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

Summary 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.     

Photorespiratory Properties of Mesophyll Protoplasts of Nicotiana plumbaginifolia

The photorespiratory activity of mesophyll protoplasts of Nicotiana plumbaginifolia has been clearly demonstrated by the presence of a Warburg-effect, the occurrence of an important CO₂-sensitive O₂ uptake and the effect of some photorespiratory inhibitors on photosynthetic activity. At a nonsaturating dissolved inorganic carbon (DIC) concentration (0.1 millimolar), we observed that the rate of CO₂ fixation was 60% lower at 50% O₂ compared to that measured at 2% O₂. Using ¹⁸O₂ and mass spectrometry, we measured O₂ exchange as a function of light intensity and of DIC concentration. Oxygen uptake measured at the CO₂ compensation point (47.4 micromoles O₂ per hour per milligram chlorophyll) was three-fold higher than that measured at a saturating CO₂ concentration. Cyanide or iodoacetamide, inhibitors of the Calvin cycle, were found to reduce the O₂ uptake to the same extent as CO₂ saturation. We conclude from these results that the major part of the CO₂-sensitive O₂ uptake is due to photorespiration. Further, we investigated the effect on net photosynthesis of some inhibitors of the glycolate pathway. At CO₂ saturation (10 millimolar DIC), 5 millimolar aminoacetonitrile (AAN), and 1 millimolar aminooxyacetate (AOA) did not cause any significant decrease in net photosynthesis. However, when these two inhibitors were added under a period of active photorespiration (10 minutes at the CO₂ compensation point at 20% O₂), we observed a decrease in the rate of net photosynthesis at 10 millimolar DIC measured afterward (respectively, 18 and 29%). This inhibition did not appear at 2% O₂, but was stronger at 50% O₂ (40% for AAN and 47% for AOA). With 0.05 millimolar butyl 2-hydroxy-3-butynoate (BHB) or 0.5 millimolar l-methionine-dl-sulfoximine (l-MSO), rates of net photosynthesis at 10 millimolar DIC were decreased by 10 to 15%. Additional decreases were observed after a period at the CO₂ compensation point at 20% O₂ (30% for BHB and 20% for l-MSO). From the sites of action of the four inhibitors tested, we suggest the inhibition of photosynthesis occurring after a period of active photorespiration to be due to the toxic accumulation of nonmetabolized phosphoglycolate.     

Somatic hybridization between potato and Nicotiana plumbaginifolia

Summary Electrofusion was carried out between mesophyll protoplasts from the transformed diploid S. tuberosum clone 413 (2n=2x=24) which contains various genetic markers (hormone autotrophy, opine synthesis, kanamycin resistance, -glucuronidase activity) and mesophyll protoplasts of a diploid wild-type clone of N. plumbaginifolia (2n=2x=20). Hybrid calli were obtained after continuous culture on selection medium containing kanamycin. Parental chromosome numbers, determined at 2 months after fusion, revealed hybrid-specific differences between the individual calli. On the basis of these differences three categories of hybrids were distinguished. Category I hybrids contained between 8 and 24 potato chromosomes and more than 20 N. plumbaginifolia chromosomes; category II hybrids had between 1 and 20 N. plumbaginifolia chromosomes and more than 24 potato chromosomes; category III hybrids contained diploid or subdiploid numbers of chromosomes from both parents. The hybrids were evenly distributed over the three categories. After a 1-year culture of 24 representative hybrid callus lines on selection medium the karyotype of 10 hybrids remained stable, whereas 8 hybrids showed polyploidization of the genome of one parent, together with no or minor changes of the chromosome numbers of the other parent. Six hybrids showed slight changes in the hybrid karyotype. The elimination of chromosomes of a particular parent was not correlated to their metaphase location. The processes of spontaneous biparental chromosome elimination leading to the production of asymmetric hybrids of different categories are discussed.     

Cloning of a Nicotiana plumbaginifolia protoplast-specific enhancer-like sequence

We have isolated a 1.5-kb plant DNA fragment (called insert 7) from Nicotiana plumbaginifolia DNA that contains a protoplast-specific enhancer-like sequence. The presence of this sequence on a plasmid carrying a chimeric nos-npt-II gene conferring kanamycin resistance to plant cells, produces an overexpression of the npt-II gene during at least eight days after protoplast transformation. This effect on the expression of the nos promoter was independent of the orientation and was observed both on circular and linearized plasmids. On the contrary, insert 7 had no influence when present on another plasmid (in trans) in cotransformation experiments. The overexpression of the nos-npt-II gene due to the presence of insert 7 on the transforming plasmid is correlated with a higher level of synthesis of the corresponding RNA. Insert 7 did not affect the level of expression of the nos-npt-II gene in stably transformed calli, or in regenerated plants. However, the overexpression was again detected in protoplasts prepared from leaves of stably transformed plants. This 1.5-kb plant DNA fragment contains highly repetitive DNA sequences, specific to N. plumbaginifolia. However, the enhancer-like activity is localized on a 600-bp unique sequence of insert 7. Insert 7 had no detectable effect on the transient expression of another gene, the nopaline synthase gene present at a longer distance on the same plasmid.     

Note on the chromosomes of Nicotiana plumbaginifolia Viviani

The centromere position on the chromosomes of three European stocks of Nicotiana plumbaginifolia (2n=20) was determined by investigating in root tip mitoses the shape of the chromosomes during congression and polar movement and during C-metaphase. One pair of chromosomes is subtelocentric and 9 pairs are acrocentric. Some chromosomes mimic (sub-)metacentrics by having noncentric constrictions. A recently published (A. Villa; Genetica 64: 145–148, 1984) idiogram of one of the stocks is commented on.     

Mendelian inheritance of streptomycin resistance in Nicotiana plumbaginifolia

In a previous study two haploid streptomycin-resistant clones of Nicotiana plumbaginifolia were isolated. The chromosome number of one of these clones has now been doubled through leaf-midvein culture and the resultant diploids were characterized genetically. Our results show that streptomycin resistance in this clone is conditioned by a recessive nuclear gene. Haploid protoplasts of this streptomycin-resistant mutant were selected for chlorate resistance. All clones obtained from the selection were deficient in nitrate reductase activity in addition to resistance to streptomycin. Genetic analysis of progeny of one of these clones revealed that the genes for streptomycin resistance and for the apoenzyme of nitrate reductase are unlinked.     

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.     

A new middle repetitive sequence of Nicotiana plumbaginifolia genome

A middle repetitive sequence NPR18 was isolated from Nicotiana plumbaginifolia nuclear genome [8]. Sequences homologous to the repeat are dispersed through genomes of several Nicotiana species. compute-assisted data analysis of NPR18 primary sequence reveals several features attributed to mobile genetic elements: an AT content higher than average for nuclear DNA of genus Nicotiana plants; a number of direct and inverted repeats. Some of the repeats displayed homology to the terminal and subterminal repeats of Ac/Ds-like plant elements.     

Abscisic-acid metabolism in a wilty mutant of Nicotiana plumbaginifolia

A mutant of Nicotiana plumbaginifolia, CKR1, isolated on the basis of its enhanced resistance to cytokinins was found to have a greater tendency to wilt than the wild type (Blonstein et al., 1991, Planta 183, 244–250). Further characterisation has shown that the wiltiness in the mutant is not caused by an insensitivity to abscisic acid (ABA) because the external application of ABA leads to stomatal closure and phenotypic reversion. The basal ABA level in the mutant is < 20% of that in the wild type. Following stress, the ABA level in wild-type leaves increases by approx 9-to 10-fold while the mutant shows only a slight increase. This deficiency in ABA is unlikely to be the consequence of accelerated catabolism as the levels of two major metabolites of ABA, phaseic and dihydrophaseic acid, are also much reduced in the mutant. The qualitative and quantitative distributions of carotenoids, the presumed presursors of ABA, are the same for the leaves of both wild type and mutant. Biosynthesis of ABA at the C15 level was investigated by feeding xanthoxin (Xan) to detached leaves. Wild-type leaves convert between 9–19% of applied Xan to ABA while the mutant converts less than 1%. The basal level of trans-ABA-alcohol (t-ABA-alc) is 3-to 10-fold greater in the mutant and increases by a further 2.5-to 6.0-fold after stress. This indicates that the lesion in the wilty mutant of N. plumbaginifolia affects the conversion of ABA-aldehyde to ABA, as in the flacca and sitiens mutants of tomato and the droopy mutant of potato (Taylor et al., 1988, Plant Cell Environ. 11, 739–745; Duckham et al., 1989, J. Exp. Bot. 217, 901–905). Wild-type tomato and N. plumbaginifolia leaves can convert trans-Xan into t-ABA-alc, and Xan into ABA, while those of flacca and the wilty N. plumbaginifolia mutant convert both Xan and t-Xan to t-ABA-alc.     

Primary structure of a hormonally regulated beta-glucanase of Nicotiana plumbaginifolia

A cDNA clone for a hormonally regulated beta-glucanase from Nicotiana plumbaginifolia has been isolated by using an oligodeoxynucleotide probe, synthesized to match the previously determined N-terminal amino acid sequence. The cDNA has the complete sequence of the mature protein and contains at least part of a hydrophobic signal peptide. At the amino acid level, the beta-glucanase of N. plumbaginifolia is 73% homologous to a beta(1,3)-glucanase from tobacco and 52% homologous to a beta(1,3;1,4)-glucanase from barley. Southern-blot analysis clearly demonstrated that N. plumbaginifolia contains at least two related genes encoding beta-glucanase. The extent of the complete signal peptide of the cloned beta-glucanase was determined by sequencing part of the corresponding gene. Northern analysis showed that the expression of the beta-glucanase gene is influenced by auxins and cytokinins.     

Mutants of Nicotiana plumbaginifolia with increased sensitivity to auxin.

Summary Two non-allelic, monogenic recessive mutations, ausl and aus2, have been isolated which result in auxin hypersensitivity in mutant Nicotiana plumbaginifolia plants. At relatively low concentrations of indole-3-acetic acid or 1-naphthaleneacetic acid, the elongation growth of mutant seedling hypocotyls is more inhibited than in the case of the wild type; at high auxin concentrations, mutant seedlings are killed. The leaves of mature mutant plants degenerate after a spray treatment with auxin that has only a mild, transient effect on the wild type. Seedling hypocotyls of ausl are more sensitive to l-tryptophan than those of the wild type but do not differ in their response to the d-isomer. The mutant is also more sensitive to ethylene and the ethylene precursor 1-aminocyclopropane-l-carboxylic acid, but not to either 6-benzyladenine or abscisic acid. Mutant seedlings display several distinct morphological characters: mild leaf epinasty, short primary root, increased root branching and no root hairs.     

A genetic linkage map of Nicotiana plumbaginifolia/ Nicotiana longiflora based on RFLP and RAPD markers

We have constructed a genetic linkage map for Nicotiana plumbaginifolia/Nicotiana longiflora (2n= 2x=20), based on the segregation of 69 RFLP and 102 RAPD loci in 99 F₂ plants from the cross N. plumbaginifolia×N. longiflora. The map consists of nine major linkage groups, each containing more than nine marker loci, and spans 1062 cM. Twenty of the RFLP markers were mapped previously to Nicotiana sylvestris (2n=2x=24) chromosomes using monosomic alien addition lines. Taxonomically, N. plumbaginifolia and N. sylvestris belong to the same section, namely the Alatae; however, cytogenetic evidence indicates that they are not closely related. Comparison of the distribution of markers common to both maps suggests that genome reorganization has occurred during the evolution of these two species. Evidence is also presented that genome reorganization may be accompanied by gain and loss of specific classes of DNA sequences in their genomes.     

Studies on Certain Aspects of Senescence in Nicotiana plumbaginifolia Sps

Senescence of Nicotiana plumbaginifolia VIV. (Solanaceae) wasstudied in normal, in deflowered, and in defruited plants. Removalof flowers and fruits delayed senescence, inhibited growth ofthe bolted axis and branches, and promoted the formation ofleaves on them. An increase of dry weight of leaves occurreddue to these operations and the rate of increase was a maximumin deflowered plants. Increase in dry weight was accompaniedby an increase in laminar area and this was most pronouncedin deflowered plants. Changes in total chlorophyll level ofrosette and axial leaves in deflowered, defruited, and controlplants could not, however, be correlated with dry-weight changes.Results of biochemical analyses showed that total basic proteinand RNA increased initially in leaves of deflowered and defruitedplants and during this period dry weight, total chlorophylllevel, and laminar area also increased. During later periodsthe basic protein and RNA levels declined and the rate of declinewas slowest in deflowered plants.     

Characterization of Cu and Zn Resistant Nicotiana plumbaginifolia Suspension Cultures

The mechanisms of Cu and Zn resistance in cultured Nicotianaplumbaginifolia cells were investigated. When crude extractsof both the Cu and Zn resistant cells were passed through SephadexG-75 gel filtration columns, two metal containing peaks wereidentified. In each case the Cu or Zn was associated with ahigh molecular weight fraction (ca. 70,000) and a low molecularweight fraction (less than 3,000) with the majority being associatedwith the low molecular weight fraction. The low molecular weightfraction in particular increased upon exposure of the cellsto the metals. Both the Cu and Zn resistant cells accumulated higher levelsof citrate and malate than the wild type cells in the presenceof the metals. The addition of citrate or malate, but not fumarateor succinate to the culture medium prevented Cu or Zn toxicityto wild type cells. These results suggest that the N. plumbaginifoliacells selected as resistant to Cu or Zn are resistant, at leastin part, because of their ability to accumulate the chelators,citrate and malate, in the presence of the metals. ¹Present address: Plantech Research Institute, MCI ResearchCenter, 1000 Kamoshida-cho, Midoriku, Yokohama 227 Japan (Received June 30, 1986; Accepted December 1, 1986)