotiana tabacum and Nicotiana plumbaginifolia by Electrofusion of Mesophyll Protoplasts

Summary Leaf mesophyll protoplasts of a streptomycin resistant nitrate reductase deficient mutant of Nicotiana tabacum were fused with leaf mesophyll protoplasts of a wild type variety of N. plumbaginifolia in a simple electrofusion apparatus. Somatic hybrid colonies, and plants were readily recovered after fusion by selection of colonies in medium with nitrate as sole nitrogen source and also containing streptomycin. The value of this simple electrofusion apparatus (Watts and King, 1984) lies in its simplicity of construction and use and also in its ability to deal with literally millions of protoplasts in any one experiment.

Bromphenol blue: nitrate reductase activity in Nicotiana plumbaginifolia: an immunochemical and genetic approach

NADH: nitrate reductase (EC 1.6.6.1) was purified from Nicotiana plumbaginifolia leaves. As recently observed with nitrate reductase from other sources, this enzyme is able to reduce nitrate using reduced bromphenol blue (rBPB) as the electron donor. In contrast to the physiological NADH-dependent activity, the rBPB-dependent activity is stable in vitro. The latter activity is non-competitively inhibited by NADH. The monoclonal antibody ZM.96(9)25, which inhibits the NADH: nitrate reductase total activity as well as the NADH: cytochrome c reductase and reduced methyl viologen (rMV): nitrate reductase partial activities, has no inhibitory effect on the rBPB: nitrate reductase activity. Conversely, the monoclonal antibody NP.17-7(6) inhibits nitrate reduction with all three electron donors: NADH, MV or BPB. Among various nitrate reductase-deficient mutants, an apoprotein gene mutant ( nia. E56) shows reduced terminal activities but a highly increased rBPB: nitrate reductase activity. rBPB: nitrate reductase thus appears to be a new terminal activity of higher plant nitrate reductase and involves specific sites which are not shared by the other activities.

Nitrogen, carbohydrate and zinc requirements for the efficient induction of shoot morphogenesis from protoplast-derived colonies of Nicotiana plumbaginifolia

Factors affecting the regeneration of shoots from Nicotiana plumbaginifolia colonies were studied. As opposed to results obtained with tobacco, phytohormone pretreatments of those colonies did not appreciably affect their subsequent aptitude to develop shoots. Hexoses generally inhibited shoot morphogenesis at concentrations higher than 20 mM, but ribose sustained efficient shoot induction at higher concentration. Shoot induction was promoted by a high nitrogen supply. Nitrate promoted greater shoot induction than ammonium succinate or glutamine. Among the micronutrients tested, zinc was stringently required for efficient shoot induction, concentrations ranging between 10 and 100 μM being optimal. Under optimized conditions of regeneration, wild type as well as nitrate reductase deficient mutants could be efficiently regenerated.

Electrofusion of biochemically well characterized nitrate reductase deficient Nicotiana plumbagnifolia mutants. Studies of optimization and complementation

Electrofusion was carried out using biochemically well characterized NR − Nicotiana plumbaginifolia mutants ( Cnx 20, Nia 26, NA 36). In analytical experiments, optimal conditions for mesophyll-mesophyll and callus-callus protoplast fusions were assessed. Subsequently, in large scale experiments NR + somatic hybrids were obtained after mesophyll protoplast fusions between Cnx 20 + Nia 26 as well as after callus protoplast fusions between Cnx 20 + Nia 26 and between Cnx 20 + NA 36. In addition, complementation of the nia mutants Nia 26 and NA 36, each characterized by a distinct biochemical phenotype, was studied using electrofusion. In these experiments no completing NR + somatic hybrid callus was obtained. As fusion conditions were optimal and fusion products were observed to be formed it was concluded that the nia mutations, although leading to distinct biochemical phenotypes, are allelic. We also studied complementation in short term experiments. NR activity in vivo was assayed 3–4 weeks after fusion. Plants could be regenerated from the majority of the NR + somatic hybrid calli, resulting from the fusions between Cnx 20 + Nia 26 and Cnx 20 + NA 36. Chromosome numbers of shoot tip cells of glass house grown plants varied between 32–58, the majority having the normal tetraploid number (2 n = 40). Most of the plants appeared to be sterile.

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.

Isolation and Initial Characterization of Constitutive Nitrate Reductase-Deficient Mutants NR328 and NR345 of Soybean (Glycine max)

Two nitrate reductase deficient mutants of soybean (Glycine max [L.] Merr. cv Bragg) were isolated from approximately 10,000 M(2) seedlings, using a direct enzymic assay in microtiter plates. Stable inheritance of NR345 and NR328 phenotypes has been demonstrated through to the M(5) generation. Both mutants were affected in constitutive nitrate reductase activity. Assayable activities of cNR in nitrate-free grown seedlings was about 3 to 4% of the control for NR345 and 14 to 16% of the control for NR328. Both mutants expressed inducible NR during early plant development and were sensitive to nitrate and urea inhibition of nodulation. These new mutants will allow an extension of the characterization of nitrate reductases and their function in soybean. Preliminary evidence indicates that NR345 is similar to the previously isolated mutant nr(1), while NR328 is different.

Expression of the Mutant Character of Chlorate-resistant Mutants of Arabidopsis thaliana in Cell Culture

Summary The expression of the mutant character of nitrate reductase deficient mutant B25 and nitrate uptake mutant B1 of Arabidopsis thaliana at the plant and cell level was compared. Callus of both B1 and B25 proliferated on ammonium nitrate. In contrast with plants, callus of B1 was not able to grow on nitrate only. B1 grew well on ammonium sulphate. Callus of B25 grew better than the wildtype on nitrate, but could not use ammonium sulphate as the nitrogen source for callus proliferation. Callus of B1 and B25 exhibited resistance to low concentrations of chlorate (1–6 mM). The levels of enzyme activities in callus were comparable with those in plants. However, in contrast with plants, in callus of mutant B25 no differences were found in the nitrate reductase activity relative to the wildtype when measured in vivo and in vitro. Xanthine dehydrogenase activity could be measured in callus without purifying the extracts from inactivating factors, which, in extracts of plants, interfere with the enzyme assay.

Somatic hybridization of Nicotiana tabacum and Petunia hybrida

Leaf mesophyll protoplasts of a nitrate reductase deficient streptomycin resistant mutant of Nicotiana tabacum were fused with cell suspension protoplasts of wild type Petunia hybrida. Somatic hybrid cell colonies were selected for streptomycin resistance and nitrate reductase proficiency. Six independent cell lines, capable of growth in selection medium, were analysed by electrophoresis of callus peroxidases and leucine aminopeptidases and also by hybridization with rDNA and a chloroplast encoded gene as molecular probes. The results show that all six lines represented nuclear somatic hybrids, possessing the chloroplast of N. tabacum, at an early stage of development. However, after 6–12 months in culture, genomic incompatibility was observed resulting in the loss of most of the tobacco nuclear genome in the majority of the cell lines. One of the latter cell lines regenerated plants which possessed the chloroplast of N. tabacum in a predominantly P. hybrida nuclear background.

Characteristics of 36ClO 3− influx into nitrate reductase deficient mutant E 1Pisum sativum seedlings: Evidence for restricted ‘induction’ by nitrate compared with wild type

The characteristics of nitrate uptake into seedlings of Pisum sativum L. cv. Rondo mutant E 1 defective for nitrate reductase (NR) and of its parent variety Rondo have been investigated using 36ClO 3 − as an analogue for nitrate. The apparent Michaelis Menten constants ( K m) for 36ClO 3 − influx measured over 10 min were similar for mutant E 1 and the wild type (Wt). There was a 28% increase in 36ClO 3 − influx into Wt seedlings following nitrate pretreatment but this was not found when mutant seedlings were used. N starvation increased 36ClO 3 − influx into both mutant and Wt seedlings, and the rate of cycling E/I was also enhanced to a similar extent. The results are discussed in terms of current ideas on the regulation of nitrate uptake and assimilation.

Biochemical characterization of some nitrate reductase deficient mutants of Nicotiana plumbaginifolia

Four nitrate reductase deficient (NR −) mutants ( Cnx 20, Cnx 103: impaired in the Mo-cofactor; Nia 26, NA 36: mutated in teh structural gene for the apoprotein) of Nicotiana plumbaginifolia were characterized biochemically. The phenotype of Cnx 20 (Mo repair in vitro, complementation of Neurospora nit-1, dimeric CcR) and Nia 26 (no Mo repair in vitro, complementation of Neurospora nit-1, absence of dimeric CcR) is similar to, respectively, other cnxA-(for Cnx 20) and nia mutants studied in Nicotiana. New Phenotypes were discovered in NA 36 and Cnx 103, the latter one belonging to complementation group cnxD. The mutant NR of Cnx 103 cannot be repaired by Mo in vitro and does not complement Neurospora nit-1. However, cytochrome- c reductase (CcR) suggests that at least some dimerization takes place. NA 36 lacks all nitrate reducing activities, irrespective of the electron donor (NADH, BVH, FADH 2) used, the Mo-cofactor is intact and CcR activity is present in the dimerized form. It is concluded, that two kinds of mutations ( cnxD, NA 36) can result in the NR − phenotype ‘dimeric nitrate-inducible CcR, not repairable by Mo’.

Nitrate Assimilation and Growth of Steptoe Barley and one of its Nitrate Reductase Deficient Mutants

Nitrate Assimilation and Growth of Steptoe Barley and one of its Nitrate Reductase Deficient Mutants

Complementation analysis by somatic hybridisation and genetic crosses of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia

Fusion complementation experiments between nitrate reductase (NR) deficient lines CNX 20, 27, 82 and 103 of Nicotiana plumbaginifolia were performed with the already characterized N. plumbaginifolia mutants nx 1, 24 and 21, belonging respectively to the complementation groups cnx A, B and C. CNX 20 and 82 were identified as belonging to the group of cnx A. CNX 27 complemented with NX 1 and NX 21 but not with NX 24 indicating another B type. The fourth line, CNX 103 showed complementation with CNX 20, NX 21 and NX 24, revealing a fourth cnx complementation group, cnx D, that until now has not been described in higher plants. Genetic crosses inside respectively the NIA and the CNX group, and between NIA and CNX confirmed the fusion complementation results, and showed allelism for the nia mutants

Electrofusion, a simple and reproducible technique in somatic hybridization of Nicotiana plumbaginifolia mutants

Electrofusion of protoplasts from two complementary nitrate reductase deficient mutants of Nicotiana plumbaginifolia has resulted in somatic hybrid lines. Mesophyll protoplasts isolated from the cofactor mutant CNX 20 and fluorescein diacetate stained protoplasts derived from a cell suspension culture of the NA 36 line, being defective in the apoenzyme, were used in the fusion experiments. In total, 594 lines were recovered which could proliferate on a selective medium with nitrate as the sole nitrogen source. This is including 141 putative hybrid lines which were obtained after transfer of 1048 heterokaryons with a micromanipulator one day after electrofusion. The hybrid character of some of the selected lines was confirmed by nitrate reductase activity measurements. Plants were grown from hybrid calli.

Presence of molybdenum cofactor in seeds of wheat and barley

Molybdenum cofactor (Mo-co) was determined in seeds of wheat and barley by its ability to restore nitrate reductase (NR) activity in extracts of nitrate reductase-deficient mutants. Its activity was compared with that of wheat roots and leaves. Conditions for assay of Mo-co from different sources in the presence of molybdate and reduced glutathione (GSH) were optimised. The effect of heat-treatment of cell-free extracts from seeds, roots and leaves was also investigated. Mutant extracts of Neurospora crassa nit-1 and Nicotiana tabacum CnxA68, used as apoprotein source for in vitro complementation, were shown to give comparable results. The Mo-co activity, extracted from wheat and barley seeds, could be separated into two peaks by gel chromatography.