Nitrate Reductase-deficient Mutants Research Articles

NIN-like protein 8 is a master regulator of nitrate-promoted seed germination in Arabidopsis.

Seeds respond to multiple different environmental stimuli that regulate germination. Nitrate stimulates germination in many plants but how it does so remains unclear. Here we show that the Arabidopsis NIN-like protein 8 (NLP8) is essential for nitrate-promoted seed germination. Seed germination in nlp8 loss-of-function mutants does not respond to nitrate. NLP8 functions even in a nitrate reductase-deficient mutant background, and the requirement for NLP8 is conserved among Arabidopsis accessions. NLP8 reduces abscisic acid levels in a nitrate-dependent manner and directly binds to the promoter of CYP707A2, encoding an abscisic acid catabolic enzyme. Genetic analysis shows that NLP8-mediated promotion of seed germination by nitrate requires CYP707A2. Finally, we show that NLP8 localizes to nuclei and unlike NLP7, does not appear to be activated by nitrate-dependent nuclear retention of NLP7, suggesting that seeds have a unique mechanism for nitrate signalling.

Genetic Manipulation of Competition for Nitrate between Heterotrophic Bacteria and Diatoms.

Diatoms are a dominant group of eukaryotic phytoplankton that contribute substantially to global primary production and the cycling of important elements such as carbon and nitrogen. Heterotrophic bacteria, including members of the gammaproteobacteria, are commonly associated with diatom populations and may rely on them for organic carbon while potentially competing with them for other essential nutrients. Considering that bacterioplankton drive oceanic release of CO2 (i.e., bacterial respiration) while diatoms drive ocean carbon sequestration vial the biological pump, the outcome of such competition could influence the direction and magnitude of carbon flux in the upper ocean. Nitrate availability is commonly a determining factor for the growth of diatom populations, particularly in coastal and upwelling regions. Diatoms as well as many bacterial species can utilize nitrate, however the ability of bacteria to compete for nitrate may be hindered by carbon limitation. Here we have developed a genetically tractable model system using the pennate diatom Phaeodactylum tricornutum and the widespread heterotrophic bacteria Alteromonas macleodii to examine carbon-nitrogen dynamics. While subsisting solely on P. tricornutum derived carbon, A. macleodii does not appear to be an effective competitor for nitrate, and may in fact benefit the diatom; particularly in stationary phase. However, allochthonous dissolved organic carbon addition in the form of pyruvate triggers A. macleodii proliferation and nitrate uptake, leading to reduced P. tricornutum growth. Nitrate reductase deficient mutants of A. macleodii (ΔnasA) do not exhibit such explosive growth and associated competitive ability in response to allochthonous carbon when nitrate is the sole nitrogen source, but could survive by utilizing solely P. tricornutum-derived nitrogen. Furthermore, allocthonous carbon addition enables wild-type A. macleodii to rescue nitrate reductase deficient P. tricornutum populations from nitrogen starvation, and RNA-seq transcriptomic evidence supports nitrogen-based interactions between diatoms and bacteria at the molecular level. This study provides key insights into the roles of carbon and nitrogen in phytoplankton-bacteria dynamics and lays the foundation for developing a mechanistic understanding of these interactions using co-culturing and genetic manipulation.

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.

Pseudo-constitutivity of nitrate-responsive genes in nitrate reductase mutants

In fungi, transcriptional activation of genes involved in NO3- assimilation requires the presence of an inducer (nitrate or nitrite) and low intracellular concentrations of the pathway products ammonium or glutamine. In Aspergillus nidulans, the two transcription factors NirA and AreA act synergistically to mediate nitrate/nitrite induction and nitrogen metabolite derepression, respectively. In all studied fungi and in plants, mutants lacking nitrate reductase (NR) activity express nitrate-metabolizing enzymes constitutively without the addition of inducer molecules. Based on their work in A. nidulans, Cove and Pateman proposed an “autoregulation control” model for the synthesis of nitrate metabolizing enzymes in which the functional nitrate reductase molecule would act as co-repressor in the absence and as co-inducer in the presence of nitrate. However, NR mutants could simply show “pseudo-constitutivity” due to induction by nitrate which accumulates over time in NR-deficient strains. Here we examined this possibility using strains which lack flavohemoglobins (fhbs), and are thus unable to generate nitrate internally, in combination with nitrate transporter mutations (nrtA, nrtB) and a GFP-labeled NirA protein. Using different combinations of genotypes we demonstrate that nitrate transporters are functional also in NR null mutants and show that the constitutive phenotype of NR mutants is not due to nitrate accumulation from intracellular sources but depends on the activity of nitrate transporters. However, these transporters are not required for nitrate signaling because addition of external nitrate (10mM) leads to standard induction of nitrate assimilatory genes in the nitrate transporter double mutants. We finally show that NR does not regulate NirA localization and activity, and thus the autoregulation model, in which NR would act as a co-repressor of NirA in the absence of nitrate, is unlikely to be correct. Results from this study instead suggest that transporter-mediated NO3- accumulation in NR deficient mutants, originating from traces of nitrate in the media, is responsible for the constitutive expression of NirA-regulated genes, and the associated phenotype is thus termed “pseudo-constitutive”.

Altered trichothecene biosynthesis in TRI6‐silenced transformants of Fusarium culmorum influences the severity of crown and foot rot on durum wheat seedlings

An RNA silencing construct was used to alter mycotoxin production in the plant pathogenic fungus Fusarium culmorum, the incitant of crown and foot rot on wheat. The transformation of a wild-type strain and its nitrate reductase-deficient mutant with inverted repeat transgenes (IRTs) containing sequences corresponding to the trichothecene regulatory gene TRI6 was achieved using hygromycin B resistance as a selectable marker. Southern analysis revealed a variety of integration patterns of the TRI6 IRT. One transformant underwent homologous recombination with deletion of the endogenous TRI6 gene, whereas, in another transformant, the TRI6 IRT was not integrated into the genome. The TRI6 IRT did not alter the physiological characteristics, such as spore production, pigmentation or growth rate, on solid media. In most transformants, a high TRI6 amplification signal was detected by quantitative reverse transcription-polymerase chain reaction, corresponding to a TRI6-hybridizing smear of degraded fragments by Northern analysis, whereas TRI5 expression decreased compared with the respective nontransformed strain. Four transformants showed increased TRI5 expression, which was correlated with a dramatic (up to 28-fold) augmentation of deoxynivalenol production. Pathogenicity assays on durum wheat seedlings confirmed that impairment of deoxynivalenol production in the TRI6 IRT transformants correlated with a loss of virulence, with decreased disease indices ranging from 40% to 80% in nine silenced strains, whereas the overproducing transformants displayed higher virulence compared with the wild-type.

Regulation of secondary metabolism by the carbon–nitrogen status in tobacco: nitrate inhibits large sectors of phenylpropanoid metabolism

Interactions between nitrogen and carbon metabolism modulate many aspects of the metabolism, physiology and development of plants. This paper investigates the contribution of nitrate and nitrogen metabolism to the regulation of phenylpropanoid and nicotine synthesis. Wild-type tobacco was grown on 12 or 0.2 mm nitrate and compared with a nitrate reductase-deficient mutant [Nia30(145)] growing on 12 mm nitrate. Nitrate-deficient wild-type plants accumulate high levels of a range of phenylpropanoids including chlorogenic acid, contain high levels of rutin, are highly lignified, but contain less nicotine than nitrogen-replete wild-type tobacco. Nia30(145) resembles nitrate-deficient wild-type plants with respect to the levels of amino acids, but accumulates large amounts of nitrate. The levels of phenylpropanoids, rutin and lignin resemble those in nitrogen-replete wild-type plants, whereas the level of nicotine resembles that in nitrate-deficient wild-type plants. Expression arrays and real time RT-PCR revealed that a set of genes required for phenylpropanoid metabolism including PAL, 4CL and HQT are induced in nitrogen-deficient wild-type plants but not in Nia30(145). It is concluded that nitrogen deficiency leads to a marked shift from the nitrogen-containing alkaloid nicotine to carbon-rich phenylpropanoids. The stimulation of phenylpropanoid metabolism is triggered by changes of nitrate, rather than downstream nitrogen metabolites, and is mediated by induction of a set of enzymes in the early steps of the phenylpropanoid biosynthetic pathway.

Expression of Rabbit Neutrophile Peptide-1 in Nitrate Reductase- deficient Mutant of Chlorella ellipsoidea

Application of transgenic Cholrella as bioreactor to express rabbit neutrophile pepetide-1 (NP-1) shows great practical value. In this paper, an NP-1 expression vector containing two selective marker genes NPTII and nitrate reductase gene was constructed. The NP-1 gene was transformed into the nitrate reductase-deficient mutant nrm-4 of Chlorella ellipsoidea via electroporation, and the transgenic alga expressed the active NP-1 were obtained.

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.

Differential regulation of the NO3- and NH4+ transporter genes AtNrt2.1 and AtAmt1.1 in Arabidopsis: relation with long-distance and local controls by N status of the plant.

Regulation of root N uptake by whole-plant signalling of N status was investigated at the molecular level in Arabidopsis thaliana plants through expression analysis of AtNrt2.1 and AtAmt1.1. These two genes encode starvation-induced high-affinity NO3- and NH4+ transporters, respectively. Split-root experiments indicate that AtNrt2.1 expression is controlled by shoot-to-root signals of N demand. Together with 15NO3- influx, the steady-state transcript level of this gene is increased in NO3--fed roots in response to N deprivation of another portion of the root system. Thus AtNrt2.1 is the first identified molecular target of the long-distance signalling informing the roots of the whole plant's N status. In contrast, AtAmt1.1 expression is predominantly dependent on the local N status of the roots, as it is mostly stimulated in the portion of the root system directly experiencing N starvation. The same behaviour was found for NH4+ influx, suggesting that the NH4+ uptake system is much less efficient than the NO3- uptake system, to compensate for a spatial restriction of N availability. Other major differences were found between the regulations of AtNrt2.1 and AtAmt1.1 expression. AtNrt2.1 is strongly upregulated by moderate level of N limitation, while AtAmt1.1 transcript level is markedly increased only under severe N deficiency. Unlike AtNrt2.1, AtAmt1.1 expression is not stimulated in a nitrate reductase-deficient mutant after transfer to NO3- as sole N source, indicating that NO3- per se acts as a signal repressing transcription of AtAmt1.1. These results reveal two fundamentally different types of mechanism involved in the feedback regulation of root N acquisition by the N status of the plant.

Transposon impala, a novel tool for gene tagging in the rice blast fungus Magnaporthe grisea.

impala, a Tc1-mariner transposable element from Fusarium oxysporum, was introduced into the rice blast fungus Magnaporthe grisea to develop transposon-based insertional mutagenesis. A construct (pNIL160) containing an autonomous impala copy inserted in the promoter of niaD encoding Aspergillus nidulans nitrate reductase was introduced by transformation into a M. grisea nitrate reductase-deficient mutant. impala excision was monitored by restoration of prototrophy for nitrate. Southern analysis of niaD+ revertants revealed that impala was able to excise and reinsert at new loci in M. grisea. As observed for its host Fusarium oxysporum, impala inserted at a TA site left a typical excision footprint of 5 bp. We have shown that a defective impala copy was inactive in M. grisea, yet it can be activated by a functional impala transposase. A transformant carrying a single copy of pNIL160 was used to generate a collection of 350 revertants. Mutants either altered for their mycelial growth (Rev2) or nonpathogenic (Rev77) were obtained. Complementation of Rev77 with a 3-kb genomic fragment from a wild-type locus was successful, demonstrating the tagging of a pathogenicity gene by impala. This gene, called ORP1, is essential for penetration of host leaves by M. grisea and has no sequence homology to known genes.

Transcription of a nitrate reductase gene isolated from the symbiotic basidiomycete fungus Hebeloma cylindrosporum does not require induction by nitrate.

Ectomycorrhizal fungi contribute to the nitrogen nutrition of their host plants, but no information is available on the molecular control of their nitrogen metabolism. The cloning and pattern of transcriptional regulation of two nitrite reductase genes of the symbiotic basidiomycete Hebeloma cylindrosporum are presented. The genomic copy of one of these genes (nar1) was entirely sequenced; the coding region is interrupted by 12 introns. The nar1 gene, which is transcribed and codes for a putative 908-amino acid polypeptide complemented nitrate reductase-deficient mutants of H. cylindrosporum upon transformation, thus demonstrating that the gene is functional. The second gene (nar2), for which no mRNA transcripts were detected, is considered to be an ancestral, non-functional duplication of nar1. In a 462-nt partial sequence of nar2 two introns were identified at positions identical to those of introns 8 and 9 of nar1, although their respective nucleotide sequences were highly divergent; the exon sequences were much more conserved. In wild-type strains, transcription of nar1 is repressed in the presence of a high concentration of ammonium. High levels of transcription are observed in the presence of either very low nitrogen concentrations or high concentrations of nitrate or organic N sources such as urea, glycine or serine. This indicates that in H. cylindrosporum, in contrast to all nitrophilous organisms studied so far, an exogenous supply of nitrate is not required to induce transcription of a nitrate reductase gene. In contrast, repression by ammonium suggests the existence of a wide-domain regulatory gene, as already characterized in ascomycete species.

Molecular and functional regulation of two NO3- uptake systems by N- and C-status of Arabidopsis plants.

Root NO3- uptake and expression of two root NO3- transporter genes (Nrt2;1 and Nrt1) were investigated in response to changes in the N- or C-status of hydroponically grown Arabidopsis thaliana plants. Expression of Nrt2;1 is up-regulated by NO3 - starvation in wild-type plants and by N-limitation in a nitrate reductase (NR) deficient mutant transferred to NO3- as sole N source. These observations show that expression of Nrt2;1 is under feedback repression by N-metabolites resulting from NO3- reduction. Expression of Nrt1 is not subject to such a repression. However, Nrt1 is over-expressed in the NR mutant even under N-sufficient conditions (growth on NH4NO3 medium), suggesting that expression of this gene is affected by the presence of active NR, but not by N-status of the plant. Root 15NO3- influx is markedly increased in the NR mutant as compared to the wild-type. Nevertheless, both genotypes have similar net 15NO3- uptake rates due to a much larger 14NO3- efflux in the mutant than in the wild-type. Expressions of Nrt2;1 and Nrt1 are diurnally regulated in photosynthetically active A. thaliana plants. Both increase during the light period and decrease in the first hours of the dark period. Sucrose supply prevents the inhibition of Nrt2;1 and Nrt1 expressions in the dark. In all conditions investigated, Nrt2;1 expression is strongly correlated with root 15NO3- influx at 0.2 mM external concentration. In contrast, changes in the Nrt1 mRNA level are not always associated with similar changes in the activities of high- or low-affinity NO3- transport systems.

Dual pathways for regulation of root branching by nitrate.

Root development is extremely sensitive to variations in nutrient supply, but the mechanisms are poorly understood. We have investigated the processes by which nitrate (NO3-), depending on its availability and distribution, can have both positive and negative effects on the development and growth of lateral roots. When Arabidopsis roots were exposed to a locally concentrated supply of NO3- there was no increase in lateral root numbers within the NO3--rich zone, but there was a localized 2-fold increase in the mean rate of lateral root elongation, which was attributable to a corresponding increase in the rate of cell production in the lateral root meristem. Localized applications of other N sources did not stimulate lateral root elongation, consistent with previous evidence that the NO3- ion is acting as a signal rather than a nutrient. The axr4 auxin-resistant mutant was insensitive to the stimulatory effect of NO3-, suggesting an overlap between the NO3- and auxin response pathways. High rates of NO3- supply to the roots had a systemic inhibitory effect on lateral root development that acted specifically at the stage when the laterals had just emerged from the primary root, apparently delaying final activation of the lateral root meristem. A nitrate reductase-deficient mutant showed increased sensitivity to this systemic inhibitory effect, suggesting that tissue NO3- levels may play a role in generating the inhibitory signal. We present a model in which root branching is modulated by opposing signals from the plant's internal N status and the external supply of NO3-.

Isolation and characterization of nitrate reductase deficient mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum

To clarify the role of the fungal nitrate assimilation pathway in nitrate reduction by mycorrhizal plants, nitrate reductase (NR)-deficient (NR- ) mutants of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum Romagnesi have been selected. These mutants were produced by u.v. mutagenesis on protoplasts originating from homokaryotic mycelia belonging to complementary mating types of this heterothallic tetrapolar species. Chlorate-resistant mutants were first selected in the presence of different nitrogen (N) sources in the culture medium. Among 1495 chlorate resistant mycelia, 30 failed to grow on nitrate and lacked a detectable NR activity. Growth tests on different N sources suggested that the NR activity of all the different mutants is specifically impaired as a result of mutations in either the gene coding for NR apoprotein or genes controlling the synthesis of the molybdenum cofactor. Furthermore, restoration of NR activity in some of the dikaryons obtained after crosses between the different mutant mycelia suggested that not all the selected mutations mapped in the same gene. Utilization of N on a NH4 15 NO3 medium was studied for two mutant strains and their corresponding wild-type homokaryons. None of the mutants could use nitrate whereas 15 N enrichment values indicated that 13-27% of N present in 13-d-old wild-type mycelia originated from nitrate. Apparently, the mutant mycelia do not compensate their inability to use nitrate by a more efficient use of ammonium. These different NR mutants still form mycorrhizas with the habitual host plant, Pinus pinaster (Ait.), making them suitable for study of the contribution of the fungal nitrate assimilation pathway to nitrate assimilation by mycorrhizal plants.

Systemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions.

Using grafting procedures, we investigated the transmission of co-suppression of nitrate reductase and nitrite reductase host genes and transgenes and of post-transcriptional silencing of a uidA transgene encoding glucuronidase in tobacco. We demonstrate that silencing is transmitted with 100% efficiency from silenced stocks to non-silenced scions expressing the corresponding transgene. Transmission is unidirectional from stock to scion, transgene specific, locus independent and requires the presence of a transcriptionally active transgene in the target scion. The transmission of co-suppression occurs when silenced stocks and non-silenced target scions are physically separated by up to 30 cm of stem of a non-target wild-type plant. Taken together, these results suggest that a non-metabolic, transgene-specific, diffusable messenger mediates the propagation of de novo post-transcriptional silencing through the plant.

Somatic Hybridization Between N.tabacum (NR−, Km+) and N.sanderae. Hybrid Genome Constitution and Expression of Resistance to Tomato Spotted Wilt Virus

ABSTRACTLeaf mesophyll protoplasts of kanamicine resistant, nitrate reductase deficient mutant Nicotiana tabacum (NR−, Km+) were fused with leaf mesophyll protoplasts of wild type Nicotiana sanderae (wt). Somatic hybrid cell colonies were selected for kanamicine resistance and nitrate reductase proficiency. Nineteen independent cell lines, capable of growth in selection medium were obtained. From 14 of them shoots were regenerated and adapted in soil. All regenerants have intermediate plant and leaf morphology. The chromosome number for regenerants from six lines was determinated, as well as analyses of four isoenzymes: peroxidase, esterase, shikimate dehydrogenase and glutamate-oxaloacetate transminase were performed. Results show that all studied plants represent nuclear somatic hybrids, possessing entire genomes of both parents. Local lesion (hypersensitivity)—type resistance to TSWV has been observed in leaves of these somatic hybrid plants after virus inoculation.

Stress-induced rearrangement of Fusarium retrotransposon sequences.

Rearrangement of fusarium oxysporum retrotransposon skippy was induced by growth in the presence of potassium chlorate. Three fungal strains, one sensitive to chlorate (Co60) and two resistant to chlorate and deficient for nitrate reductase (Co65 and Co94), were studied by Southern analysis of their genomic DNA. Polymorphism was detected in their hybridization banding pattern, relative to the wild type grown in the absence of chlorate, using various enzymes with or without restriction sites within the retrotransposon. Results were consistent with the assumption that three different events had occurred in strain Co60: genomic amplification of skippy yielding tandem arrays of the element, generation of new skippy sequences, and deletion of skippy sequences. Amplification of Co60 genomic DNA using the polymerase chain reaction and divergent primers derived from the retrotransposon generated a new band, corresponding to one long terminal repeat plus flanking sequences, that was not present in the wild-type strain. Molecular analysis of nitrate reductase-deficient mutants showed that generation and deletion of skippy sequences, but not genomic amplification in tandem repeats, had occurred in their genomes.

Characterization of four molybdenum cofactor mutants of rice, Oryza sativa L.

Four nitrate reductase-deficient (NR −) mutants, C25, C27, C32 and C33, were isolated from 100 000 M 2 seedlings of rice ( Oryza sativa L., ssp. indica, cv. IR30) by means of selection for chlorate-resistance. All mutations were monogenic and recessive. Biochemical analysis showed that both NADH- and NADPH-dependent activities of nitrate reductase (NR) in these four mutants were less than 25% of the wild type IR30. However, nitrite reductase activities were more than the wild type. Also, xanthine dehydrogenase (XDH) activities and molybdenum cofactor biosynthesis abilities (MoCo activities) were deficient indicating that these mutants were cnx and not nia type. The cnx mutants could be divided into two groups: one could recover NADH-NR activity by adding 0.5 mM molybdate to the growth medium (C27, C32 and C33) while the other could not (C25). The result of allelism test coincided with the phenotypic classification by adding molybdate. The growth response to tungstate was also investigated in the mutants. None of the mutants classifies to the tungstate-sensitive group as reported in Arabidopsis cnx mutants.

Induction of nitrate reductase host gene expression has a negative effect on the expression of transgenes driven by the nitrate reductase promoter

In two previous studies the expression of the tobacco nitrate reductase genes ( Nia1 or Nia2) introduced into nitrate reductase deficient mutants or of glucuronidase reporter genes linked to the promoter of a tobacco Nia gene were characterized. In both cases no expression of the introduced genes was found in the leaves of approximately 85% of the transformants and only a low level of expression was detected in the remaining transformants ranging from 0.1 to 7% of the expression of host Nia genes. In this paper we show that reduced levels of transgene expression in leaves result from a negative effect of host Nia gene expression. Transformants have been generated carrying a neomycin phosphotransferase reporter gene ( Npt) linked to a 1.4 kb promoter fragment (−1350 to +40) of the Nia1 gene by electroporation. The level of resistance to the antibiotic declined in transformed calli after 1 month of culture whereas a high level of resistance was recovered in cells freshly prepared from leaf protoplasts of the regenerated transgenic plants, conditions in which the expression of host Nia genes is severely down-regulated. The level of resistance in seedlings also declined after 1 month of growth when plants were grown on a medium containing nitrate whereas a high level of resistance was observed when plants were grown on a medium supplemented with ammonium as a sole nitrogen source, conditions that do not allow the induction of host Nia genes expression. The analysis of a series of progressive deletions covering the 5′ end of the promoter down to position −92 showed that such a negative correlation between the expression of pNia1-Npt transgenes and host Nia genes is found in approximately 70% of the transformants, irrespective of the size of the deletion. A model is proposed to explain how the expression of host Nia genes can impede the expression of pNia-driven transgenes.

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)