NR-deficient Mutants Research Articles

New insights into natural rubber biosynthesis from rubber-deficient lettuce mutants expressing goldenrod orguayule cis-prenyltransferase.

Lettuce produces natural rubber (NR) with an average Mw of > 1 million Da in laticifers, similar to NR from rubber trees. As lettuce is an annual, self-pollinating, and easily transformable plant, it is an excellent model for molecular genetic studies of NR biosynthesis. CRISPR/Cas9 mutagenesis was optimized using lettuce hairy roots, and NR-deficient lettuce was generated via bi-allelic mutations in cis-prenyltransferase (CPT). This is the first null mutant of NR deficiency in plants. In the CPT mutant, orthologous CPT counterparts from guayule (Parthenium argentatum) and goldenrod (Solidago canadensis) were expressed under a laticifer-specific promoter to examine how the average Mw of NR is affected. No developmental defects were observed in the NR-deficient mutants. The lettuce mutants expressing guayule and goldenrod CPT produced 1.8 and 14.5 times longer NR, respectively, than the plants of their origin. This suggests that, although goldenrod cannot synthesize a sufficiently lengthy NR, goldenrod CPT has the catalytic competence to produce high-quality NR in the cellular context of lettuce laticifers. Thus, CPT alone does not determine the length of NR. Other factors, such as substrate concentration, additional proteins, and/or the nature of protein complexes including CPT-binding proteins, influence CPT activity in determining NR length.

The difference in N metabolism between NAD(P)H-specific NR-deficient mutant and wild-type barley (Hordeum vulgare L.)

Barley (Hordeum vulgare L.) is an important crop for cereal research. In this study, two barley genotypes the wild-type (Steptoe) and the mutant (Az12) were used. An experiment was conducted using 15N-tracing method to NADH-specific nitrate reductase (NR)-deficient mutant seedling of barley. The N-depleted seedlings were exposed to a nutrient solution containing nitrate and nitrite, and were labeled with 15N for 38 h under (14 L/10D) cycles. The two genotypes utilized 15NO3− and accumulated it as reduced 15N, predominately in the shoots. However, nitrate reduction in the Az12 shoots was 9% lower than that in the Steptoe shoots at 38 h. As a result, in the Az12, nitrate accumulation in shoots was 78% higher than that in the Steptoe. Accumulation of reduced 15N in the Az12 roots was nearly similar to that of the Steptoe roots, but 8% lower in the Az12 shoots than in the Steptoe shoots at the end of the experiment. Also for both genotypes, root contribution increased during L/D cycles and decreased during the subsequent light cycle. Upward transport of reduced 15N via the xylem in the Az12 was nearly two times higher than that in Steptoe during the second light period (24–38 h). In both genotypes, xylem transport of reduced 15N was far exceeded the downward phloem transport.AbbreviationsAnlaccumulation of reduced 15N from 15NO3− in non-labeled roots of split rootsAraccumulation in roots of reduced 15N from 15NO3−Asaccumulation in shoots of reduced 15N from 15NO3−Rr15NO3− reduction in rootsRs15NO3− reduction in shootsTptranslocation to root of shoot reduced 15N from 15NO3− in phloemTxtranslocation to shoot of root-reduced 15N from 15NO3− in xylemFWfresh weight

Characterization of novel nitrate reductase-deficient mutants for transgenic Dunaliella salina systems.

The aim of the present study was to isolate and characterize novel nitrate reductase (NR)-deficient mutants, which may be useful for the transgenic manipulation of Dunaliella salina. Three NR-deficient mutants of D. salina, J-1, J-2, and J-3, were successfully isolated by screening for chlorate resistance after chemical mutagenesis with ethylnitrosourea. NR activity was not detected in the mutants and the expression of NR mRNA was significantly decreased. Growth analysis of D. salina strains grown in media containing different nitrogen sources revealed that these mutants were capable of utilizing nitrite and urea, but not nitrate as a nitrogen source, indicating that these mutants are indeed NR-deficient. Mutation analysis of NR cDNA sequences revealed that there were 11 point mutations shared by the J-1, J-2, and J-3 mutants. Furthermore, the results of the functional complementation experiment showed that NR activity of transformant T-1 derived from J-1 was recovered to 48.1 % of that of the wild-type D. salina. The findings of the present study indicate that nitrate may be used as a selective agent rather than antibiotics or herbicides for the isolated NR-deficient mutants in future transgenic D. salina systems.

Nitrate reductase is required for the transcriptional modulation and bactericidal activity of nitric oxide during the defense response of Arabidopsis thaliana against Pseudomonas syringae

Nitrate reductase (NR) has emerged as a potential NO source in plants. Indeed, the Arabidopsis thaliana NR double-deficient mutant (nia1 nia2) produces low NO and develops abnormal susceptibility to bacterial infection. We have employed quantitative real-time polymerase chain reactions to analyze the effects of NO gas on the expression of defense-related genes in wild-type and nia1 nia2 A. thaliana plants that were inoculated with an avirulent strain of Pseudomonas syringae pv. tomato. The pathogenesis-related gene 1 (PR1) was up-regulated by bacterial infection, and its expression was higher in the wild type than in nia1 nia2. Fumigation with NO attenuated the expression of PR1 and other salicylic acid-related genes in plants that had been inoculated with P. syringae. Nevertheless, NO inhibited the most intense bacterial growth and disease symptoms in nia1 nia2 leaves. The NO fumigation also directly modulated lignin biosynthesis-related gene expression (CAD1) and parts of the auxin (TIR1, ILL1, GH3) and ethylene (ACCS7) pathways, among other defense-related genes, and their modulation was more intense in the NR-deficient mutant. Pathogen inoculation induced delayed but intense H2O2 production in mutant leaves in comparison with the wild type. Hydrogen peroxide potentiated the microbicidal effects of NO against bacterial cultures. These results suggest that NO has a direct microbicidal effect in combination with H2O2 to allow for the attenuation of the SA-mediated defense response, thereby reducing the energy expenditure associated with defense-related gene transcription. Overall, these results highlight the importance of NR-dependent NO production in the establishment of disease resistance.

Tungstate: is it really a specific nitrate reductase inhibitor in plant nitric oxide research?

Nitrate reductase (NR) is an enzymatic source of nitric oxide (NO) in plants, and it needs Mo for the Mo-cofactor to be activated. Because NR-deficient mutants are not always available in some species, a cheap and simple pharmacological application of tungstate, which substitutes for Mo in the Mo-cofactor as a competitive antagonist, is widely used as a NR inhibitor in plant NO research. However, evidence indicates that tungstate not only inactivates NR but also inhibits other molybdate-dependent enzymes in plants. In addition, a number of investigations have shown that tungstate also inhibits root growth, affects cortical microtubule formation, and induces programmed cell death (PCD) in plants, just like other heavy metals do. Therefore, tungstate has been shown to exert many other effects that are not connected with the inhibition of NR activity. The origin and mechanism of using tungstate as a NR inhibitor in plants is reviewed here and the progress regarding tungstate toxicity to plants and the possible problems involved in using tungstate as a NR inhibitor in plant NO research are analysed. In summary, the use of tungstate as a NR inhibitor in plant NO research must be treated with caution, keeping in mind that it is not completely specific. It is necessary to search for more NR-deficient mutants and new, specific NR inhibitors. A combination of pharmacological and biochemical analysis with a genetic approach will be necessary in order to investigate the roles of NO in plants.

A duplicated carbonic anhydrase 1 (DCA1) promoter mediates the nitrate reductase gene switch of Dunaliella salina

The unicellular Dunaliella salina, the most extremely halotolerant eukaryote, is able to survive in salt concentrations from 0.05 to 5 M NaCl, whose halotolerant property encouraged us to develop salt-inducible promoters for research on transgenic bioreactors. A duplicated carbonic anhydrase 1 (DCA1) promoter from D. salina has been identified to drive the inducible expression of the bar gene under the induction of different NaCl concentrations. The purpose of this study was to identify whether the DCA1 promoter could drive and modulate the expression of the nitrate reductase (NR) gene in NR-deficient mutants of D. salina. Here, the wild-type NR gene under the control of the DCA1 promoter was transformed into the NR-deficient mutant (J1) of D. salina by electroporation, and the complemented transformant T1-J1 was screened by nitrate, in which NR activity was restored up to 48% of the wild-type (WT) cells. Different from the natural NR gene switch in WT cells where NR activity disappeared in ammonium medium, NR activity was detected in the T1-J1 cells despite changes of the nitrogen sources. Furthermore, both the NR transcripts and NR activity were increased with rising NaCl concentration from 0.5 to 2.0 M in the T1-J1 cells but not in the WT cells. These findings strongly support that the DCA1 promoter not only drives the inducible expression of the NR gene effectively, but also mediates lost of the natural NR gene switch in D. salina.

Verticillium dahliae toxins-induced nitric oxide production in Arabidopsis is major dependent on nitrate reductase

The source of nitric oxide (NO) in plants is unclear and it has been reported NO can be produced by nitric oxide synthase (NOS) like enzymes and by nitrate reductase (NR). Here we used wild-type, Atnos1 mutant and nia1, nia2 NR-deficient mutant plants of Arabidopsis thaliana to investigate the potential source of NO production in response to Verticillium dahliae toxins (VD-toxins). The results revealed that NO production is much higher in wild-type and Atnos1 mutant than in nia1, nia2 NR-deficient mutants. The NR inhibitor had a significant effect on VD-toxins-induced NO production; whereas NOS inhibitor had a slight effect. NR activity was significantly implicated in NO production. The results indicated that as NO was induced in response to VD-toxins in Arabidopsis, the major source was the NR pathway. The production of NOS-system appeared to be secondary.

Exogenous auxin-induced NO synthesis is nitrate reductase-associated in Arabidopsis thaliana root primordia

Nitric oxide (NO) functions in various physiological and developmental processes in plants. However, the source of this signaling molecule in the diversity of plant responses is not well understood. It is known that NO mediates auxin-induced adventitious and lateral root (LR) formation. In this paper, we provide genetic and pharmacological evidence that the production of NO is associated with the nitrate reductase (NR) enzyme during indole-3-butyric acid (IBA)-induced lateral root development in Arabidopsis thaliana L. NO production was detected using 4,5-diaminofluorescein diacetate (DAF-2DA) in the NR-deficient nia1, nia2 and Atnoa1 (former Atnos1) mutants of A. thaliana. An inhibitor for nitric oxide synthase (NOS) N(G)-monomethyl-l-arginine (l-NMMA) was applied. Our data clearly show that IBA increased LR frequency in the wild-type plant and the LR initials emitted intensive NO-dependent fluorescence of the triazol product of NO and DAF-2DA. Increased levels of NO were restricted only to the LR initials in contrast to primary root (PR) sections, where NO remained at the control level. The mutants had different NO levels in their control state (i.e. without IBA treatment): nia1, nia2 showed lower NO fluorescence than Atnoa1 or the wild-type plant. The role of NR in IBA-induced NO formation in the wild type was shown by the zero effects of the NOS inhibitors l-NMMA. Finally, it was clearly demonstrated that IBA was able to induce NO generation in both the wild-type and Atnoa1 plants, but failed to induce NO in the NR-deficient mutant. It is concluded that the IBA-induced NO production is nitrate reductase-associated during lateral root development in A. thaliana.

Isolation and characterization of a nitrate reductase deficient mutant of Chlorella ellipsoidea (Chlorophyta)

Using X-ray mutagenesis and a chlorate-resistance selection method, a nitrate reductase (NR)-deficient mutant of Chlorella ellipsoidea nrm-4 was isolated, which is incapable of using NO3− as a nitrogen source. NR activity was not detected in nrm-4, suggesting this is a null mutation. Molecular analysis revealed that nrm-4 has a two base deletion at position 2348–49 of its DNA sequence, which produced a frame-shift mutation. The nrm-4 mutation is stable and nrm-4 algae could only use NH4+ and NO2− as nitrogen sources. Expression of a wild-type NR gene could complement this NR-deficient mutant. Furthermore, this transgenic strain was able to grow in NO3− medium, when the growth rate of the nrm-4 strain was equal to that of the wide type alga. The nrm-4 strain could potentially be used as a bioreactor that uses nitrate as a selectable marker instead of an antibiotic or herbicide.

Nitrate reductase and growth of Arabidopsis thaliana in solution culture

As a principal limiting nutrient, the metabolism of nitrogen is central to understanding plant growth and performance. Nitrate reductase (NR) is the first, and thus a critical, enzyme in the overall pathway for N incorporation. As such, and because it is highly regulated at the transcriptional, translational and post-translational levels, NR has received a large amount of experimental attention. On the other hand, there have been a number of reports that plant growth can be maintained with much less, or much more, NR activity than is normally present, and without the normal controls. The actual relationship between NR activity and growth is, however, poorly understood. The objective of this study was to examine the integration of growth, protein accumulation and patterns of nitrate reductase (NR) activity in wild-type Arabidopsis thaliana L. plants and a mutant lacking NR. Our results show that WT plants, during their exponential growth phase, had as much as 15-times more NR activity than needed to support the recorded growth. NR-deficient mutants, on the other hand, achieved equivalent exponential growth rates, lagging WT by about two weeks, but had less than 20% of the NR needed to support that growth. Both WT and mutants grew equally well when nitrite was supplied instead of nitrate, so long as the nitrite was continuously present, suggesting involvement of N-conversions in the growth medium. When N was supplied only in short pulses, nitrite toxicity was indicated, but the mutants still survived and grew on nitrate. We conclude that the absolute requirements for NR in plants may be highly flexible. The mechanism by which NR-deficient plants incorporate nitrate and support growth remains unclear, however.

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.

Gene expression of the NO3- transporter NRT1.1 and the nitrate reductase NIA1 is repressed in Arabidopsis roots by NO2-, the product of NO3- reduction.

NRT1.1 and NIA1 genes, which encode a nitrate (NO3-) transporter and the minor isoform of NO3- reductase (NR), respectively, are overexpressed in roots of NR-deficient mutants of Arabidopsis grown on nutrient solution containing NO3- and reduced N. The overexpression is found only in mutants with reduced NIA2 activity, and disruption of the NIA1 gene alone has no effect on NRT1.1 expression. Because the up-regulation of NRT1.1 and NIA1 is observed in N-sufficient NR mutant plants, it cannot be related to a release of the general feedback repression exerted by the N status of the plant. Our data do not support the hypothesis of overinduction of these genes by an increased concentration of NO3- in tissues. Furthermore, although a control by external pH might contribute to the regulation of NRT1.1, changes in external pH due to lack of NR activity cannot alone explain the up-regulation of both genes. The stimulation of NRT1.1 and NIA1 in NR mutants in these conditions suggests that NR activity is able to repress directly the expression of both genes independently of the availability of reduced N metabolites in wild-type plants. Accordingly, nitrite (NO2-) strongly represses NRT1.1 and NIA1 transcript accumulation in the roots. This effect is rapid, specific, and reversible. Furthermore, transport studies on plants exposed to NO2- show that down-regulation of the NRT1.1 gene is associated with a decrease in NO3- influx. These results indicate that feedback regulation of genes of NO3- assimilation relies not only on the repression exerted by reduced N metabolites, such as NH4+ or amino acids, but may also involve the action of NO2- as a regulatory signal.

A conserved acidic motif in the N-terminal domain of nitrate reductase is necessary for the inactivation of the enzyme in the dark by phosphorylation and 14-3-3 binding.

It has previously been shown that the N-terminal domain of tobacco (Nicotiana tabacum) nitrate reductase (NR) is involved in the inactivation of the enzyme by phosphorylation, which occurs in the dark (L. Nussaume, M. Vincentz, C. Meyer, J.P. Boutin, and M. Caboche [1995] Plant Cell 7: 611-621). The activity of a mutant NR protein lacking this N-terminal domain was no longer regulated by light-dark transitions. In this study smaller deletions were performed in the N-terminal domain of tobacco NR that removed protein motifs conserved among higher plant NRs. The resulting truncated NR-coding sequences were then fused to the cauliflower mosaic virus 35S RNA promoter and introduced in NR-deficient mutants of the closely related species Nicotiana plumbaginifolia. We found that the deletion of a conserved stretch of acidic residues led to an active NR protein that was more thermosensitive than the wild-type enzyme, but it was relatively insensitive to the inactivation by phosphorylation in the dark. Therefore, the removal of this acidic stretch seems to have the same effects on NR activation state as the deletion of the N-terminal domain. A hypothetical explanation for these observations is that a specific factor that impedes inactivation remains bound to the truncated enzyme. A synthetic peptide derived from this acidic protein motif was also found to be a good substrate for casein kinase II.

Post-transcriptional regulation of nitrate reductase by light is abolished by an N-terminal deletion.

Higher plant nitrate reductases (NRs) carry an N-terminal domain whose sequence is not conserved in NRs from other organisms. A gene composed of a full-length tobacco NR cDNA with an internal deletion of 168 bp in the 5' end fused to the cauliflower mosaic virus 35S promoter and appropriate termination signals was constructed and designated as delta NR. An NR-deficient mutant of Nicotiana plumbaginifolia was transformed with this delta NR gene. In transgenic plants expressing this construct, NR activity was restored and normal growth resulted. Apart from a higher thermosensitivity, no appreciable modification of the kinetic parameters of the enzyme was detectable. The post-transcriptional regulation of NR by light was abolished in delta NR transformants. Consequently, deregulated production of glutamine and asparagine was detected in delta NR transformants. The absence of in vitro delta NR activity modulation by ATP suggests the impairment of delta NR phosphorylation and thereby suppression of delta NR post-translational regulation. These data imply that post-transcriptional control of NR expression is important for the flow of the nitrate assimilatory pathway.

Co-suppression of nitrate reductase host genes and transgenes in transgenic tobacco plants.

Constructs carrying the entire or part of the tobacco nitrate reductase cDNA (NIA) cloned between the promoter and terminator sequences of the 35S RNA of the cauliflower mosaic virus were introduced into tobacco, in an attempt to improve nitrate assimilation. Several transgenic plants that had elevated NIA mRNA and nitrate reductase (NR) activity were obtained. In addition, a few plants that exhibited a chlorotic phenotype characteristic of NR-deficient mutants were also obtained. One of these plants contained no NIA mRNA, no NR activity and accumulated nitrate. This phenotype was therefore assumed to result from co-suppression of 35S-NIA transgenes and host NIA genes. NR-deficient plants were also found among the progeny of transformants overexpressing NIA mRNA. Genetic analyses indicated that these NR-deficient plants were homozygous for the 35S-NIA transgene, although not all homozygous plants were deficient for NR. The ratio of normal to NR-deficient plants in the progeny of homozygous plants remained constant at each generation, irrespective of the state of expression of the NIA genes (active or inactive) in the previous generation. This ratio also remained unchanged when field trials were performed in two areas of France: Versailles and Bergerac. The analysis of homozygous plants revealed that co-suppression was reversible at some stage of sexual reproduction. Indeed, host genes and transgenes reactivated at each generation, and co-suppression always appeared after a lag period of normal growth, suggesting that the phenomenon is developmentally regulated.(ABSTRACT TRUNCATED AT 250 WORDS)

T-DNA-insert-independent mutations induced in transformed plant cells duringAgrobacterium co-cultivation

Transformation frequencies were determined for 1n, 2n, and 4n Nicotiana plumbaginifolia protoplast cultures in Agrobacterium-mediated gene transfer experiments. An unexpected large drop (50%) in plating efficiencies was observed in the non-selected (control) 1n populations after transformation treatment with virulent strains. This effect was not observed in the 2n or 4n cultures or in the 1n cultures when treated with avirulent bacteria. The mortality was disproportionally high and could not be explained by the low (0.1-0.5%) transformation efficiency in the 1n population, indicating mutagenesis of the cell populations independently from the T-DNA insertions. Mutagenesis was also indicated in gene tagging experiments where nitrate reductase-deficient (NR-) mutants were selected from haploid Nicotiana plumbaginifolia protoplasts, as well as from leaf disc cultures or protoplasts of diploid plants that were heterozygotic for a mutation either in the NR apoenzyme gene (nia/wt) or one of the molybdenum-containing cofactor genes (cnxA/wt), after Agrobacterium co-cultivation. The chlorate-resistant isolates were tested for the T-DNA-specific kanamycin resistance trait only after NR-deficiency had been established. Thirty-nine independent NR-deficient mutants were analysed further by Southern blot hybridization. There was no indication of integrated T-DNA sequences in the mutated NR genes, despite the fact that NR-deficient cells were found more frequently in cell populations which became transformed during the treatment than in the populations which did not.(ABSTRACT TRUNCATED AT 250 WORDS)

Molybdenum cofactor in nitrate reductase-deficient tobacco mutants. III. Induction of cofactor synthesis by nitrate

The molybdenum cofactor (Mo-co) content of cultured cells of Nicotiana tabacum was estimated by the ability of the acid and heat treated extract to restore NADPH-nitrate reductase (NR) of Neurospora crassa nit-1. Addition of nitrate to wild-type cells grown on amino acids caused a rapid increase in Mo-co activity concomitant with induction of NADH-NR. Time course experiments revealed both the constitutive and the induced levels of Mo-co activity to be dependent on culture age, the maximum occurring during log phase. According to their Mo-co activity, the 12 NR-deficient mutant cell lines tested were shown to fall into 3 groups: Group I nia mutants are characterized by a low Mo-co level, which does not increase either during log phase or after addition of nitrate. Group II nia mutants are similar to wild type. The four cnx mutants tested display a constitutive-high Mo-co level, being not further inducible by nitrate. Possible mechanisms for the common regulation of Mo-co and NR synthesis are discussed.

Antigenicity of nitrate reductase-deficient mutants in Hordeum vulgare L.

Ten nitrate reductase (NR)-deficient mutants have been characterized for their cross-reactivity against specific barley (Hordeum vulgare L.) nitrate reductase antibodies. The rabbit antibodies raised against the purified barley wild type (cv. Steptoe) enzyme quantitatively inactivate nitrate reductase in crude extracts. All nitrate-grown (induced) mutants show positive precipitin reaction against the antiserum by Ouchterlony double diffusion test and all have the ability to neutralize antisera in a NR protection assay. Under induced growth conditions, mutants Az 12, Az 23, Az 29 and Az 30 which have low NR associated catalytic activities also have the lowest level of antigenicity; mutants Az 13, Az 31, Az 33 and Az 34 have intermediate level of both NR associated catalytic activities and antigenicity, while mutants Az 28 and Az 32 have the highest level of both NR associated catalytic activities and antigenicity. Under noninduced growth conditions, all mutants except Az 12 contain detectable but very low levels of NR antigenicity. These results support the concept that these NR-deficient mutants with various levels of NR associated catalytic activities represent different mutation events at the loci coding the NR structural components.