Chromosomes in somatic hybrids between Nicotiana plumbaginifolia and a monoploid potato

Summary 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.     

Improved in vitro selection of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia

Summary 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.     

Effect of nitrate pulses on the nitrate-uptake rate,synthesis of mRNA coding for nitrate reductase,and nitrate-reductase activity in the roots of barley seedlings

Using pulses of nitrate, instead of the permanent presence of external nitrate, to induce the nitrate-assimilating system in Hordeum vulgare L., we demonstrated that nitrate can be considered as a trigger or signal for the induction of nitrate uptake, the appearance of nitratereductase activity and the synthesis of mRNA coding for nitrate reductase. Nitrate pulses stimulated the initial rate of nitrate uptake, even after subsequent cultivation in N-free medium, and resulted in a higher acceleration of the uptake rate in the presence of nitrate than in its absence.Abbreviations NR nitrate reductase     

Short-term modulation of nitrate reductase activity by exogenous nitrate in Nicotiana plumbaginifolia and Zea mays leaves

Maize (Zea mays L.) grown on low (0.8 mM) NO ₃ ^- , as well as untransformed and transformed Nicotiana plumbaginifolia constitutively expressing nitrate reductase (NR), was used to study the effects of NO ₃ ^- on the NR activation state. The NR activation state was determined from the relationship of total activity extracted in the presence of ethylenediaminetetracetic acid to that extracted in the presence of Mg²⁺. Light activation was observed in both maize and tobacco leaves. In the tobacco lines, NO ₃ ^- did not influence the NR activation state. In excised maize leaves, no correlation was found between the foliar NO ₃ ^- content and the NR activation state. Similarly, the NR activation state did not respond to NO ₃ ^- . Since the NR activation state determined from the degree of Mg²⁺-induced inhibition of NR activity is considered to reflect the phosphorylation state of the NR protein, the protein phosphatase inhibitor microcystin LR was used to test the importance of protein phosphorylation in the NO ₃ ^- -induced changes in NR activity. In-vivo inhibition of endogenous protein phosphatase activity by microcystin-LR decreased the level of NR activation in the light. This occurred to the same extent in the presence or absence of exogenous NO ₃ ^- . We conclude that NO ₃ ^- does not effect the NR activation state, as modulated by protein phosphorylation in either tobacco (a C₃ species) or maize (a C₄ species). The short-term regulation of NR therefore differs from the NO ₃ ^- -mediated responses observed for phosphoenolpyruvate carboxylase and sucrose phosphate synthase.Abbreviations Chl chlorophyll - MC microcystin-LR - PEP-Case phosphoenolpyruvate carboxylase - SPS sucrose-phosphate synthase We are indebted to Madeleine Provot and Nathalie Hayes for excellent technical assistance. This work was funded by EEC Biotechnology Contract No. BI02 CT93 0400, project of technical priority, Network D — Nitrogen Utilisation and Efficiency.     

The fate of recombinant chromosomes and genome interaction in Nicotiana asymmetric somatic hybrids and their sexual progeny

Genomic in-situ hybridization (GISH) was used to monitor the behaviour of parental genomes, and the fate of intergenomic chromosome translocations, through meiosis of plants regenerated from asymmetric somatic hybrids between Nicotiana sylvestris and N. plumbaginifolia. Meiotic pairing in the regenerants was exclusively between chromosomes or chromosome segments derived from the same species. Translocation (recombinant) chromosomes contained chromosome segments from both parental species, and were detected at all stages of meiosis. They occasionally paired with respectively homologous segments of N. sylvestris or N. plumbaginifolia chromosomes. Within hybrid nuclei, the meiotic division of N. plumbaginifolia lagged behind that of N. sylvestris. However, normal and recombinant chromosomes were eventually incorporated into dyads and tetrads, and the regenerants were partially pollen fertile. Recombinant chromosomes were transmitted through either male or female gametes, and were detected by GISH in sexual progeny obtained on selfing or backcrossing the regenerants to N. sylvestris. A new recombinant chromosome in one plant of the first backcross generation provided evidence of further chromosome rearrangements occurring at, or following, meiosis in the original regenerants. This study demonstrates the stable incorporation of chromosome segments from one parental genome of an asymmetric somatic hybrid into another, via intergenomic translocation, and reveals their transmission to subsequent sexual progeny.     

Characterization of three hormone mutants of Nicotiana plumbaginifolia: evidence for a common ABA deficiency

Summary Various auxin-resistant Nicotiana plumbaginifolia mutants have already been isolated, including 1217 which shows cross-resistance to paclobutrazol. Recently, a cytokinin-resistant mutant, CKR1, has been characterized and has been shown to be affected in abscisic acid (ABA) biosynthesis. We have isolated a new mutant, Esg152, which was selected on the basis of its early germination. In each of these mutants, resistance is due to a recessive nuclear mutation at a single locus. Complementation analysis indicated that mutants I217, CKR1 and Esg152 belong to the same complementation group. They have a similar phenotype, which includes a reduction in seed dormancy and an increased tendency to wilt. These mutants display an increased auxin tolerance and enhanced root formation when leaf or hypocotyl sections are cultivated on auxin. By immunoenzymatic methods, we show that the endogenous levels of ABA are significantly lower than in the wild-type. We have assigned the symbol aba1 to the recessive alleles of the locus affected in the three mutants. The complexity of hormonal interactions is discussed briefly emerging from a consideration of this class of mutants.     

Isolation and characterization of valine-resistant mutants of Nicotiana plumbaginifolia

Summary Haploid mesophyll protoplasts of Nicotiana plumbaginifolia were mutagenized by UV-irradiation. Protoplast-derived colonies were then selected for valine resistance on a medium containing 5 or 10 mM valine. From the resistant calli, plants were regenerated. Resistance was inherited as a recessive Mendelian character in seven clones. Mutations conferring valine resistance were shown to be allelic. Protoplast-derived cells of L-valine-resistant plants were also resistant to L-threonine. Resistance to valine was based on a reduced valine uptake rate.     

Ethanol-resistant mutants of Nicotiana plumbaginifolia are deficient in the expression of pollen and seed alcohol dehydrogenase activity

Summary Six independent mutant lines ofNicotiana plumbaginifolia resistant to ethanol, designated E3, E8, E101, E112, E144 and E251, were isolated as germinating seedlings on selective medium. In all cases, resistance to ethanol was conferred by a single recessive nuclear mutation at the same locus. Mutant seeds and pollen lacked detectable ADH activity, with the exception of E251 where a residual activity was detected. An antiserum directed againstArabidopsis thaliana ADH detected an ADH-related polypeptide of 44 kDa present in wild-type seeds and, to a lesser extent, in the seeds of the leaky mutant E251. No ADH-related polypeptide could be detected in seeds of the other mutants. However, all of them had a nearly normal level of ADH mRNA except one which did not synthesize any mRNA. These results suggest that these ethanol-resistant mutants are impaired in one of the structural genes coding for alcohol dehydrogenase. The corresponding locus has been designatedAdh1.Abbreviations ADH alcohol dehydrogenase - EMS ethyl methane-sulfonate - MTT dimethyl thiazol tetrazolium - NAD nicotinamide adenine dinucleotide - NBT nitro blue tetrazolium - p-cells protoplast-derived cells - PMS phenazine methosulfate - SDS sodium dodecyl sulfate     

Isolation and characterization of streptomycin-resistant mutants in Nicotiana plumbaginifolia

Summary Streptomycin-resistant colonies were isolated from protoplast cultures of haploid Nicotiana plumbaginifolia based on their ability to green in medium containing 1 mg/ml streptomycin sulfate. The frequency of resistant colonies was 0.9×10^–5 in nonmutagenized culture, and increased ten-fold following treatment of culture with 10 g/ml N-methyl-N-nitro-N-nitrosoguanidine. Of a total of 52 resistant clones isolated, 2 gave rise to haploid, 15 to diploid, and 3 to tetraploid plants upon transfer of calli to differentiation medium. Leaf-segment and protoplast assays showed that all diploid regenerates were resistant to streptomycin but sensitive to chloramphenicol, kanamycin, lincomycin, neomycin, and spectinomycin. Plants in most diploid clones were fertile and able to set seeds when self-fertilized and crossed reciprocally to wild-type plants. Inheritance of streptomycin resistance was studied in the diploid clones and, without exception, the resistance was transmitted maternally. Comparative studies of the ultrastructure of organelles and protein synthesis in isolated chloroplasts between wild-type and resistant clones in the presence of streptomycin suggest that streptomycin resistance is controlled by chloroplasts.     

Study of the divergence of moderately repetitive sequences in Nicotiana species and in protoclones of Nicotiana plumbaginifolia Viviani

Summary Molecular DNA markers can be very useful to assess the amount of genetic variation and are thus important for taxonomic studies. Two moderately repetitive sequences were isolated from N. plumbaginifolia leaf DNA and used to screen various Nicotiana species. A huge variability was detected among species belonging to the same subgenus or the same section, which could be utilized for a molecular taxonomy of the genus Nicotiana. Although variation at the DNA level between somaclonal lines was reported, we did not find evidence for variability of both repetitive sequences in established callus culture obtained from protoplasts of Nicotiana plumbaginifolia.     

Characterization and regeneration of salt- and water-stress mutants from protoplast culture of Nicotiana plumbaginifolia (Viviani)

Summary Protoplast-derived colonies of haploid N. plumbaginifolia leaves were used to select for resistance to NaCl, KCl and polyethylene glycol 6000 (PEG). Salt-and PEG-tolerant cell lines were isolated on the basis of growth in a culture medium containing inhibitory concentrations of either NaCl or KCl (200 mM) or PEG (25%). The frequency of resistant lines ranged from 10^-5 to 10^-6. One resistant line from each treatment was regenerated into plants. All resistant lines produced 10–25 times more proline than the wild type when grown on a non-selective medium. Similar values were also observed in the leaves of resistant progeny plants. In each mutant line, salt or PEG resistance was transmitted as a single dominant nuclear gene as shown by segregation ratios in progenies of crosses between resistant and wild-type plants. The latter observation demonstrates clearly the existence of a genetic basis for increased salt tolerance.     

Fusion of plant protoplasts: a study using auxotrophic mutants of Nicotiana plumbaginifolia,Viviani

Summary Protoplast fusion studies between various auxotrophic mutants of Nicotiana plumbaginifolia were performed to optimize conditions for PEG-mediated fusion and to identify factors influencing the plant protoplast fusion process. Numerous parameters in the isolation, culture, and fusion of protoplasts were tested, and established fusion protocols were compared. Fusion rates, calculated on the basis of colony growth on selection medium (genetic complementation), ranged from 10^–4 to 10^–2. Conditions that allow rapid and reproducible fusions at the highest rates were established. Particular emphasis was given to fusion of mesophyll-derived protoplasts, for which the ability to regenerate fertile plants from fusion products was shown to be particularly high. Preliminary experiments using electric-field mediated fusion suggest that electrofusion may offer significant advantages over the traditional chemical fusion.     

Reduced intracellular content of methotrexate in an isolated MTX-resistant cell line of Nicotiana plumbaginifolia

Summary In plant cells methotrexate (MTX) may exert its toxic effect through several mechanisms, including inhibition of its target protein dihydrofolate reductase. Resistance based on a mechanism operating before MTX binds to proteins should confer protection to plant cells. A methotrexate-resistant cell line of Nicotiana plumbaginifolia was isolated by a stepwise selection procedure. This cell line survived in the presence of 10 M MTX which is 50–100 fold higher than the lethal dose for the wild type cells. Neither alteration in kinetic characteristics of dihydrofolate reductase, nor elevated binding capacity of ³H-MTX to target protein(s), were observed. However, in comparison with wild type cells, markedly lower amounts of intracellular ³H-MTX were found after the selected cell line was incubated with ³H-MTX, indicating that either reduced uptake or enhanced efflux of MTX is the major reason for MTX-resistance in this cell line.     

Effects of constitutive expression of nitrate reductase in transgenic Nicotiana plumbaginifolia L. in response to varying nitrogen supply

Transformed Nicotiana plumbaginifolia plants with constitutive expression of nitrate reductase (NR) activity were grown at different levels of nitrogen nutrition. The gradients in foliar NO ₃ ^– content and maximum extractable NR activity observed with leaf order on the shoot, from base to apex, were much decreased as a result of N-deficiency in both the transformed plants and wild type controls grown under identical conditions. Constitutive expression of NR did not influence the foliar protein and chlorophyll contents under any circumstances. A reciprocal relationship between the observed maximal extractable NR activity of the leaves and their NO ₃ ^– content was observed in plants grown in nitrogen replete conditions at low irradiance (170 mol photons·m^–2 ·s^–1). This relationship disappeared at higher irradiance (450 mol photons·m^–2·S^–1) because the maximal extractable NR activity in the leaves of the wild type plants in these conditions increased to a level that was similar to, or greater than that found in constitutive NR-expressors. Much more NO ₃ ^– accumulated in the leaves of plants grown at 450 mol photons·m^–2·s^–1 than in those grown at 170 mol photons·m^–2·s^–1 in N-replete conditions. The foliar NO ₃ ^– level and maximal NR activity decreased with the imposition of N-deficiency in all plant types such that after prolonged exposure to nitrogen depletion very little NO ₃ ^– was found in the leaves and NR activity had decreased to almost zero. The activity of NR decreased under conditions of nitrogen deficiency. This regulation is multifactoral since there is no regulation of NR gene expression by NO ₃ ^– in the constitutive NR-expressors. We conclude that the NR protein is specifically targetted for destruction under nitrogen deficiency. Consequently, constitutive expression of NR activity does not benefit the plant in terms of increased biomass production in conditions of limiting nitrogen.Abbreviations Chl chlorophyll - N nitrogen - NR NADH-nitrate reductase - WT wild type     

Effect of repeated DNA sequences on direct gene transfer in protoplasts of Nicotiana plumbaginifolia

Summary Highly repeated nuclear DNA sequences from leaves of Nicotiana plumbaginifolia were cloned in pBR322 and tested for their effect on direct gene transfer in protoplasts of the same organism. Protoplasts were prepared from suspension cultures and were incubated in the presence of the plasmid pHP23 carrying the kanamycin resistance gene APH(3)II and in the presence of the plasmids carrying the cloned sequence. DNA uptake was induced by a polyethyleneglycol (PEG) treatment. Out of the 22 tested clones, 3 significantly stimulated the frequency of appearance of transformed colonies. DNA was extracted from some of the kanamycin-resistant calli obtained by co-transformations. Dot-blots have shown that the stimulatory effect on transformation frequency is often accompanied by a consistent increase in integrated genes sequences.     

Role of light and CO2 fixation in the control of nitrate-reductase activity in barley leaves

Nitrate reductase (NR, NADH:nitrate oxidoreductase, EC 1.6.6.1) from barley (Hordeum vulgare L. cv. Hassan) leaves was inactivated during a light-dark transition, losing approx. 50% of activity after 30 min of darkness. The dark inactivation was reversed by illumination of the seedlings, the kinetics of reactivation being similar to those of inactivation. High extractable NR activity and significant differences between illuminated and darkened leaves were observed in media containing EDTA and inorganic phosphate (P_i). Addition of Ca²⁺ ions during extraction and assay decreased NR activity from illuminated and darkened leaves, enhancing the light-dark difference. While no clear correlation could be found between irradiance and NR activity, a hyperbolic correlation appeared between extractable NR activity and in-vivo rates of CO₂ fixation, indicating that NR activation follows saturation kinetics with respect to CO₂ fixation. Furthermore, hexoses and hexose-phosphates fed to the leaves via the transpiration stream protected against the dark-inactivation of NR. The results indicate that carbon-assimilation products are regulatory factors of NR activity in barley leaves, mediating both the light-dark modulation of NR and its dependence upon CO₂ fixation.     

Identification and expression analyses of two genes encoding putative low-affinity nitrate transporters from Nicotiana plumbaginifolia

Higher plants have both high- and low-affinity nitrate uptake systems (HATS and LATS respectively). Here we report the isolation and characterization of two genes, NpNRT1.1 and NpNRT1.2, from Nicotiana plumbaginifolia whose structural features suggest that they both belong to the NRT1 gene family, which is involved in the LATS. Amino acid sequence alignment showed that the N. plumbaginifolia proteins have greater similarity to their corresponding tomato homologues than to each other. Genomic Southern blot analysis indicates that there are probably more than two members of this family in N. plumbaginifolia. Northern blot analysis shows that NpNRT1.2 expression is restricted strictly to roots, whereas NpNRT1.1, in addition to roots, is expressed at a basal level in all other plant organs. Likewise, differential expression in response to external treatments with various N sources was observed for these two genes: NpNRT1.1 can be considered as a constitutively expressed gene whereas NpNRT1.2 expression is dependent strictly on high nitrate concentrations. Finally, over-expression of a gene involved in the HATS does not lead to any modification of LATS gene expression.     

Evidence for commitment of flowers on the mother plant to floral regeneration inNicotiana plumbaginifolia VIV

The tobacco explants did not regenerate any floral shoots when they were excised from the mother plant whose flower buds were removed before they developed, while those from the equivalent-aged intact mother plant regenerated floral shoots more than a quarter of the total regenerated shoots.     

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

Summary 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 suggeests 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 methylviologenitrate 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.