Ralf R. Mendel

Nitrate reductase deficient cell lines from haploid protoplast cultures ofNicotiana plumbaginifolia

SummaryNitrate reductase deficient (NR-) cell lines were selected indirectly by their resistance to 40 mM chlorate in protoplast cultures of haploidNicotiana plumbaginifolia. Frequency of the chlorate resistant clones was 5.8×10-5 in non-mutagenized cultures, which could be increased up to 25 times by treatment with N-ethyl-N-nitrosourea (NEU) or gamma irradiation.Out of 136 chlorate resistant clones 29 were fully deficient in nitrate reductase. The rest of the clones contained decreased or normal levels of NR activity (91 and 16 clones, respectively).Further characterization was carried out in 9 clones which were fully deficient in NR and in 2 clones containing resisdual (0–5%) NR activity. The clones were tentatively classified as defective in the apoenzyme (7 clones including the 2 with residual NR activity) or the cofactor (4 clones) of NR by the xanthine dehydrogenase activity and in vitro enzyme complementation. The cofactor defectives could be further classified into two groups. In one of these (2 clones) the NR activity could be partially restored by unphysiologically high (0.2–1 mM) molybdate in the culture medium. The other two are new types which have not been described in flowering plants.Plant regeneration was obtained only in the clones which contained residual NR activity.

Presence of the molybdenum-cofactor in nitrate reductase-deficient mutant cell lines of Nicotiana tabacum

SummaryNicotiana tabacum mutant cell cultures lacking nitrate reductase activity were assayed for the presence of the molybdenum-cofactor using its ability to restore NADPH-nitrate reductase activity in extracts of Neurospora crassa nit-1 mycelia. The molybdenum-cofactor of the tobacco wild-type line was shown to complement efficiently the N. crassa nit-1 mutant in vitro. The molybdenum-cofactor seems to exist in a bound form, as acid-treatment was required for release of cofactor activity. Molybdate (5–10 mM), ascorbic acid, and anaerobic conditions greatly increased the activity of the cofactor, demonstrating its high lability and sensitivity to oxygen. Similar results were obtained with two tobacco nia mutants, which are defective in the apoprotein of nitrate reductase. The four cnx mutants studied were shown to contain exclusively an inactive form of the molybdenum-cofactor. This inactive cofactor could be reactivated in vitro and in vivo by unphysiologically high concentrations of molybdate (1–10 mM), thereby converting the cnx cells into highly active cofactor sources in vitro, and restoring nitrate reductase and xanthine dehydrogenase in vivo to partial acitivity. Thus the defect of the cnx mutants resides in a lack of molybdenum as a catalytically active ligand metal for the cofactor, while the structural moiety of the cofactor seems not to be impaired by the mutation. The subunit assembly of the nitrate reductase was found to be independent of the molybdenum content of the cofactor.

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.

Reconstitution of NADH-nitrate reductase in vitro from nitrate reductase-deficient Nicotiana tabacum mutants

SummaryTwo types of nitrate reductase-deficient mutant cell lines (nia and cnx) of Nicotiana tabacum have been used for in vitro reconstitution of NADH-nitrate reductase. The cnx mutants 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 interpreted to be defective in the molybdenum-containing cofactor necessary for nitrate reductase activity. In the nia lines xanthine dehydrogenase activity is unaffected, and the loss of NADH-nitrate reductase is accompanied by a loss of all partial activities of nitrate reductase, including NADH-cytochrome c reductase. When cnx cells (induced by nitrate) were homogenized together with nia cells (induced by nitrate or uninduced), NADH-nitrate reductase activity was detectable in the cell extract. No nitrate reductase was observed when the cnx mutants were homogenized together, or after cohomogenization of the nia mutants. Thus, the inactive nitrate reductase molecule formed in the cnx mutants has been complemented in vitro with the molybdenum-containing cofactor supplied by nia extracts, thus giving rise to NADH-nitrate reductase activity. This result gives additional support to the interpretation that the active nitrate reductase of Nicotiana tabacum is composed of at least the NADH-cytochrome c reductase moiety and a molybdenum-containing cofactor which is formed by the action of the cnx gene product(s).

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.

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.

Molybdenum cofactor in nitrate reductase-deficient tobacco mutants. III. Induction of cofactor synthesis by nitrate

Abstract The molybdenum cofactor (Mo-co) content of cultured cells of Nicotiana tabacum was estimated by the ability of the acid and heat treated extract to restore NADPH-nitrate reductase (NR) of Neurospora crassa nit -1. Addition of nitrate to wild-type cells grown on amino acids caused a rapid increase in Mo-co activity concomitant with induction of NADH-NR. Time course experiments revealed both the constitutive and the induced levels of Mo-co activity to be dependent on culture age, the maximum occurring during log phase. According to their Mo-co activity, the 12 NR-deficient mutant cell lines tested were shown to fall into 3 groups: Group I nia mutants are characterized by a low Mo-co level, which does not increase either during log phase or after addition of nitrate. Group II nia mutants are similar to wild type. The four cnx mutants tested display a constitutive-high Mo-co level, being not further inducible by nitrate. Possible mechanisms for the common regulation of Mo-co and NR synthesis are discussed.

Characterization of a new type of molybdenum cofactor-mutant in cell cultures of Nicotiana tabacum

SummaryFour allelic putative cnx (molybdenum-cofactor defective) cell lines (O42, P12, P31 and P47) of Nicotiana tabacum var. Xanthi, biochemically and genetically distinct from N. tabacum var. Gatersleben cnxA mutants, were examined further. Their molybdenum-cofactor could efficiently reconstitute NADPH-nitrate reductase activity from Neurospora crassa mutant nit-1 extract only in the presence of exogenous molybdenum unlike that of the wild-type cofactor which could reconstitute NADPH-nitrate reductase activity in either the absence or presence of exogenous molybdenum. Loss of cofactor activity in vivo was not due to a defect in molybdenum uptake into the cells. In vitro nitrate reductase complementation between extracts of each of these four lines and a nia mutant showed that they possessed a functional nitrate reductase haemoflavoprotein subunit. Both constitutive molybdenum cofactor and NADH cytochrome c reductase activity were derepressed in the four cell lines. These results show that the four cell lines are indeed altered at a cnx locus, called cnxB, that the defect is probably in molybdenum processing and that there is a link between synthesis of functional molybdenum cofactor and nitrate reductase aporprotein.

Molybdenum cofactor in nitrate reductase-deficient tobacco mutants. II. Release of cofactor by heat treatment

Abstract An improved method for release of molybdenum cofactor (Mo-co) activity from cell-free extracts of Nicotiana tabacum cell cultures is presented. Subsequent to acid treatment (45 s at pH 2), cell extracts were subjected to heat treatment (5–10 min at 80°C), giving rise to a 3–10-fold higher cofactor activity than achieved by acid treatment alone. The release effect of high temperature, which was observed in wild type as well as in nitrate reductase-deficient mutant cell lines ( nia and cnx ), was found to be fully dependent on the presence of 1–4 mM ascorbic acid as a protective agent in the extract. The activity of cofactor released in this way could be stimulated up to 3-fold by addition of 5–10 mM molybdate (wild type; nia mutants), or was fully dependent on additional molybdate ( cnx mutant).