Nitrate Reductase Deficiency Research Articles

The effect of nitrate assimilation deficiency on the carbon and nitrogen status of Arabidopsis thaliana plants

Carbon (C) and nitrogen (N) metabolism are integrated processes that modulate many aspects of plant growth, development, and defense. Although plants with deficient N metabolism have been largely used for the elucidation of the complex network that coordinates the C and N status in leaves, studies at the whole-plant level are still lacking. Here, the content of amino acids, organic acids, total soluble sugars, starch, and phenylpropanoids in the leaves, roots, and floral buds of a nitrate reductase (NR) double-deficient mutant of Arabidopsis thaliana (nia1 nia2) were compared to those of wild-type plants. Foliar C and N primary metabolism was affected by NR deficiency, as evidenced by decreased levels of most amino acids and organic acids and total soluble sugars and starch in the nia1 nia2 leaves. However, no difference was detected in the content of the analyzed metabolites in the nia1 nia2 roots and floral buds in comparison to wild type. Similarly, phenylpropanoid metabolism was affected in the nia1 nia2 leaves; however, the high content of flavonol glycosides in the floral buds was not altered in the NR-deficient plants. Altogether, these results suggest that, even under conditions of deficient nitrate assimilation, A. thaliana plants are capable of remobilizing their metabolites from source leaves and maintaining the C-N status in roots and developing flowers.

THE USE OF PROTOPLAST FUSION AND TRANSFORMATION FOR THE PRODUCTION OF ASYMMETRIC SOMATIC HYBRIDS

ABSTRACT In asymmetric hybridization experiments, a Nicotiana paniculata cell line transformed with the Agrobacterium tumefaciens strain B6S3 was used as the donor plant material. This cell line exhibits two dominant marker genes, hormone-independent growth behavior and synthesis of octopine, as a result of the incorporation of T-DNA from Agrobacterium. The recipient cell line was the cnx-68 N. tabacum cell line which has two recessive markers: nitrate reductase deficiency and presence of an intracellular calcium-oxalate druse. Isolated protoplasts of the donor cell line N. paniculata B6S3 were mitotically inactivated by X-rays and fused with protoplasts of the cell line cnx-68. Asymmetric somatic hybrids were selected on hormone-free agar medium supplemented with 50 mM KClO3. Eighteen cell lines survived the selection treatment; seven of them exhibited the two dominant and the two recessive markers. In a second experiment, the marker gene hygromycin resistance was introduced into Brassica nigra by using ...

Nitrate Reductase- and Nitric Oxide-Dependent Activation of Sinapoylglucose:malate sinapoyltransferase in Leaves of Arabidopsis thaliana

Nitrate reductase (NR) activity is necessary for the synthesis of nitric oxide (NO), a key signaling molecule in plants. Here, we investigated the effect of NR deficiency on NO production and phenylpropanoid metabolism of Arabidopsis thaliana leaves. HPLC-mass spectrometry analysis showed that the NR double mutant (nia1 nia2) is deficient in the synthesis of sinapoylmalate (SM), the main phenylpropanoid end-product in wild-type leaves, resulting in accumulation of its precursor sinapoylglucose (SG). While real-time PCR analysis revealed no significant difference at the transcript level, sinapoylglucose:malate sinapoyltransferase (SMT) activity in leaf extracts was reduced in the mutant compared with the wild type. The low levels of SM in nia1 nia2 leaves do not result from the deficient nitrogen incorporation into amino acids, since the recovery of the amino acid content of nia1 nia2 by irrigating the plants with glutamine did not change the metabolic profile of this mutant. In contrast, an increased supply of nitrate stimulated NR activity and NO production, and enhanced SM and decreased SG levels in both genotypes. Nevertheless, sinapic acid esters in nia1 nia2 were not recovered when compared with those detected in the leaves of the wild-type plant. Mutant plants grown in medium supplemented with malate and an NO donor recovered SM to the levels of wild-type leaves. Overall, the results suggest that SMT activity is dependent on the NR-dependent steady-state levels of NO during plant development.

Effect of nitrate reductase deficiency on the accumulation of phenylpropanoids in Arabidopsis thaliana leaves

Effect of nitrate reductase deficiency on the accumulation of phenylpropanoids in Arabidopsis thaliana leaves

Contribution of shoots and roots to in vivo nitrate reduction in NADH-specific nitrate reductase-deficient mutant seedlings of barley (Hordeum vulgare L.)

Barley (Hordeum vulgare L.) has two nitrate reductase (NR) isozymes, namely NADH-specific NR and NAD(P)H-bispecific NR. To determine the effect of NADH-specific NR deficiency on in vivo nitrate reduction and distribution of reduced N to shoots and roots, the 15N-incorporation model was applied to NADH-specific NR-deficient mutant (AzI2) seedlings of barley. The N-deprived seedlings were treated with a nutrient solution containing 2.3 mM N03 - and 0.2 mM N02 -, and were labeled with different amount of 15N for 48 h under continuous illumination. In Az12, the total in vitro NR activity derived from the NAD(P)H-bispecific NR was only 10% of that of the wild type (Cv. Steptoe). In Az12, the total 15N03 - incorporation and translocation of absorbed 15N03 - to shoots were about 25% higher than those of Steptoe. Nitrate reduction in the Az12 roots was 2 times higher than that in Steptoe during the first period (0-24 h) and 1.3 times higher during the second period (24-48 h). However, nitrate reduction in the Az12 shoots was 10 to 30% lower than that in the Steptoe shoots. As a result, nitrate markedly accumulated in the Az12 shoots. Accumulation of reduced 15N in the Az12 roots was 2 times higher than that in the Steptoe roots, but 10% lower in the Az12 shoots than in the Steptoe shoots at the end of the experiment. Upward transport of reduced 15N via the xylem in Az12 was nearly 2 times more active than that of Steptoe throughout the experiment. This result suggested that the derepression of the NAD(P)H-NR isozyme in the Az12 shoots could compensate for the absence of the NADH-NR isozyme. Furthermore, increased levels of root nitrate reduction seemed to make up for the limited nitrate assimilation in the Az12 shoots.

A novel high efficiency, low maintenance, hydroponic system for synchronous growth and flowering of Arabidopsis thaliana

BackgroundArabidopsis thaliana is now the model organism for genetic and molecular plant studies, but growing conditions may still impair the significance and reproducibility of the experimental strategies developed. Besides the use of phytotronic cabinets, controlling plant nutrition may be critical and could be achieved in hydroponics. The availability of such a system would also greatly facilitate studies dealing with root development. However, because of its small size and rosette growth habit, Arabidopsis is hardly grown in standard hydroponic devices and the systems described in the last years are still difficult to transpose at a large scale. Our aim was to design and optimize an up-scalable device that would be adaptable to any experimental conditions.ResultsAn hydroponic system was designed for Arabidopsis, which is based on two units: a seed-holder and a 1-L tank with its cover. The original agar-containing seed-holder allows the plants to grow from sowing to seed set, without transplanting step and with minimal waste. The optimum nitrate supply was determined for vegetative growth, and the flowering response to photoperiod and vernalization was characterized to show the feasibility and reproducibility of experiments extending over the whole life cycle. How this equipment allowed to overcome experimental problems is illustrated by the analysis of developmental effects of nitrate reductase deficiency in nia1nia2 mutants.ConclusionThe hydroponic device described in this paper allows to drive small and large scale cultures of homogeneously growing Arabidopsis plants. Its major advantages are its flexibility, easy handling, fast maintenance and low cost. It should be suitable for many experimental purposes.

Nitrate Reductase Activities in Rice Genotypes in Irrigated Lowlands

Nitrate reductase (NR) is a key enzyme involved in nitrate assimilation in crops. In irrigated lowlands, specific activities of NADH‐ and NADPH‐NR in rice (Oryza saliva L.) have not been investigated. The objectives of this study were to evaluate the nitrate reductase activity (NRA) patterns of rice genotypes with different NRA levels under irrigated lowland conditions, and to determine the effects of NR deficiency on nitrate use, grain yield, biomass, and other agronomic characteristics. Seven cultivars and seven NR mutants were grown under irrigated lowland conditions; bioassayed in vitro for NRA at different growth stages; and evaluated for grain yield, biomass, and other agronomic characters. NADH‐NRA was present at minimal levels in all cultivars before transplanting. At 15 d after transplanting (DAT), NADH‐NRA in all cultivars reached optimum levels and gradually declined until maturity. A similar pattern was observed in most of the NR mutants but with lower levels of activity. NADPHNRA in cultivars and mutants gradually increased from 15 to 45 DAT and then fluctuated between 5 to 10 nmol NO−2 min−1 g−1 fresh weight until maturity. Nitrate availability in the soil may significantly influence NRA and nitrate accumulation in rice in irrigated lowlands. Effects of low or deficient levels of NR on traits evaluated varied among mutants. A decrease of 27 to 73% in grain yield was observed in mutants relative to original cultivars. This study demonstrates that rice assimilates nitrate in anaerobic and flooded conditions, which may have implications for genetic improvement and nitrate or nitrogen management strategies in rice.

Registration of Five Near‐Isogenic Genetic Stocks of ‘Juneau’ Pea with Altered Nodulation and Nitrate Reductase Deficiency: A317I, nod 3 I, A317nod 3 I, E135I, and R25I

Registration of Five Near‐Isogenic Genetic Stocks of ‘Juneau’ Pea with Altered Nodulation and Nitrate Reductase Deficiency: A317I, nod ₃ I, A317nod ₃ I, E135I, and R25I

Amplification of repetitive DNA from Nicotiana plumbaginifolia in asymmetric somatic hybrids between Nicotiana sylvestris and Nicotiana plumbaginifolia

Asymmetric somatic hybrids were obtained between a chlorophyll-deficient mutant of Nicotiana sylvestris (V42) and a nitrate-reductase (NR)-deficient line of N. plumbaginifolia (cnx20 or Nia26), using each of the parents alternately as the irradiated donor. Irradiation doses applied ranged from 10 to 1,000 Gy of gamma-rays. Hybrid selection was based on complementation of NR deficiency with wild-type NR genes. To aid in the analysis of somatic hybrids, species-specific repetitive DNA sequences from N. plumbaginifolia (NPR9 and NPR18) were cloned. NPR18 is a dispersed repetitive sequence occupying about 0.4% of the N. plumbaginifolia genome. In turn, NPR9, which is part of a highly repetitive DNA sequence, occupies approximately 3% of the genome. The species-specific plant DNA repeats, together with cytological analysis data, were used to assess the relative amount of the N. plumbaginifolia genome in the somatic hybrids. In fusion experiments using irradiated N. plumbaginifolia, an increase in irradiation dose prior to fusion led to a decrease in N. plumbaginifolia nuclear DNA content per hybrid genome. For some hybrid lines, an increase in the quantity of repetitive sequences was detected. Thus, hybrid lines 1NV/21, 100NV/7, 100NV/ 9, and 100NV/10 (where N. plumbaginifolia was the irradiated donor) were characterized by amplification of NPR9. In the reverse combination (where N. sylvestris was the irradiated donor), an increase in the copy number of NPR18 was determined for hybrid clones 1VC/2, 1VC/3, 100VC/2 and oct100/7. Possible reasons for the amplification of the repeated sequences are discussed.

Microprotoplast isolation, enrichment and fusion for partial genome transfer in plants

Somatic hybridization using protoplasts with whole genomes has often resulted in complex hybrids with many unwanted chromosomes or genes. Several researchers have attempted to reduce the number of undesired chromosomes through irradiation of the donor protoplasts, but so far without much success. Alternatively, micropro‐toplasts containing one or a few chromosomes can be used for partial genome transfer, as has been demonstrated in human and other mammalian cell systems using microcells. Recently, we have optimized the ‘microprotoplast system’ for several donor cell lines (potato, Nicotiana, sugar beet) carrying various genetic markers, such as kanamycin resistance, β‐glucuronidase, nitrate reductase deficiency, hormone autotrophy, opines, etc. Protocols were developed to obtain higher yields of micro‐protoplasts as well as to enrich sub‐diploid microprotoplasts containing one or a few chromosomes. These microprotoplasts were fused with whole mesophyll protoplasts of recipient lines using polyethylene glycol. Various requirements for lines used as donor and recipient partners in microprotoplast‐protoplast fusions are described. The results are discussed in the context of partial genome transfer.

Spontaneous and induced loss of chromosomes in slow-growing somatic hybrid calli of Solanum tuberosum and Nicotiana plumbaginifolia

Rate and extent of spontaneous and induced chromosome loss have been determined at the callus level of somatic hybrids of mutants of Solanum tuberosum and Nicotiana plumbaginifolia. AEC (amino ethyl cystein) resistance in potato and Nitrate-Reductase deficiency in N. plumbaginifolia have been used as genetic markers and chromosome morphology as a cytological marker. In this combination, development of hybrid callus was late and slow. Only a limited number of non-regenerable hybrid calli have become available. Chromosome loss could clearly be established in these hybrids from loss of markers and from chromosome cytology. Loss of markers occurred independantly.

M2 seed screening for nitrate reductase deficiency in Nicotiana plumbaginifolia

Nitrate reductase-deficient mutants can be isolated from protoplast-derived cells on the basis of chlorate resistance. Since nitrate assimilation is a highly regulated and tissue specific process, it was expected that mutant selection at the plant level could lead to the identification of new genes involved in the control of nitrate assimilation. Three selective procedures were compared for the selection of mutants impaired in nitrate assimilation, from M2 seeds: chlorate selection, and rescue of plants specifically unable to grow on low, or high, nitrate concentrations. The rescue of some of the plantlets unable to grow on nitrate was achieved through a shoot culture step by treatment with growth regulators. All three procedures led to comparable frequencies of around 5 × 10 −3 in the isolation of mutants totally impaired for nitrate assimilation. The majority of these mutants were of the cnx type, as opposed to mutants selected for chlorate resistance at the cell level, which are predominantly of the nia type. nia mutants and mutants with leaky phenotypes were also identified, but unexpectedly no other class of mutants totally impaired in nitrate assimilation was identified among isolated mutants. The implications of these data are that M2 seed screening can be used as an alternative route to mutant selection at the cell level for the isolation of mutants totally impaired in nitrate assimilation. This approach can be envisaged for the isolation of nitrate reductase-deficient mutants from species recalcitrant to in vitro manipulation at the cell level and subsequent regeneration into fertile plants.

Asymmetric hybridization in Nicotiana by “gamma fusion” and progeny analysis of self-fertile hybrids

Mesophyll protoplasts of the nitrate-reductase (NR)-deficient Nicotiana plumbaginifolia mutant, "Nia26", were fused with γ-irradiated mesophyll protoplasts of Nicotiana sylvestris, V-42. Hybrid selection was based on complementation of NR deficiency by transfer of the donor NR gene to N. plumbaginifolia. Regenerated hybrids had different numbers of donor chromosomes in a tetraploid background of N. plumbaginifolia. The transfer and expression of different isozymes from the donor were also observed. Six self-fertile regenerants were obtained from 21 independently isolated cell colonies. Progeny analyses revealed: (1) the linkage of NR and shikimate dehydrogenase (ShDh); (2) a stabilization of the transmission rate of NR; and (3) the obtainment of mono- and disomic addition lines in the first and second progeny of the original regenerants. Southern hybridization analyses demonstrated unequivocally the presence of the NR gene from the donor partner in progeny plants.

Combination of kanamycin resistance and nitrate reductase deficiency as selectable markers in one nuclear genome provides a universal somatic hybridizer in plants

The combination in the nuclear genome of a dominant resistance marker (to select against unfused wild-type cells) and a recessive deficiency marker (to select against unfused mutant cells) in a cell line should provide a system for selecting fusion hybrids between the mutant line and any wild-type line. To test this idea, we fused protoplasts from a non-morphogenic cell line of Nicotiana tabacum which was kanamycin resistant (by transformation) and deficient in nitrate reductase (NR-K+) with protoplasts from N. tabacum cv. Petit Havana clone SR1, which provided resistance against streptomycin as an additional selectable marker (NR+K-SR+). Putative hybrids were selected using a culture medium containing no available reduced nitrogen source and 50 mg/l kanamycin sulphate. After regeneration into plants, the hybrid character was demonstrated from: (i) the morphological variation of the regenerants; (ii) the chromosome number; (iii) the ability to grow on medium without a reduced nitrogen source and containing kanamycin sulphate at 50 mg/l; (iv) the presence of nitrate reductase activity; (v) the presence of the gene coding for neomycin phosphotransferase, which provides resistance to kanamycin sulphate; (vi) callus formation from leaves on medium containing 1 g/l streptomycin or 50 mg/l kanamycin sulphate; (vii) F1 plants containing nitrate reductase and the gene for neomycin phosphotransferase. Fusions between the mutant cell line (NR-K+) and three wild-type tobacco species and subsequent cultivation on medium containing no available nitrogen source but 50 mg/l kanamycin sulphate resulted in callus formation with all combinations, while hybrid plants were only regenerated when N. sylvestris was the fusion partner.

Immunological evidence for transfer of the barley nitrate reductase structural gene to Nicotiana tabacum by protoplast fusion

A protoplast fusion experiment was designed in which the selectable marker, nitrate reductase (NR), also served as a biochemical marker to provide direct evidence for intergeneric specific gene transfer. NR-deficient tobacco (Nicotiana tabacum) mutant ‘Nia30’ protoplasts were the recipients for the attempted transfer of the NR structural gene from 50 krad γ-irradiated barley (Hordeum vulgare L.) protoplasts. Barley protoplasts did not form colonies and Nia30 protoplasts could not grow on nitrate medium; therefore, selection was for correction of NR deficiency allowing tobacco colonies to grow on nitrate medium. Colonies were selected from protoplast fusion treatments at an approximate frequency of 10-5. This frequency was similar to the Nia30 reversion frequency, and thus provided little evidence for transfer of the barley NR gene to tobacco. Plants regenerated from colonies had NR activity and were analyzed by western blotting using barley NR antiserum to determine the characteristics of the NR conferring growth on nitrate. Ten plants exhibited tobacco NR indicating reversion of a Nia30 mutant NR locus. Twelve of 26 regenerated tobacco plants analyzed had NR subunits with the electrophoretic mobility and antigenic properties of barley NR. These included plants regenerated from colonies selected from 1) co-culturing a mixture of Nia30 protoplasts with irradiated barley protoplasts without a fusion treatment, 2) a protoplast fusion treatment of Nia30 and barley protoplasts, and 3) a fusion treatment of Nia30 protoplasts with irradiated barley protoplasts. No barley-like NR was detected in plants regenerated from a colony that grew on nitrate following selfed fusion of Nia30 protoplasts. Because tobacco plants expressing barley-like NR were recovered from mixture controls as well as fusion treatments, explanations for these results other than protoplast fusionmediated gene transfer are discussed.

Co-segregation of nitrate-reductase activity and normal regeneration ability in selfed sibs of Nicotiana plumbaginifolia somatic hybrids, heterozygotes for nitrate-reductase deficiency

The nitrate-reductase (NR) defective cell lines of Nicotiana plumbaginifolia isolated in our laboratory could not be regenerated into plants on the standard medium (Márton et al. 1982 a). The normal regeneration potential, however, was restored in somatic hybrids obtained by fusing the NR(-) (green) lines with a pigment deficient (P(-)), but NR(+) line, A28. Somatic hybrid plants were fertile in two combinations (A28 + NA9 and A28 + NX9). As expected, segregation for NR(-) and P(-) was found after selfing the somatic F1 (SF1) obtained by protoplast fusion, and in the F2. The variable segregation ratios are explained by chromosome abnormalities. Co-segregation of the NR(-) phenotype and the altered response to shoot induction on standard medium suggest the involvement of the nitrate-assimilatory pathway in determining shoot regeneration ability.

A rapid assay for genetic complementation of nitrate reductase deficiency via bulk protoplast fusion

Genetic complementation of different nitrate reductase-deficient (NR-) Nicotiana plumbaginifolia cell lines could be recognized in the described fusion disc technique as early as 5–10 days after protoplast fusion. Protoplasts of previously characterized mutant cell lines, belonging to four different complementation groups, were mixed in pairwise combinations and fused by the drop-wise fusion technique at very high protoplast densities (106 protoplasts in an 8–10 mm spot) which resulted in the formation of a ‘fusion disc’ on the bottom of the petri dish. After 5–10 days of incubation in K3 medium the restoration of NR activity could be detected directly in the original culture by the in vivo NR assay. Such a rapid test giving information about the genetic complementation of different auxotrophic cell lines has not previously been published for plants.

Genetic analysis of nitrate reductase-deficient tobacco plants regenerated from mutant cells

Fifteen nitrate reductase (NR)-deficient mutants that had been selected from amphihaploid cell cultures of Nicotiana tabacum and regenerated to fertile amphidiploid plants were genetically analyzed through crosses. All the 15 mutants proved to be allelic. Segregation among F2 progeny from crosses between mutant and wild-type plants and among testcross progeny showed the regenerated plants to be homozygous double mutants. The NR deficiency is conferred by two unlinked recessive nuclear mutations, which thus define a pair of duplicate loci (nia1 nia2). These loci were identified as the structural genes for the apoprotein of NADH-NR. — Two mutants (Nia28, Nia30) were characterized further. Both Nia28 and Nia30 plants had less than 2% of wild-type NR activity, were resistant to chlorate and incapable of sustained growth on nitrate or in soil. However, they grew normally on media containing ammonium succinate (with or without nitrate). Growth test showed that Nia28 seedlings do not utilize nitrate, whereas Nia30 seedlings utilize nitrate at a very small rate. — The examination of single mutants (nia1-28/28 and nia2-28/28) revealed that either of the two loci is able to produce wild-type levels of NR activity. NR activity and chlorate sensitivity respond to nia + gene dosage only at the very early seedling stage. At later developmental stages, both the basal and the nitrate induced levels of NR activity were found to be independent of the number of nia + genes, indicating complete compensation of gene dosage effects by regulatory mechanism.

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.

Effect of Nitrate Reductase Deficiency upon Growth, Yield, and Protein in Barley1

Two nitrate reductase (NR)‐deficient mutants, Az 12 and Az 13, and the ‘Steptoe’ control were grown in the field to determine the effect of NR upon growth, N utilization, yield, and grain protein in barley (Hordeum vulgate L.). Although seasonal in vitro and in vivo NR activities in the mutants were less than 10% of the control, total vegetative dry weight, total reduced N, and percent grain protein in the mutants and Steptoe were not significantly different at maturity. Grain yields of both mutants, however, were significantly lower than the control. A greenhouse study indicated that Az 12 could utilize nitrate as a nitrogen source, although not as effectively as the control. These results indicate that harley mutants with very low levels of apparent NR can utilize nitrate as a N source and that NR activity is not closely associated with percent grain protein. At present it is not known whether the low level of NR in the mutants is sufficient to account for the nitrate reduced or whether there is another mechanism for reducing nitrate in plants that is not detected by conventional NR assay techniques.