Andreas J. Müller

THE FUSCA GENES OF ARABIDOPSIS : NEGATIVE REGULATORS OF LIGHT RESPONSES

More than 200 fusca mutants of Arabidopsis have been isolated and characterised, defining 14 complementation groups. Mutations in at least nine FUSCA genes cause light-dependent phenotypic changes in the absence of light: high levels of anthocyanin accumulation in both the embryo and the seedling, inhibition of hypocotyl elongation, apical hook opening, and unfolding of cotyledons. In double mutants, the fusca phenotype is epistatic to the hy phytochromedeficiency phenotype, indicating that the FUSCA genes act downstream of phytochrome. By contrast, the accumulation of anthocyanin is suppressed by mutations in TT and TTG genes, which affect the biosynthesis of anthocyanin, placing the FUSCA genes upstream of those genes. Regardless of the presence or absence of anthocyanin, fusca mutations limit cell expansion and cause seedling lethality. In somatic sectors, mutant fus1 cells are viable; expressing tissue-specific phenotypes: reduced cell expansion and accumulation of anthocyanin in subepidermal tissue, formation of ectopic trichomes but no reduced cell expansion in epidermal tissue. Our results suggest a model of FUSCA gene action in light-induced signal transduction.

A Common Genetic Determinant of Xanthine Dehydrogenase and Nitrate Reductase in Nicotiana tabacum

Summary Xanthine dehydrogenase activity has been found in callus cultures of Nicotiana tabacum and in 8 nitrate reductase-deficient mutant lines derived from the former. Another mutant cell line lacks both xanthine dehydrogenase and nitrate reductase activity. This finding supports the hypothesis that in Nicotiana tabacum xanthine dehydrogenase and nitrate reductase share a common molybdenum-containing co-factor.

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

SummaryTwo 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.

High meiotic stability of a foreign gene introduced into tobacco by Agrobacterium-mediated transformation.

SummaryTwo lines of transgenic Nicotiana tabacum transformed to kanamycin resistance by means of a binary Agrobacterium vector containing a nos-npt gene were investigated over three generations. Southern hybridization and crossing analyses revealed that a single copy of T-DNA had integrated in each line and that the kanamycin resistance was regularly transmitted to the progeny as a monogenic dominant trait. Homozygous transgenic plants were fully fertile, morphologically normal and did not significantly differ from wild-type plants in the quantitative characters examined (plant height, flowering time). The two lines showed very low, but significantly different levels of meiotic instability: kanamycin-sensitive plants occurred among backcross progeny from homozygous transgenic plants with frequencies of 6/45,000 and 25/45,000, respectively. The sensitive plants arose independently of each other and thus resulted from meiotic rather than mitotic events. These findings demonstrate for the first time that integrated foreign genes can be transmitted to progeny with the high degree of meiotic stability required for commercial varieties of crop plants. They emphasize the importance of non-homologous integration and of avoiding co-integration of inactive gene copies for achieving meiotically stable transformants.

Nitrate reductase-deficient mutant cell lines of Nicotiana tabacum

SummaryThe wild-type line and 14 nitrate reductase-deficient mutant cell lines of Nicotiana tabacum were tested for the presence of nitrate reductase partial activities, and for nitrite reductase and xanthine dehydrogenase activity. Data characterizing the electron donor specificity of nitrate reductase (EC 1.6.6.1., NADH:nitrate oxidoreductase) and nitrite reductase (EC 1.7.7.1., ferredoxin:nitrite oxidoreductase) of the wild-type line are presented. Three lines (designated cnx) simultaneously lack NADH-, FADH2-, red. benzyl viologen-nitrate reductase, and xanthine dehydrogenase activities, but retain the nitrate reductase-associated NADH-cytochrome c reductase activity. These mutants are, therefore, interpreted to be impaired in gene functions essential for the synthesis of an active molybdenum-containing cofactor. For cnx-68 and cnx-101, the sedimentation coefficient of the defective nitrate reductase molecules does not differ from that of the wild-type enzyme (7.6S). In 11 lines (designated nia) xanthine dehydrogenase activity is unaffected, and the loss of NADH-nitrate reductase is accompanied by a loss of all partial activities, including NADH-cytochrome c reductase. However, one line (nia-95) was found to possess a partially active nitrate reductase molecule, retaining its FADH2- and red. benzyl viologen nitrate reductase activity. It is likely that nia-95 is a mutation in the structural gene for the apoprotein. Both, the nia and cnx mutant lines exhibit nitrite reductase activity, being either nitrate-inducible or constitutive. Evidence is presented that, in Nicotiana tabacum, nitrate, without being reduced to nitrite, is an inducer of the nitrate assimilation pathway.

Genetic analysis of nitrate reductase deficient mutants of Nicotiana plumbaginifolia: Evidence for six complementation groups among 70 classified molybdenum cofactor deficient mutants

SummaryA total of 70 cnx mutants have been characterized from a collection of 211 nitrate reductase deficient (NR-) mutants isolated from mutagenized Nicotiana plumbaginifolia protoplast cultures after chlorate selection and regeneration into plants. They are presumed to be affected in the biosynthesis of the molybdenum cofactor since they are also deficient for xanthine dehydrogenase activity but contain NR apoenzyme. The remaining clones were classified as nia mutants. Sexual crosses performed between cnx mutants allowed them to be classified into six independent complementation groups. Mutants representative of these complementation groups were used for somatic hybridization experiments with the already characterized N. plumbaginifolia mutants NX1, NX24, NX23 and CNX103 belonging to the complementation groups cnxA, B, C and D respectively. On the basis of genetic analysis and somatic hybridization experiments, two new complementation groups, cnxE and F, not previously described in higher plants, were characterized. Unphysiologically high levels of molybdate can restore the NR activity of cnxA mutant seedlings in vivo, but cannot restore NR activity to any mutant from the other cnx complementation groups.

Comparative characterisation of nitrate reductase from wild-type and molybdenum cofactor-defective cell cultures of Nicotiana tabacum

Affinity-purified nitrate reductase (EC 1.6.6.1) from wild-type and the molybdenum cofactor-defective cnx-68/2 cell line of Nicotiana tabacum has been comparatively characterised with respect to enzymological properties. Km values and pH optima for the 4 nitrate reductase-associated activities, including diaphorase, are presented. Exposure to different inhibitors and moderate heat (45°C) reveal the tobacco nitrate reductase to be functionally divided into two distinct parts. Both the wild-type and cnx mutant nitrate reductase contain a heme group of cytochrome b type, possess an identical sedimentation coefficient of 7.6S and an identical gel filtration derived molecular weight of 200 000. Both enzymes were found to exhibit similar enzymatic parameters, inhibitor specificities and heat stabilities. Upon affinity chromatography, varying portions of the 7.6S holoenzyme dissociate into slower sedimenting (about 4S) subunits with cytochrome c reductase activity, which has been established for the wild-type as well as the cnx mutant enzyme. From the data obtained it can be concluded that (1) the apoprotein of the cnx mutant nitrate reductase is unaffected by the mutation while the defect of this enzyme type resides exclusively in the molybdenum-cofactor; (2) the cnx mutant nitrate reductase possesses a molybdenum-cofactor, defective in its catalytic properties, but still able to mediate the assembly of the cytochrome c reductase subunits to form the 7.6S holoenzyme.

Comparative biochemical characterization of mutants at the nitrate reductase/molybdenum cofactor loci cnxA, cnxB, and cnxC of Nicotiana plumbaginifolia

Abstract Mutations in any of the three gene loci cnxA, cnxB, cnxC can lead to a total loss of nitrate reductase activity in Nicotiana species. The cnx loci are involved in synthesis and processing of the molybdenum cofactor, which is an essential structural constituent of nitrate reductase. The biochemical properties of cnxA, cnxB and cnxC mutant cell lines of Nicotiana plumbaginifolia were examined further. The cnxA line (N×9) was found to possess a catalytically defective but dimerization-active and under in vivo/in vitro-conditions repairable molybdenum cofactor, thus, resembling the properties of N. tabacum cnxA lines. The cnxB (N×24) and cnxC (N×21) mutants. however, show a phenotype very different from cnxA. This new phenotype is characterised by an irreversible loss of both the catalytic function and dimerization ability of the molybdenum cofactor which makes it likely that the molybdopterin moiety of the cofactor is defective or lacking in these mutants. In this report we summarize and compare the phenotypic data presently available for the Nicotiana loci cnxA, cnxB and cnxC. Possible functions of the gene products of these loci will be discussed.

Repair in vitro of nitrate reductase-deficient tobacco mutants (cnxA) by molybdate and by molybdenum cofactor

Two nitrate reductase-deficient mutant cell lines (CnxA68/2, CnxA101) of Nicotiana tabacum are shown to be repairable under in-vitro conditions by (i) molybdate or (ii) by preparations of active molybdenum cofactor of homologous or heterologous origin, thereby yielding about 20% and 80%, respectively, of the corresponding wild-type NADH-nitrate reductase (EC 1.6.6.1) activity. In-vitro repair of nitrate reductase activity is dependent on sulphydryl-group protecting reagents and ethylenediaminetetraacetic acid (EDTA) in the extraction medium, the nitrogen source in the growth medium and the age of the cells. The results support the conclusion that the cnxA gene controls the insertion of molybdenum into the molybdenum cofactor. They are consistent with the idea of two interlinked pathways for the metabolic processing of molybdenum acquisition, one involving the synthesis of the structural moiety of the molybdenum cofactor and the other involving processing of the molybdate anion.

Expression of a Vicia faba Legumin B Gene in Transgenic Tobacco Plants: Gene dosage-dependent Protein accumulation

Summary A legumin B gene of the field bean Vicia faba L. var. minor was transferred into tobacco using a Ti plasmid derived binary vector system. The foreign gene shows seed specific expression and the synthesized polypeptides are correctly processed in the heterologous background. A correlation was found between transferred copy number and amount of accumulated Vicia polypeptide in the transgenic seeds indicating a relatively low influence of the different integration sites on the expression of the transferred gene.