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.     

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.     

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.     

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.     

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.     

The expression of a chimeric Phaseolus vulgaris nodulin 30-GUS gene is restricted to the rhizobially infected cells in transgenic Lotus corniculatus nodules

In Phaseolus vulgaris there is a nodulin family, Npv30, of ca. 30 kDa, as detected in an in vitro translation assay [2]. We isolated a gene (npv30-1) for one of the members of this family. The nucleotide sequence of the promoter of npv30-1 contains nodule-specific motifs common to other late nodulin genes. The promoter was fused to the GUS reporter gene; this chimeric fusion was introduced into Lotus corniculatus via Agrobacterium rhizogenes transformation. GUS activity was only detected in the infected cells of the nodules of transgenic plants. By contrast, the expression of a 35S-GUS construct was restricted to the uninfected cells and the vascular tissue.     

The SELF-PRUNING gene family in tomato

The SELF PRUNING (SP) gene controls the regularity of the vegetative-reproductive switch along the compound shoot of tomato and thus conditions the 'determinate' (sp/sp) and 'indeterminate' (SP_) growth habits of the plant. SP is a developmental regulator which is homologous to CENTRORADIALIS (CEN) from Antirrhinum and TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS T (FT) from Arabidopsis. Here we report that SP is a member of a gene family in tomato composed of at least six genes, none of which is represented in the tomato EST collection. Sequence analysis of the SP gene family revealed that its members share homology along their entire coding regions both among themselves and with the six members of the Arabidopsis family. Furthermore, members of the gene family in the two species display a common genomic organization (intron-exon pattern). In tomato, phylogenetically close homologues diverged considerably with respect to their organ expression patterns while SP2I and its closest homologue from Arabidopsis (MFT) exhibited constitutive expression. This research focusing on a plant of sympodial growth habit sets the stage for a functional analysis of this weakly expressed gene family which plays a key role in determining plant architecture.     

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.     

The nitrate reductase promoter of birch directs differential reporter gene expression in tissues of transgenic tobacco

A 1535 bp promoter of the nitrate reductase gene (nia) from birch (Betula pendula) and a series of 5′ deletions were fused to the β-glucuronidase (GUS) gene and introduced into Nicotiana plumbaginifolia. In transgenic plants the NR promoter sequences directed strong GUS expression in the root epidermal hair cells, and in phloem cells of leaf and stem vascular tissue. The NR promoter confers also a significant stimulation of the GUS gene expression by nitrate. These findings might indicate that nitrate flow is one of the signals involved into tissue and cell specific expression of the NR promoter GUS fusions. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Increase of endogenous zeatin riboside by introduction of the ipt gene in wild type and the lateral suppressor mutant of tomato

We studied axillary meristem formation of the lateral suppressor (ls) mutant of tomato after elevating the endogenous cytokinin levels through introduction of the isopentenyltransferase (ipt) gene from Agrobacterium tumefaciens. Growth and development of several transformants were examined during in vitro culture. Transformants exhibited phenotypes varying in severity and were divided into four classes. A number of the ipt transformants had a normal phenotype, as non-transformed plants. Others showed a mild to severe cytokinin-like phenotype. Transformants with a mild phenotype exhibited reduced internode length and reduced root development. Transformants with a severe phenotype showed even shorter internodes, loss of apical dominance, reduction of leaf size, production of callus at the basis of the shoots and absence of root development or development of green non-branching roots. The severity of the phenotype correlated well with the level of ipt gene expression, as measured by northern analysis. Transformants with a severe phenotype also exhibited increased levels of zeatin riboside, but zeatin levels were not elevated. The increase in endogenous zeatin riboside levels in the ls mutant did not restore axillary meristem formation, but sometimes bulbous structures were formed in the initially empty leaf axils. Several adventitious meristems and shoots developed from below the surface of these structures. It is concluded that a reduced level of cytokinins in the ls mutant shoots is not responsible for the absence of axillary meristem formation.     

Transposition pattern of a modified Ds element in tomato

Several aspects of transposition of an in vitro modified Ds element are described. This Ds element, designated ds-r, is equipped with bacterial plasmid sequences and can, therefore, be rescued from the plant genome. Our results indicate that the Ds-r element has a late timing of transposition from T-DNAs. This feature of the element might be advantageous for tagging experiments because it leads to independently transposed germinally transmitted elements. Furthermore, it is shown that Ds-r transposition generates clusters of insertions, indicating that genes to be tagged should be located in genomic regions covered by insertions.