Nitrate reduction in the wildtype and a nitrate reductase deficient mutant of Arabidopsis thaliana

A chlorate-resistant mutant B25 of Arabidopsis thaliana (L.) Heinh. was isolated, which has very little or no in vitro nitrate reductase activity and grows poorly on a substrate with nitrate as the sole nitrogen source. The mutation of B25 ( rgn ) is monogenic and recessive, tightly linked to the marker gene an on chromosome 1. Nitrate induces cytochrome- c reductase activity in the mutant but to a lower level than in the wildtype. After sucrose gradient centrifugation the greatest part of the cytochrome- c reductase from induced wildtype is found as 8s type whereas cytochrome- c reductase from B25 under the same conditions is found as 4s type. Nitrate reductase is found at the 8s position. It is suggested that B25 has lost the ability to assemble two 4s subunits showing cytochrome- c reductase activity and a Mo-bearing co-factor into the functional nitrate reductase. Nitrate rather than nitrite is the inducing agent for nitrite reductase, since in B25 nitrite reductase is even more rapidly induced than in the wildtype after addition of nitrate. Both the wildtype and B25 contain a nitrate reductase inhibiting factor when grown on ammonium. This inhibiting factor is a small protein, possibly similar to the nitrate reductase inactivating enzyme reported for other plants.     

Identification and characterization of a chlorate-resistant mutant of Arabidopsis thaliana with mutations in both nitrate reductase structural genes NIA1 and NIA2

Mutant plants defective in the assimilation of nitrate can be selected by their resistance to the herbicide chlorate. In Arabidopsis thaliana, mutations at any one of nine distinct loci confer chlorate resistance. Only one of the CHL genes, CHL3, has been shown genetically to be a nitrate reductase (NR) structural gene (NIA2) even though two NR genes (NIA1 and NIA2) have been cloned from the Arabidopsis genome. Plants in which the NIA2 gene has been deleted retain only 10% of the wildtype shoot NR activity and grow normally with nitrate as the sole nitrogen source. Using mutagenized seeds from the NIA2 deletion mutant and a modified chlorate selection protocol, we have identified the first mutation in the NIA1 NR structural gene. nia1, nia2 double mutants have only 0.5% of wild-type shoot NR activity and display very poor growth on media with nitrate as the only form of nitrogen. The nial-1 mutation is a single nucleotide substitution that converts an alanine to a threonine in a highly conserved region of the molybdenum cofactor-binding domain of the NR protein. These results show that the NIA1 gene encodes a functional NR protein that contributes to the assimilation of nitrate in Arabidopsis.     

Characterization of a chlorate-hypersensitive,high nitrate reductase Arabidopsis thaliana mutant

Summary A population of A. thaliana, produced by self-fertilization of ethylmethane sulfonate treated plants, was exposed to chlorate in the watering solution, and plants showing early susceptibility symptoms were rescued. Among the progeny lines of these plants five were shown to be repeatably chlorate-hypersusceptible. One of these lines (designated C-4) possessed elevated activity of nitrate reductase (NR). The NR activity of mutant C-4 was higher than that of normal plants throughout the life cycle. Nitrite reductase and glutamine synthetase activities of C-4 were normal, as were chlorate uptake rate and tissue nitrate content. The elevated NR activity apparently was responsible for the chlorate hypersusceptibility of C-4. Inheritance studies of NR indicated that the elevated activity of C-4 was probably controlled by a single recessive allele.     

Isolation and characterization of nitrate reductase-deficient mutants of Arabidopsis thaliana

Summary Chlorate resistant mutants of Arabidopsis thaliana were isolated, of which 10 exhibited a lowered nitrate reductase activity and 51 were chlorate-resistant because of an impaired uptake of chlorate. The 51 mutants of this type are all affected in the same gene. The mutants with a lowered nitrate reductase activity fall into 7 different complementation groups. Three of these mutants grow poorly on media with nitrate as the sole nitrogen source, while the others apparently can reduce sufficient nitrate to bring about growth. In all cases a low nitrate reductase activity coincides with an enhanced nitrite reductase activity. After sucrose gradient centrifugation of wildtype extracts nitrate reductase is found at the 8S position, whereas cytochrome-c reductase is found both at 4 and 8S positions. It is suggested that the functional nitrate reductase is a complex consisting of 4S subunits showing cytochrome-c reductase activity and a Mo-bearing cofactor. All mutants except B25 are capable of assembling the 4S subunits into complexes which for most mutants have a lower S value and exhibit a lower nitrate reductase activity than the wildtype complexes. Since the mutants B25 and B73 exhibit a low xanthine dehydrogenase activity, the Mo-bearing cofactor is probably less available in these mutants than in the wildtype. B73 appears to be the only mutant which is partly repaired by excessive Mo. The possible role of several genes is discussed.     

Isolation of a nitrate reductase deficient mutant of Pisum sativum by means of selection for chlorate resistance

Summary After EMS treatment of seeds of the Pisum variety Rondo a chlorate resistant mutant was isolated which showed a decrease in the in vitro activity of the enzyme nitrate reductase of roughly 95%. The mutation is monogenic and recessive. The mutant shows a decrease in protein content, and an increase in the amount of nitrate accumulated and in the activity of the enzyme nitrite reductase. On a liquid nutrient medium containing nitrate as the sole nitrogen source and in soil, the mutant grows very poorly due to necrosis of the leaves. On liquid medium containing ammonium, either with or without nitrate, growth is as good as that of the parent variety.     

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     

Nitrate reductase is not accumulated in chloroplast-ribosome-deficient mutants of higher plants

The activities of nitrite reductase (EC 1.7.7.1) are 60–70% of wild-type activity in pigment-deficient leaves of the chloroplast-ribosomedeficient mutants albostrians (Hordeum vulgare) and iojap (Zea mays). The activity and apoprotein of nitrate reductase (EC 1.6.6.1.) are lacking in the barley mutant. Only very low activities of nitrate reductase can be extracted from leaves of the maize mutant. The molybdenum cofactor of nitrate reductase and xanthine dehydrogenase (EC 1.2.3.2) is present in maize and barley mutant plants. However, it is not inducible by nitrate in pigment-deficient leaves of albostrians. From these results we conclude: (i) Nitrite reductase (a chloroplast enzyme) is synthesized in the cytoplasm and does not need the presence of nitrate reductase for the induction and maintenance if its activity. (ii) The loss or low activity of nitrate reductase is a consequence of the inability of the mutants to accumulate the apoprotein of this enzyme. (iii) The chloroplasts influence the accumulation (i.e. most probably the synthesis) of the nonchloroplast enzyme, nitrate reductase. The accumulation of nitrate reductase needs a chloroplast factor which is not provided by mutant plastids blocked at an early stage of their development.Abbreviations CRM cross-reacting material - Mo-co molybdenum cofactor - NiR nitrite reductase - NR nitrate reductase     

Homology modeling of Ferredoxin-nitrite reductase from Arabidopsis thaliana

Different subtypes of Influenza A virus are associated with species specific, zoonotic or pandemic Influenza. The cause of its severity underlies in complicated evolution of its segmented RNA genome. Although genetic shift and genetic drift are well known in the evolution of this virus, we reported the significant role of unique RNA palindromes in its evolution. Our computational approach identified the existence of unique palindromes in each subtype of Influenza A virus with its absence in Influenza B relating the fact of virulence and vigorous genetic hitchhiking in Influenza A. The current study focused on the re-assortment event responsible for the emergence of pandemic-2009 H1N1 virus, which is associated with outgrow of new palindrome and in turn, changing its RNA structure. We hypothesize that the change in RNA structure due to the presence of palindrome facilitates the event of re-assortment in Influenza A. Thus the evolutionary process of Influenza A is much more complicated as previously known, and that has been demonstrated in this study.     

Characterization of purified nitrate reductase A and chlorate reductase C from Proteus mirabilis

Nitrate reductase A has been solubilized from purified cytoplasmic membranes by extraction with terl-amyl alcohol. The resulting aqueous solution contained monomeric reductase which polymerized slowly to dimers and tetramers with sedimentation coefficients of respectively 10.5, 16 and 23 Svedbergunits. The polymerization could be stopped to some extent by addition of a small amount of Triton X-100. These distinct entities of nitrate reductase A were separable on electro-focusing, DEAE-column chromatography and polyacrylamide gel electrophoresis, and have been proved to consist of similar subunits with molecular weights of 104000, 63000, and 56000 daltons. The molecular weights of monomeric nitrate reductase A was found to be about 240000 daltons.Chlorate reductase C has been solubilized by a similar procedure, resulting in only monomeric enzyme. Chlorate reductase C exhibited a sedimentation coefficient of 7.7 Svedbergunits, an isoelectric point of pH=4.55 and a molecular weight of approx. 180000 daltons. It was found to consist of three subunits with molecular weights of 75000, 63000 and 56000 daltons. The latter two subunits are most probably common in nitrate reductase A and chlorate reductase C.     

In vitro morphogenesis of arrested embryos from lethal mutants of Arabidopsis thaliana

Summary Arrested embryos from lethal (emb) mutants of Arabidopsis thaliana were rescued on a nutrient medium designed to promote plant regeneration from immature wild-type cotyledons. The best response was observed with mutant embryos arrested at the heart to cotyledon stages of development. Embryos arrested at a globular stage produced callus but failed to turn green or form normal shoots in culture. Many of the mutant plants produced in culture were unusually pale with abnormal leaves, rosettes, and patterns of reproductive development. Other plants were phenotypically normal except for the presence of siliques containing 100% aborted seeds following self-pollination. These results demonstrate that genes with essential functions during plant embryo development differ in their pattern of expression at later stages of the life cycle. Most of the 15 genes examined in this study were essential for embryogenesis but were required again for subsequent stages of development. Only EMB24 appeared to be limited in function to embryo development. These differences in the response of mutant embryos in culture may facilitate the classification of embryonic lethals and the identification of genes with developmental rather than housekeeping functions.     

Developmental anatomy of cotyledons and leaves in has mutant of Arabidopsis thaliana

Summary. In this work, we analyzed the developmental anatomy of cotyledons and leaves in the has mutant of Arabidopsis thaliana. It is a recessive T-DNA insertion mutation that causes changes in the size, shape, and tissue organization of the cotyledons and leaves of has plants. Analysis of has cotyledons revealed a prominent decrease in the cell number and an increase in the area of cotyledon cells and intercellular spaces of has plants. At early stages of development, has leaves are fingerlike structures, but later they develop small, lobed blades with rare trichomes. An important characteristic of the mutant leaf anatomy is the absence of mesophyll tissue differentiation. In addition, both cotyledons and leaves display a disrupted pattern of vascular bundles. Furthermore, mutant plants are defective in root and shoot morphology, indicating that the has mutation affects a number of aspects in plant development. Correspondence and reprints: Institute of Botany and “Jevremovac” Botanical Garden, Faculty of Biology, Belgrade University, Takovska 43, 11 000 Belgrade, Serbia.     

Identification of two tungstate-sensitive molybdenum cofactor mutants, chl2 and chl7, of Arabidopsis thaliana.

Summary The characterization of mutants that are resistant to the herbicide chlorate has greatly increased our understanding of the structure and function of the genes required for the assimilation of nitrate. Hundreds of chlorate-resistant mutants have been identified in plants, and almost all have been found to be defective in nitrate reduction due to mutations in either nitrate reductase (NR) structural genes or genes required for the synthesis of the NR cofactor molybdenum-pterin (MoCo). The chlorate-resistant mutant ofArabidopsis thaliana, ch12, is also impaired in nitrate reduction, but the defect responsible for this phenotype has yet to be explained.chl2 plants have low levels of NR activity, yet the map position of thechl2 mutation is clearly distinct from that of the two NR structural genes that have been identified inArabidopsis. In addition,chl2 plants are not thought to be defective in MoCo, as they have near wild-type levels of xanthine dehydrogenase activity, which has been used as a measure of MoCo in other organisms. These results suggest thatchl2 may be a NR regulatory mutant. We have examinedchl2 plants and have found that they have as much NR (NIA2) mRNA as wild type a variable but often reduced level of NR protein, and one-eighth the NR activity of wild-type plants. It is difficult to explain these results by a simple regulatory model; therefore, we reexamined the MoCo levels inchl2 plants using a sensitive, specific assay for MoCo: complementation ofNeurospora MoCo mutant extracts. We found thatchl2 has low levels of MoCo — about one-eighth the wild-type level and less than the level in anotherArabidopsis MoCo mutantchl6 (B73). To confirm this result we developed a new diagnostic assay for MoCo mutants, growth inhibition by tungstate. Bothchl2 andchl6 are sensitive to tungstate at concentrations that have no effect on wildtype plants. The tungstate sensitivity as well as the chlorate resistance, low NR activity and low MoCo levels all cosegregate, indicating that all are due to a single mutation that maps to thechl2 locus, 10 centimorgans fromerecta on chromosome 2. We also report on the isolation of a new chlorate-resistant mutant ofArabidopsis, ch17, which is a MoCo mutant with the same phenotypes aschl2 andchl6.     

A spermine-resistant mutant of Arabidopsis thaliana displays precocious germination

From an ethylmethane sulphonate-mutagenized M₂ population of Arabidopsis thaliana L. var Landsberg erecta, a mutant was isolated on the basis of its ability to germinate in the presence of a germination inhibitory concentration (0.35 mM) of spermine. The mutant produced yellowish green seeds that lacked a mucilaginous sheath, exhibited reduced dormancy and were generally viviparous under ambient conditions. Dose-response assays indicated increased resistance of the mutant to spermine but normal sensitivity to spermidine, putrescine and abscisic acid. The spermine resistance and the associated phenotype of the mutant was inherited as a single recessive nuclear mutation. Following the genetic analysis, spermine-resistant mutant has been designated as spr2. The results suggest a role for spermine in seed dormancy.     

Translation initiation by non-AUG codons in Arabidopsis thaliana transgenic plants

The efficiency of translation initiation at codons differing at one or two nucleotides from AUG was tested as initiation codons for the phosphinotricin-acetyltransferase gene in T-DNA plant transformation in Arabidopsis thaliana. With the exception of UUA codon that differs from AUG at two nucleotides and does not permit any detectable activity, all the other codons (AUC, GUG, ACG, and CUG) present a phosphinotrycin acetyltransferase activity that varies between 5 and 10% of the AUG activity. This low activity is sufficient to confer glufosinate resistance to some of the plants. These results indicate that, in plants as is the case in animals, non-AUG initiating codons may be used for translation initiation, namely when a low expression rate is needed.     

Toxicity of and mutagenesis by chlorate are independent of nitrate reductase activity in Chlamydomonas reinhardtii

Summary Spontaneous chlorate-resistant (CR) mutants have been isolated from Chlamydomonas reinhardtii wildtype strains. Most of them, 244, were able to grow on nitrate minimal medium, but 23 were not. Genetic and in vivo complementation analyses of this latter group of mutants indicated that they were defective either at the regulatory locus nit-2, or at the nitrate reductase (NR) locus nit-1, or at very closely linked loci. Some of these nit-1 or nit-2 mutants were also defective in pathways not directly related to nitrate assimilation, such as those of amino acids and purines. Chlorate treatment of wild-type cells resulted in both a decrease in cell survival and an increase in mutant cells resistant to a number of different chemicals (chlorate, methylammonium, sulphanilamide, arsenate, and streptomycin). The toxic and mutagenic effects of chlorate in minimal medium were not found when cells were grown either in darkness or in the presence of ammonium, conditions under which nitrate uptake is drastically inhibited. Chlorate was also able to induce reversion of nit ^– mutants of C. reinhardtii, but failed to produce His ⁺ revertants or Ara^r mutants in the BA-13 strain of Salmonella typhimurium. In contrast, chlorate treatment induced mutagenesis in strain E1F1 of the phototrophic bacterium Rhodobacter capsulatus. Genetic analyses of nitrate reductase-deficient CR mutants of C. reinhardtii revealed two types of CR, to low (1.5 mM) and high (15 mM) chlorate concentrations. These two traits were recessive in heterozygous diploids and segregated in genetic crosses independently of each other and of the nit-1 and nit-2 loci. Three her loci and four lcr loci mediating resistance to high (HC) and low (LC) concentrations of chlorate were identified. Mutations at the nit-2 locus, and deletions of a putative locus for nitrate transport were always epistatic to mutations responsible for resistance to either LC or HC. In both nit ⁺ and nit ^– chlorate-sensitive (CS) strains, nitrate and nitrite gave protection from the toxic effect of chlorate. Our data indicate that in C. reinhardtii chlorate toxicity is primarily dependent on the nitrate transport system and independent of the existence of an active NR enzyme. At least seven loci unrelated to the nitrate assimilation pathway and mediating CR are thought to control indirectly the efficiency of the nitrate transporter for chlorate transport. In addition, chlorate appears to be a mutagen capable of inducing a wide range of mutations unrelated to the nitrate assimilation pathway.     

Identification of Arabidopsis thaliana variants with differential glyphosate responses

To facilitate future investigations of glyphosate-resistance mechanisms, three approaches were taken to obtain Arabidopsis thaliana variants that differed in glyphosate response. Recurrent selection by spraying with sub-lethal glyphosate concentrations was performed with Columbia-0 seedlings. After seven cycles of treatment, no resistance was found. A population of 800,000 ethylmethanesulfonate-mutagenized M(2) seedlings was screened on agar containing 0.2mM glyphosate, a lower concentration than that previously used in other studies, and no resistant mutants were recovered. Seventy-two Arabidopsis ecotypes were screened with glyphosate and a range of responses was observed. In a follow-up experiment on a subset of these ecotypes, reduction of seed yield by 11.5 g/ha glyphosate (about 1% the typical field use rate) ranged among ecotypes from 0% to >90%, relative to untreated controls. However, even the least sensitive ecotypes were severely injured by relatively low glyphosate rates. Overall, attempts to select Arabidopsis seedlings that were significantly glyphosate-resistant were unsuccessful and consistent with previous reports. Arabidopsis ecotypes identified with differential glyphosate responses could be used for further studies though the inherently high sensitivity of Arabidopsis to glyphosate could limit their utility in studying glyphosate-resistance mechanisms.     

Effect of light on the NADPH-protochlorophyllide oxidoreductase of Arabidopsis thaliana

A cDNA encoding the NADPH-protochlorophyllide oxidoreductase (Pchlide reductase) of Arabidopsis thaliana has been isolated and sequenced. The cDNA contains the complete reading frame for the precursor of the Pchlide reductase. The deduced amino acid sequence of the Arabidopsis enzyme closely resembles the corresponding sequences of barley and oat. The cDNA has been used as a template for the synthesis of the enzyme protein in Escherichia coli. An antiserum was raised against this enzyme protein and both the antiserum and the cDNA were used as experimental tools to study the effects of light on the Pchlide reductase in A. thaliana.When etiolated seedlings of Arabidopsis were exposed to light the enzyme activity and the concentration of the enzyme protein rapidly declined. Similar light effects have been described previously for other angiosperms. In contrast to most of these species, however, in Arabidopsis only minor changes in Pchlide reductase mRNA content could be observed when etiolated seedlings were exposed to light.     

Reduction of mineral nutrient availability accelerates flowering of Arabidopsis thaliana

The time of flowering is regulated by various environmental cues, and in some plant species, it is known to be affected by abiotic stresses. We investigated the effect of nutrient stress caused by an abrupt reduction of mineral nutrition on flowering of Arabidopsis thaliana. We used a hydroponic culture system that enabled us to precisely control nutrient levels. When plants were grown in full-strength nutrient solution for several weeks and then transferred to a diluted medium, the time from sowing to bud appearance was significantly shortened. This acceleration of flowering was more pronounced in short days than in long days, and stronger in the ecotype Landsberg erecta than in Columbia and San Feliu-2. The response was also affected by the age of plants at the beginning of nutrient stress and by the concentration of the diluted medium: earlier treatment and more diluted solutions strengthened the effect. Flowering was affected by nutrient stress, not by a change in the osmotic potential of the medium: addition of mannitol to a 1000-fold diluted solution had no effect on the promotion of flowering. When 3-week-old Landsberg erecta plants were exposed to 1000-fold diluted nutrient solution in an 8-h day length, flower bud appearance was strongly and reproducibly advanced by 10.8–12.8 d compared with control plants (which developed buds 41.1–46.2 d after sowing). This treatment can serve as an optimized protocol for future studies concerning physiological, molecular and ecological aspects of flower induction by nutrient stress in A. thaliana.