Non-coordinate expression of the neighbouring genes rplL and rpoB,C of Escherichia coli.

Summary Two 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).     

Isolation of molybdenum cofactor defective cell lines of Nicotiana tabacum

Summary Thirty-nine chlorate resistant cell lines were isolated after plating ethylmethane sulphonate treated allodihaploid cells of Nicotiana tabacum cv. Xanthi on agar medium containing 20 mM chlorate. Thirty-two of these cell lines grew as well on nitrate medium as on amino acid medium and three other cell lines grew well on amino acid medium but poorly on nitrate medium. Four other cell lines, 042, P12, P31 and P47 which could grow on amino acid medium, but not on nitrate medium, were examined further. They lacked in vitro nitrate reductase activity but were able to accumulate nitrate. All lines possessed nitrite reductase activity. Lines 042, P12, and P31 had a cytochrome c reductase species which was the same size as the wild type nitrate reductase associated cytochrome c reductase species, whilst the cytochrome c reductase species in line P47 was slightly smaller. All four lines lacked xanthine dehydrogenase activity and neither nitrate reductase nor xanthine dehydrogenase activity was restored by subculture of the four lines into either nitrate medium or glutamine medium supplemented with 1 mM sodium molybdate. These four lines are different from other molybdenum cofactor defective cell lines so far described in N. tabacum and possess similar properties to certain other cnx mutants described in Aspergillus nidulans.     

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

Summary Nicotiana 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.     

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

Summary Four 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.     

GENETIC STUDIES OF NITRATE ASSIMILATION IN ASPERGILLUS NIDULANS

(1) In Aspergillus nidulans, at least 16 genes can mutate to affect the reduction of nitrate to ammonium, a process requiring two enzymes, nitrate reductase and nitrite reductase. (2) niaD is the only gene whose effects on enzyme structure are confined to nitrate reductase alone. It specifies a core polypeptide, one or more of which form the basic subunit of nitrate reductase, molecular weight 50000. (3) At least five cnx genes together specify a molybdenum co-factor, necessary for the activity of nitrate reductase, and of xanthine dehydrogenases I and II. The cnxH gene specifies a polypeptide component of this co-factor, and the cnxE and F gene products are involved in co-factor elaboration, The role of the remaining cnx genes is at present unknown. (4) Functional nitrate reductase has a molecular weight of 200000 and is likely to consist of four subunits, together with one or more molecules of the cnx-specified co-factor. (5) The co-factor plays a catalytic role in the aggregation of nitrate-reductase subunits. (6) The niiA gene is the structural gene for nitrite reductase. (7) Other genes affecting nitrate assimilation are either regulatory or bring about their effects indirectly. (8) Of the genes affecting nitrate assimilation, close linkage is found only between the niiA and niaD genes. (9) Nitrate and nitrite reductases are subject to control by nitrate induction and ammonium repression. (10) Nitrate induction is mediated by the nirA gene whose product must be active for the niiA and niaD genes to be expressed. Since most niaD mutants produce nitrite reductase constitutively, it is likely that the nirA gene product is normally inactivated by nitrate reductase, but only when the latter is not complexed with nitrate, (11) Ammonium repression is mediated by the areA gene, whose product must be active for the expression of the niiA and niaD genes, and which is inactive in the presence of ammonium. (12) The tamA gene may function similarly to the areA gene, both gene products being necessary for the expression of the niiA and niaD genes. (13) Although the niiA and niiD genes are probably contiguous, they are not likely to be organized into a structure equivalent to a bacterial operon. (14) Whereas the areA and nirA genes regulate the synthesis of nitrate and nitrite reductases, it is probable that at least nitrate reductase is also subject to post-translational control, the presence of active enzyme being correlated with high levels of NADPH. (15) The regulation of the pentose-phosphate pathway, of mannitol-I-phosphate dehydrogenase and of certain activities required for the catabolism of some nitrogen-containing compounds appears to be connected with that of nitrate assimilation. In all cases, it is probable that the nirA gene and nitrate reductase itself are involved.     

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.     

Formation of NADPH-nitrate reductase activity in vitro from Aspergillus nidulans niaD and cnx mutants

Summary Mutants of A. nidulans at several loci lack detectable NADPH-nitrate reductase activity. These loci include niaD, the structural gene for the nitrate reductase polypeptide, and five other loci termed cnxABC, E, F, G and H which are presumed to be involved in the formation of a molybdenum-containing component (MCC) necessary for nitrate reductase activity. When frozen mycelia from A. nidulans deletion mutant niaD26 were homogenized in a Ten Broeck homogenizer together with frozen mycelia from either enzA6, cnxE29, cnxF12, enxG4 or cnxH3 strains grown on urea+nitrate as the nitrogen source, nitrate reductase activity was detectable in the extract. Similar results were obtained by co-homogenizing niaD mycelia with Neurospora crassa nit-1 mycelia induced on nitrate. Thus, all A. nidulans cnx mutants are similar to the N. crassa nit-1 strain in their capacity to yield NADPH-nitrate reductase in the presence of the presumed MCC. As judged by the amounts of nitrate reductase formed, niaD26 mycelia grown on urea±nitrate contained much more available MCC than ammonium-grown mycelia. No NADPH-nitrate reductase activity was found in extracts prepared by co-homogenizing mycelia from all five A. nidulans cnx strains. Wild-type A. nidulans NADPH-nitrate reductase acid dissociated by adjustment to pH 2.0–2.5 and re-adjusted to pH 7 could itself re-assemble to form active nitrate reductase and thus was not a sueful source of MCC for these experiments. These results are consistent with the conclusion that the active nitrate reductase complex is composed of polypeptide components which are the niaD gene product, plus the MCC which is formed through the combined action of the cnx gene products. Further, the production of MCC may be regulated in response to the nitrogen nutrition available to the organism.     

Correction of nitrate reductase defect in auxotrophic plant cells through protoplast-mediated intergeneric gene transfers

Summary Nitrate reductase-deficient cells of Nicotiana tabacum cv Gatersleben (coded cnx-68) lacking active molybdenum-cofactor were corrected by introducing the genes from Physalis minima and Datura innoxia into NR^- genomes. In these itergeneric reconstruction experiments, X-irradiated inactive mesophyll protoplasts of Physalis and Datura were fused separately with the cultured cell protoplasts of cnx-68 Nicotiana. A total of 45 cell colonies, 37 transformed by Physalis and 8 by Datura, were selected from about 1.7×10³ heteroplasmic fusion products. The selection of transformants was made by their ability to grow on a medium containing nitrate as the sole nitrogen source. Some of these transformants were further characterized with respect to nitrate reductase, xanthine dehydrogenase and glutamate dehydrogenase activities, chlorate sensitivity, and chlorophyll synthesis. The restoration of nitrate reductase and xanthine dehydrogenase activities confirm the presence of an active form of the molybdenum-cofactor by the expression of introduced genes of Physalis and Datura into the genome of cnx-68 Nicotiana. Such stable transformations via fusion of normal and highly irradiated protoplasts may have a considerable application in higher plants for introducing desirable characters from diverse genomes.     

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

Summary A 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.     

Genetic analysis of revertants for the nitrate reductase function of Nicotiana plumbaginifolia

Summary Spontaneous revertants of nitrate reductase (NR)-less mutants were isolated by screening for nitrate utilization in diploid NR^– protoplast cultures of Nicotiana plumbaginifolia. The revertants contained in vivo NR activity in the case of apoenzyme mutants (nia) as well as of a cofactor-deficient (cnx) mutant. Revertants of the NIA type proved to be tetraploid, and genetic analysis showed that only one out of the four NR structural genes had reverted to a functional allele.     

In vitro restoration of nitrate reductase: Investigation of Aspergillus nidulans and Neurospora crassa nitrate reductase mutants

Extracts of Aspergillus nidulans wild type (bi-1) and the nitrate reductase mutant niaD-17 were active in the in vitro restoration of NADPH-dependent nitrate reductase when mixed with extracts of Neurospora crassa, nit-1. Among the A. nidulans cnx nitrate reductase mutants tested, only the molybdenum repair mutant, cnxE-14 grown in the presence of 10⁻³ M Na₂MoO₄ was active in the restoration assay.Aspergillus extracts contained an inhibitor(s) which was measured by the decrease in NADPH-dependent nitrate reductase formed when extracts of Rhodospirillum rubrum and N. crassa, nit-1 were incubated at room temperature. The inhibition by extracts of A. nidulans, bi-1, cnxG-4 and cnxH-3 was a linear function of time and a logarithmic function of the protein concentration in the extract.The molybdenum content of N. crassa wild type and nit-1 mycelia were found to be similar, containing approx. 10 μg molybdenum/mg dry mycelium. The NADPH-dependent cytochrome c reductase associated with nitrate reductase was purified from both strains. The enzyme purified from wild-type N. crassa contained more than 1 mol of molybdenum per mol of enzyme, whereas the enzyme purified from nit-1 contained negligible amounts of molybdenum.     

Complementation analysis by somatic hybridisation and genetic crosses of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia

Summary Fusion complementation experiments between nitrate reductase (NR) deficient lines CNX 20, 27, 82 and 103 of Nicotiana plumbaginifolia were performed with the already characterized N. plumbaginifolia mutants nx 1, 24 and 21, belonging respectively to the complementation groups cnx A, B and C. CNX 20 and 82 were identified as belonging to the group of cnx A. CNX 27 complemented with NX 1 and NX 21 but not with NX 24 indicating another B type. The fourth line, CNX 103 showed complementation with CNX 20, NX 21 and NX 24, revealing a fourth cnx complementation group, cnx D, that until now has not been described in higher plants. Genetic crosses inside respectively the NIA and the CNX group, and between NIA and CNX confirmed the fusion complementation results, and showed allelism for the nia mutants     

Biochemical genetics of further chlorate resistant, molybdenum cofactor defective, conditional-lethal mutants of barley

Summary Three plants, R9201 and R11301 (from cv. Maris Mink) and R12202 (from cv. Golden Promise), were selected by screening M₂ populations of barley (Hordeum vulgare L.) seedlings (mutagenised with azide in the M₁) for resistance to 10 mM potassium chlorate. Selections R9201 and R11301 were crossed with the wild-type cv. Maris Mink and analysis of the F₂ progeny showed that one quarter lacked shoot nitrate reductase activity. These F₂ plants also withered and died in the continuous presence of nitrate as sole nitrogen source. Loss of nitrate reductase activity and withering and death were due in each case to a recessive mutation in a single nuclear gene. All F1 progeny derived from selfing selection R12202 lacked shoot nitrate reductase activity and also withered and subsequently died when maintained in the continuous presence of nitrate as sole nitrogen source. All homozygous mutant plants lacked not only shoot nitrate reductase activity but also shoot xanthine dehydrogenase activity. The plants took up nitrate, and possessed wild-type or higher levels of shoot nitrite reductase activity and NADH-cytochrome c reductase activity when treated with nitrate for 18 h. We conclude that loss of shoot nitrate reductase activity, xanthine dehydrogenase activity and withering and death, in the three mutants R9201, R11301 and R12202 is due to a mutation affecting the formation of a functional molybdenum cofactor. The mutants possessed wild-type levels of molybdenum and growth in the presence of unphysiologically high levels of molybdate did not restore shoot nitrate reductase or xanthine dehydrogenase activity. The shoot molybdenum cofactor of R9201 and of R12202 is unable to reconstitute NADPH nitrate reductase activity from extracts of the Neurospora crassa nit-1 mutant and dimerise the nitrate reductase subunits present in the respective barley mutant. The shoot molybdenum cofactor of R11301 is able to effect dimerisation of the R11301 nitrate reductase subunits and can reconstitute NADPH-nitrate reductase activity up to 40% of the wild-type molybdenum cofactor levels. The molybdenum cofactor of the roots of R9201 and R11301 is also defective. Genetic analysis demonstrated that R9201, but not R11301, is allelic to R9401 and Az34 (nar-2a), two mutants previously shown to be defective in synthesis of molybdenum cofactor. The mutations in R9401 and R9201 gave partial complementation of the nar-2a gene such that heterozygotes had higher levels of extractable nitrate reductase activity than the homozygous mutants.We conclude that: (a) the nar-2 gene locus encodes a step in molybdopterin biosynthesis; (b) the mutant R11301 represents a further locus involved in the synthesis of a functional molybdenum cofactor; (c) mutant Rl2202 is also defective in molybdopterin biosynthesis; and (d) the nar-2 gene locus and the gene locus defined by R11301 govern molybdenum cofactor biosynthesis in both shoot and root.     

Occurrence of xanthine dehydrogenase in Chlamydomonas reinhardii: A common cofactor shared by xanthine dehydrogenase and nitrate reductase

Wild-type Chlamydomonas reinhardii cells have xanthine dehydrogenase activity when grown with nitrate, nitrite, urea, or amino acid media. Mutant strains 102, 104, and 307 of Chlamydomonas, lacking both xanthine dehydrogenase and nitrate reductase activities, were incapable of restoring the NADPH-nitrate reductase activity of the mutant nit-1 of Neurospora crassa, whereas wild type cells and mutants 203 and 305 had xanthine dehydrogenase and were able to reconstitute the nitrate reductase activity of nit-1 of Neurospora. Therefore, it is concluded that in Chlamydomonas a common cofactor is shared by xanthine dehydrogenase and nitrate reductase. Xanthine dehydrogenase is repressed by ammonia and seems to be inessential for growth of Chlamydomonas.     

A convener role for the cnxH gene specified component in the NADPH-nitrate reductase from Aspergillus nidulans

Summary The role of the cnxH⁺ gene specified polypeptide in the formation and function of the NADPH-nitrate reductase in Aspergillus nidulans was examined with the use of two complementing mutant strains which were grown as forced heterocaryons in the presence of nitrate. The niaD-421 structural gene mutant and the cnxH-318 co-factor gene mutant produce two components of the NADPH-cytochrome c reductase co-activity which can be distinguished by their enzymatic and physical behavior. This combination enabled us to isolate the de novo synthesis of niaD⁺ gene specified protomers from the constitutively formed co-factor at two stages of development. The proportion of induced and constitutively formed protomers in the isolated holoenzyme was measured after pulsing with [³H]-histidine or [¹⁴C]-histidine prior to induction with nitrate. The newly formed nitrate reductase was resolved by agarose gel electrofocusing and activity staining. In vivo assembly of a 7.8s enzyme in the heterocaryotic mycelium of the above strains is apparently achieved by the convener action of the cnxH⁺ gene directed polypeptide from the niaD^– strain on the niaD⁺ gene directed protomers of the cnxH^– partner. This occurs with or without Mo as a co-factor.     

Molybdate metabolism in Aspergillus nidulans. I. Mutations affecting nitrate reductase and-or xanthine dehydrogenase

Summary Further evidence supports the hypothesis that nitrate reductase and xanthine dehydrogenase are molybdo-enzymes inAspergillus nidulans, probably sharing a molybdenum-containing cofactor. This evidence includes (1) five-fold greater toxicity of tungstate on nitrate and hypoxanthine than on other nitrogen sources, (2) locus-specific molybdate reparability of both nitrate reductase and xanthine dehydrogenase at one (cnxE) of five (cnx) loci where mutation can result in pleiotropic loss of both enzyme activities, and (3) an additional class of mutants (molB) which are both molybdate resistant and partially defective in utilization of nitrate and hypoxanthine as nitrogen sources. Moreover, the phenotypes on molybdate-containing media of various mutants altered in the regulation of nitrate reductase synthesis and the ability of nitrate to protect against molybdate toxicity suggest that incorporation of molybdenum into nitrate reductase or into something having the same control properties as nitrate reductase can detoxify molybdate. However, mutations affecting regulation of xanthine dehydrogenase synthesis do not affect growth responses to molybdate. The properties of another class of molybdate resistance mutations (molA) suggest that there is another nitrate-inducible intracellular molybdate detoxification mechanism in addition to the one having identical control properties to nitrate reductase.     

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

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.     

The Mechanism of Nitrate Accumulation in Pakchoi [Brassica Campestris L.ssp. Chinensis(L.)]

Decreased nitrate in vegetables can improve crop nitrogen utilization efficiency and lessen the human health risk caused by the reduction of nitrate to nitrite in vegetables. This paper studied the mechanisms of differences in nitrate accumulation and distribution within organs of two cultivars of pakchoi (Brassica campestris L.ssp. Chinensis (L.) previously screened in hydroponic experiments from 12 cultivars popularly grown in China at present. The two typical cultivars used in this experiment were Shanghaiqing with low nitrate accumulation and Liangbaiye 1 with high nitrate accumulation. There was no significant difference of total nitrate uptake but a significant difference in nitrate content existed between the two cultivars. Compared with Liangbaiye 1, Shanghaiqing showed a significantly higher photosynthetic rate and nitrate reductase activity. Determination of nitrate concentration (activity) in vacuoles with double-barrelled nitrate-selective microelectrodes showed that Shanghaiqing had lower vacuolar nitrate activity than Liangbaiye 1. Two putative nitrate reductase genes, nia1 and nia2, were amplified from the leaf blades of these two cultivars. Nia1 mRNA fragments (887 bp, accession numbers DQ082868 and DQ082869) were amplified using degenerate primer and nia2 mRNA fragment was amplified using one pair of generate primers designed according to DQ001901. Sequence analysis of DQ082868 and DQ082869 both showed 97% and 87% similarity with two nitrate reductase mRNA sequences of Brassica napus, accession numbers D38219 and D38220, respectively. The results of real time PCR to compare the relative expression of the putative nitrate reductase genes (nia1 and nia2) showed that Shanghaiqing had significantly higher expression level than Liangbaiye 1 and nia2 was significantly higher than nia1 in leaf blade and petiole. Both the nitrate reductase activity and the relative expression level of nia1 were in the order of leaf blade > root > petiole, while that of nia2 was leaf blade > petiole > root. There was no statistically significant difference of nitrate activity stored in vacuoles between the different organs of the two cultivars. It can be concluded that Shanghaiqing took up slightly less nitrate, but had significantly higher nitrate reductase activity in cytosol and had a higher relative expression of the putative nitrate reductase genes than Liangbaiye 1; this leads to the fact that Shanghaiqing has a lower nitrate content than Liangbaiye 1.     

Characterization of a rice (Oryza sativa L.) mutant deficient in the heme domain of nitrate reductase

Summary Biochemical and genetical characterization of a rice nitrate reductase (NR)-deficient mutant, M819, which had been isolated as a chlorate-resistant mutant, was carried out. In M819, leaf NADH-NR activity was found to be about 10% of that of the wild-type cv Norin 8, while NADPH-NR activity was higher than that in the wild-type; FMNH₂-NR and MV-NR activities were also 10% of those of the wild type; BPB-NR activity was higher than that of the wild type; and xanthine dehydrogenase activity was revealed to be present in both. These results suggest that the mutant line M819 lacks the functional heme domain of the NADH-NR polypeptide due to a point mutation or a small deletion within the coding region of the structural gene. Chlorate resistance in M819 was transmitted by a single recessive nuclear gene.Abbreviations NR Nitrate reductase - NiR nitrite reductase - FMNH₂ reduced flavin nucleotide - MV reduced methyl viologen - BPB reduced bromphenol blue - XDH xanthine dehydrogenase     

Consequence of Absence of Nitrate Reductase Activity on Photosynthesis in Nicotiana plumbaginifolia Plants

Chlorate-resistant Nicotiana plumbaginifolia (cv Viviani) mutants were found to be deficient in the nitrate reductase apoprotein (NR⁻nia). Because they could not grow with nitrate as sole nitrogen source, they were cultivated as graftings on wild-type Nicotiana tabacum plants. The grafts of mutant plants were chlorotic compared to the grafts of wild type. Mutant leaves did not accumulate nitrogen and nitrate but contained less malate and more glutamine than wild leaves. They exhibited a slight increase of the proportion of the light-harvesting chlorophyll a/b protein complexes and a lowering of the efficiency of energy transfer between these complexes and the active centers. After a 3 second ¹⁴CO₂ pulse, the total ¹⁴C incorporation of the mutant leaves was approximately 20% of that of the control. The ¹⁴C was essentially recovered in ribulose bisphosphate in these plants. It was consistent with a decline of ribulose bisphosphate carboxylase activity observed in the mutant. After a 3 second ¹⁴CO₂ pulse followed by a 60 second chase with normal CO₂, ¹⁴C was mainly accumulated in starch which was labeled more in the mutant than in the wild type. These results confirm the observation that in the nitrate reductase deficient leaves, chloroplasts were loaded with large starch inclusions preceding disorganization of the photosynthetic apparatus.